Handbook of Food Science Technology and Engineering

HANDBOOK OF FOOD SCIENCE, TECHNOLOGY, AND ENGINEERING Volume 1 FOOD SCIENCE AND TECHNOLOGY A Series of Monographs, T...

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HANDBOOK OF FOOD SCIENCE, TECHNOLOGY, AND

ENGINEERING Volume 1

FOOD SCIENCE AND TECHNOLOGY A Series of Monographs, Textbooks, and Reference Books Editorial Advisory Board Gustavo V. Barbosa-Cánovas Washington State University–Pullman P. Michael Davidson University of Tennessee–Knoxville Mark Dreher McNeil Nutritionals, New Brunswick, NJ Richard W. Hartel University of Wisconsin–Madison Lekh R. Juneja Taiyo Kagaku Company, Japan Marcus Karel Massachusetts Institute of Technology Ronald G. Labbe University of Massachusetts–Amherst Daryl B. Lund University of Wisconsin–Madison David B. Min The Ohio State University Leo M. L. Nollet Hogeschool Gent, Belgium Seppo Salminen University of Turku, Finland John H. Thorngate III Allied Domecq Technical Services, Napa, CA Pieter Walstra Wageningen University, The Netherlands John R. Whitaker University of California–Davis Rickey Y. Yada University of Guelph, Canada

76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94.

Food Chemistry: Third Edition, edited by Owen R. Fennema Handbook of Food Analysis: Volumes 1 and 2, edited by Leo M. L. Nollet Computerized Control Systems in the Food Industry, edited by Gauri S. Mittal Techniques for Analyzing Food Aroma, edited by Ray Marsili Food Proteins and Their Applications, edited by Srinivasan Damodaran and Alain Paraf Food Emulsions: Third Edition, Revised and Expanded, edited by Stig E. Friberg and Kåre Larsson Nonthermal Preservation of Foods, Gustavo V. Barbosa-Cánovas, Usha R. Pothakamury, Enrique Palou, and Barry G. Swanson Milk and Dairy Product Technology, Edgar Spreer Applied Dairy Microbiology, edited by Elmer H. Marth and James L. Steele Lactic Acid Bacteria: Microbiology and Functional Aspects, Second Edition, Revised and Expanded, edited by Seppo Salminen and Atte von Wright Handbook of Vegetable Science and Technology: Production, Composition, Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam Polysaccharide Association Structures in Food, edited by Reginald H. Walter Food Lipids: Chemistry, Nutrition, and Biotechnology, edited by Casimir C. Akoh and David B. Min Spice Science and Technology, Kenji Hirasa and Mitsuo Takemasa Dairy Technology: Principles of Milk Properties and Processes, P. Walstra, T. J. Geurts, A. Noomen, A. Jellema, and M. A. J. S. van Boekel Coloring of Food, Drugs, and Cosmetics, Gisbert Otterstätter Listeria, Listeriosis, and Food Safety: Second Edition, Revised and Expanded, edited by Elliot T. Ryser and Elmer H. Marth Complex Carbohydrates in Foods, edited by Susan Sungsoo Cho, Leon Prosky, and Mark Dreher Handbook of Food Preservation, edited by M. Shafiur Rahman

95.

96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129.

International Food Safety Handbook: Science, International Regulation, and Control, edited by Kees van der Heijden, Maged Younes, Lawrence Fishbein, and Sanford Miller Fatty Acids in Foods and Their Health Implications: Second Edition, Revised and Expanded, edited by Ching Kuang Chow Seafood Enzymes: Utilization and Influence on Postharvest Seafood Quality, edited by Norman F. Haard and Benjamin K. Simpson Safe Handling of Foods, edited by Jeffrey M. Farber and Ewen C. D. Todd Handbook of Cereal Science and Technology: Second Edition, Revised and Expanded, edited by Karel Kulp and Joseph G. Ponte, Jr. Food Analysis by HPLC: Second Edition, Revised and Expanded, edited by Leo M. L. Nollet Surimi and Surimi Seafood, edited by Jae W. Park Drug Residues in Foods: Pharmacology, Food Safety, and Analysis, Nickos A. Botsoglou and Dimitrios J. Fletouris Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, edited by Luis M. Botana Handbook of Nutrition and Diet, Babasaheb B. Desai Nondestructive Food Evaluation: Techniques to Analyze Properties and Quality, edited by Sundaram Gunasekaran Green Tea: Health Benefits and Applications, Yukihiko Hara Food Processing Operations Modeling: Design and Analysis, edited by Joseph Irudayaraj Wine Microbiology: Science and Technology, Claudio Delfini and Joseph V. Formica Handbook of Microwave Technology for Food Applications, edited by Ashim K. Datta and Ramaswamy C. Anantheswaran Applied Dairy Microbiology: Second Edition, Revised and Expanded, edited by Elmer H. Marth and James L. Steele Transport Properties of Foods, George D. Saravacos and Zacharias B. Maroulis Alternative Sweeteners: Third Edition, Revised and Expanded, edited by Lyn O’Brien Nabors Handbook of Dietary Fiber, edited by Susan Sungsoo Cho and Mark L. Dreher Control of Foodborne Microorganisms, edited by Vijay K. Juneja and John N. Sofos Flavor, Fragrance, and Odor Analysis, edited by Ray Marsili Food Additives: Second Edition, Revised and Expanded, edited by A. Larry Branen, P. Michael Davidson, Seppo Salminen, and John H. Thorngate, III Food Lipids: Chemistry, Nutrition, and Biotechnology: Second Edition, Revised and Expanded, edited by Casimir C. Akoh and David B. Min Food Protein Analysis: Quantitative Effects on Processing, R. K. Owusu-Apenten Handbook of Food Toxicology, S. S. Deshpande Food Plant Sanitation, edited by Y. H. Hui, Bernard L. Bruinsma, J. Richard Gorham, Wai-Kit Nip, Phillip S. Tong, and Phil Ventresca Physical Chemistry of Foods, Pieter Walstra Handbook of Food Enzymology, edited by John R. Whitaker, Alphons G. J. Voragen, and Dominic W. S. Wong Postharvest Physiology and Pathology of Vegetables: Second Edition, Revised and Expanded, edited by Jerry A. Bartz and Jeffrey K. Brecht Characterization of Cereals and Flours: Properties, Analysis, and Applications, edited by Gönül Kaletunç and Kenneth J. Breslauer International Handbook of Foodborne Pathogens, edited by Marianne D. Miliotis and Jeffrey W. Bier Food Process Design, Zacharias B. Maroulis and George D. Saravacos Handbook of Dough Fermentations, edited by Karel Kulp and Klaus Lorenz Extraction Optimization in Food Engineering, edited by Constantina Tzia and George Liadakis Physical Properties of Food Preservation: Second Edition, Revised and Expanded, Marcus Karel and Daryl B. Lund

130. Handbook of Vegetable Preservation and Processing, edited by Y. H. Hui, Sue Ghazala, Dee M. Graham, K. D. Murrell, and Wai-Kit Nip 131. Handbook of Flavor Characterization: Sensory Analysis, Chemistry, and Physiology, edited by Kathryn Deibler and Jeannine Delwiche 132. Food Emulsions: Fourth Edition, Revised and Expanded, edited by Stig E. Friberg, Kare Larsson, and Johan Sjoblom 133. Handbook of Frozen Foods, edited by Y. H. Hui, Paul Cornillon, Isabel Guerrero Legarret, Miang H. Lim, K. D. Murrell, and Wai-Kit Nip 134. Handbook of Food and Beverage Fermentation Technology, edited by Y. H. Hui, Lisbeth Meunier-Goddik, Ase Solvejg Hansen, Jytte Josephsen, Wai-Kit Nip, Peggy S. Stanfield, and Fidel Toldrá 135. Genetic Variation in Taste Sensitivity, edited by John Prescott and Beverly J. Tepper 136. Industrialization of Indigenous Fermented Foods: Second Edition, Revised and Expanded, edited by Keith H. Steinkraus 137. Vitamin E: Food Chemistry, Composition, and Analysis, Ronald Eitenmiller and Junsoo Lee 138. Handbook of Food Analysis: Second Edition, Revised and Expanded, Volumes 1, 2, and 3, edited by Leo M. L. Nollet 139. Lactic Acid Bacteria: Microbiological and Functional Aspects: Third Edition, Revised and Expanded, edited by Seppo Salminen, Atte von Wright, and Arthur Ouwehand 140. Fat Crystal Networks, Alejandro G. Marangoni 141. Novel Food Processing Technologies, edited by Gustavo V. Barbosa-Cánovas, M. Soledad Tapia, and M. Pilar Cano 142. Surimi and Surimi Seafood: Second Edition, edited by Jae W. Park 143. Food Plant Design, Antonio Lopez-Gomez; Gustavo V. Barbosa-Cánovas 144. Engineering Properties of Foods: Third Edition, edited by M. A. Rao, Syed S.H. Rizvi, and Ashim K. Datta 145. Antimicrobials in Food: Third Edition, edited by P. Michael Davidson, John N. Sofos, and A. L. Branen 146. Encapsulated and Powdered Foods, edited by Charles Onwulata 147. Dairy Science and Technology: Second Edition, Pieter Walstra, Jan T. M. Wouters and Tom J. Geurts 148. Food Biotechnology, Second Edition, edited by Kalidas Shetty, Gopinadhan Paliyath, Anthony Pometto and Robert E. Levin 149. Handbook of Food Science, Technology, and Engineering - 4 Volume Set, edited by Y. H. Hui 150. Thermal Food Processing: New Technologies and Quality Issues, edited by Da-Wen Sun 151. Aflatoxin and Food Safety, edited by Hamed K. Abbas 152. Food Packaging: Principles and Practice, Second Edition, Gordon L. Robertson

HANDBOOK OF FOOD SCIENCE, TECHNOLOGY, AND

ENGINEERING Volume 1 Edited by

Y. H. HUI Associate Editors J. D. Culbertson, S. Duncan, I. Guerrero-Legarreta, E. C. Y. Li-Chan, C. Y. Ma, C. H. Manley, T. A. McMeekin, W. K. Nip, L. M. L. Nollet, M. S. Rahman, F. Toldr , Y. L. Xiong

Boca Raton London New York

A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.

Published in 2006 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2006 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-9847-9 (Set) International Standard Book Number-10: 1-57444-551-0 (Vol 1) International Standard Book Number-10: 0-8493-9848-7 (Vol 2) International Standard Book Number-10: 1-57444-552-9 (Vol 3) International Standard Book Number-10: 0-8493-9849-5 (Vol 4) International Standard Book Number-13: 978-0-8493-9847-6 (Set) International Standard Book Number-13: 978-1-57444-551-0 (Vol 1) International Standard Book Number-13: 978-0-8493-9848-3 (Vol 2) International Standard Book Number-13: 978-1-57444-552-7 (Vol 3) International Standard Book Number-13: 978-0-8493-9849-0 (Vol 4) Library of Congress Card Number 2005050551 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Handbook of food science, technology, and engineering / edited by Y.H. Hui. p. cm. -- (Food science and technology ; 148) Includes bibliographical references and index. ISBN 1-57444-551-0 (v. 1 : alk. paper) -- ISBN 0-8493-9848-7 ( v. 2 : alk. paper) -- ISBN 1-57444-552-9 (v. 3 : alk. paper) -ISBN 0-8493-9849-5 (v. 4 : alk. paper) 1. Food industry and trade--Handbooks, manuals, etc. 2. Food--Analysis--Handbooks, manuals, etc. 3. Food--Composition-Handbooks, manuals, etc. I. Hui, Y. H. (Yiu H.) II. Food science and technology (Taylor & Francis) ; 148. TP370.4.H38 2005 664--dc22

2005050551

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com Taylor & Francis Group is the Academic Division of Informa plc.

and the CRC Press Web site at http://www.crcpress.com

Preface for Volumes 1 and 2 In the last 30 years, progress in food science, food technology, and food engineering has advanced exponentially. As usual, information dissemination for this progress is expressed in many media, both printed and electronic. Books are available for almost every specialty area within these three disciplines, numbering in the hundreds. Collective works on the disciplines are also available, though in smaller number. Examples are encyclopedias (food science, food engineering, food packaging) and handbooks (nutrition, food processing, food technology). Because handbooks on these topics are limited, this four-volume treatise is released by Taylor & Francis to fill this gap. The title of these four volumes is Handbook of Food Science, Technology, and Engineering with individual volume title as follows: ● ● ● ●

Volume 1: Food Science: Properties and Products Volume 2: Food Science: Ingredients, Health, and Safety Volume 3: Food Engineering and Food Processing Volume 4: Food Technology and Food Processing

This preface introduces Volumes 1 and 2. Each volume contains about 1,000 printed pages of scientific and technical information. Volume 1 contains 55 chapters and Volume 2 contains 46 chapters. Volume 1 presents the following categories of topics, with the number of chapters indicated: ● ● ● ●

Food components and their properties, 14 Food categories, 26 Food analysis, 9 Food microbiology, 6

Volume 2 presents the following categories of topics, with the number of chapters indicated: ● ● ● ● ●

Food attributes, 7 Food fermentation, 8 Food and workers safety, food security, 12 Functional food ingredients, 15 Nutrition and health, 4

A brief discussion of the coverage for each volume is described below. In Volume 1, the first group of topics covers the components and properties of food such as carbohydrate, protein, fat, vitamins, water, and pigments. The second group of topics covers the different categories of food products including, but not limited to, beverages, bakery, cereals, legumes, vegetables, fruits, milk, meat, poultry, fats, oils, seafood, and wine. The third group of topics describes the analysis of food such as basic principles and various techniques (chemical method, spectroscopy, chromatography, mass spectrometry, and other analytical methodology). The last group of topics covers food microbiology such as basic considerations, spoilage, land and marine animals, and analytical methodology. In Volume 2, the first group of topics covers the attributes of food such as sensory science, data base concepts, flavor, texture, and color. The second group of topics covers food fermentation including basic principles, quality, flavor, meat, milk, cultured products, cheese, yeasts, and pickles. The third group of topics covers food from the perspective of safety, workers health, and security, especially in the United States, such as food standards, food protection methods, filth, pathogens, migratory chemicals, food plant sanitation, retail food sanitation, establishment safety, animal feeds and drugs, and bio-terrorism. The fourth group of topics covers major functional food ingredients including, but not limited to, antioxidants, colors, aroma, flavor, spice, enzyme, emulsifiers, phytates, sorbates, artificial sweeteners, eggs, gums. The last group of topics covers special topics in nutrition and health such as food allergy, Chinese edible botanicals, dietary supplements, and health related advertisement in the United States.

When studying the information in this two-volume text, please note two important considerations: 1. Although major topics in the discipline are included, there is no claim that the coverage is comprehensive. 2. Although the scientific information is applicable worldwide, a small number of topics with legal implications are especially pertinent in the United States. These two volumes are the result of the combined effort of more than 150 professionals from industry, government, and academia. They are from more than 15 countries with diverse expertise and background in the discipline of food science. These experts were led by an international editorial team of 13 members from 8 countries. All these individuals, authors and editors, are responsible for assembling 2,000 printed pages of scientific topics of immense complexity. In sum, the end product is unique, both in depth and breadth, and will serve as an essential reference on food science for professionals in government, industry, and academia. The editorial team thanks all the contributors for sharing their experience in their fields of expertise. They are the people who make this book possible. We hope you enjoy and benefit from the fruits of their labor. We know how hard it is to develop the content of a book. However, we believe that the production of a professional book of this nature is even more difficult. We thank the editorial and production teams at Taylor & Francis for their time, effort, advice, and expertise. You are the best judge of the quality of this book. Y. H. Hui J. D. Culbertson S. Duncan I. Guerrero-Legarreta E. C. Y. Li-Chan C. Y. Ma C. H. Manley T. A. McMeekin W. K. Nip L. M. L. Nollet M. S. Rahman F. Toldrá Y. L. Xiong

The Editor Dr. Y. H. Hui holds a Ph.D. in food and nutritional biochemistry from the University of California at Berkeley. He is semi-retired and has been a consultant to the food industry since 2000. He is currently a senior scientist with the consultant firm Science Technology System in West Sacramento, CA. He has authored, coauthored, edited, and coedited more than 35 books in food science, food technology, food engineering, food law, nutrition, health, and medicine, including the Encyclopedia of Food Science and Technology, Bailey’s Industrial Oils and Fat Products, Foodborne Disease Handbook, Food Plant Sanitation, and Food Processing: Principles and Applications.

ASSOCIATE EDITORS Dr. Jeff Culbertson is a professor of food science at the University of Idaho. He earned his B.S. and M.S. degrees at Oregon State University and his Ph.D. at Washington State University—all in Food Science. He previously taught at the University of Wisconsin–River Falls and Central Michigan University. For a number of years he was the manager of Corporate Quality at the Kellogg Corporation. He maintains an active consulting business with many Fortune 500 clients from the food and beverage industry. Dr. Susan E. Duncan is a professor in the Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg, VA. She earned her Ph.D. in food technology and science, The University of Tennessee, Knoxville. She is the director of the Macromolecular Interfaces with Life Sciences Program, a multidisciplinary graduate program integrating polymer chemistry and life sciences. Dr. Duncan is a sensory specialist with a focus on quality issues of dairy, lipids, nutraceutical, and water/beverage products, emphasizing interactions with packaging materials. She has authored 50 peer-reviewed research publications and 7 book chapters. She is a member of the Institute of Food Technologists and American Dairy Science Association. Dr. Eunice C. Y. Li-Chan is a professor of food science at the University of British Columbia, Faculty of Agricultural Sciences, Food Nutrition & Health Program. Her significant research contributions include pioneering studies that launched the use of Raman spectroscopy and fluorescent hydrophobic probes as tools to study food protein systems, research that established the potential and protocols for using egg yolk antibodies in lieu of mammalian polyclonal antibodies in immunochemistry and immunoaffinity techniques, and the isolation and characterization of value-added proteins and peptides as functional food ingredients. Her publication record includes authorship or coauthorship in over 75 original articles in peer-reviewed scientific journals, more than 25 chapters in books, and a book entitled Hydrophobic Interactions in Food Systems (1988, CRC Press). Dr. Isabel Guerrero Legarreta is a profesor of food science, Department of Biotechnology, Universidad Autónoma Metropolitana, Iztapalapa, México. She received a B.Eng. degree (1972) in chemical engineering from the Universidad Nacional Autónoma de Mexico, Mexico City, an M.Sc. degree (1975) in food science from the University of Reading, England, and a Ph.D. (1983) in food science from the University of Guelph, Canada. Her research and teaching work has been focused on meat and fish preservation and utilization in subtropical areas. She has also studied the obtainment of products from marine resources, stressing the utilization of marine underutilized material and its by-products. Her professional contributions include over 100 papers, book chapters, and a patent on industrial carotenoid pigment separation from shrimp wastes. Dr. C. Y. Ma obtained his Ph.D. in food chemistry from the University of British Columbia, Canada. After working as a research scientist in Agriculture and Agri-Food Canada for 16 years, he is now a professor of food science at the University of Hong Kong. His current research activities include the study of structure-function relationships of food proteins and bioactive peptides. The molecular structure and conformation of selected proteins with potential uses as food ingredients and peptides possessing biological/pharmaceutical activities are studied by various physical and chemical techniques. Professor Ma also studies the potential uses of under-utilized protein sources from cereal and legume seeds, and the improvements of functional properties of these proteins by various chemical and physical methods.

Dr. Charles Manley received his Ph.D. from the University of Massachusetts–Amherst for research in the area of food and flavor chemistry. He received a B.S. degree in chemistry at University of Massachusetts–Dartmouth. He has worked as a research chemist for the Givaudan Company, and in various research and management positions within a number of Unilever Companies, including manager of Beverage Development and Technology for Thomas J. Lipton, director of Flavor Operations for the National Starch and Chemical Company, and as vice president, International Business Development for Quest International. Currently he serves as vice president of science and technology for Takasago International Corporation (U.S.A.). Takasago is one of the leading Global Flavor and Fragrance Companies with sales volumes in the top five. His major corporate responsibilities have been in managing commercialization of scientific research efforts and departments at both Unilever and Takasago. He has made major professional contributions, including over 150 publications, patents, and presentations in the field of flavor ingredient safety, food processing and science, and natural product chemistry. He has served as the president of the Institute of Food Technologists (IFT) and the Flavor and Extract Manufacturers’ Association (FEMA). Professor Tom McMeekin holds a personal Chair of Microbiology at the University of Tasmania and is co-director of the Australian Food Safety Centre of Excellence. He is a Fellow of the Australian Academy of Technological Sciences and Engineering, Scientific Fellow of Food Standards Australia New Zealand, and Chair of the Food Safety Information Council. Professor McMeekin has contributed to more than 200 publications, including the monograph “Predictive Microbiology: Theory and Application,” and has made greater than 30 invited international conference and workshop presentations. He is an executive board member of the International Committee of Food Microbiology and Hygiene and an editor of the International Journal of Food Microbiology. Awards include the JR Vickery Medal (International Institute of Refrigeration, 1987), the Annual Award of Merit (Australian Institute of Food Science and Technology, 1998), and International Leadership Award (International Association of Food Professionals, 2002). Dr. Wai-Kit Nip is a food technologist emeritus from the Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu. Dr. Nip received his B.S. degree (Food Technology, 1962) from National Chung-Hsing University, Taiwan, and an M.S. degree (Food Technology, 1965) and Ph.D. (1969) from Texas A&M University, College Station, Texas, U.S.A. He has taught classes in food processing, food safety, and experimental foods. Research activities include handling and processing of tropical fruits and vegetables, and aquatic foods. He has published numerous refereed articles, proceeding papers, and book chapters, and coedited several books in the food science and techology area. He is also the senior contributor of a patent. He has served at various capacities in local and national scientific organizations. Dr. Leo M. L. Nollet is a professor of biotechnology at Hogeschool Ghent, Ghent, Belgium. The author and coauthor of numerous articles, abstracts, and presentations, Dr. Nollet is the editor of the Handbook of Water Analysis, Food Analysis by HPLC, and Handbook of Food Analysis (3 volumes) (all titles Marcel Dekker). His research interests include food analysis techniques, HPLC, and environmental analysis techniques. He received an M.S. degree (1973) and a Ph.D. (1978) in biology from the Katholieke Universiteit, Leuven, Blegium. Dr. Mohammad Shafiur Rahman is an associate professor at the Sultan Qaboos University, Sultanate of Oman. He is the author or coauthor of over 150 technical articles and the author of the internationally acclaimed and award-winning Food Properties Handbook published by CRC Press, Boca Raton, FL. He is editor of the Handbook of Food Preservation published by Marcel Dekker, New York, which was translated into Spanish by Acribia, Spain in 2003. He is one of the editors for the Handbook of Food and Bioprocess Modeling Techniques, which will be published by Taylor & Francis. Dr. Rahman has initiated the International Journal of Food Properties (Marcel Dekker) and has been serving as the founding editor for more than 6 years. He is one of the section editors for the Sultan Qaboos University Journal of Agricultural Sciences (1999). In 1998 he was invited to serve as a food science adviser for the International Foundation for Science (IFS) in Sweden. He received B.Sc.Eng. (chemical) (1983) and M.Sc.Eng. (chemical) (1984) degrees from Bangladesh University of Engineering and Technology, Dhaka, an M.Sc. degree (1985) from Leeds University, England, and a Ph.D. (1992) in food engineering from the University of New South Wales, Sydney, Australia. Dr. Rahman has received numerous awards and fellowships in recognition of research/teaching achievements, including the HortResearch Chairman’s Award, the Bilateral Research Activities Program (BRAP) Award, CAMS Outstanding Researcher Award 2003, and the British Council Fellowship. Dr. Fidel Toldrá holds a B.Sc. degree in chemistry (1980), M.Sc. degree in food technology (1981), and a Ph.D. in chemistry (1984). Currently, he is research professor and head of the Laboratory of Meat Science, Department of Food Science,

at the Instituto de Agroquímica y Tecnología de Alimentos (CSIC) in Burjassot, Valencia (Spain). He is also an associate professor of food technology at the Polytechnical University of Valencia. Professor Toldrá has received several awards such as the 2002 International Prize for Meat Science and Technology, given by the International Meat Secretariat during the 14th World Meat Congress held in Berlin. Professor Toldrá has filed 7 patents, authored 1 book and 45 chapters of books, coedited 9 books, and published more than 121 manuscripts in worldwide recognized scientific journals. His research interests are based on food chemistry and biochemistry with special focus on muscle foods. He has served in several committees for international societies and, since May 2003, is also a member of the Scientific Commission on Food Additives, Flavorings, Processing Aids and Materials in contact with foods of the European Food Safety Authority (EFSA). Dr. Youling L. Xiong is professor of food chemistry at the Department of Animal and Food Sciences, University of Kentucky. He obtained a Ph.D. from Washington State University (1989) and received postdoctoral training at Cornell University. Professor Xiong also holds joint appointments with the Graduate Center for Nutritional Sciences and the Center for Membrane Sciences at the university. Dr. Xiong’s research focuses primarily on food protein chemistry and biochemistry, functionality, and applications, with an emphasis on muscle food processing. His fundamental work in food protein oxidation and the study of enzymic modification of soy, whey, wheat, and potato proteins to obtain physicochemically and biologically functional peptides has earned him several prestigious national awards. Dr. Xiong has published more than 130 research papers, contributed to 18 book chapters, and coedited two food science books. He also teaches undergraduate and graduate food chemistry, food protein, and meat science courses.

Contributors Sufian F. Al-Khaldi Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration College Park, Maryland

Yizhong Cai Cereal Science Laboratory Department of Botany The University of Hong Kong Hong Kong, China

Paw Dalgaard Danish Institute for Fisheries Research Technical University of Denmark Lyngby, Denmark

Christine Z. Alvarado Department of Animal and Food Sciences Texas Tech University Lubbock, Texas

C.G. Carter Tasmanian Aquaculture and Fisheries Institute University of Tasmania Tasmania, Australia

Srinivasan Damodaran Department of Food Science University of Wisconsin–Madison Madison, Wisconsin

Pedro Alvarez Department of Food Science and Agricultural Chemistry McGill University Quebec, Canada

Chung Chieh Department of Chemistry University of Waterloo Waterloo, Ontario, Canada

Johan Debevere Department of Food Technology, Chemistry, Microbiology and Human Nutrition Ghent University Ghent, Belgium

Sameer F. Al-Zenki Department of Biotechnology Kuwait Institute for Scientific Research Safat, Kuwait

Robert Cocciardi Department of Food Science and Agricultural Chemistry McGill University Quebec, Canada

Joannie Dobbs Department of Human Nutrition Food and Animal Sciences University of Hawaii at Manoa Honolulu, Hawaii

Fletcher M. Arritt III Department of Food Science and Technology Virginia Polytechnic Institute and State University Blacksburg, Virginia

Harold Corke Cereal Science Laboratory Department of Botany The University of Hong Kong Hong Kong, China

Joseph D. Eifert Department of Food Science and Technology Virginia Polytechnic Institute and State University Blacksburg, Virginia

Eveline J. Bartowsky The Australian Wine Research Institute Adelaide, Australia James N. BeMiller Whistler Center for Carbohydrate Research Purdue University West Lafayette, Indiana Daniel W. Bena PepsiCo International Purchase, New York

Nanna Cross Consultant Chicago, Illinois

Ronald R. Eitenmiller Department of Food Science and Technology University of Georgia Athens, Georgia

Steve W. Cui Food Research Program Agriculture and Agri-Food Canada Guelph, Ontario, Canada

John Flanagan Riddet Centre Massey University Palmerston North, New Zealand

Jeff D. Culbertson Department of Food Science and Toxicology University of Idaho Moscow, Idaho

Frederick S. Fry Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration College Park, Maryland

Ifigenia Geornaras Center for Red Meat Safety Department of Animal Sciences Colorado State University Fort Collins, Colorado

Maria Beatriz Abreu Glória Departamento de Alimentos Universidade Federal de Minas Gerais Belo Horizonte, MG Brazil

Lone Gram Danish Institute for Fisheries Research Technical University of Denmark Lyngby, Denmark

Douglas G. Hayward Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration College Park, Maryland Francisco J. Hidalgo Instituto de la Grasa y sus Derivados Consejo Superor de Investigaciones Cientificas Sevilla, Spain Y.-H. Peggy Hsieh Department of Nutrition, Food and Exercise Sciences Florida State University Tallahassee, Florida Kerry C. Huber Department of Food Science and Toxicology University of Idaho Moscow, Idaho Ashraf A. Ismail Department of Food Science and Agricultural Chemistry McGill University Quebec, Canada

Shann-Tzong Jiang Department of Food Science National Taiwan Ocean University Keelung, Taiwan, R.O.C. David Kang Department of Food Science and Technology Virginia Polytechnic Institute and State University Blacksburg, Virginia A.L. Kelly Department of Food and Nutritional Sciences University College Cork Cork, Ireland Konstantinos P. Koutsoumanis Department of Food Science and Technology Aristotle University of Thessaloniki Thessaloniki, Greece JaeHwan Lee Department of Food Science and Technology Seoul National University of Technology Seoul, Korea Tung-Ching Lee Department of Food Science Rutgers University New Brunswick, New Jersey Tomasz Lesiów Department of Quality Analysis University of Economics Wroclaw, Poland Eunice C.Y. Li-Chan Food, Nutrition and Health Program Faculty of Agricultural Sciences The University of British Columbia Vancouver, British Columbia, Canada Li Lite Department of Food Science and Nutritional Engineering China Agricultural University Beijing, China

Hsiao-Feng Lo Department of Horticulture Chinese Culture University Taipei, Taiwan, R.O.C. Miguel A. de Barros Lopes The Australian Wine Research Institute Adelaide, Australia R. Malcolm Love Consultant East Silverburn, Kingswells Aberdeen, Scotland Ching-Yung Ma Department of Botany The University of Hong Kong Hong Kong, China Armando McDonald Department of Forest Products University of Idaho Moscow, Idaho P.L.H. McSweeney Department of Food and Nutritional Sciences University College Cork Cork, Ireland Natalie A. Moltschaniwskyj Tasmanian Aquaculture and Fisheries Institute University of Tasmania Tasmania, Australia Magdi M. Mossoba Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration College Park, Maryland Lorraine L. Niba Department of Human Nutrition, Foods and Exercise Virginia Polytechnic Institute and State University Blacksburg, Virgina

S. Suzanne Nielsen Department of Food Science Purdue University West Lafayette, Indiana Gregory O. Noonan Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration College Park, Maryland Casey M. Owens Department of Poultry Science University of Arkansas Fayetteville, Arkansas Richard Owusu-Apenten Department of Food Science Pennsylvania State University University Park, Pennsylvania Jan Pokorný Department of Food Chemistry and Analysis Prague Institute of Chemical Technology Prague, Czech Republic Isak S. Pretorius The Australian Wine Research Institute Adelaide, Australia Mark P. Richards Muscle Biology & Meat Science Laboratory University of Wisconsin–Madison Madison, Wisconsin Manoj K. Rout Department of Botany The University of Hong Kong Hong Kong, China Robert B. Rucker Department of Agricultural and Environmental Science and Nutrition University of California Davis, California

Christine H. Scaman Food, Nutrition and Health Program Faculty of Agricultural Sciences The University of British Columbia Vancouver, British Columbia, Canada Steven J. Schwartz Department of Food Science and Technology The Ohio State University Columbus, Ohio

Peggy Stanfield Dietetic Resources Twin Falls, Idaho Francene Steinberg Department of Agricultural and Environmental Science and Nutrition University of California Davis, California

Jacqueline Sedman Department of Food Science and Agricultural Chemistry McGill University Quebec, Canada

C. Alan Titchenal Department of Human Nutrition, Food and Animal Sciences University of Hawaii at Manoa Honolulu, Hawaii

Jiwan S. Sidhu College for Women Kuwait University Safat, Kuwait

Fidel Toldrá Instituto de Agroquímica y Tecnología de Alimentos (CSIC) Burjassot (Valencia), Spain

Harjinder Singh Riddet Center Massey University Palmerston North, New Zealand Antoine-Michel Siouffi Université Paul Cezanne Campus St. Jerôme Marseille, France John N. Sofos Center for Red Meat Safety Department of Animal Sciences Colorado State University Fort Collins, Colorado Frank W. Sosulski GrainTech Consulting Inc. Saskatoon, Canada

Jocelyn Shing-Jy Tsao Department of Horticulture National Taiwan University Taipei, Taiwan, R.O.C. Sherri B. Turnipseed Animal Drug Research Center U.S. Food and Drug Administration Denver, Colorado Mieke Uyttendaele Department of Food Technology, Chemistry, Microbiology and Human Nutrition Ghent University Ghent, Belgium

Krystyna Sosulski GrainTech Consulting Inc. Saskatoon, Canada

Baowu Wang Department of Food and Nutritional Sciences Tuskegee University Tuskegee, Alabama

Bernd Spangenberg Umweltanalytik Fachhochschule Offenburg Offenburg, Germany

Qi Wang Food Research Program Agriculture and Agri-Food Canada Guelph, Ontario, Canada

P. J. Wood Food Research Program Agriculture and Agri-Food Canada Guelph, Ontario, Canada

Lin Ye Department of Food Science and Technology University of Georgia Athens, Georgia

Rosario Zamora Instituto de la Grasa y sus Derivados Consejo Superor de Investigaciones Cientificas Sevilla, Spain

Contents PART A Components Chapter 1 Carbohydrate Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1 Kerry C. Huber, Armando McDonald, and James N. BeMiller Chapter 2 Carbohydrates: Physical Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1 Qi Wang and P.J. Wood Chapter 3 Carbohydrates: Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1 Lorraine L. Niba Chapter 4 Functional Properties of Carbohydrates: Polysaccharide Gums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1 Steve W. Cui and Qi Wang Chapter 5 Food Protein Analysis: Determination of Proteins in the Food and Agriculture System. . . . . . . . . . . . . . . . . . . . . . . 5-1 Richard Owusu-Apenten Chapter 6 Protein: Denaturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1 Srinivasan Damodaran Chapter 7 Food Protein Functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1 Jeff D. Culbertson Chapter 8 Lipid Chemistry and Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-1 Mark P. Richards Chapter 9 Fats: Physical Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1 Francisco J. Hidalgo and Rosario Zamora Chapter 10 The Water-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-1 Francene Steinberg and Robert B. Rucker

Chapter 11 Fat-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-1 Lin Ye and Ronald R. Eitenmiller Chapter 12 Fundamental Characteristics of Water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12-1 Chung Chieh Chapter 13 Bioactive Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13-1 Maria Beatriz Abreu Glória Chapter 14 Pigments in Plant Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14-1 JaeHwan Lee and Steven J. Schwartz PART B Food Categories Chapter 15 Carbonated Beverages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15-1 Daniel W. Bena Chapter 16 Muffins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16-1 Nanna Cross Chapter 17 Cereals–Biology, Pre- and Post-Harvest Management. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17-1 Yizhong Cai and Harold Corke Chapter 18 Legumes: Horticulture, Properties, and Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18-1 Frank W. Sosulski and Krystyna Sosulski Chapter 19 Asian Fermented Soybean Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19-1 Li Lite Chapter 20 Vegetables: Types and Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20-1 Jocelyn Shing-Jy Tsao and Hsiao-Feng Lo Chapter 21 Nutritional Value of Vegetables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21-1 C. Alan Titchenal and Joannie Dobbs

Chapter 22 Canned Vegetables: Product Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22-1 Peggy Stanfield Chapter 23 Frozen Vegetables: Product Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23-1 Peggy Stanfield Chapter 24 Fruits: Horticultural and Functional Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24-1 Jiwan S. Sidhu and Sameer F. Al-Zenki Chapter 25 Frozen Fruits: Product Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25-1 Peggy Stanfield Chapter 26 Milk Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26-1 Harjinder Singh and John Flanagan Chapter 27 Enzymes of Significance to Milk and Dairy Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27-1 A.L. Kelly and P.L.H. McSweeney Chapter 28 Meat: Chemistry and Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28-1 Fidel Toldrá Chapter 29 Chemical Composition of Red Meat. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29-1 Baowu Wang Chapter 30 Meat Species Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30-1 Y.-H. Peggy Hsieh Chapter 31 Poultry: Chemistry and Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31-1 Christine Z. Alvarado and Casey M. Owens Chapter 32 Chemical Composition of Poultry Meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32-1 Tomasz Lesiów Chapter 33 Poultry Processing Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33-1 Christine Z. Alvarado

Chapter 34 Fats and Oils: Science and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34-1 Jan Pokorný Chapter 35 Fish Biology and Food Science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35-1 R. Malcolm Love Chapter 36 Edible Shellfish: Biology and Science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36-1 Natalie A. Moltschaniwskyj Chapter 37 Aquaculture of Finfish and Shellfish: Principles and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37-1 C.G. Carter Chapter 38 Frozen Seafood Products: Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38-1 Peggy Stanfield Chapter 39 Freezing Seafood and Seafood Products: Principles and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39-1 Shann-Tzong Jiang and Tung-Ching Lee Chapter 40 The Application of Gene Technology in the Wine Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40-1 Miguel A. de Barros Lopes, Eveline J. Bartowsky, and Isak S. Pretorius PART C Food Analysis Chapter 41 Food Analysis: Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41-1 S. Suzanne Nielsen Chapter 42 Analysis of the Chemical Composition of Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42-1 Eunice C. Y. Li-Chan Chapter 43 Spectroscopy Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43-1 Christine H. Scaman Chapter 44 Infrared and Raman Spectroscopy in Food Science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44-1 Ashraf A. Ismail, Robert Cocciardi, Pedro Alvarez, and Jacqueline Sedman

Chapter 45 Application of Gas Chromatography to the Identification of Foodborne Pathogens and Chemical Contaminants in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45-1 Magdi M. Mossoba, Frederick S. Fry, Sufian F. Al-Khaldi, Gregory O. Noonan, and Douglas G. Hayward Chapter 46 Modern Thin-Layer Chromatography in Food Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46-1 Bernd Spangenberg Chapter 47 High Performance Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47-1 Antoine-Michel Siouffi Chapter 48 The Use of Mass Spectrometry in Food Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48-1 Sherri B. Turnipseed Chapter 49 Food Analysis: Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49-1 Manoj K. Rout and Ching-Yung Ma PART D Food Microbiology Chapter 50 Microbiology of Food Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50-1 Joseph D. Eifert, Fletcher M. Arritt III, and David Kang Chapter 51 Microbial Food Spoilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51-1 Lone Gram Chapter 52 Microbiology of Land Muscle Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52-1 Konstantinos P. Koutsoumanis, Ifigenia Geornaras, and John N. Sofos Chapter 53 Microbiology of Marine Muscle Foods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53-1 Paw Dalgaard Chapter 54 Microbial Analysis of Foods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54-1 Mieke Uyttendaele and Johan Debevere Chapter 55 Rapid Methods in Food Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55-1 Mieke Uyttendaele and Johan Debevere

Part A Components

1

Carbohydrate Chemistry

Kerry C. Huber

Department of Food Science and Toxicology, University of Idaho

Armando McDonald

Department of Forest Products, University of Idaho

James N. BeMiller

Whistler Center for Carbohydrate Research, Purdue University

CONTENTS I. Introduction to Carbohydrates ................................................................................................................................1-1 II. Monosaccharides ....................................................................................................................................................1-2 III. Reactions of Carbohydrates ....................................................................................................................................1-5 A. Hydrolysis ........................................................................................................................................................1-5 B. Oxidation/Reduction ........................................................................................................................................1-5 C. Thermal Reactions ..........................................................................................................................................1-5 D. Ester/Ether Formation......................................................................................................................................1-6 IV. Oligosaccharides......................................................................................................................................................1-7 A. Disaccharides ..................................................................................................................................................1-7 B. Fructooligosaccharides ....................................................................................................................................1-8 V. Polysaccharides........................................................................................................................................................1-9 A. Classification of Polysaccharides ....................................................................................................................1-9 B. Structural Regimes of Polysaccharides ........................................................................................................1-10 C. Impact of Polysaccharide Molecular Features on Physical Properties ........................................................1-12 D. Polysaccharide Stability and Reactivity ........................................................................................................1-15 VI. Polysaccharide Structures and Functions ..............................................................................................................1-15 A. Starch and Its Derivatives ..............................................................................................................................1-15 B. Cellulosics......................................................................................................................................................1-17 C. Galactomannans: Locust Bean and Guar Gums............................................................................................1-18 D. Alginate..........................................................................................................................................................1-19 E. Pectin..............................................................................................................................................................1-20 F. Carrageenans..................................................................................................................................................1-20 G. Agar................................................................................................................................................................1-20 H. Xanthan ..........................................................................................................................................................1-21 I. Gum Arabic....................................................................................................................................................1-21 References ......................................................................................................................................................................1-23

I.

INTRODUCTION TO CARBOHYDRATES

Carbohydrates, which in their basic form exhibit the general chemical formula Cn(H2O)n, are a class of organic compounds that were historically designated “hydrates of carbon” due to their observed elemental composition. As

the most abundant class of organic compounds on Earth, carbohydrates are the primary constituents of plants and exoskeletons of crustaceans and insects. Therefore, carbohydrates are virtually an unavoidable element of daily life, as they are encountered in food (glucose, sucrose, starch, etc.), wood, paper, and cotton (cellulose). Carbohydrates 1-1

1-2

Handbook of Food Science, Technology, and Engineering, Volume 1

themselves can be sub-grouped according to the number of sugar building blocks comprising their respective structures from monomers (monosaccharides) right through to polymers (polysaccharides). In addition, the diversity of carbohydrates occurring within nature arises from the number of carbon atoms comprising sugar monomer units (monosaccharides of 3 to 9 carbon atoms), the varied chemical structure of monosaccharides (including substituent groups), and the nature of linkages joining monosaccharide units.

II. MONOSACCHARIDES Monosaccharides, which represent the most basic carbohydrate elements, are polyhydroxy aldehydes and ketones commonly referred to as aldoses and ketoses, respectively. In addition, the number of carbon atoms present in the

FIGURE 1.1 Acyclic form of the D-aldose series.

molecule also aids classification of monosaccharides. For sugars comprised of 3, 4, 5, 6, and 7 carbon atoms, the analogous aldose sugars are referred to as trioses, tetroses, pentoses, hexoses, and heptoses, respectively, while the same ketoses are correspondingly and officially named triuloses, tertruloses, pentuloses, hexuloses, and heptuloses, respectively. They may also be unofficially grouped with names such as ketopentose and ketohexose. The simplest aldose and ketose monosaccharides are the two entantiomers of glyceraldehyde (D and L) (Figure 1.1) and 1,3-dihydroxyacetone (Figure 1.2), respectively. Aldoses exhibit one additional chiral center compared to ketoses for the same number of carbon atoms. With the addition of an extra carbon atom to a growing monosaccharide chain, the number of possible stereoisomers increases. For the total number of chiral or asymmetric centers (n) possessed by a


1-3

FIGURE 1.2 Acyclic form of the D-ketose series.

monosaccharide, there are 2n possible arrangements. The reference monosaccharide is considered to be D-glyceraldehyde, which provides a template for generation of acyclic carbon skeletons (from 3 to 6 carbon atoms) as outlined in Figure 1.1 (Fischer projection format). For D-sugars, the hydroxyl group of the highest numbered asymmetric carbon atom (the one furthest from the carbonyl group) is situated on the right-hand side of the Fischer projection, while for L-sugars, the same hydroxyl group is positioned on the left. Thus, the analogous L-aldose series (for brevity not shown) is represented by the exact mirror image structures presented for the D-aldose series. Most sugars found in nature are of the D-configuration, though some common exceptions include L-arabinose, L-rhamnose, L-fucose, L-guluronic acid and L-iduronic acid. Monosaccharide units that differ only in the configuration about a single chiral carbon atom are referred to as epimers (diastereomers). For example, D-glucose and D-galactose are C-4 epimers. Similar to the pattern previously presented for the aldoses, the ketose acyclic series begins with 1,3-dihydroxyacetone; however, the chiral template series starts at D-erythrulose (Figure 1.2) [1,2]. The carbonyl group of aldoses and ketoses is reactive and readily forms an intramolecular cyclic hemiacetal.

Therefore, most monosaccharides (except glyceraldehydes, 1,3-dihydroxyacetone and tetrulose) form energetically stable 5- (furan) and 6- (pyran) membered ring structures. Through cyclization, an additional chiral center is formed (compared to the acyclic form) at C-1 (aldoses) or C-2 (ketoses), which is designated the anomeric carbon atom. At the new chiral center, there are two possible anomeric configurations, α and β, which denote the hydroxyl group below and above the ring plane, respectively (true for D-sugars, while the opposite designation is true for L-sugars). The cyclic hemiacetal formation for both pyranose and furanose ring structures (Haworth projections) is illustrated in Figure 1.3 for D-glucose. The actual conformation of the glucopyranosyl structure exists predominantly in the form of a chair-shaped ring (not all ring atoms within the same plane) with the bulky hydroxyl groups in an equatorial arrangement to minimize steric (1,3-syn-diaxial) interactions and lessen bond angle strain. For example, β-D-glucopyranose is shown in the 4C1 conformation (Figure 1.3). The superscript and subscript numbers of the conformational notation denote the numbers of the carbon atoms above and below the plane of the ring, respectively [1,2]. Aldoses and ketoses (both hemiacetals) can readily react with alcohols to produce acetals called glycosides. The

1-4


FIGURE 1.3 Cyclic hemiacetal formation of D-glucose and ring conformation.

HO

OH OH

OH COOH O

H3C HO

O

HO

OH

OH

HOH2C HO HO

OH

L-rhamnose (6-deoxy-L-mannose)

HO HO

OH COOH L-iduronic acid

HO OH

O OH

CH3O HO

OH 3-deoxy-D-glucose

OH

2-amino-2-deoxy-D-glucose (D-glucosamine)

HOH2C

O

OH OH

NH2

OH D-galacturonic acid

OH

O

Myo-inositol

O

COOH O OH

OH

CH2OH

OH

OH

4-O-methyl-D-glucuronic acid

OH OH D-apiose

FIGURE 1.4 Structures of other common monosaccharides and inositol.

suffix -ide indicates an acetal linkage. For example, D-xylose reacting with methanol produces a mixure of methyl α-D-xylopyranoside and methyl β-D-xylopyranoside [1]. The alcohol (methanol in the above example) portion of the glycoside is called the aglycon. In nature, the aglycon (alcohol) is most often another monosaccharide unit, and the covalent bond joining two monosaccharide units is termed a glycosidic bond. This concept can be used to describe two (disaccharide) or more monosaccharide units attached through glycosidic linkages, including extensive polymeric chains (e.g., polysaccharides) comprised of many monosaccharide units.

In addition to the stereoisomeric configurations of sugars, the chemical diversity of monosaccharides can include chemical functionalities such as: carboxyl groups at the primary hydroxyl group position (uronic acids), amino groups in place of hydroxyl groups (amino sugars), hydroxyl groups replaced with hydrogen atoms (deoxy sugars), double bonds (unsaturated derivatives), branch chain sugars, ether substituents, and ester substituents. Examples of these diverse structures are shown in Figure 1.4. A uronic acid is an aldose in which the primary alcohol group (e.g., C-6) has been converted to a carboxylic acid (e.g., α-D-galacturonic acid). A deoxy monosaccharide involves the replacement of


a hydroxyl group with a substituent such as a hydrogen atom (e.g., 6-deoxy-L-mannopyranose, commonly known as L-rhamnose; 2-deoxy-D-erythro-pentose, also known as 2-deoxy-D-ribose; 3-deoxyl-D-ribo-hexose, also known as 3-deoxy-D-glucose). An amino sugar is a monosaccharide, in which a hydroxyl group is replaced by an amino group (e.g., 2-amino-2-deoxy-β-D-glucopyranose). A branch chain sugar is C-substituted at a non-terminal carbon (e.g., 3-C-hydroxymethyl-D-erythro-tetrose, also known as Dapiose). Ether and ester carbohydrate derivatives will be discussed later. Polyhydroxycyclohexanes, also known as cyclitols or inositols, are discussed here due to their similarities to pyranoses. Nine stereoisomers are possible, and the most widespread in nature is myo-inositol (Figure 1.4). Methyl ether derivatives of inositols are also common.

III. REACTIONS OF CARBOHYDRATES A.

HYDROLYSIS

Glycosides, including disaccharides and polymeric chains (oligosaccharides and polysaccharides), undergo hydrolysis in aqueous acids to yield free sugars. The process somewhat randomly cleaves glycosidic bonds to reduce large carbohydrate chains into smaller fragments, which can in turn be further depolymerized to monosaccharide units. Hydrolysis is initiated in glycosides by protonation of the exocyclic oxygen atom followed by breakdown of the conjugate acid (cleavage of the bond between the anomeric carbon atom and the glycosidic oxygen atom) resulting in the formation of a cyclic carbocation, which is attacked by water to yield the hemiacetal product (Figure 1.5). Glycosidic bonds can also be cleaved by enzymes, which are very specific to the

1-5

type of sugar residue (e.g., D-galactosyl vs. D-glucosyl), anomeric configuration (α or β), and the glycosidic linkage site (e.g., 1→3). Both acid- and enzyme-catalyzed hydrolysis are commonly employed in the manufacture of maltodextrins and corn syrups, as well as in the commercial production schemes of polysaccharides.

B.

OXIDATION/REDUCTION

Aldoses can be readily oxidized to aldonic acids. Because during the oxidation there is a concurrent reduction of the oxidizing agent, aldoses are called reducing sugars (Figure 1.6). Aldonic acids can readily cyclize to form a stable lactone under neutral or acidic conditions. This oxidation reaction has been successfully exploited either chemically (Fehling solution, Cu(OH)2; bromine solution; Tollens reagent) or enzymatically (glucose oxidase) to quantitatively determine sugars [1,2]. In contrast, ketoses must first be isomerized to an aldose (under alkaline conditions), which can then undergo oxidation. Reduction of an aldose or ketose results in the formation of an alditol or sugar alcohol (denoted by the -itol suffix). Commercial-scale operations typically use high-pressure hydrogenation in conjunction with nickel catalyst for such reductions. Sorbitol (D-glucitol) is a commonly occurring alditol in fruits, and is 50% as sweet as sucrose. Sugar alcohols, such as D-glucitol, D-mannitol, and D-xylitol, are frequently used as alternative sweeteners (noncariogenic) in chewing gum and confectionary applications.

C.

THERMAL REACTIONS

Heating of reducing sugars results in a complex series of reactions called caramelization. The process is a cascade of

FIGURE 1.5 Abbreviated mechanism of acid-catalyzed hydrolysis of a glycoside.

-

-

FIGURE 1.6 Oxidation of an aldose to an aldonic acid with subsequent formation of D-gluconolactone.

1-6


dehydration reactions that form semi-volatile anhydrides (e.g., 1,6-anhydro-␤-D-glucopyranose (levo-glucosan)) and unsaturated compounds (e.g., 5-hydroxy-methylfuraldehyde (HMF) and furaldehyde) as shown in Figure 1.7 [1]. Catalysts such as salts and acids are added to promote the reaction. Reducing sugars in the presence of amines (such as proteins and amino acids) undergo a thermal reaction called the Amadori rearrangement. In the case of D-glucose, reaction with an amine (R-NH2) will form a derivative of 1-amino-1-deoxy-D-fructose and D-glucosylamine (Figure 1.8a). If the reaction continues under acidic conditions, it will undergo dehydration reactions to form HMF. Above pH 5, reactive Amadori intermediates yield complex polymerized dark-colored products via the poorly understood non-enzymatic browning or Maillard reaction, which contributes both color and flavor components to a wide range of food systems (e.g., bread crust, chocolate, caramels, etc.) Recently, acrylamide has been detected in a myriad of high-temperature processed foods (French fries, bread, breakfast cereal, popcorn, etc.), and seems to be primarily derived by the reaction between D-glucose and asparagine. The reaction likely proceeds via the glucosyl-asparagine derivative, and then undergoes decarboxylative deamination to form acrylamide (Figure 1.8b) [4]. To date, it is not known whether the low levels (ppb) detected in food pose any significant health risk to humans.

D. ESTER/ETHER FORMATION Hydroxyl groups of sugars can form esters with organic and inorganic acids. Reaction of hydroxyl groups with acyl chlorides or acid anhydrides in the presence of a catalyst (base) produces esters. Industrially, starches are esterified (acetates, phosphates, succinates, adipates, etc.) to improve their food-use properties. Acetates, sulfates, and phosphates are commonly found as native constituents of carbohydrates. For example, acetyl groups are present in certain polysaccharides such as the plant hemicelluloses (xylan and glucomannan), certain pectins, and xanthan, while sugar phosphates are common intermediates in the biosynthesis of monosaccharides and polysaccharides. The polysaccharide carrageenan contains sulfate half-ester substituents. In addition to esters from sugar hydroxyl groups, esterified uronic acid units are found in polysaccharides. The best example is pectin, in which some of its D-galacturonic acid units exist in the methyl ester form. The hydroxyl groups of carbohydrates can also form ethers. In nature, ether groups are not common, though some D-glucuronic acid units, particularly in hemicelluloses, such as glucurononxylan, are methylated at the O-4 position (4-O-methyl-D-glucuronic acid). Industrially, starches and celluloses are methylated (cellulose), hydroxypropylated (starch, cellulose), and carboxymethylated (cellulose) to improve the properties of these polysaccharides for a variety of food applications.

(a)

HOH2C -D-glucopyranoside

O

HO HO

O

H2C OH

−H2O

O

Levo-glucosan

OH

(b)

- elimination

HC H

HC

O

OH

HC

HO

H

H

O

HC

OH

OH

OH Enolization

HO

OH

OH

OR

−H 2 O

HC

O

O

O

H

H

−H 2 O

CH

H

OH

H

OH

H

OH

H

OH

H

OH

H

OH

H

OH

H

OH

CH2OH

CH2OH

CH2OH

5-hydroxymethyl-furaldehyde

O

CHO

−H 2O

HOH2C H

FIGURE 1.7 Reaction mechanism for the formation of (a) levo-glucosan and (b) HMF.

O

CH CH H

OH CH2 OH

CH2OH

D-glucose

HOH2C

O

CHO OH


1-7

(a)

(b)

FIGURE 1.8 (a) Amadori reaction scheme and (b) formation of acrylamide.

IV. OLIGOSACCHARIDES Oligosaccharides are comprised of 2 to 20 glycosidicallylinked monosaccharide units [3]. In nature, enzymes called glycosyltransferases catalyze the biosynthesis of both oligosaccharides and larger polymeric carbohydrates (e.g., polysaccharides). These very specific enzymes link specific monosaccharide units together according to a defined anomeric configuration and linkage position (e.g., C-3) on the aglycon sugar. Commercially, oligosaccharides also can be generated through enzyme- or acidcatalyzed hydrolysis of polysaccharides. The following section will briefly discuss common disaccharides, trisaccharides, and fructo-oligosaccharides.

A.

DISACCHARIDES

Disaccharides are composed of two monosaccharide units joined by a glycosidic bond. Disaccharides can either be reducing (e.g., maltose and lactose, Figure 1.9) or nonreducing (e.g., sucrose, Figure 1.10), depending on whether one or both anomeric carbon atoms are involved in the disaccharide glycosidic bond. Maltose (Figure 1.9), a disaccharide formed by enzymatic hydrolysis of starch, is produced commercially from the malting of barley, and

is the primary fermentable sugar used in the production of beer [3]. The structure of maltose (α-D-glucopyranosyl(1→4)-D-glucopyranose) can be written in shorthand notation as αGlcp(1→4)Glcp. The shorthand abbreviation for a monosaccharide unit is based on its first three letters, except for glucose, which is designated as Glc. The position of the linkage is designated as (1→4) from carbon atom 1 of the glycosyl unit to carbon atom 4 of the agylcon unit. The sugar ring size is denoted as p for pyranose or f for furanose, while the anomeric configuration is designated as either α or β. In the case of D or L configuration, it is only necessary to stipulate L-sugars (D-sugars are assumed unless noted otherwise). This shorthand notation can be used to define both oligosaccharide and more complex polymeric (polysaccharide) carbohydrate structures. Lactose (βGalp(1→4)Glcp; Figure 1.9) is found in milk at concentrations between 4 and 9%, and is the primary carbohydrate source for developing mammals. For energy utilization, it is necessary that lactose be hydrolyzed by the enzyme lactase (β-galactosidase) to D-galactose and D-glucose in the small intestine to facilitate absorption into the bloodstream. In some individuals, lactose is not (or is only partially) hydrolyzed (lactase deficiency), which

1-8


OH CH2OH O

CH2OH

O

HO HO

CH2 OH

OH

O

O HO

OH

O

O HO

CH2 OH

HO

OH OH

OH

Maltose

Lactose

OH

FIGURE 1.9 Structures of maltose and lactose. OH CH2OH

O HO OH O HO

OH CH2OH

O

O

HO

HO OH

CH2OH

O

O

HO HO

OH

H2 C

O

O

HO HO

OH CH2OH O O

OH

O OH CH2OH O O

HO CH2OH

H2 C

HO HO

OH CH2OH O O

HO

Sucrose

CH2

HO CH2OH

Raffinose

OH

CH2OH Stachyose

OH

FIGURE 1.10 Structures of sucrose, raffinose, and stachyose.

condition is clinically termed lactose intolerance, and results in the bacterial, anaerobic fermentation of lactose in the large intestine to lactic acid and gaseous products [3]. Sucrose (αGlcp(1↔2)βFruf; Figure 1.10) is composed of an α-D-glucopyranosyl unit linked (reducing end to reducing end) to a β-D-fructofuranosyl unit, and therefore is non-reducing, because it has no free carbonyl (aldehyde) group. Sucrose (table sugar) is one of the most common low-molecular-weight carbohydrates in the human diet. It is found in plants (e.g., sugar beets, sugarcane, and fruit), where it represents an easily transportable energy and carbon source and an intermediate in starch and cellulose biosynthesis. Another attribute of sucrose is its solubility in water to form highly concentrated solutions, which result in the lowering of the freezing point of water (anti-freeze) and resistance against dehydration in plants and fruits [3]. As a food ingredient, sucrose is utilized due to its water-solubility, desirable sweet taste, effects on colligative properties (e.g., boiling and freezing point regulation), preservative function (osmotic effect), and texturizing effects. In certain plants, some sucrose molecules are α-galactosylated to form the non-reducing trisaccharide, raffinose

(αGalp(1→6)αGlcp(1↔2)βFruf), the tetrasaccharide, stachyose (αGalp(1→6)αGalp(1→6)αGlcp(1↔2)βFruf) as shown in Figure 1.10, and the pentasaccharide, verbascose. These oligosaccharides are found especially in beans, onions, and sugarcane. They are non-digestible and are responsible for causing the flatulence (due to microbial fermentation in the colon) associated with the eating of beans and onions [3].

B.

FRUCTOOLIGOSACCHARIDES

Fructans, which are polymers (polysaccharides) consisting of β-D-fructofuranosyl units, are found in higher plants, and are composed of two types, inulins and levans (Figure 1.11). Inulins consist of (2→1)-linked β-D-fructofuranosyl units and are found in Jerusalem artichoke, chicory, and dahlia tubers, while levans, consisting of (2→6)-linked β-D-fructofuranosyl units, are found in grasses. Both types of fructans are terminated at the reducing end with a sucrose unit [2]. Fructo-oligosaccharides, which are smaller versions of fructans are used in prebiotic food applications, and are believed to serve as a


1-9

CH2OH

O

HO HO

OH CH2OH O O

CH2OH

HO H2C OH

H2 C

CH2OH O O

CH2OH O O

HO

HO

O

H2C

OH

H2 C

O

O

O

HO

HO

CH2OH OH

OH

O

HO HO

CH2OH OH

CH2 OH n

OH

n

CH2OH O O Inulin

Levan

HO CH2OH OH

FIGURE 1.11 Structures of inulin and levan oligosaccharides.

TABLE 1.1 Categorization of Select Polysaccharides according to Origin1 Origin/Source

Polysaccharide Examples

Higher plants Cell wall associated Energy stores (seeds, roots, tubers) Exudates Marine plants (seaweed extracts) Microorganisms (bacterial fermentation) Chemical derviatives (of varied native origin)

Cellulose, hemicellulose, pectin Starch, guar gum, locust bean gum Gum arabic, gum karaya Carrageenan, alginate, agar Xanthan, gellan Hydroxypropylstarch, starch acetate, starch phosphate, carboxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose Polydextrose

Synthetic 1

Adapted from Ref. [3].

preferred substrate to promote colonization of beneficial gut microflora (e.g., bifidobacteria).

V. POLYSACCHARIDES By definition, polysaccharides (glycans) are long-chain, carbohydrate polymers comprised of, at minimum, 20 glycosidically linked monosaccharide (monomer) units [3]. The number of individual monosaccharide units that comprise a particular polysaccharide is referred to as the degree of polymerization (DP). Most indigenous polysaccharides possess DPs far in excess of the stated minimum (200–3000 DP is typical), though extremes are observed in nature at both ends of the DP spectrum [3]. While polysaccharides are present in a wide range of plant and animal biological systems, most glycans of commercial significance occur in higher plants (though a few are produced by

bacteria). Collectively, polysaccharides from varied origins offer a multitude of structural and functional diversity consistent with their respective intended roles (e.g., structure, energy storage, hydration, etc.) within biological systems. Of the various carbohydrate classes, polysaccharides are by far the most abundant in nature [3], and, as a class of compounds, represent the greatest single component of biomass on the planet. Their relative abundance combined with their diverse structural and functional characteristics make them a superb source of biopolymers for utilization in a wide range of food applications.

A.

CLASSIFICATION

OF

POLYSACCHARIDES

Though commonly classified by source (Table 1.1), polysaccharides may also be categorized according to the number of different monosaccharide types contained

1-10


TABLE 1.2 Categorization of Select Polysaccharides according to Multiple Classification Schemes Related to Structure and Behavior1 Origin/Source By Shape Linear

Branched Branch-on-branch By Number of Types of Monomeric Units Homoglycan (Di)Heteroglycan (Tri)Heteroglycan (Tetra)Heteroglycan By Charge Neutral

Anionic By Rheological Properties Gelling

Non-gelling

Polysaccharide Examples Cellulose, starch (amylose2), pectin,3 alginate, agar, carrageenan, gellan, cellulose derivatives (carboxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose) Guar gum, locust bean gum, xanthan Starch (amylopectin), gum arabic Cellulose, starch (amylose, amylopectin) Guar gum, locust bean gum, alginate, agar, carrageenan, pectin3 Xanthan, gellan Gum arabic Cellulose, starch (amylose, amylopectin4), agar,5 guar gum, locust bean gum, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylstarch, starch acetate Xanthan, gellan, alginate, carageenan, pectin, gum arabic, gum karaya, carboxymethylcellulose, starch phosphate Starch and starch derivatives, alginate, agar, carrageenan (κ- and ι-types), pectin, gellan, hydroxypropylmethylcellulose, methylcellulose Celulose, xanthan,6 locust bean gum,7 guar gum, carrageenan (λ-type), gum arabic,8 carboxymethylcellulose, polydextrose

1

Adapted from Ref. [3].

2

Depending on botanical source, amylose can contain some minor short branches toward the molecular reducing end [3].

3

Categorization does not account for native pectin hairy regions (regions of extensive branching composed of multiple monosaccharide units), most of which are lost during processing to commercial grade pectin [26].

4

Some starch amylopectin molecules (i.e., potato) may possess small amounts of native starch monophosphate [3].

5

Agar does possess small amounts of sulfate [30], but is considered to be largely neutral.

6

Though xanthan solutions do not gel, xanthan does form synergistic gels with locust bean gum, agar, and κ-carrageenan [3].

7

Though primarily a thickener, locust bean gum exhibits synergistic gelling behavior with xanthan, agar, and κ-carrageenan [3].

8

Forms gels at very high concentrations [3].

within their molecular structure (e.g., homoglycan: one type vs. heteroglycan: more than one type), molecular shape (e.g., branched vs. linear), electrostatic charge (e.g., neutral vs. anionic) and properties (e.g., gelling vs. nongelling) (Table 1.2). In addition, polysaccharides differ from proteins and nucleic acids in that they are both polydisperse and polymolecular [3]. With regard to polydispersity, a particular polysaccharide type (e.g., pectin) is not defined by a specific number of monomeric units or a defined molecular weight, but rather possesses a range of DPs and molecular weights. Further, the majority of polysaccharides are not chemically homogeneous (cellulose and bacterial polysaccharides are exceptions); they are polymolecular in the sense that individual molecules within a polysaccharide type (e.g., pectin) may differ from one another with respect

to fine structure (monosaccharide sequence, proportion of monosaccharide constituents, linkage type, branching frequency). Thus, it is important to keep in mind that the described structure of a polysaccharide type often is not absolute; rather it is an idealized, statistical representation for a population of macromolecules. For every polysaccharide, the reported molecular weight is also an average value.

B.

STRUCTURAL REGIMES

OF

POLYSACCHARIDES

Nevertheless, structural aspects of polysaccharides may be defined on several different organizational levels (analogous to protein primary, secondary, tertiary, and quaternary structural regimes) [5]. Polysaccharide primary structure refers to the sequence of monosaccharide units and the configuration of accompanying glycosidic


1-11

hydrogen bonding between the C-2 and C-3 hydroxyl groups of neighboring glucosyl units. Finally, the α(1→6) glycosidic linkage inherent to dextran introduces an additional bond (C-5–C-6), about which free rotation can occur. This additional bond also increases the distance between adjacent glycosyl units such that hydrogen bonding cannot occur. The resulting consequence is that dextran molecules do not generally possess an ordered three-dimensional conformation, but instead adopt the structure of a random coil (possess no defined shape). The ability to form ordered secondary structure is favored by a high degree of chain uniformity (regularity of monosaccharide sequence and glycosidic linkage) [3], while a random coil results from the lack thereof. In summary, the ribbon, helix, and random coil conformations described for cellulose, amylose, and dextran, respectively, effectively demonstrate the range of secondary structure typical of polysaccharide systems. An example of polysaccharide tertiary structure is observed with starch amylose molecules, which can associate to form sections of ordered, double-helical arrangements [5]. Triple-helical tertiary structures have also been reported to exist for various polysaccharides [7,8]. Most polysaccharide tertiary structures are typically stabilized through intermolecular hydrogen bonds. Temperature and physical state also influence the tendency for a polysaccharide to adopt an ordered secondary

linkages. However, it is the glycan primary structure that ultimately dictates the nature and extent of intramolecular and intermolecular associations within a polysaccharide system that lead to development of three-dimensional molecular order (secondary, tertiary, and quaternary structures). Of the two defining elements of primary structure, linkage type generally exerts a greater influence on molecular conformation than monosaccharide type [6]. While there is free rotation about glycosidic bonds, the extent of rotation is limited to a narrow range of thermodynamically favored conformations that coincide with potential energy minima (as a function of hydrogen bonding, van der Waals, polar, and torsional interactions) [5]. These preferred conformations define the proximity of adjacent glycosyl units one to another, and dictate the polysaccharide long-range, three-dimensional shape. This principle is illustrated by the classic comparison of cellulose, amylose, and dextran polysaccharides, which are all linear chains of polyglucose, differing only in the nature of their glycosidic linkages (Figures 1.12a–c) [3]. The equatorial-equatorial β(1→4) glycosidic linkage of cellulose, which facilitates a strong hydrogen bonding interaction between the ring oxygen atom and the C-3 hydroxyl group of adjacent glycosyl units, gives rise to a flat, ribbon-like molecular conformation. On the contrary, the axial-equatorial α(1→4) linkage of amylose leads to a more open, coiled, helical structure, based on favorable

(a)

HO2HC

H

O

O HO OH

O HO2HC

O

O HO

O CH2OH

(b)

HO

O O

CH2OH O

O H O H

OH

O 2HC

HO HO (c)

O

O H

O

HO HO

CH2

O OH

FIGURE 1.12 Rotation about glycosidic bonds (φ and ψ) exhibited by polyglucose chains of (a) cellulose, (b) amylose, and (c) dextran (also exhibits free rotation about C5–C6 bond, ω) that provide the basis for long-range, three-dimensional conformational structure (ribbon, helix, and random coil, respectively). Dotted lines between adjacent glucosyl units depict stabilizing hydrogen bonds.

1-12


(a)

Helix

Random coil

(b)

or

Double helix

Partially dissociated coils

Fully dissociated coils

FIGURE 1.13 Depiction of the conformational changes associated with the thermoreversible order to disorder transition for (a) single- and (b) double-helical structures.

or tertiary structure. A polysaccharide in an ordered conformation typically undergoes a reversible order (helix) to disorder (random coil) transition with an increase in temperature sufficient to disrupt hydrogen bonds that stabilize the ordered conformation (Figure 1.13a) [9]. Under these circumstances, double-stranded tertiary structures generally unfold (Figure 1.13b). Upon cooling below the transition temperature, polysaccharide molecules are again able to regain their respective ordered secondary and/or tertiary arrangements. For polysaccharides capable of forming ordered secondary structures, the crystalline state generally favors the existence of the ordered conformation, while the solution state (in water) often results in adoption of a random coil [5]. In the solution state, competing hydrogen bonds between solute (polysaccharide) and solvent (water) molecules tend to minimize the stabilizing effects of intramolecular (solute-solute) hydrogen bonds that would otherwise stabilize an ordered polysaccharide secondary structure. Nevertheless, the solution state does not necessarily impede the formation of doublehelical tertiary structures, though solvent conditions necessary for development of such structures may vary with polysaccharide type. The ability to form some degree of ordered secondary or tertiary structure is generally a prerequisite (but not a guarantee) for polysaccharides to participate in advanced quaternary supramolecular structures. Quaternary structure develops through alignment and aggregation of secondary- and/or tertiary-ordered polysaccharide molecules

[5], and is typically stabilized by non-covalent interactions (electrostatic, non-polar, hydrogen bond associations) under requisite solvent conditions. Such quaternary order is responsible for the intermolecular associations that lead to development of both gel (junction zone) and other crystalline structures, which are important to processed foods and native plant cell wall systems. However, in discussing any level of polysaccharide three-dimensional structure, it is important to note that polysaccharide molecules in solution are in a constant state of dynamic flux, and likely exist in a wide range of physical forms (helix, double helix, random coil, etc.) at any point in time (even though a statistically favored conformation may be dominant) [3,5]. Nevertheless, the three-dimensional structures discussed here provide a basis for many of the observed properties of polysaccharide systems. A more detailed description of molecular features impacting polysaccharide conformation and physical properties is presented next.

C. IMPACT OF POLYSACCHARIDE MOLECULAR FEATURES ON PHYSICAL PROPERTIES While polysaccharides possess ring oxygen and hydroxyl groups capable of interacting with water through hydrogen bonds [3], physical properties such as solubility, viscosity, and gelling capability are additionally influenced by other molecular features inherent to a polysaccharide. Water solubility of a polysaccharide is generally enhanced by molecular features that prevent formation of


1-13

TABLE 1.3 General Description1 of Polysaccharide Molecular Features and Conditions That Promote Water-Solubility, Viscosity Development, Gelling Behavior Polysaccharide Feature

Water-Solubility

Viscosity Development

Gelling Behavior/Stability

Backbone linkage and/or monosaccharide repeat

Irregular

Regular (rigid structures)

Mixed (both regular and irregular segments)

Backbone shape

Branch-on-branch structure

Linear, extended structures

Linear, extended structures

Degree of branching and/or substitution

Regular, even distribution of sidechains or substituents along polymer chains

Regular, even distribution of short sidechains or substituents along polymer chains

Sporadic or irregular distribution of side chains or substituents along polymer chains

Molecular charge (if charged)

Even distribution of charge (repulsive) along polymer chains

Even distribution of charge (repulsive) along polymer chains

Uneven distribution of charge (repulsive) along polymer chains

Degree of solvation

Maximum

High

Balanced (segments of both polymer-polymer and polymerwater interactions)

Molecular size

Low

Intermediate to high

Low to intermediate

1

It is important to note that polysaccharides do not necessarily need to possess all suggested molecular features or conditions to exhibit a particular property, though the greater number of molecular features present will increase the likelihood for a particular property to be exhibited. Exceptions do also exist.

an ordered three-dimensional structure (e.g., irregular backbone structure) or that present physical barriers to intermolecular interactions (e.g., uniform sidechains, backbone repulsive charge) (Table 1.3). An irregular polysaccharide glycosidic linkage or monosaccharide repeat tends to promote polymer flexibility, which can reduce opportunity for intermolecular association and aid solubility. The presence of regular sidechains or derivatized polysaccharide hydroxyl groups can introduce steric hindrance and molecular repulsion (if substituents are charged), which minimize polysaccharide intermolecular associations, leading to increased solubility [3]. The basis for the increased viscosity of polysaccharide solutions (relative to pure water) varies according to polysaccharide concentration. The viscosity of a polysaccharide system within the dilute regime arises from the restructuring of water at the polysaccharide-water interface, and represents the collective (additive) effect of individual polysaccharide molecules in solution [10]. At more intermediate concentrations, typical of industrial applications, intermolecular effects become more predominant. As a result of being in constant dynamic motion, a polysaccharide molecule in solution sweeps out or occupies a theoretical volume or domain of spherical shape [10]. With increasing polysaccharide concentration, the probability for individual polysaccharide molecular domains to collide or overlap becomes increasingly likely, leading to entanglements, internal friction, and increased viscosity [3,11]. The polysaccharide concentration at which interpenetration of polymer domains occurs is referred to as the overlap concentration, and coincides with a concurrent

rise in the slope of the viscosity increase in response to an increasing polysaccharide concentration [11]. Aside from concentration effects, molecular characteristics of polysaccharides greatly influence solution viscosity. The greater the theoretical volume swept out by a polysaccharide molecule in motion, the greater the resulting viscosity (assuming a constant concentration). Thus, in principle, the volume swept out by a polysaccharide in solution is a function of both molecular size (DP) and shape (three-dimensional structure) [3]. While a polysaccharide of high molecular weight or DP might generally be expected to sweep out a greater volume compared to a glycan of relatively smaller size, the factor of molecular shape must also be considered. A random coil (highly flexible) structure will occupy a smaller spherical solution domain than that of a stiff, rod-like extended structure of equal molecular size (Figures 1.14a and 1.14b) [3]. Likewise, with the continued assumption of equal molecular weight, a highly branched polysaccharide is anticipated to exhibit a more compact shape and smaller volume in solution compared to that of a highly linear, extended glycan (Figures 1.14b and 1.14c) [3]. Thus, linear, high-molecular-weight polysaccharides capable of forming ordered secondary (helical) and/or tertiary (double-helical) rod-like, extended structures generally produce highly viscous solutions (at relatively low concentrations). As previously described, formation of ordered secondary or tertiary structures is generally favored by extended regions of chain uniformity (regularity of monosaccharide sequence and glycosidic linkage). Nevertheless, some degree of chain disruption (presence of sidechain, charged, or derivatized moieties

1-14

(a) Random coil


(b) Extended rod

(c) Branched structure

FIGURE 1.14 Comparison of theoretical solution volumes occupied or swept out by (a) a random coil, (b) a somewhat rigid rod, and (c) a branched macromolecule with the assumption of identical molecular weight.

along backbone, etc.) is often necessary to retain polysaccharide solubility (Table 1.2) [3,5]. In particular, charged groups along the polysaccharide backbone tend to keep

FIGURE 1.15 Schematic representation of a generalized polysaccharide gel structure consisting of segments of aggregated, ordered polysaccharide molecules (double helices) that comprise junction zones (intermolecular cross-links) stabilizing a porous, continuous three-dimensional network or suprastructure. Void spaces are occupied by entrapped solvent (water) and unordered (fully solvated) portions of polysaccharide molecules to yield a viscoelastic material.

polysaccharides in extended form by way of intramolecular repulsion, and enhance solubility and increase viscosity. The ability to form viscoelastic (combination of both liquid-like (viscous) and solid-like (elastic) behavior) gels represents another significant physical property inherent to many polysaccharide systems. A polysaccharide gel typically consists of some form of an open, continuous, three-dimensional network of aggregated solute macromolecules (polysaccharides) capable of entrapping significant volumes of solvent molecules (water) (Figure 1.15) [3]. The polysaccharide network is generally reinforced through limited aggregation of secondary- and/or tertiaryordered polysaccharide molecules that form regions of supramolecular quaternary structure termed junction zones (intermolecular cross-links) [3,5]. Junction zones may be anchored by a range of stabilizing forces (hydrogen bonds, hydrophobic interactions, electrostatic forces, van der Waals attractions, molecular entanglement, etc.) defined by the polysaccharide structure and solvent conditions. Regions of polysaccharide molecules not involved in junction zone structure maintain strong interaction with water molecules to achieve a delicate balance between the solute-solute (junction zone structure) and solute-solvent (soluble polysaccharide) interactions that constitute a gel. In general, the polysaccharide structural features that promote gel formation (junction zone development) are similar to those previously described to favor development of secondary- and/or tertiary-ordered structures (characteristics that encourage chain regularity). Nevertheless, to achieve gel stability, most gelling polysaccharides also possess some degree of structural perturbation or disruption that breaks up or limits the formation of the ordered arrangement at sites along the length of polysaccharide chains (Table 1.3) [5]. Such disruptions prevent excessive growth or development of junction zones that would otherwise lead to syneresis (loss of water-holding capacity) and gradual precipitation of polysaccharide molecules [3]. Specific structural features that serve this purpose include: occasional irregularity within the chain primary structure (e.g., carrageenan); occurrence of mixed blocks of monsaccharides within the primary chain (e.g., alginate); and presence of short, sporadic sidechains (e.g., locust bean gum in mixed gel systems with xanthan or carrageenan), substituent groups (e.g., hydroxypropylated starch), or charged moieties (e.g., high-methoxyl pectin). Formation of a stable gel structure also requires manipulation of solvent conditions to meet gelling requirements imposed by the specific structural features of a polysaccharide. Addition of lowmolecular-weight solutes (acids, salts, sugar, etc.) or adjustment of temperature may also be used to encourage polysaccharide interaction (reduction of solvation), and regulate the balance of attractive and repulsive forces that coincide with the formation of a stable gel system.


D. POLYSACCHARIDE STABILITY AND REACTIVITY Polysaccharides are subject to a range of environments and conditions in food systems that have the potential to alter not only their conformations, but also their chemical structures and behaviors. A primary means by which molecular structure is significantly altered occurs through the cleavage of glycosidic bonds (depolymerization), which transpires by two primary means, hydrolysis and β-elimination reactions. The mechanism of chain cleavage by hydrolysis, which may be initiated by acids or enzymes, was described in an earlier section (Section II, Figure 1.5). While the rate of acid-catalyzed hydrolysis is influenced by pH (lower = faster rate), temperature, and time of exposure, it also varies with the nature of the glycosidic linkage [3]. For example, the rate of acid-catalyzed hydrolysis for uronic acid-based polysaccharides (e.g., alginate) is significantly slower than for corresponding neutral polysaccharides. For enzyme-catalyzed hydrolysis, polysaccharides such as starch can be readily hydrolyzed into maltose and branched oligosaccharides by treatment with β-amylase (exo-glucanase), which cleaves terminal maltosyl residues from starch polysaccharides. In contrast, α-amylase (endoglucanase) cleaves α(1→4)-linked bonds at random points along the polysaccharide chain affording oligosaccharide products. Thus, for various polysaccharides, the pattern of enzymatic hydrolysis may differ according to the specific enzymes employed. Lastly, polysaccharide depolymerization by means of beta-elimination is favored under alkaline conditions, and requires oxidation at O-2, O-3, or O-6 for the reaction to proceed as depicted below (Figure 1.16). Aside from conditions encountered within food systems, it is important to note that depolymerization reactions are often intentionally employed in the production schemes of many commercial polysaccharides [3]. The reactivity of polysaccharides is also frequently manipulated to improve and extend their physical properties. The reactions described earlier in relation to monosaccharides (Section II) are also pertinent to polysaccharides, and generally involve derivatization of polysaccharide

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hydroxyl groups. The extent of chemical modification is most commonly described by the degree of substitution (DS). Most individual monosaccharide units within a polysaccharide structure possess an average of three hydroxyl groups available for reaction. The DS, which may exhibit a maximum value of three, depicts the average number of modified hydroxyl groups per glycosyl unit [3]. For reactions in which it is possible for a substituent group resulting from reaction with a polysaccharide hydroxyl group to further react with another reagent molecule, the degree of reaction is described in terms of molar substitution (MS), which is defined as the average number of moles of reactant per glycosyl unit [3].

VI. POLYSACCHARIDE STRUCTURES AND FUNCTIONS Polysaccharides of commercial significance will be discussed in terms of their structural constituents that are ultimately responsible for their observed properties. The discussion of specific polysaccharides is anticipated to highlight the diversity of structures and functions common to food systems, but is not intended to represent a comprehensive list of polysaccharides present in foods either naturally or as added ingredients.

A.

STARCH AND ITS DERIVATIVES

As the primary storage medium in higher plants, starch in its simplest form consists of two diverse homopolymers, amylose (linear structure) and amylopectin (branch-onbranch structure), both of which are comprised exclusively of D-glucosyl units (Figure 1.17a and 1.17b). The linear fraction, amylose, consists of (1→4)-linked α-D-glucopyranosyl units, and has a molecular weight in the range of 30,000 to greater than 106, depending on source [3]. While the amylopectin backbone exhibits a primary structure identical to that of amylose, it also possesses sidechains of (1→4)-linked α-D-glucosyl units (average chain length

FIGURE 1.16 A possible mechanism for depolymerization of pectin, which possesses native carboxylate and carboxy methyl ester groups, via β-elimination.

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(a)

(b)

FIGURE 1.17 Idealized diagrams depicting the linear and branch-on-branch structures of starch molecules, (a) amylose and (b) amylopectin, respectively (∅ depicts the molecular reducing end).

of 20–30 units) attached to the main chain through α(1→6) linkages. The sidechains themselves give rise to further branches to yield large, yet compact, branch-onbranch structures of significant molecular weight (approaching 109) (Figure 1.17b) [3,12]. Starch is unique in the sense that amylose and amylopectin molecules are biosynthesized and assembled in the form of semi-crystalline aggregates, called granules, which vary in size (1–100 µm) and shape (spherical, elliptical, angular, lenticular, etc.) according to the botanical source. Starch granules, which are stabilized by regions of complex molecular order (double-helical association of polymer chains), are insoluble in room temperature water. Slurries of starch granules in water require heating sufficient to

disrupt the native granular structure to achieve solubility and realize the functionality of starch [3]. Heating of starch granules in water brings about gelatinization or the irreversible loss of granular order, which is accompanied by increased granule hydration, swelling, and leaching of soluble components (primarily amylose) [3,12–14]. In the presence of shear, the fragile, swollen granules are reduced to a paste composed of granule remnants dispersed within a continuous phase of solubilized starch. As the paste is cooled, the linear amylose molecules retrograde (crystallize), adopting regions of doublehelical structure, which through aggregation, form junction zones that comprise a continuous three-dimensional gel network (Figure 1.18) [15]. The dispersed phase of a starch gel network consists of amylopectin-rich regions and granule remnants. The branched nature of amylopectin limits its intermolecular association, and favors initial water solubility, though amylopectin chains do slowly interact (crystallize) in time [3,12]. Thus, waxy starches, which contain only amylopectin, lack the ability to form strong gel networks, but are nevertheless capable of generating highly viscous solutions over time at starch levels above the overlap concentration via the development of weak intermolecular associations. Due to their properties and abundance, starches of varied biological origin are frequently exploited as thickeners, gelling agents, binding agents, texture modifiers, and substrates in diverse food applications. However, most food starch (≈75%) added as an ingredient is first chemically and/or physically modified [16], while yet in the granular form, to enhance the physical properties of starch polymers in accordance with the intended end-use. Several categories of starch derivatives will be discussed briefly, though in reality most commercial starch derivatives undergo multiple modifications.

FIGURE 1.18 Schematic representation of the structural changes associated with the gelatinization and pasting of native starch granules. Gelatinization (loss of granular molecular order) is accompanied by granule swelling and leaching of soluble starch components (amylose) during aqueous heating. With the application of shear, swollen granules undergo further disintegration to yield a paste, which is composed of a continuous phase of solubilized starch and a dispersed phase of granule remnants. Upon cooling, amylose retrogradation (depicted by the cross-hatching between molecules within the paste) results in the formation of a gel network.


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O

(a) Starch

O

P

OH

O O

(b) Starch

O

P

O

Starch

O

FIGURE 1.19 Chemical structures of a (a) stabilized (starch monophosphate) and (b) cross-linked (distarch phosphate) starch derivatives.

Starch stabilization generally involves conversion of starch hydroxyl groups to phosphate monoesters (DS ⱕ 0.002), acetate esters (DS ⱕ 0.09), or hydroxypropyl ethers (MS ⱕ 0.1) [3]. Modification is employed to overcome the tendency for syneresis of native starch pastes, which occurs due to excessive junction zone growth. The periodic incorporation of bulky (hydroxypropyl) and/or charged (phosphate monoester) substituent groups onto starch molecules (particularly amylose) introduces a physical and/or electrostatic impediment to intermolecular association and formation of ordered structures (Figure 1.19a) [3,17]. By regulating junction zone growth, stabilized starches exhibit improved paste clarity and syneresis/freeze-thaw stability in comparison to their native counterparts [3,17]. Cross-linked food starches are most frequently generated through reaction with phosphorus oxychloride or sodium trimetaphosphate, and exhibit of low levels of distarch phosphate ester cross-links (one per 1000–2000 glycosyl units) between adjacent starch molecules and/or chains (Figure 1.19b) [17]. While the presence of crosslinks generally reduces the swelling of granules during gelatinization, it also contributes stability and rigidity to the swollen granule structure (less breakdown with shear), leading to a higher ultimate paste viscosity (compared to the unmodified starch) [17]. Due to the reinforced granule structure, cross-linked starches display good stability to shear, acidic conditions, and extended heating, and are utilized in a broad array of food systems (retorted, extruded, frozen, baked, and dehydrated applications) [3]. Acid-modified starch results from treatment of granular starch with dilute acid to effect partial hydrolysis of starch molecules within granule amorphous (disordered) regions [17]. While retaining their granular shape, acid-modified starch granules display minimal swelling and almost complete disintegration upon heating in water. Most importantly, hot pastes of acid-modified starches exhibit very low viscosities (breakdown of swollen granules), and are easily pumped while hot, but form stiff, opaque gels upon cooling [17]. Acid treatment of starch increases the proportion of

linear starch molecules (due to hydrolysis of branched starch chains), which facilitate development of tertiary- and quaternary-ordered structures that comprise a gel network. Primary applications of acid-thinned starches involve production of gelled candy products [3]. Generation of pregelatinized or cold-water swelling starches requires the partial or complete disruption of the native granule structure (molecular order) by pre-processing (heating) a starch slurry under prescribed conditions [3]. The resulting starch products exhibit either ambient temperature solubility (pregelatinized) or granule swelling (cold-water swelling) to achieve viscosity development in aqueous environments without the requirement of additional heating. Pregelatinized and cold-water swelling starches are incorporated as both thickening and gelling agents in dehydrated and/or instant food products that do not require heat preparation. Lastly, starch is the substrate for an assortment of carbohydrate ingredients classified as starch hydrolyzate products, which include maltodextrins, dextrose (commercial name for glucose), corn syrups, and high fructose syrups (HFS) [18]. Generation of these products involves variable degrees of acid and/or enzyme conversion of starch to lower-molecular-weight polysaccharides, oligosaccharides, and glucose. With the exception of some maltodextrins (bulking agent), all other noted starch hydrolyzate products (sweeteners) are reduced in molecular size to the point they are no longer classified as polysaccharides.

B.

CELLULOSICS

As the most abundant component of biomass on the planet, cellulose is the key structural constituent of plant primary cell walls. It consists of long, linear chains composed solely of (1→4)-linked β-D-glucopyranosyl units (Figure 1.12a) [3]. As previously described, the nature of the cellulose glycosidic linkage, its regular monosaccharide sequence, and its linear backbone causes cellulose molecules to adopt flat, rigid, ribbon-like secondary structures that readily aggregate to form crystalline, waterinsoluble superstructures [3]. Thus, in the native state, while cellulose represents a good source of dietary fiber in indigenous whole foods or in isolated form (referred to as powdered cellulose), it generally requires further processing or derivatization to enhance functionality for broader food use. Several such cellulose derivatives will be highlighted below. Microcrystalline cellulose (MCC), which is generated by acid-catalyzed hydrolysis of native crystalline cellulose fibers, can be categorized into two primary types, powdered and colloidal, based on processing scheme and function. Both are insoluble in water. For powdered MCC, hydrolysis is conducted to generate small crystalline

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fragments, which are spray-dried and agglomerated to yield open, porous, aggregates of crystals of desired size (20–100 µm typical) [3]. Powdered MCC is used as a bulking agent and flow aid in food systems. On the other hand, colloidal MCC is produced by applying mechanical shear to crystalline fragments (obtained by acid hydrolysis) sufficient to further reduce crystallite size to the colloidal range (0.2 µm) [3]. A second polysaccharide (generally one with a backbone negative charge) is added to stabilize the dispersed phase (cellulose crystals) by providing a physical and or electrostatic barrier to aggregation [3,19]. Functioning as a protective colloid, the second polysaccharide interacts with cellulose crystals along uncharged segments of its backbone, while its charged regions provide electrostatic repulsion to prevent excessive association of cellulose particles. The dried dispersion, known as colloidal MCC, functions in food as an emulsion stabilizer, thickener, or fat replacer depending upon the properties of the protective colloid. Production of carboxymethylcellulose (CMC) involves reaction of cellulose with chloroacetic acid, and converts native hydroxyl groups to carboxymethyl ethers (Figure 1.20a). For food applications, typical DS levels range from 0.4–0.95 [20]. The introduction of charged substituents along the cellulose backbone greatly enhances solubility (relative to that of native cellulose) by way of intermolecular repulsion [3]. At pH values above the carboxyl pKa, CMC molecules occur as extended linear structures and sweep out large molecular domains to form high viscosity solutions. Commercially, CMC is available in a range of molecular weights (viscosity grades) as are most food gums. It is utilized primarily as a thickener in a wide range of food applications [20]. Methylcellulose (MC) and hydroxypropylmethylcellulose (HPMC) are additional ether derivatives that offer unique properties to food systems. Methylcellulose is achieved through reaction of cellulose with methyl chloride (MS levels 1.6–1.9) (Figure 1.20b), while production of HPMC involves additional derivatization with propylene oxide (DS levels 0.07–0.34) (Figure 1.20c) [3]. Relative to CMC, significantly higher derivatization levels are required to achieve water-solubility of methylcellulose, which is only marginally enabled by the presence of ClCH2CO2− Na+

(a)

Rcell-OH

(b)

Rcell-OH + CH3Cl

+

NaOH

NaOH

Rcell-OH + H2C

C. GALACTOMANNANS: LOCUST BEAN AND GUAR GUMS Galactomannans of significance include guar and locust bean (carob) gums, which commercially are the ground crude flours of their respective seed endosperm [3]. The primary polysaccharide component of both guar and locust bean gums possesses a backbone structure comprising of (1→4)-linked β-D-mannopyranosyl units with the occurrence of solitary α-D-galactopyranosyl units attached glycosidically at C-6 of main-chain mannosyl units (Figure 1.21) [3,21]. While guar and locust bean gums have only low to moderate molecular weights (200,000 and 80,000, respectively) [21], the mannan backbone (extended ribbon-like structure) contributes molecular rigidity that facilitates a large hydrodynamic volume and development of high viscosity solutions [3]. The presence of sidechains (impede aggregation) enhances the solubility of both guar and locust bean gums relative to unsubstituted mannan, which forms insoluble, crystalline, intermolecular aggregates (akin to native cellulose) [21]. Though the two gums have similar structures, substitution with D-galactosyl units is more frequent in guar gum (about 1 of 2 backbone units substituted) and more evenly distributed over the length of the polysaccharide chains as compared to locust bean gum Rcell-O-CH2CO2− Na+ + NaCl + H2O

Rcell-O-CH3 + NaCl + H2O

O

(c)

bulky (but nonpolar) substituent groups distributed along the length of cellulose chains. The marginal solubility of MC becomes further reduced at increased temperatures (due to loss of water molecules of solvation, which facilitates intermolecular association of polymer chains, through hydrophobic interactions). The result is thermoreversible gelation over the temperature range of 50–90°C [3,20]. Due to the ability to form thermal gels, MC may provide a physical barrier against moisture loss and fat uptake during high-temperature frying operations. While HPMC also exhibits thermal gelation behavior, gels are typically weaker (relative to those of MC), and increase in softness with an increasing degree of hydroxypropylation (decreases hydrophobic nature and provides a physical barrier to intermolecular associations) [20]. In addition, HPMC exhibits good surface activity as a foam stabilizer [3].

OH C H

CH3

NaOH

Rcell-O-CH2-CH-CH3

FIGURE 1.20 Reactions used for generation of (a) carboxymethyl-, (b) methyl-, and (c) hydroxypropylcellulose derivatives.


HO

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κ-carrageenan (Figure 1.22) [3,21]. Thus, it is the differing patterns of sidechain substitution that primarily account for the basic differences in the properties of locust bean and guar gum.

OH CH2OH O OH O

D. ALGINATE OH O

H2C

O HO

O HO

Extracted from brown seaweeds, alginates are complex, linear, block copolymers composed of (1→4)-linked β-D-mannopyranosyluronic acid and α-L-gulopyranosyluronic acid (occurs in 1C4 chair conformation) units [3,5,22]. Three major types of primary structure generally describe the polymer backbone of alginate: 1) uninterrupted sections of D-mannuronate units (M blocks), 2) uninterrupted regions of L-guluronate units (G blocks), and 3) intermingled sequences of D-mannuronate and L-guluronate units (mixed or MG blocks) (Figure 1.23a) [23]. The occurrence of multiple, primary structural regimes within a single molecule has significant consequences on alginate three-dimensional structure and properties. Due to backbone charge, alginate molecules adopt an extended solution structure consisting of sections of M blocks (ribbon-like structure), G blocks (buckled shape), and MG blocks (irregular coil). In the presence of divalent cations, alginate forms gel structures that are described by the egg-box model (Figure 1.23b) [24]. In this model, junction zones are stabilized by divalent cations, which provide electrostatic cross-bridges between oriented G block regions of adjacent molecules. While the M and MG blocks do not participate in junction zone formation, they do serve to balance intermolecular associations by breaking up G block regions and limiting excessive junction zone growth. At excessively low pH values (below the pKa of the carboxylate group), intermolecular electrostatic repulsion is lost, and precipitation can occur [3].

OH O CH2OH

FIGURE 1.21 Generalized structural repeat of galactomannans.

FIGURE 1.22 Schematic representation of the junction zone gel structure between locust bean gum “naked regions” and xanthan or carrageenan double-helical segments.

(about 1 of 4 backbone units substituted with irregular sidechain distribution) [3,21]. The regular substitution pattern of guar gum minimizes intermolecular associations, and explains the excellent water solubility and nongelling behavior of this polysaccharide. The “naked regions” (large polymer sections devoid of sidechains) of locust bean gum afford open segments of the main chain capable of intermolecular interaction, and account for the gel-forming capabilty with xanthan gum and (a)

O HO

O

HO

COO− O

HO

COO− OH O

M

O HO

HO

O

O

HO

O

O HO

O

COO − OH O

OH

O OH COO−

O

G

O−

(b) Ca++

Ca++

Ca++

M

M HO O

O

HO

G

O − OOC

COO− OH O

M

COO−

HO

OH

G

HO

O

O− Ca++

G

G

O HO

O

O HO

COO− OH O

M

FIGURE 1.23 Depiction of alginate (a) G block, M block, and MG (mixed) block conformational structures and (b) the contribution of each respective conformation to junction zone gel structure characterized by the egg-box model.

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E.


PECTIN

Pectin, a cell-wall associated polysaccharide of higher plants, is a predominantly linear glycan consisting of αD-galactopyranosyluronic acid units, some of which are present in a methyl ester form [3,25,26]. The polygalacturonate chain may also be disrupted by the occasional insertion of an α-L-rhamnopyranosyl unit [3,25,26] and the presence of sporadic, highly branched segments (hairy regions) [25,26], both of which introduce backbone irregularity (though hairy regions are mostly removed during preparation of commercial pectin). Commercially, pectins are categorized according to their degree of esterification as either low-methoxyl (LM; 50% esterified), which designation also defines the optimum conditions in which they gel [3]. LM pectins form gels in the presence of divalent cations, and align to form an “egg box” junction zone structure similar to that previously depicted for alginate (LM pectin and alginate G blocks possess almost mirror image secondary structures). For HM pectin, solvent conditions must be adjusted to reduce both polysaccharide solvation and intermolecular repulsion (due to ionized carboxylate groups) to facilitate junction zone development. In food systems, the addition of competing solute (usually sugar; 55% minimum) and acid (to achieve a pH ι > λ (non-gelling), which is inversely related to the degree of polysaccharide molecular charge.

G.

AGAR

Agar, which is also derived from specific species of red seaweed, exhibits a chemical structure similar to that of κ-carrageenan, except that the second unit of the characteristic disaccharide repeat is a 3,6-anhydro-α-L-galactopyranosyl unit (in carrageenans, D-entantiomer is present instead) (Figure 1.24d) [29]. Similar to the carrageenans, the backbone structure is interrupted by an occasional kink (presence of sulfate hemiester at C-6 of the α-L-galactosyl unit),


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FIGURE 1.25 Representation of the gelation mechanism and junction zone structure for κ-carrageenan gels, in which potassium ions (•) shield backbone negative charges to allow intermolecular interaction of double-helical polysaccharide segments. The presence of “kinks” (occasional backbone structural irregularity) lends stability to the gel by limiting junction zone size and growth. Agar gels are believe to possess a similar gel mechanism and structure, except that potassium ions are not required to bring about gelation.

though the sulfate content of agar is nominal (1.5–2.5%) compared to that of carrageenan (>20%) [30]. While heating (85°C) is required to bring about water solubility, the lack of consistent negative charge along the length of the agar backbone results in a fairly flexible, non-extended polymer chain of relatively low viscosity (in comparison to carrageenan) [29]. However, upon cooling (40°C), agar molecules undergo a transition to an ordered double-helical tertiary structure, which leads to intermolecular aggregation of sections of ordered polymer chains and development of a quaternary gel structure [23]. Agar junction zone and gel structure are thought to mimic that of gelling carrageenans, with the exception that counterions are not required to promote gel formation in agar (lack significant negative charge that would require shielding for intermolecular association to occur) (Figure 1.25). Similar to carrageenan, occasional kinks in backbone structure disrupt the double-helical arrangement, which in turn prevents excessive growth of junction zones and aids gel stability [29].

H.

XANTHAN

Xanthan is the common name for the heteroglycan isolated from the bacterium Xanthomonas campestris. While xanthan has a backbone primary structure identical to that of cellulose, it differs from cellulose in that it possesses a trisaccharide sidechain glycosidically attached to O-3 of alternating backbone units [3,31]. The sidechain consists of two mannosyl units separated by a glucuronic acid unit (Figure 1.26). Approximately half of the terminal

mannose units of the sidechain contain pyruvic acid, linked at C-4 and C-6 via a cyclic acetal structure, while the nonterminal mannosyl units contain an acetyl substituent attached at C-6. The presence of the trisaccharide sidechain, which reduces intermolecular associations (due to electrostatic repulsion and steric hindrance), is thought to account for the excellent water solubility of xanthan relative to that of native cellulose (water-insoluble) [32]. Xanthan forms highly viscous, pseudoplastic (shear-thinning) solutions at low concentrations, which solutions are stable to viscosity change over a wide range of pH (1–12), salt concentration (up to 0.7%), and temperature (0–95°C) [3]. The relatively high-viscosity solutions are attributable to a high molecular weight (2–10 ⫻ 106) and molecular rigidity derived from its ordered conformation, which is thought to consist of an extended double-stranded helix [33]. At a temperature of 120°C, xanthan solutions lose up to 98% of their original viscosity due to loss of molecular order and rigidity [34]. At reduced temperature, xanthan solutions regain up to 80% of their original viscosity as molecules appear to reform the ordered conformation [34]. Due to its unique solution behavior, xanthan is used as a multipurpose thickener in a wide range of food applications.

I. GUM ARABIC Gum arabic, also known as acacia gum, is the exudate material of the acacia tree common to the Sahel zone of

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O HO

O O

Backbone

HO2HC HO HO

OH OH O

HO O

HO H3C O

O

− OOC

OH

OH

n

* O CH2OH m

O O

O

O O HO

O HO

CH2

CH3 − OOC

HO

O O O CH2OH

OH O

CH2OH O

HO

CH2OH O

OH OH O

HO O

− OOC

OH

HO H3C O

O

CH2 O

O

FIGURE 1.26 Chemical structural repeat of xanthan.

FIGURE 1.27 Representation of the branched chemical structure and composition of gum arabic.

Africa [35,36]. The gum contains 2–3% protein (gives rise to emulsification capability), which is covalently bound to the polysaccharide component [3]. Chemically, gum arabic has a (1→3)-linked backbone of β-D-galactopyranosyl units, which constitute approximately 40% of the total monosaccharide content of the gum (Figure 1.27) [3,36]. Further, the gum arabic backbone is highly substituted with sidechains (which themselves may give rise to further branching), producing a highly branched structure. It contains at least four additional types of monosaccharide units (L-arabinofuranosyl, L-rhamnopyranosyl, D-glucopyranosyluronic acid, and 4-O-methyl-D-glucopyranosyluronic acid units) attached to the branched backbone [3,35].

Due to its highly branched nature, gum arabic, though of substantial molecular weight (580,000), possesses a very compact three-dimensional structure, which provides the basis for its most unique physical properties, its astronomical solubility, and low viscosity (up to 50% gum solutions may be prepared) [3]. The compact nature of gum arabic molecules is best comprehended by the fact that gum solutions of up to 10% (w/v) display Newtonian flow behavior, and that it is not until 30% (w/v) solutions are achieved that steric overlap of individual molecular domains begins to occur accompanied by a more substantial rise in viscosity as a function of increasing gum concentration [36].


REFERENCES 1. P Collins, R Ferrier. Monosaccharides: Their Chemistry and Their Roles in Natural Products. Chichester: John Wiley & Sons Ltd, 1995. 2. J Lehmann. Carbohydrates Structure and Biology. Stuttgart: Georg Thieme Verlag, 1998. 3. RL Whistler, JN BeMiller. Carbohydrate Chemistry for Food Scientists. St. Paul, MN: Eagan Press, 1997. 4. M Friedman. Chemistry, Biochemistry, and Safety of Acrylamide. A review. J Agric Food Chem 51: 4504–4526, 2003. 5. D Oakenfull. Polysaccharide molecular structures. In: RH Walter. ed. Polysaccharide Association Stuctures in Food. New York: Marcel Dekker, Inc., 1998, pp. 15–36. 6. ER Morris. Polysaccharide structure and conformation in solutions and gels. In: JMV Blanshard, JR Mitchel. eds. Polysaccharides in Food. London: Butterworths, 1979, pp. 15–31. 7. CT Chuah, A Sarko, Y Deslandes, RH Marchessault. Triple helical crystal curdlan and paramylon hydrates. Macromolecules 16:1375–1382, 1983. 8. Y Deslandes, RH Marchessault, A Sarko. Triple helical structure of (1-3)-β-D-glucan. Macromolecules 13:1466– 1471, 1980. 9. D Oakenfull. Gelling agents. Crit Rev Food Sci Nutrit 26:1–26, 1987. 10. RH Walter. Origin of polysaccharide supramolecular assemblies. In: RH Walter. ed. Polysaccharide Association Structures in Food. New York: Marcel Dekker, Inc., 1998, pp. 1–13. 11. ER Morris. Polysaccharide rheology and in-mouth perception. In: AM Stephen. ed. Food Polysaccharides and Their Applications. New York: Marcel Dekker, Inc., 1995, pp. 517–546. 12. CG Biliaderis. Structures and phase transitions of starch polymers. In: RH Walter. ed. Polysaccharide Association Structures in Food. New York: Marcel Dekker, Inc., 1998, pp. 57–168. 13. P Colonna, A Buleon. New insight on starch structure and properties. In: P Feillet. ed. Cereal Chemistry and Technology: A Long Past and a Bright Future. Paris: Ninth International Cereal and Bread Congress, 1992, pp. 25–42. 14. DJ Gallant, B Bouchet, PM Baldwin. Microscopy of starch: evidence of a new level of granule organization. Carbohydr Polym 32:177–191, 1997. 15. JJG van Soest, D de Wit, H. Turnois, JFG Vliegenthart. Retrogradation of potato starch as studied by Fourier transform infrared spectroscopy. Starch 46:453–457, 1994. 16. RJ Alexander. Carbohydrates used as fat replacers. In: RJ Alexander, HF Zobel. eds. Developments in Carbohydrate Chemistry. St. Paul, MN: American Association of Cereal Chemists, 1992, pp. 343–370. 17. OB Wurzburg. Modified starches. In: AM Stephen. ed. Food Polysaccharides and Their Applications. New York, Marcel Dekker, Inc., 1995, pp. 67–97. 18. PH Blanchard, FR Katz. Starch hydrolysates. In: AM Stephen. ed. Food Polysaccharides and Their Applications. New York, Marcel Dekker, Inc., 1995, pp. 99–122.

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19. GS Buliga, GW, Ayling, GR Krawczyk, EJ McGinley. Microcrystalline cellulose technology. In: RH Walter. ed. Polysaccharide Association Stuctures in Food. New York: Marcel Dekker, Inc., 1998, pp. 169–225. 20. DG Coffey, DA Bell. Cellulose and cellulose derivatives. In: AM Stephen. ed. Food Polysaccharides and Their Applications. New York: Marcel Dekker, Inc., 1995, pp. 123–153. 21. JE Fox. Seed gums. In: A Imeson. ed. Thickening and Gelling Agents for Food. 3rd ed. Gaithersburg, MD: Aspen Publishers, Inc., 1999, pp. 262–283. 22. E Onsoyen. Alginates. In: A Imeson. ed. Thickening and Gelling Agents for Food. 3rd ed. Gaithersburg, MD: Aspen Publishers, Inc., 1999, pp. 22–44. 23. D. Oakenfull. Gelation mechanisms. Food Ingredients J Jpn 167:48–68, 1996. 24. GT Grant, ER Morris, DA Rees, PJC Smith, D Thom. Biological interactions between polysaccharides and divalent cations: The egg-box model. FEBS Lett 32:195–198, 1973. 25. AGJ Voragen, W Pilnik, J-F Thibault, MAV Axelos, CMGC Renard. Pectins. In: AM Stephen. ed. Food Polysaccharides and Their Applications. New York: Marcel Dekker, Inc., 1995, pp. 287–339. 26. CD May. Pectins. In: A Imeson. ed. Thickening and Gelling Agents for Food. 3rd ed. Gaithersburg, MD: Aspen Publishers, Inc., 1999, pp. 230–261. 27. D Oakenfull, A Scott. Hydrophobic interaction in the gelation of high methoxyl pectins. J Food Sci 49:1093, 1984. 28. L Piculell. Gelling carrageenans. In: AM Stephen. ed. Food Polysaccharides and Their Applications. New York: Marcel Dekker, Inc., 1995, pp. 205–244. 29. NF Stanley. Agars. In: AM Stephen. ed. Food Polysaccharides and Their Applications. New York: Marcel Dekker, Inc., 1995, pp. 187–204. 30. R Armisen. Agar. In: A Imeson. ed. Thickening and Gelling Agents for Food. 3rd ed. Gaithersburg, MD: Aspen Publishers, Inc., 1999, pp. 1–21. 31. S Kitamura, K Takeo, T Kuge, BT Stokke. Thermally induced conformational transition of double-stranded xanthan in aqueous salt solutions. Biopolymers 31:1243–1255, 1991. 32. G Robinson, CE Manning, ER Morris, ICM Dea. Sidechain-mainchain interactions in bacterial polysaccharides. In: GO Phillips, DJ Wedlock, PA Williams. eds. Gums and Stabilisers for the Food Industry 4. Washington, DC: IRL Press Ltd, 1987. 33. A Gamini, M Mandel. Physicochemical properties of aqueous xanthan solutions: static light scattering. Biopolymers 34:783–797, 1994. 34. M Glicksman. Food Hydrocolloids, Vol 1. Boca Raton, FL: CRC Press, Inc., 1982. 35. AM Stephen, SC Churms. Gums and mucilages. In: AM Stephen. ed. Food Polysaccharides and Their Applications. New York: Marcel Dekker, Inc., 1995, pp. 377–440. 36. MV Wareing. Exudate gums. In: A Imeson. ed. Thickening and Gelling Agents for Food. 3rd ed. Gaithersburg, MD: Aspen Publishers, Inc., 1999, pp. 86–118.

2

Carbohydrates: Physical Properties

Qi Wang and P.J. Wood

Food Research Program, Agriculture and Agri-Food Canada

CONTENTS I. II.

Introduction..............................................................................................................................................................2-2 Conformation of Carbohydrates ..............................................................................................................................2-2 A. Monosaccharides ..............................................................................................................................................2-2 B. Oligosaccharides ..............................................................................................................................................2-3 C. Polysaccharides ................................................................................................................................................2-3 1. Ordered Structures in the Solid State..........................................................................................................2-3 2. Secondary and Tertiary Structures in Solutions and Gels ..........................................................................2-4 D. Physical Techniques Used to Study Carbohydrate Conformation ..................................................................2-4 1. X-Ray Diffraction........................................................................................................................................2-4 2. Light, X-Ray, and Neutron Scattering ........................................................................................................2-5 3. Chiroptical Methods....................................................................................................................................2-6 4. Microscopy Techniques ..............................................................................................................................2-6 5. Nuclear Magnetic Resonance......................................................................................................................2-7 III. Molecular Weight and Molecular Weight Distribution ..........................................................................................2-8 A. Polydispersity and Molecular Weight Averages ..............................................................................................2-8 B. Physical Methods for Molecular Weight Determination ..................................................................................2-9 1. Osmometry ..................................................................................................................................................2-9 2. Static Light Scattering ................................................................................................................................2-9 3. Sedimentation............................................................................................................................................2-10 4. Viscometry ................................................................................................................................................2-10 5. Gel Permeation Chromatography..............................................................................................................2-11 6. Other Methods ..........................................................................................................................................2-11 IV. Hydration and Solubility of Carbohydrates ........................................................................................................2-11 A. Low-Molecular-Weight Carbohydrates ..........................................................................................................2-11 B. Polysaccharides ..............................................................................................................................................2-11 C. Dissolution Kinetics........................................................................................................................................2-12 V. Rheological Properties of Polysaccharides ..........................................................................................................2-13 A. Concentration Regime ....................................................................................................................................2-13 B. Dilute Solutions ..............................................................................................................................................2-13 1. Steady Shear Viscosity ............................................................................................................................2-13 2. Intrinsic Viscosity......................................................................................................................................2-13 C. Semi-Dilute and Concentrated Solutions........................................................................................................2-13 1. Steady Shear Viscosity ..............................................................................................................................2-13 2. Concentration and Molecular Weight Effects ..........................................................................................2-14 3. Temperature and Ionic Strength Effects....................................................................................................2-14 4. Dynamic Properties ..................................................................................................................................2-14 D. Polysaccharide Gels ........................................................................................................................................2-15 1. Gelation Mechanism ................................................................................................................................2-15 2. Physical Properties of Polysaccharide Gels ..............................................................................................2-15 References ......................................................................................................................................................................2-15

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intermonomeric linkages. Abundant evidence has shown that most of the physical properties of carbohydrates depend on the size, shape, charge, and polarity of the individual molecules. The study of structure-function relationships has been an important topic of carbohydrate research, and advances in physical techniques continue to improve our understanding and provide more insight into these relationships.

I. INTRODUCTION Carbohydrates include monosaccharides, oligosaccharides, and polysaccharides as well as substances derived from them by various reactions such as reduction, oxidation, esterification, etc. Monosaccharides are the basic units from which all carbohydrates are built. Linking of monosaccharides via glycosidic bonds leads to the formation of oligosaccharides (2 to 20 monomers) and polysaccharides (more than 20 monomers). The term “sugars” is often used to refer to the monosaccharides and some disaccharides (e.g., sucrose). Polysaccharides are grouped into two major classes: (1) simple polysaccharides, which contain only monosaccharides and their derivatives (esters and ethers), and (2) conjugate polymers made up of a polysaccharide linked to another polymer, such as polypeptide. It is the purpose of this chapter to focus on the physical properties of simple carbohydrates and associated characterization techniques that are important to food sciences. As one of the three major food components, carbohydrates have enormous functions and applications. They not only supply most of the energy in the diet of humans, but also have various functionalities which are used to confer desired texture in foods. In these latter applications, the physical properties of carbohydrates, such as solubility, water holding capacity, and solution rheology, play important roles. Although containing similar building blocks, mono-, oligo-, and poly-saccharides have different physical properties. An extreme example of this is the contrast between the highly soluble monomeric glucose and the completely insoluble cellulose, which is a polymer of glucose. It has long been known that the configuration and conformation of sugars are the determinants of their chemical and physical properties, and those of oligosaccharides and polysaccharides inevitably depend on the constituent monosaccharide as well as the 1

3

CONFORMATION OF CARBOHYDRATES

A. MONOSACCHARIDES Most monosaccharides and their derivatives encountered in foods are polyhydric alcohols carrying a “reducing” keto or aldehydo unit, and they exist primarily in cyclic tetrahydropyran and tetrahydrofuran forms, with the latter occurring less frequently than the former. However, the common ketosugars are more likely than aldosugars to exist as furanoses. Seven-membered rings occur but are not common in foods. Free reducing sugars in solution may exist in different cyclic forms, which are in equilibrium via the acyclic aldehydo or keto form. There are three potentially stable shapes for the sixmembered saturated sugar rings, namely chair, boat, and skew (Figure 2.1). The chair conformation predominates in most cases because the widest separation of the electronegative oxygen atoms is usually achieved through equatorial orientations of most of the hydroxyl and CH2OH groups. The anomeric hydroxyl unit differs in that it may adopt two orientations (α or β), which are strongly influenced by the ring oxygen. Similarly, there are two principal conformations for saturated furanoid rings, described as envelope (E) and twist (T) (Figure 2.1), each with four or three coplanar atoms, respectively. Because of the low energy barriers between the E and

4

1

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5

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5 2

O

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3

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2 4

C

B

S 3

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O

5

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3

E

T

4

FIGURE 2.1 Examples of chair (C), boat (B), and skew (S) forms for pyranoid rings and envelope (E) and twist (T) forms for furanoid rings.


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T conformers, interconversions of these occur more readily than between the pyranoid forms. The shapes of acyclic aldehydo and keto carbohydrates and their reduced forms are usually described as either a linear (zigzag) or a sickle shape. The advent of diffraction and NMR techniques has allowed the determination of the configuration and conformation of almost all the important monosaccharides (1). In crystals, most molecules adopt a single conformation, whereas in solution there is generally more than one conformation undergoing fast interconversion. For a more detailed treatment of monosaccharide chemistry and nomenclature, the readers are referred to standard textbooks (2).

C. POLYSACCHARIDES

B. OLIGOSACCHARIDES

1. Ordered Structures in the Solid State

The conformation of oligosaccharides is less well documented than that of monosaccharides, although the naturally occurring common oligosaccharides are well characterized. Most data from x-ray diffraction and NMR analysis are limited to oligosaccharides having less than four monomeric units. There is considerable experimental difficulty encountered when applying these techniques to large oligosaccharides (3). However, although based on limited amount of data, some general features about the conformation of oligosaccharides can be drawn. Once incorporated into an oligosaccharide or polysaccharide chain, the monosaccharide ring is relatively rigid and the ring geometry becomes effectively fixed. Thus, the overall shape of oligosaccharides become more determined by the two torsion (dihedral) angles φ and ψ across the two single bonds of their connecting glycosidic linkage than by the unit geometries. Wells of minimum potential energy may be calculated, which limit the values adopted by φ and ψ but not rigidly so. Generally speaking, disaccharides should have a preference for staggered conformations about the two linkage bonds, unless there are geometric constraints imposed by, for example, a hydrogen bond between the two rings. The crystal structures of many oligosaccharides have been elucidated (3). Monosaccharides and certain oligosaccharides possess definite crystalline structures, and thus have well-defined melting points and solubilities.

A repeated sequence of monomers or oligomers leads to an ordered and periodic conformation of polysaccharide molecules. The different linkage types, arising from the anomeric nature of glycosidic linkage and the orientation of OH units through which it is attached, impose certain general features on oligosaccharide and polysaccharide conformations because of the limitations placed on the dihedral angles. Fundamentally there are four different types of chain shapes: ribbons, hollow helices, loosely jointed, and crumpled types (4). For example, for β-(1→4) linked D-glucopyranosyl units, the two bonds from the ring to its two bridging oxygens define a zig-zag form, which promotes a tendency to adopt a flat, extended, ribbonlike conformation, in the polymer (Figure 2.2a). In contrast, when the links between the D-glucopyranosyl units are β-(1→3) or α-(1→4), they define a U-turn form (Figure 2.2b); this geometry extended over multiple units often produces a hollow helical conformation, which becomes stabilized in multiple helices. The linkage through the primary hydroxyl units, such as between (1→6) linked hexopyranose units, leads to a loosely jointed type of conformation and marked molecular flexibility in the resultant polysaccharides. This arises from the extra single bond and torsion angle (ω) between the two sugar rings that separates the rings, reducing inter-unit interactions and allowing a greater range of conformational

Similar to polypeptides, polysaccharides also have different levels of structures, although higher level structures are less well defined. The primary structure describes the covalent sequence of monosaccharide units and the respective glycosidic linkages. The secondary structure describes the characteristic shapes of individual chains such as ribbons and helices, which arise from repetition of units adopting a particular average orientation in shape. Polysaccharide chains with well-defined secondary structure (or sufficient areas of such) may interact with each other, leading to further ordered organizations incorporating a group of molecules. This is known as the tertiary structure. Further association of these ordered entities results in large quaternary structures.

O HO

H H

H

H

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OH H

H

OH H H

HO O

H

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H

H

OH

O HO H H

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H

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OH

O H

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O (a)

OH

H

OH

H

(b)

FIGURE 2.2 Examples of geometrical relationships across sugar rings. (a) Zig-zag relationship across 1,4-linked β-D-glucopyranose; (b) U-turn relationship across 1,3-linked β-D-glucopyranose.

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possibilities. A further type of conformation known as “crumpled,” such as in β-(1→2) linked glucopyranoxyl units, is less common in food carbohydrates. The regular conformation of polysaccharides can always be described as a helix, which may be defined by just two parameters, the number of units per turn of the helix, and the translation of each repeating residue along the helical axis. The resultant single helix may associate to form multiple helices, which are then further packed in various ways to form higher ordered structures in the solid state. The majority of polysaccharides in their native form exist in an amorphous structure, examples being the antiparallel, extended twofold ribbon-like organized chain structure in the family of mannans and galactomannans (5). A relatively small number of polysaccharides are organized into a repeating crystalline or partly crystalline structure, examples being cellulose, starches, chitin, and some β-D-glucans. The crystalline element is usually capable of existing in different polymorphic forms. The ordered structures of polysaccharides have been extensively studied by x-ray and electron diffraction (6), and the x-ray structures of more than 50 well-defined polysaccharides are known (7). 2. Secondary and Tertiary Structures in Solutions and Gels The extensively ordered conformation of a polysaccharide in the solid state may not be retained following hydration in solutions and gels. Polysaccharide chains tend to adopt a more or less coiled shape in solutions and fluctuate continuously between different local and overall conformations. A large group of non-gelling polysaccharides, or gelling polysaccharides in non-gelling conditions, exist in solutions with a conformation known as disordered random coils. Since polysaccharide molecules contain a large number of hydroxyl groups, they have a high tendency to associate into supramolecular aggregates through hydrogen bonding in aqueous solutions. For example, combining static and dynamic light scattering, a fringed micelle model was proposed for the aggregates formed in solutions by a number of neutral polysaccharides including tamarind xyloglucan (8) and cereal (1→3)(1→4)-β-glucans (9). The association of molecules in such a form markedly increases the stiffness of the single chains, leading to enhanced solution viscosity. More ordered structures may be developed, in solution through the so-called cooperative interactions, especially for polysaccharides in which identical repeat units result in a regularity of sequence. Conformational transitions in solution between random coils and helices have been well recognized and characterized for a number of polysaccharides such as curdlan, xanthan, and gellan (10). Under favorable conditions, these ordered structures may further associate, leading to the formation of three-dimensional gel networks.

D. PHYSICAL TECHNIQUES USED TO STUDY CARBOHYDRATE CONFORMATION A wide range of physical techniques has been used to study the structures of carbohydrates at different levels, i.e., molecular, macromolecular, and supramolecular structures (11). The use of such means as mass spectroscopy and molecular spectroscopy to elucidate the primary structure of carbohydrates will not be covered in this chapter. The purpose is to include only those physical techniques used for studying the conformation of carbohydrates in general, and for probing the higher level structures of polysaccharides. Generally there is a need to combine several physical techniques to provide complementary information about the structure of carbohydrates. 1. X-Ray Diffraction

a. Background X-ray diffraction and other types of diffraction methods (electron and neutron) have contributed to our understanding of the molecular geometry of carbohydrates. Diffraction is essentially a scattering phenomenon. When a monochromatic x-ray beam travels through a test specimen, a small proportion of the radiation is scattered with mutual reinforcement of a large number of scattered rays, and the resultant x-ray intensity in specific directions depends on the arrangement of the scattering atoms within the sample. X-ray scattering techniques are divided into two categories: wide-angle x-ray scattering (WAXS) and small-angle x-ray scattering (SAXS). Typically, SAXS gives information on a scale of ~ a few nanometers and smaller, while WAXS gives information on a scale of 1–1000 nm. WAXS is used to measure crystal structure and related parameters, which is the topic of this section. SAXS will be discussed in the next section together with light and neutron scattering techniques. The diffraction pattern, commonly recorded on photographic film, consists of an array of spots (reflections) of varying intensities, from which structural information for a chemical repeat may be deduced. If a large enough size of crystal can be prepared, it is usually possible to determine the crystal structure and hydrogen bonding to a high degree of accuracy. Information such as repeating unit cell dimensions, lattice type, space group symmetry and bond lengths, and valence angles can be derived from the analysis. b. Monosaccharides and oligosaccharides For almost all monosaccharides and many oligosaccharides with low degrees of polymerization, it is not a major problem to prepare single crystals for x-ray measurement. X-ray characterized structures are available for most of these molecules (3, 12–16). As an example, in the study of mannotriose (O-β-D-mannopyranosyl-(1→4)-O-β-Dmannopyranosyl-(1→4)-O-α-D-mannopyranose) (14), the unit cell was determined as monoclinic with dimensions of


a ⫽ 0.1183 nm, b ⫽ 0.1222 nm, and c ⫽ 0.9223 nm, and β ⫽ 112.34°; the space group was P21. The crystal structure includes three water molecules, two of which are involved in hydrogen bonding such that the mannotriose molecules occur effectively as sheets of long parallel chains, with each consecutive sheet having chains lying at approximately right angle to those in a neighboring sheet.

c. Polysaccharides Large oligosaccharides rarely and polysaccharides never form single crystals that are good enough for classical x-ray crystallography. They tend to form fibers that are amorphous, or at best only partly crystalline, starch being a typical example of the latter. X-ray study of starches mostly measures the degree of crystallinity and identifies different polymorphic forms. To obtain useful x-ray diffraction data from other more amorphous non-starch polysaccharides, oriented fibers or films are used (6). These polycrystalline fibers or films are prepared in such a way that the polysaccharide helices are preferentially oriented with their long axes nearly parallel. The x-ray diffraction intensities then provide information about the helical structures such as repeat spacing of the helix and helix screw symmetry, and if the diffraction pattern is sufficiently “crystalline,” the unit cell dimensions and lattice type. However, the x-ray data alone are inadequate to solve a fiber structure, and interpretation requires supplementation with molecular modeling analysis using existing stereochemical information derived from surveys of crystal structures of related mono- or oligosaccharides (7, 17). X-ray fiber diffraction is of great value in the determination of the conformations of polysaccharides. Studies of the (1→3)-β-D-glucan family, curdlan, schizophyllan, and scleroglucan, are good examples. Curdlan is a linear (1→3)-β-D-glucan, whereas schizophyllan and scleroglucan also contain some β-(1→6)-glucosyl branches. These (1→3)-β-D-glucans usually form triple-stranded helices (18). The structure of curdlan (in both hydrated and anhydrous forms), determined from oriented fibers, assume a right-handed, parallel, six-fold triple-helical conformation. There are interstrand O2…O2 hydrogen bonds in the hexagonal unit cell, with parameters a ⫽ b ⫽ 1.441 nm and c ⫽ 0.587 nm. The space group is P63 and there is one helix per unit cell (19). The short-branch substitutions on the main chain primary hydroxyls in schizophyllan and scleroglucan do not seem to affect the fundamental triplehelical structure (20). 2. Light, X-Ray, and Neutron Scattering

a. Background The principles on which light, x-ray, and neutron scattering depend are basically similar and can be treated by the same fundamental sets of equations. For all three modes of scattering, angular dependence of the normalized scattering

2-5

intensity provides information on the size and shape of the macromolecules. The resolving power of scattering techniques is related to the wavelength of the scattered radiation (21). The wavelengths are 0.1–0.3 nm for SAXS, 0.2–1.0 nm for small angle neutron scattering (SANS), and ~500 nm for light scattering. Conventional light scattering typically reveals only the global dimensions of a macromolecule, which may be tens to hundreds of nanometers for a typical polysaccharide. SAXS and SANS can probe molecular structures at closer ranges of about 2–25 nm (22). SANS may additionally observe the Gaussian behavior of polymer chains in their own bulk (solid), which conventional light scattering cannot. Light scattering is effective in measuring the angular dependence of intensity typically in the range 30° to 135°. SAXS can be carried out at very small angles, typically less than 1°, and is thus superior for the determination of the size and shape of macromolecules, but it is less convenient for the determination of molecular weight and second virial coefficient.

b. Application to polysaccharides Scattering measurements can be carried out in two modes, static and dynamic. The former measures the average scattering intensity within a selected time period, whereas the latter measures the fluctuation of the intensity over time. From static measurements, the weight average molecular weight (Mw), z-average radius of gyration (Rg), and the second virial coefficient can be extracted. From dynamic measurement, the translational diffusion coefficient is obtained from which the hydrodynamic radius (Rh) can be determined. The parameter, ρ = Rg/Rh, may provide information on the architecture of the macromolecules and their aggregates (23). From the combination of static and dynamic scattering data, other information may be derived including the linear mass density, Kuhn segment length, and polydispersity index. To obtain as much structural information as possible, experimental data from scattering are usually processed and presented through various plots, and need to be interpreted using molecular model such as the worm-like chain model (23). Light scattering was applied to study the solution properties of amyloses and the retrogradation of amyloses as early as the 1960s (24–26). A typical flexible chain behavior was observed for high-molecular-weight amyloses in freshly prepared aqueous solutions. With decreasing molecular weight, the tendency to aggregate increased considerably so that a stable aqueous solution could not be prepared. The many studies on amylopectin and glycogen demonstrated how scattering techniques may be used for investigating the branching behavior of polysaccharides (27). The branching nature of amylopectin and glycogen can be detected clearly by light scattering from the Zimm plot, which shows an upturn (28, 29). Scattering techniques can be used to probe the conformational transition of polysaccharides in solution. For

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example, the thermal transition evident in low ionic strength xanthan solutions was followed by light scattering (30). It was observed that the apparent hydrodynamic radius significantly decreases with increasing temperature in the vicinity of the helix-coil transition temperature. As discussed above (Section II.C.2), light scattering is also useful in investigating aggregation properties of polysaccharides. 3. Chiroptical Methods

a. Background Optical activity is one of the most readily and often measured physical properties of carbohydrates. Carbohydrates contain several similarly substituted asymmetric carbon atoms and are therefore all optically active. The optical activity can be determined by optical rotation (OR), optical rotatory dispersion (ORD), and circular dichroism (CD). OR is measured by a polarimeter at a single wavelength, usually the sodium D line (589 nm), and expressed as specific (or molecular) rotations [α]D. A number of approaches, all of them empirical in nature, have been devised to interpret the relationship between the measured optical rotations and structural features of carbohydrates (31). Specific rotations are used extensively to characterize new derivatives and to recognize known ones. Instead of using a single wavelength, optical rotatory dispersion measures the optical rotation angle (ϕ) over a wide range of wavelengths, and circular dichroism measures the differential absorption of right- and left-circularly polarized light as a function of wavelength. Both ORD and CD spectra can exhibit marked changes in slope in the vicinity of the absorption maximum of a chromophore attached to the chiral center, known as the Cotton effect. b. Optical rotation In a monosaccharide molecule, several chiral carbons contribute to the overall optical rotation, but the configuration of the carbon atoms attached to the ring oxygen atom have the greatest influence on the overall rotation value. For many monosaccharides and reducing oligosaccharides, the initial optical rotation in aqueous solutions changes with time until reaching a constant value. This phenomenon is known as mutarotation, most often the outcome of interconversion between α and β ring isomers, until reaching an equilibrium. Similar to monosaccharides, oligo- and polysaccharides have optical activity. With advances in the understanding of carbohydrate stereochemistry, it has become generally recognized that the overall optical rotation is determined more by the relative orientation of adjacent monosaccharide residues (defined by dihedral angles) than by the additive contributions from each asymmetric center. The optical activity of these is therefore beyond those arising from the simple monosaccharides, but is rather associated with the conformation of larger molecules or

macromolecules. Stevens and co-workers developed a chiroptical technique to investigate disaccharide conformation (32), based upon the estimates of variation in the optical activity of a particular disaccharide as a function of its glycosidic conformation. A number of disaccharides, including sucrose, maltose and cellobiose, have been characterized using this method (33). The optical rotation of polysaccharides at long wavelengths is usually dominated by the optical activity of the polymer backbone. Measurement of optical rotation at long wavelengths remains a standard and practical technique for polysaccharide systems despite the advent of ORD and CD instruments. For example, OR is used frequently for monitoring the progress of cooperative conformational transitions of polysaccharides (34).

c. Circular dichroism and optical rotatary dispersion Monosaccharides of most food carbohydrates exist in cyclic forms, thus do not possess the unsaturated chromophores necessary to display a Cotton effect at long wavelengths. In the absence of unsaturated chromophores, two very short wavelength transitions associated with conformational transitions of carbohydrate backbone may be used (35–37). These can be observed by modern vacuum UV polarimetry. One such transition is centered near 175 nm, attributed to the n→σ* transitions of the acetal oxygen atoms. The second is usually found around 150 nm and is closely related to the optical rotation at long wavelengths. CD and ORD experiments show that the variation in intensity of these two bands in polysaccharides is correlated to their composition and conformation (38). Thus, CD and ORD offer powerful tools to study structural and conformational transitions. Some polysaccharides contain chromophores that absorb at substantially longer wavelengths than the polymeric backbone and thus give significant CD and ORD bands at wavelengths above ~185 nm. Examples are acyl and pyruvate ketal constituents and the carboxyl groups. In these cases, the CD spectra are close to those of the isolated monosaccharides, with little direct influence from the chain geometry. Since CD is very sensitive to the local environment of chromophores, conformational changes caused by, for example, specific site binding of uronate segments are usually accompanied by dramatic changes in CD spectra (39). This provides an alternative approach to study the gelation mechanisms of polysaccharides containing carboxyl groups such as alginate, pectin, xanthan, and gellan (40, 41). 4. Microscopy Techniques

a. Background Direct imaging of polysaccharides using microscopy provides an important additional method for physical


characterization of polysaccharides. Two types of microscopy are especially of interest. First, electron microscopy (EM) is the traditional type, like light microscopy, but instead uses an electron beam to probe smaller structures than possible with light. Atomic force microscopy (AFM) senses forces such as electrostatic, magnetic, capillary, or van der Waals forces, as the molecular surface is approached by a probe. EM has considerable power to study supramolecular assemblies such as starch granules and mixed structures such as composite gels, whereas AFM has wide potential applications in investigating the structures of single molecules, as well as supramolecular assemblies and gel networks.

b. Electron microscopy In EM, an electron beam produced from an electron gun is employed as an illuminating source instead of visible light. In transmission electron microscopy (TEM), when a fine electron beam hits the specimen, the electrons are transmitted after a series of interactions with the specimen, and then magnified to produce the image on a fluorescent screen or a photographic film. In scanning electron microscopy (SEM), the secondary electrons originating from ionization of the specimen atoms by the incident primary electrons are collected by an electron detector. The incident beam is scanned over a small area corresponding to the area of the micrograph. EM gives a better resolution than light microscopy because the wavelength of an electron beam is shorter than that of visible light. A critical part of electron microscopy is adequate preparation of the specimen to minimize structural changes and to avoid artifacts. In most cases, the samples are exposed to a series of treatments prior to observation such as dehydration (or solidification), sectioning, and coating with electrical conducting materials. Thus, the image shapes obtained from the specimens may differ from their true shapes in the hydrated state. Information can be obtained from EM on how macromolecules associate into supramolecular assemblies, and under favorable conditions, form gel networks (42). EM was used to monitor the conformational changes of polysaccharides that often initiate gelation such as coil-helix transitions (42). Direct visualizing of the structure of gel networks using EM has helped the understanding of structure-function relationships of polysaccharide gelation. In addition to these qualitative assessments of structural features, it is also possible to quantify properties like contour length, persistence length, linear mass density, and thickness of strands, using advanced image analysis systems (42, 43). Polysaccharides like xanthan and various β-D-glucans, all with a persistence length in the order of 100 nm, are ideally suited for such EM investigations. Since EM only provides a two-dimensional projection of the specimen, it is important to compare the parameters derived from EM with those obtained from other physical

2-7

techniques, or from specimens prepared by different techniques.

c. Atomic force microscopy AFM is still a relatively new form of microscopy and has only been applied to the study of biopolymers since the late 1990s. It generates images by sensing the changes in force between a probe and the sample surface as the sample is scanned. Using a variety of probing methods (44), a three-dimensional image with sub-nanometer resolution of the surface topography of tested samples can be produced (45). Thus, AFM affords an opportunity to directly image individual molecules and the helical structures of polysaccharides with minimal sample preparation (44, 46, 47). The polysaccharides are simply deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged directly under appropriate liquid (45). For highly flexible polysaccharides such as dextrans, the AFM images show globular structures representing time-averaged pictures of the random coil structure. For more extended polysaccharides, such as xanthan and β-D-glucans, the AFM images may be quantified to yield persistence length, contour length, and its distributions (48). The dimensions observed by AFM are often larger than those derived from conventional techniques (44). This is believed to be due to the polymer-surface interactions which occur when the molecules are absorbed onto the mica surface prior to observation. AFM can be used to investigate the nature of association in junction zones, and also the overall structure of gel networks (49–51). The use of EM and AFM has led to an improved understanding of the functional properties of polysaccharides at a molecular level. Furthermore, the ability to provide direct information about heterogeneity makes microscopy not simply complementary to other physical techniques, but also indispensable for obtaining additional detailed structural information. 5. Nuclear Magnetic Resonance

a. Background Nuclear magnetic resonance (NMR) spectroscopy provides detailed structural information of carbohydrates, such as identification of monosaccharide composition, elucidation of α or β configurations, and establishment of linkage patterns and sequence of the sugar units in oligosaccharides and polysaccharides. Recent advances in two-dimensional NMR techniques allow the elucidation of some polysaccharides without chemical analysis (52). NMR can also be used to determine the conformation and chain stiffness/mobility of oligosaccharides and some polysaccharides in solution and to monitor coil-helix transitions and gel formation (53). The principle of NMR spectroscopy is based on the magnetic property of the nucleus in atoms associated with

2-8


spins. The most useful nuclei in carbohydrate research are 1 H and 13C, which by absorbing radio frequency energy in a strong magnetic field, jump to higher energy levels. Spins at the higher energy levels tend to relax to lower energy levels, and the transitions are dependent on the magnetic field strength in the local environment of the nucleus. Therefore, every nuclear spin in a molecule is influenced by the small magnetic fields of the nuclei of its nearest neighbors. Hence, the signal released by the nucleus reveals structural information of the nucleus in specific environment. The analysis of these individual signals relative to a standard, expressed by chemical shift and spin-coupling between nuclei, can yield detailed information on the structure and shape of molecules. One-dimensional NMR experiments are limited to the portrayal of response intensity as a function of the observation frequency under the applied field. Two-dimensional NMR techniques utilize a second frequency domain, which greatly expands the information contained in the spectrum. The introduction of this second domain allows correlations to be established and hence connectivity information can be obtained. These are very useful in determining molecular structures, particularly of complex oligosaccharides and polysaccharides. For example, COSY (COrrelation SpectroscopY) and TOCSY (TOtal Correlation SpectroscopY) are used to establish connectivities around monosaccharide rings. Long-range correlation experiments, such as Nuclear Overhauser Effect (NOE), a through-space phenomenon, can be used in the study of shape and conformation. Long-range heteronuclear correlation experiments can establish interresidue connectivity, and the sequences of complex oligosaccharides and polysaccharides can therefore be determined.

b. NMR in molecular dynamics and conformational analysis NMR relaxation data (T1 and T2* ) provide information on the dynamics of oligosaccharides and polysaccharides involving several different types of internal motion (53). NOESY provides information on inter-glycosidic spatial constraints, which helps define linkage conformations. Since they are able to provide conformational analysis of oligosaccharides in solution, NMR techniques are important means to obtain information on the three-dimensional structures free of crystal lattice constraints. NMR measurements of vicinal long-range homonuclear couplings

* T1 relaxation, or spin-lattice relaxation, is characterized by the longitudinal return of the net magnetization to its ground state of maximum length in the direction of the main magnetic field through energy loss to the surrounding lattice. T2 relaxation, or spin-spin relaxation, is characterized by the exchange of energy of spins at different energy levels, and does not lose the energy to the surrounding lattice.

(3JH,H) and long-range heteronuclear couplings (3JC,H) provide information on both intra- and inter-residue conformation(s) by measuring the parameters controlled by the dihedral angles between constituent monosaccharides of oligosaccharides and polysaccharides. In a recent application of NMR spectroscopy, long-range heteronuclear coupling constants were measured across the glycosidic linkages of a series of eight α- or β-linked disaccharides in solution (54). The 3JC,H values were determined by multiple 13C site-selective excitation experiments using 1 H decoupling under pulsed field gradient-enhanced spectroscopy. The experimentally determined long-range three-bond heteronuclear coupling constants were converted to calculate values of the glycosidic dihedral angles of each disaccharide using a Karplus-type equation. Wide applications of NMR in solution dynamics, conformational analysis, and prediction of helical structure of oligosaccharides and polysaccharides can be found in the literature (55–57). In summary, NMR spectroscopy is a very powerful tool not only for analyzing the primary structures of carbohydrates to provide information such as anomeric configuration, linkage sites, and sequences of monosaccharides, but also to determine the dynamics and shape of carbohydrates in solutions. The information can be further enhanced by combining with molecular modeling techniques. In this way, a deeper understanding of the dynamic properties and three-dimensional conformation of oligosaccharides and polysaccharides, and hence, the structure-property relationships, are obtained.

III. MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION A. POLYDISPERSITY AND MOLECULAR WEIGHT AVERAGES Monosaccharides and oligosaccharides have well-defined chemical structures, and specific molecular weights. However, polysaccharides contain molecules with different numbers of monosaccharide units (thus different molecular weights) and are said to be polydisperse. The distribution of molecular weight (MWD) varies, depending on the synthetic pathway and environments, as well as the extraction conditions to isolate the polysaccharides. The distribution may be described as mono-, bi-, or polymodal. Before we discuss how to quantitatively describe this polydispersity in molecular weight, we have to introduce the concept of molecular weight averages. There are four statistically described molecular weight averages in common use, number average molecular weight (Mn), weight average molecular weight (Mw), z-average molecular weight (Mz), and viscosity average molecular weight (Mv). The mathematical descriptions of


2-9

these averages in terms of the numbers of molecules Ni having molecular weight Mi are: ∞

Mi Ni 冱 i⫽1

Mn ⫽ ᎏ ∞ 冱Ni

(2.1)

i⫽1

∞

Mi2Ni 冱 i⫽1

Mw ⫽ ᎏ ∞ 冱Mi Ni

(2.2)

∞

Mi3Ni 冱 i⫽1

Mz ⫽ ᎏ ∞ 冱Mi2Ni

(2.3)

i⫽1

冤

∞

冱Mi1⫹αNi i⫽1

ᎏᎏ ∞ 冱Mi Ni i⫽1

冥

1/α

(2.4)

In Equation 2.4, α is the Mark Houwink exponent (Section V.B.2). Most of the thermodynamic properties are dependent on Mn and bulk properties such as viscosity are particularly affected by Mw. Mw and Mz emphasize the heavier molecules to a greater extent than does Mn. Mv is usually between Mw and Mn and closer to Mw; when α = 1, Mw = Mv. For very stiff polysaccharides with α > 1, Mv exceeds Mw. A convenient measure of the range of molecular weights present in a distribution is the ratio Mw/Mn, called the polydispersity index (PI). In a random MWD produced by condensation syntheses, as with polysaccharides, PI is typically around 1.5~2.

B. PHYSICAL METHODS DETERMINATION

FOR

Polymer solutions exert osmotic pressure across a porous boundary because the chemical potentials of a pure solvent and the solvent in a polymer solution are unequal. There is a thermodynamic drive toward dilution of the polymer-containing solution with a net flow of solvent through a separating membrane, toward the side containing the polymer. When sufficient pressure is built up on the solution side of the membrane, equilibrium is restored. The osmotic pressure π depends on Mn and polymer concentration c as follows (58): c π ⫽ RT ᎏ ⫹ A2c2 ⫹ A3c3 ⫹ … (2.5) Mn where R is the molar universal gas constant, T is the absolute temperature, and A2 and A3 are the second and the third virial coefficients, respectively. In very dilute solutions, it is usually sufficient to consider only the first two terms in the equation, which can then be rearranged as: π RT (2.6) ᎏ ⫽ ᎏ ⫹RTA2c Mn c where π/c is called the reduced osmotic pressure. According to the above equation, Mn may be determined by a plot of π/c versus c extrapolated to zero concentration. The intercept gives RT/Mn, and the slope of the plot yields A2. For neutral polysaccharides, osmotic pressure measurements can be made in water. However, for charged polysaccharides, salt solutions should be used to suppress the charge effects on apparent molecular weights. Usually 0.1–1 M NaCl or LiI is of sufficient ionic strength. Since osmotic pressure is dependent on the number of molecules present in solution, it is less sensitive to high MW polysaccharides. In practice, this method is only useful for polysaccharides having MW less than 500,000 g/mol (59).

冢

i⫽1

Mv ⫽

1. Osmometry

MOLECULAR WEIGHT

Absolute techniques for MW determination include membrane osmometry, static light scattering and equilibrium sedimentation. These techniques require no assumptions about molecular conformation and do not require calibration employing standards of known MW. Relative techniques include gel permeation chromatography (GPC), dynamic light scattering, velocity sedimentation and viscometry, and require either knowledge/assumptions concerning macromolecular conformation or calibration using standards of known MW. Combined techniques use information from two or more methods, such as velocity sedimentation combined with dynamic light scattering, velocity sedimentation combined with intrinsic viscosity measurements, and GPC combined with on-line (or off-line) static light scattering or equilibrium sedimentation.

冣

2. Static Light Scattering Static light scattering is widely used for determining the MW of macromolecules and measures Mw. For a highly dilute solution, the normalized intensity of scattered light R(q) as a function of scattering wave vector (q) and concentration (c) is given as (60): 1 Kc ᎏ ⫽ ᎏ ⫹ 2A2c Mw P(q) R(q)

(2.7)

where K is a contrast constant and P(q) is the particle scattering factor. For a random coil, P(q) is expressed by: q2R2g P(q) ⫽ 1 ⫺ ᎏ ⫹ … 3

θ 4π and q ⫽ ᎏ sin ᎏ 2 λ

冢冣

(2.8) (2.9)

where λ is the wavelength, θ is the scattering angle, and Rg is the radius of gyration. Equations 2.7–2.9 form the

2-10


−1

Kc/R (mol g )

2.392e-06

6.570e-07 0.0

11.9 sin2(/2) + 10000 c

FIGURE 2.3 Zimm plot of tricarbanilate of β-D-(1→3) (1→4)-glucan measured in dioxan.

basic theory for MW determination using static light scattering. In practice, this is done by measuring the angular dependence of scattered light from a series of dilute solutions. The scattering data are then processed in the form of a Zimm plot or other associated plots (Berry and Gunniur plots). In a typical Zimm plot, Kc/R(q, c) is plotted against q2 ⫹ kc, where k is an arbitrary constant to separate the angle-dependent curves from different concentrations. The double extrapolation to c ⫽ 0 and q ⫽ 0 (i.e., θ⫽0) results in two limiting curves intersecting the ordinate at the same point. This point gives 1/Mw. The initial slope of the curve at θ ⫽ 0 is 2A2, and from the initial slope of the curve at c ⫽ 0, Rg is obtained. Figure 2.3 is a Zimm plot of (1→3) (1→4)-β-D-glucan tricarbanilate measured in dioxan by static light scattering. The measurement of the MW of polysaccharides by light scattering has not been an easy task when compared to many other macromolecules. The major difficulty is the preparation of optically clear solutions that are free of dust and molecular aggregates. A detailed procedure for the preparation and clarification of polymer solutions is given by Tabor (61) and Harding et al. (59). The measurement of MW is especially complicated by the existence of aggregates. Extreme caution has to be taken in interpreting the data. Poor reproducibility is often an indication of the presence of aggregates. Extensive efforts have been made to eliminate aggregates by the selection of appropriate solvents (9, 62, 63) or by chemically transforming the polysaccharides to reduce H-bonding, using derivatives such as carbanilates (64).

solute. Analysis of the distribution of the solute concentration along the centrifugal field at such an equilibrium provides a means to study the MWD and the average MW. For polysaccharides, such an equilibrium distribution is generally achieved in 24–48 hours depending on the nature of the solute and experimental conditions (59). The basic equation describing the distribution of solute concentration J(r) at sedimentation equilibrium is given for an ideal system as (65): Mw(r)(1 ⫺ υρ)ω 2 dlnJ(r) ᎏ ⫽ ᎏᎏ 2 d(r ) 2RT

(2.10)

where r is the distance of a given point in the cell from the center of the rotor, ω is the rotor speed (rad/s), υ is the partial specific volume (ml/g), and ρ is the solution density. The solute concentration profile is recorded, usually by a Rayleigh interference optical system, and transformed into plots of log J(r) versus r2, from which the (point) weight average molecular weight can be obtained. The whole-cell Mw can then be calculated as 2RT J(b) ⫺ J(a) Mw ⫽ ᎏᎏ 2 2 2 ᎏᎏ J0(b ⫺ a ) ω (1 ⫺ υρ)

(2.11)

where a and b are the distance from the center of the rotor to the cell meniscus and cell bottom, respectively, and J0 is the initial loading concentration. Sedimentation equilibrium can cover a very wide range of molecular weights compared to light scattering and osmotic pressure methods. However, since the procedure is inherently time consuming and the thermodynamic non-ideality of polysaccharides can complicate interpretation of the measurements, the technique is not frequently applied in polysaccharide research. As with equilibrium sedimentation, velocity sedimentation is based on the principle that the sedimentation rate of a polymer under a centrifugal field is directly proportional to its MW and shape. Velocity sedimentation monitors the boundary movement during ultracentrifugation by an optical method, from which the sedimentation coefficient, and hence MW, can be estimated provided the conformation of the molecule is known. By the use of high angular velocities, initial sedimentation may occur before diffusion effects become important. Compared to equilibrium sedimentation, velocity sedimentation is less time consuming, but can only provide qualitative information on average MW and MWD.

3. Sedimentation Sedimentation methods are of two types, sedimentation equilibrium and sedimentation velocity. The equilibrium technique employs a centrifugal field to create concentration gradients in a polymer solution contained in a special centrifuge cell. For a solute under appropriate conditions (sedimentation equilibrium), sedimentation and diffusion become comparable so that there is no net transport of the

4. Viscometry Because of the simple experimental setup and ease of operation, viscometry is extensively used to determine the MW of polysaccharides. The method simply requires the measurement of the relative viscosity ηr and polymer concentration of dilute solutions. Experimentally, ηr can be measured either by a capillary viscometer, a rotational viscometer, or


a differential viscometer (66). The MW of the polysaccharides is then calculated via the Mark-Houwink relationship (Equation 2.18). The Mark-Houwink constants K and α are usually determined experimentally using a series of ideally monodisperse substances with known molecular weights. More discussion of this method will follow (Section V.B). Caution is needed when applying this relative method to polysaccharides with chemical heterogeneity. Any factors that may change chain extension lead to changes in K and α values; examples are degree of branching (as with amylopectin and dextrans) and the distribution and/or substitution of certain monosaccharide units (as with alginates and galactomannans). The chemical composition and structure of the material under test should resemble those of the calibration substances. 5. Gel Permeation Chromatography Gel permeation chromatography (GPC) or size exclusion chromatography (SEC) is widely used for the determination of MW and MWD of polysaccharides. In GPC, the polymer chains are separated according to differences in hydrodynamic volume by the column packing material. Separation is achieved by partitioning the polymer chains between the mobile phase flowing through the column and the static liquid phase that is present in the interior of the packing material, which is constructed to allow access of smaller molecules and exclude larger ones. Thus, larger molecules are eluted before smaller ones. Conversion of the retention (or elution) volume of a polymer solute on a given column to MW can be accomplished in a number of ways. Narrow MWD standards with known MW, such as pullulan and dextran, may be used to calibrate the column. As with viscometry, the difference in structure between the calibration standards and the tested sample may lead to over- or underestimating the MW. To overcome this, a universal calibration approach may be applied in which the product of intrinsic viscosity [η] and MW, being proportional to hydrodynamic volume, is used (67). For different polysaccharides, a plot of log [η] MW versus elution volume emerges to a common line, the so-called “universal calibration curve.” The calibration is usually obtained using narrow MWD standards from which the MW of a test sample can be read, provided the intrinsic viscosity is known. In the last two decades or so, methods for the determination of MWD have been facilitated by combining GPC with a laser light scattering detector (68, 69). These methods provide absolute measurement of average MW and information on MWD and molecular conformations.

2-11

are referred to the review by Harding (59) for a detailed discussion of alternative methods on MW determination of carbohydrates. In addition, recent development in AFM has shown that it is a potential means for MW determination of polysaccharides. The power of this approach is that it permits MW measurements of single polysaccharide molecules rather than mixtures of single molecules and aggregates. All the other methods described above determine the apparent MW of samples that often include molecular aggregates. Round et al. (46) found that Mn and Mw obtained from AFM is 2–3 times smaller than that for similar samples measured by conventional techniques.

IV. HYDRATION AND SOLUBILITY OF CARBOHYDRATES A. LOW-MOLECULAR-WEIGHT CARBOHYDRATES Carbohydrates contain both polar -OH groups and non-polar -CH groups. In an aqueous system, the numerous hydroxyl groups of carbohydrates may hydrogen bond strongly with water molecules. Also, the ring oxygen atom and the glycosidic bridging oxygen atom can form hydrogen bonds with water. Franks and coworkers discussed the thermodynamic data of small carbohydrates in the context of NMR and dielectric relaxation data (70). They found no solute-solute interactions in aqueous solutions even at fairly high concentrations. Both the sites of hydration and their relative conformations are important factors in the resultant hydration properties. Molecular dynamics studies have revealed that hydroxyl groups make on average between two and three hydrogen bonds with solvent (71, 72). Because of the proximity of adjacent hydroxyl groups, many water molecules were found to simultaneously hydrogen bond to two hydroxyl groups (71). The geometric requirements of these solute-solvent hydrogen bonds favor one conformation over another, leading to some solutes experiencing less favorable interactions with water, and hence being less soluble (73, 74). Nevertheless, low-molecular-weight carbohydrates, with degrees of polymerization less than 15~20, are generally very soluble in water and other polar solvents (75). The solubility decreases with increasing degree of polymerization because of increased solute-solute interactions. Addition of polar organic solvents to solutions of carbohydrates results in the precipitation of an amorphous or crystalline form of the carbohydrates. Increasing the concentration of alcohol decreases the solubility of monoand oligosaccharides, and they are only slightly soluble when the alcohol concentration is higher than 80% (76).

6. Other Methods There are a number of other less frequently used methods for MW determination of carbohydrates, such as mass spectrometry, end group analysis, and NMR. The readers

B. POLYSACCHARIDES Polysaccharides display a wide range of solubilities conventionally described as easily soluble, intermediately

2-12


soluble, and insoluble. There is no clear boundary between the three groups but the general consensus is: easily soluble polysaccharides are readily dissolved in cold water; intermediately soluble ones are only soluble in hot water; and insoluble ones cannot be dissolved even in boiling water. Structure and molecular weight are the two primary factors that determine solubility. Polysaccharides with a highly regular conformation that can form crystalline or partial crystalline structures (Section II.C.1) are usually insoluble in water. Linear polysaccharides with high regularity in structure, such as 1→4 or 1→3 linked β-Dglucans, and 1→4 linked β-D-mannans, are examples of this group. Although (1→4)-β-D-mannan can be dissolved in 5% alkaline solution, neutralization leads to reassociation and precipitation. Cellulose is insoluble, but swells in strong alkaline solutions such as 18% sodium hydroxide (77). Only cellodextrins with DP of about 15–80 can be dissolved or dispersed in such alkaline solutions; for DP less than 15, there is solubility in neutral aqueous solutions (75). Amylose, an α-(1→4)-homoglycan, is insoluble in cold water but can be dissolved in hot water. A decrease in uniformity/regularity of molecular structure is always accompanied by an increase in solubility. The irregularity of the molecular chains prevents the formation of a closely packed structure, allowing many polysaccharides to readily hydrate and dissolve when water is available. The mixed linkage (1→3) (1→4)-β-D-glucans from cereals differ from cellulose only by the introduction of occasional single (1→3) linkages. The insertion of these linkages introduces “kink” points into the otherwise stiff cellulosic backbone, rendering the polymer soluble in water. Branching or substitution of the polysaccharide chain also reduces the possibility of intermolecular association and usually increases solubility. Examples are easily seen by comparing the solubility of galactomannans with that of (1→4)-β-D-mannan. By introducing single α-Dgalactopyranosyl constituents (1→6) linked to the mannan backbone, the resulting galactomannans are fairly soluble in water. Any structures which contain especially flexible units such as (1→6) linkages will lead to higher solubility because of a larger favorable entropy of solution. Highly branched polysaccharides are almost always very soluble in water as in the case of amylopectin which has a much better solubility compared to its linear counterpart, amylose.

C. DISSOLUTION KINETICS The ability of a substance to be solvated is governed by the fundamental thermodynamic equation: ∆G ⫽ ∆H ⫺ T∆S

(2.12)

where ∆G, ∆H, and ∆S are the changes of Gibbs free energy, enthalpy, and entropy of mixing, respectively. T is

the absolute temperature of the system. A homogeneous solution is obtained when the Gibbs free energy is negative. For an ideal system, ∆H is usually small, so dissolution is an entropically driven process. For low-molecular-weight carbohydrates, dissolution of the molecules is promoted by a large increase in entropy on mixing. The dissolution rate is mainly controlled by the diffusion or convective transport of solute from the interfacial boundaries to the bulk solution, which in turn is determined by the difference between the solute concentration and the saturated concentration at a given temperature. The dissolution process is generally fast as long as the solution is not close to the saturation point. Increase in the hydrodynamic field, such as stirring, promotes dissolution. For polysaccharides, the contribution of entropy changes during dissolution is limited because of conformational constraints of the polymer chains. Most linear polysaccharides only form colloidal dispersions in aqueous systems that are not in thermodynamic equilibrium. In the initial stage of dissolution, amorphous polymer starts to swell as a result of water diffusing into the particle with a simultaneous transition from a glassy state to a rubbery gel-like state. Consequently, a gel layer forms on the surface of the polymer particle. The dissolution rate may be determined by a number of factors, either individually or combined together, including the rate of water penetration into the polymer, the rate of disentanglement of the polymer from the gel layer, and the diffusion or convective transport of solute from the interfacial boundaries to the bulk solution. In the case of high MW polysaccharides, the disentanglement of molecules is often the limiting step of dissolution. Thus the dissolution rate is expected to decrease with increasing MW because disentanglement of large molecules from the gel layer takes a longer time. The dissolution rate of guar gum was shown to be inversely related to the MW of the galactomannan (78). Diffusion or transport of solutes may also be the controlling factor in combination with disentanglements, such as in the case of a low MW polymer in low hydrodynamic environment (low temperature, low agitation), or when the viscosity of the solvent phase has built up significantly (78). The initial solvent content may also affect the dissolution of certain polysaccharides, but in various ways. Theoretical work and experiments suggest that dissolution rate increases with the level of residual solvent in the solid polymer (79, 80). However, if the presence of low levels of solvent leads to an increase in structure ordering, the suggested enhanced dissolution may not occur. For example, it has been observed that purified (1→3) (1→4)-β-D-glucan is very difficult to dissolve in water when it is precipitated from an aqueous solution and air dried. Solvent exchange using isopropanol before drying greatly improves solubility and dissolution. This is presumably due to the presence of water in the polymer, resulting in increased ordering of


2-13

the polymer and poorer solubility. Other factors such as particle size and porosity of the polymer may also influence dissolution rate.

V. RHEOLOGICAL PROPERTIES OF POLYSACCHARIDES A. CONCENTRATION REGIME Rheology is the study of flow and deformation of materials, and for any given polysaccharide, concentration is of course of primary importance. A dilute polymer solution is one in which each polymer coil and the solvent associated with it occupies a discrete hydrodynamic domain within the solution. The isolated macromolecules provide their individual contribution to the rheological properties of the system almost independently of the imposed shear rate. As the concentration of polymer increases, a stage is reached at which the individual molecular domains begin to touch one another frequently. The corresponding concentration is called the overlap concentration c*. At polymer concentration c>c*, the solution is called semi-dilute and when c>>c* the solution is concentrated.

B. DILUTE SOLUTIONS 1. Steady Shear Viscosity

2. Intrinsic Viscosity In dilute solutions, viscosity usually increases with concentration according to the Huggins and the Kramer equations:

ηsp ⫽ [η]c ⫹ K⬘[η]2c2

(2.15)

ln(ηr) ⫽ [η]c ⫹ (K⬘ ⫺ 0.5)[η]2c2

(2.16)

where K⬘ is the Huggins coefficient. [η] is known as intrinsic viscosity and is the limit of reduced viscosity (ηsp/c) as c→0: [η] ⫽ lim(ηsp兾c) c→0

(2.17)

Experimentally, [η] is usually determined from the measurement of ηr or ηsp over a series of dilute solutions. By plotting ηsp/c or ln (ηr)/c versus c, [η] is obtained as the average of the two intercepts at the ordinate via graphic extrapolations of c→0. Intrinsic viscosity is not actually a viscosity but is a characteristic property of an isolated polymeric molecule in a given solvent, and is a measure of its hydrodynamic volume. It has a unit of volume per unit weight. Mark (82) and Houwink (83) independently correlated the intrinsic viscosity with the viscosity average molecular weight Mv

.

The ratio of applied shearing stress (τ) to rate of shear (γ ) for an ideal viscous fluid is called the coefficient of viscosity, or simply viscosity (η), which is a measure of the resistance to flow. The term “fluidity,” which is the reciprocal of viscosity, is sometimes used in the food industry. The viscosity increase due to the contribution of dissolved or dispersed solutes over the solvent is described by the relative viscosity (ηr) and specific viscosity (ηsp):

η ηr ⫽ ᎏ ηs

(2.13)

η⫺η ηsp ⫽ ᎏs ⫽ ηr ⫺1 ηs

(2.14)

where ηs is the solvent viscosity and η the overall solution viscosity. For most polysaccharides, especially of the random coil type, dilute solutions under shear flow show essentially Newtonian behavior. That means the viscosity of the solution is a constant independent of share rate. However, non-Newtonian flow behavior is observed for dilute solutions of some rigid polysaccharides, such as xanthan and some other β-glucans (81). For these systems, the apparent viscosity falls as the shear rate increases –– a phenomenon called shear thinning. The shear thinning behavior of such polysaccharide solutions is a result of progressive orientation of the stiff molecules in increasing field of shear.

[η] ⫽ KMαv

(2.18)

where both K and α are constants for a given polysaccharide-solvent pair at a given temperature. The exponent α is a conformation-sensitive parameter and usually lies in the range of 0.5–0.8 for random coil polymers, and increases with increasing chain stiffness. It can be as high as 1.8 for polysaccharides with a stiff rod conformation. Low values of α ( c* (85). Precisely, the viscosity generated by disordered polymer coils is dependent on the degree of space-occupancy by the polymer, which is determined by both concentration and molecular weight. In general, for linear polysaccharides in

100

a given solvent, solution viscosity increases proportionally to their molecular weight and concentration. The space occupancy is characterized by the dimensionless product of concentration and intrinsic viscosity c[η], since [η] is a measure of volume occupancy of the isolated coil in the solvent. Morris et al. (86) found that the double-logarithmic plots of ηsp vs. c[η] for a number of different disordered polysaccharides and the same polysaccharides with different molecular weights are virtually identical.

10

100

1000

FIGURE 2.4 Shear rate (γ ) dependence of viscosity (η) for xyloglucan from Detarium senegalense Gmelin in aqueous solutions at different concentrations. From Wang et al., 1997 (84).

4. Dynamic Properties Polysaccharide solutions are viscoelastic substances, i.e., have both solid and liquid characteristics. An important experimental approach to the study of the viscoelasticity of a polymer solution is to use a dynamic oscillatory measurement. A sample is subjected to a small sinusoidal oscillating strain (γ); this generates two stress components in viscoelastic materials, an elastic component which is in phase with the applied strain and a viscous component which is 90° out of phase with the strain:

σ0 ⫽ G⬘γ0 sinω t ⫹ G⬘⬘γ0 cosω t

(2.20)

G⬘⬘ tanδ ⫽ ᎏ G⬘

(2.21)

where G′ is the elastic or storage modulus, G″ is the viscous or loss modulus, tanδ is the loss tangent, and ω is the frequency of oscillation. The loss tangent is the ratio of the energy dissipated to that stored per cycle of deformation. The frequency dependency of these viscoelastic quantities allows specific features of different classes of polysaccharides to be distinguished. Based on the relative magnitudes of G′ and G″ in a frequency sweep experiment within the linear viscoelastic strain range, three types of polysaccharide systems may be distinguished: solutions, weak gels, and gels (85). For dilute solutions of polysaccharides, G″ values are higher than G′, with G″ ∝ ω and G′ ∝ ω2 at low frequency. When the frequency or concentration is increased, there is a crossover between G′ and G″, implying that the system passes from being a more and


more viscous liquid to being a viscoelastic solid. Also, both G′ and G″ become less frequency dependent as the frequency is increased; a “rubbery” plateau of G′ is seen at high frequencies. Gels have a very different spectrum, with G′ remaining almost constant and G″ only increasing slightly as frequency increases; and G′ values are higher than G″ at all frequencies, with tanδ around 10⫺1 for a weak gel and 10⫺2 for a true gel.

D. POLYSACCHARIDE GELS 1. Gelation Mechanism Under certain conditions, the association of hydrated polysaccharides results in a three-dimensional polymeric network (a gel) that fills the liquid available rather than precipitation of the polysaccharide. In these resultant gels, polysaccharide molecules or portions of these are aggregated in the junction zones through interactions such as hydrogen bonding, hydrophobic association, and cationmediated cross-linking. To induce gelation, polysaccharides usually have to be first dissolved or dispersed in a solution, in order to disrupt mostly the hydrogen bonds from the solid state. The subsequent transformation of sols to gels is achieved by treatments such as heating and cooling, addition of cations, and change of pH. The adoption of an ordered secondary and tertiary structure such as a helix or flat ribbon is a primary mechanism for the gelation of polysaccharides. The familiar gelation of algal polysaccharides agarose and κ-carrageenan, and bacterial polysaccharide gellan, involves the formation of helices (87). These helices may further associate to form a quaternary structure (gel network) through intermolecular hydrogen bonding or incorporation of counterions in the case of some charged polymers. The gelation of some other polysaccharides is through the formation of pleated sheets, sometimes described as an egg-box structure. Familiar examples of this are gels of low-methoxyl pectin and alginate. In this structure, the polysaccharides associate into matched aggregates in a twofold ribbon-like conformation, with the metal ions cooperatively bound during the process, sitting inside the electronegative cavities like eggs in an egg box. 2. Physical Properties of Polysaccharide Gels Polysaccharides are able to form a vast range of gel structures which can be controlled by the properties of polysaccharides themselves and by the gelling conditions. A list of gelling food polysaccharides and a comparison of their relative textural characteristics are given by Williams and Phillips (88). Some polysaccharides form thermoreversible gels and examples exist where gelation occurs on either the cooling or heating cycle. Thermal hysteresis may exist in some of the thermo-reversible gels; the melting temperature of the gel is significantly higher than the setting temperature. Thus, gelation occurs when hot agarose

2-15

solutions are cooled to below 40°C, but this gel does not melt until the temperature is raised to above ~90°C. Some polysaccharides form thermally irreversible gels, which are usually formed by cross-linking polysaccharide chains with divalent cations. Gel formation occurs above a critical minimum concentration for each polysaccharide, and gel strength normally increases with increasing concentration. Molecular weight is also important. Intermolecular associations of polysaccharides are stable only above a minimum critical chain length necessary for the cooperative nature of the interaction, typically in the range of 15–20 residues (75). Gel strength normally increases significantly as MW increases up to a certain point, then becomes MW independent at higher values. The gelation of anionic polysaccharides is also dependent on the type and concentration of associated cations because the association of the charged tertiary structures may be promoted by specific counterions whose radii and charges are suitable for incorporation into the structure of the junction zones. Mixed gels from two or three polysaccharides may impart novel and improved rheological characteristics to food products. Synergy is observed for a number of binary systems including pectin-alginate, xanthan-galactomannan or glucomannan, and agarose or carrageenan–galactomannan or glucomannan. In these mixtures, synergism confers either enhanced gelling properties at a given polysaccharide concentration, or gelation under conditions in which the individual components will not gel. Although the gelation mechanisms for mixed polysaccharides are still controversial, there is evidence that some form of binding and structure compatibility has to be present between the two polysaccharides (87).

REFERENCES 1. GA Jeffrey, M Sundaralingam. Bibliography of crystal structures of carbohydrates, nucleosides, and nucleotides. Adv Carbohydr Chem Biochem 43: 203–421, 1985. 2. RL Whistler, JN BeMiller. Carbohydrate Chemistry for Food Scientists. St. Paul, MN: Eagan Press, 1997, pp. 1–17. 3. J Brady. Oligosaccharides geometry and dynamics. In: P Finch. ed. Carbohydrates: Structures, Syntheses and Dynamics. London: Kluwer Academic Publishers, 1999, pp. 228–257. 4. DA Rees. Polysaccharides Shapes. New York: John Wiley & Sons, 1977, pp. 42–43. 5. RH Marchessault, A Buleon, Y Deslandes, T Goto. Comparison of x-ray diffraction data of galactomannans. J Colloid Interface Sci 71: 375–382, 1979. 6. AH Clark. X-ray scattering and diffraction. In: SB Ross-Murphy. ed. Physical Techniques for the Study of Food Biopolymers. New York: Blackie Academic & Professional, 1994, pp. 65–150.

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7. R Chandrasekharan. Molecular architecture of polysaccharide helices in oriented fibres. Adv Carbohydr Chem Biochem 52: 311–439, 1997. 8. P Lang, W Burchard. Structure and aggregation behaviour of tamarind seed polysaccharide in aqueous solution. Makromol Chem Macromol Chem Phys 194: 3157–3166, 1993. 9. A Grimm, E Kruger, W Burchard. Solution properties of β-D-(1,3) (1,4)-glucan isolated from beer. Carbohydr Polym 27: 205–214, 1995. 10. R Lapasin, S Pricl. Rheology of Industrial Polysaccharides. London: Blackie Academic and Professional, 1995, pp. 250–494. 11. SB Ross-Murphy. Introduction. In: SB Ross-Murphy. ed. Physical Techniques for the Study of Food Biopolymers. New York: Blackie Academic & Professional, 1994, pp. 1–12. 12. GA Jeffrey, M Sundaralingam. Bibliography of crystal structures of carbohydrates, nucleosides, and nucleotides. Adv Carbohydr Chem Biochem 43: 203–421, 1985. 13. GA Jeffrey, D Huang. The hydrogen bonding in the crystal structure of raffinose pentahydrate. Carbohydr Res 206: 173–182, 1990. 14. W Mackie, B Sheldrick, D Akrigg, S Perez. Crystal and molecular structure of mannotriose and its relationship to the conformations and packing of mannan and glucomannan chains and mannobiose. Int J Bio Macromol 8: 43–51, 1986. 15. R Gilardi, JL Flippen-Anderson. The tetrasaccharide stachyose. Acta Crys C43: 806–808, 1986. 16. S Raymond, A Heyraud, DT Qui, A Kvick, H Chanzy. Crystal and molecular structure of β-D-cellotetraose hemihydrate as a model of cellulose II. Macromol 28: 2096–2100, 1995. 17. S Arnott, WE Scott. Accurate x-ray diffraction analysis of fibrous polysaccharides containing pyranose rings. l. linked-atom approach. J Chem Soc Perkin Trans 2: 324–335, 1972. 18. RH Marchessault, Y Deslandes, K Ogawa, PR Sundarajan. X-Ray diffraction data for β-(1,3)-D-glucan. Can J Chem 55: 300–303, 1977. 19. Y Deslandes, RH Marchessault, A Sarko. Triple-helical structure of (1,3)-β-D-glucan. Macromolecules 13: 1466–1471, 1980. 20. T Yanaki, T Norisuye, H Fujita. Triple helix of schizophyllum commune polysaccharide in dilute solution. 3. Hydrodynamic properties in water. Macromolecules 13: 1462–1466, 1980. 21. K Kajiwara, T Miyamoto. Progress in structural characterization of functional polysaccharides. In: S Dumitriu. ed. Polysaccharides: Structural Diversity and Functional Versatility. New York: Marcel Dekker, 1998, pp. 1–55. 22. DA Brant. Novel approaches to the analysis of polysaccharide structures. Current Opinion in Structural Biology 9: 556–562, 1999. 23. W Burchard. Light scattering. In: SB Russ-Murphy. ed. Physical Techniques for the Study of Food Biopolymers. New York: Blackie Academic & Professional, 1994, pp. 151–214.

24. E Husemann, B Pfannemüller, W Burchard. Streulichtmessungen und Viskositätsmessungen an wässrigen Amyloselösungen, I and II. Makromol Chem 59: 1–27, 1963. 25. HL Doppert, AJ Staverman. Kinetics of amylose retrogradation. J Polym Sci A-1 4: 2353–2366, 1966. 26. M Kodama, H Noda, T Kamata. Conformation of amylose in water. I. Light scattering and sedimentationequilibrium measurements. Biopolymers 17: 985–1002, 1978. 27. W Burchard. Static and dynamic light scattering from branched polymers and biopolymers. Adv Polym Sci 48: 1–120, 1983. 28. A Thurn, W Burchard. Heterogeneity in branching of amylopectin. Carbohydr Polym 5: 441–460, 1985. 29. W Burchard, JMG Cowie. Selected topics in biopolymeric systems. In: Light Scattering from Polymer Solutions. New York: Academic Press, 1972, pp. 725–787. 30. JG Southwick, AM Jamieson, J Blackwell. Quasielastic light scattering studies of xanthan in solution. In: DA Brant. ed. Solution Properties of Polysaccharides. Washington, D.C.: American Chemical Society, 1980, pp 1–13. 31. ES Stevens, CA Duda. Solution conformation of sucrose from optical rotation. J Am Chem Soc 113: 8622–8627, 1991. 32. ES Stevens, BK Sathyanarayana. A semiempirical theory of the optical activity of saccharides. Carbohydr Res 166: 181–193, 1987. 33. ES Stevens. The potential energy surface of methyl 3-O(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside in aqueous solution: Conclusions derived from optical rotation. Biopolymers 34: 1395–1401, 1994. 34. DA Rees, ER Morris, D Thom, JK Madden. Shapes and interactions of carbohydrate chains. In: GO Aspinall. ed. The Polysaccharides. Vol. 1. Toronto: Academic Press, 1982, pp 195–290. 35. RG Nelson, WC Johnson Jr. Optical properties of sugars. I. Circular dichroism of monomers at equilibrium. J Am Chem Soc 94: 3343–3345, 1972. 36. RG Nelson, WC Johnson Jr. Optical properties of sugars. 3. Circular dichroism of aldo- and ketopyranose anomers. J Am Chem Soc 98: 4290–4295, 1976. 37. JS Balcerski, ES Pysh, GC Chen, JT Yang. Optical rotatory dispersion and vacuum ultraviolet circular dichroism of a polysaccharide, ι-Carrageenan. J Am Chem Soc 97: 6274–6275, 1975. 38. WC Johnson Jr. The circular dichroism of carbohydrates. Adv Carbohydr Chem Biochem 45: 73–124, 1987. 39. I Listowsky, S Englard, G Avigad. Conformational aspects of acidic sugars: circular dichroism studies. Trans NY Acad Sci 34: 218–226, 1972. 40. ER Morris, DA Rees, GR Sanderson, D Thom. Conformation and critical dichroism of uronic acid residues in glycosides and polysaccharides. J Chem Soc Perkin Trans II: 1418–1425, 1975. 41. D Thom, GT Grant, ER Morris, DA Rees. Characterisation of cation binding and gelation of polyuronates by circular dichroism. Carbohydr Res 100: 29–42, 1982.


42. BT Stokke, A Elgsaeter. Conformation, order-disorder conformational transitions and gelation of non-crystalline polysaccharides studied using electron microscopy. Micron 25: 469–491, 1994. 43. AM Hermansson, M Langton. Electron microscopy. In: SB Ross-Murphy. ed. Physical Techniques for the Study of Food Biopolymers. New York: Blackie Academic & Professional, 1994, pp. 277–341. 44. VJ Morris, AR Kirby, AP Gunning. Atomic Force Microscopy for Biologists. London: Imperial College Press, 1999. 45. AR Kirby, AP Gunning, VJ Morris. Imaging polysaccharides by atomic force microscopy. Biopolymers 38: 355–366, 1996. 46. AN Round, AJ MacDougall, SG Ring, VJ Morris. Unexpected branching in pectin observed by atomic force microscopy. Carbohydr Res 303: 251–253, 1997. 47. AW Decho. Imaging an alginate polymer gel matrix using atomic force microscopy. Carbohydr Res 315: 330–333, 1999. 48. MJ Ridout, GJ Brownsey, AP Gunning, VJ Morris. Characterisation of the polysaccharide produced by Acetobacter xylinum strain CR1/4 by light scattering and atomic force microscopy. Int J Bio Macromol 23: 287–293, 1998. 49. AP Gunning, AR Kirby, MJ Ridout, GJ Brownsey, VJ Morris. Investigation of gellan networks and gels by atomic force microscopy. Macromolecules 29: 6791– 6796, 1996. 50. AP Gunning, AR Kirby, MJ Ridout, GJ Brownsey, VJ Morris. ‘Investigation of gellan networks and gels by atomic force microscopy vol. 29, pp. 6791–6796, 1996.’ Macromolecules 30: 163–164, 1997. 51. VJ Morris, AR Mackie, PJ Wilde, AR Kirby, ECN Mills, PA Gunning. Atomic force microscopy as a tool for interpreting the rheology of food biopolymers at the molecular level. Lebensm-Wiss Technol 34: 3–10, 2001. 52. W Cui. Application of two dimensional (2D) NMR spectroscopy in the structural analysis of selected polysaccharides. In: PA Williams, GO Phillips. eds. Gums and Stabilizers for the Food Industry. Vol. 11, Cambridge: Royal Chemical Society, 2001, pp. 27–38. 53. CA Bush, M Martin-Pastor, A Imberty. Structure and conformation of complex carbohydrates of glycoproteins, glycolipids, and bacterial polysaccharides. Ann Rev Biophys Biomol Struct 28: 269–293, 1999. 54. NWH Cheetham, P Dasgupta, GE Ball. NMR and modelling studies of disaccharide conformation. Carbohydr Res 338: 955–962, 2003. 55. P Dais. Carbon-13 nuclear magnetic relaxation and motional behavior of carbohydrate molecules in solution. Adv Carbohydr Chem Biochem 51: 63–131, 1995. 56. L Catoire, C Derouet, AM Redon, R Goldberg, CH du Penhoat. An NMR study of the dynamic single-stranded conformation of sodium pectate. Carbohydr Res 300: 19–29, 1997. 57. B Coxon, N Sari, G Batta, V Pozsgay. NMR spectroscopy, molecular dynamics, and conformation of a synthetic octasaccharide fragment of the O-Specific

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75. RL Whistler. Solubility of polysaccharides and their behavior in solution. Adv Chem Series 117: 242– 255, 1973. 76. M Levine, JF Foster, RM Hixon. Structure of the dextrins isolated from corn sirup. J Am Chem Soc 64: 2331–2337, 1942. 77. JO Warwicker, AC Wright. Function of sheets of cellulose chains in swelling reactions on cellulose. J Appl Polym Sci 11: 659–671, 1967. 78. Q Wang, PR Ellis, SR Ross-Murphy. Dissolution kinetics of guar gum powders, II. Effects of concentration and molecular weight. Carbohydr Polym 53: 75–83, 2003. 79. I Devotta, MV Badiger, PR Rajamohanan, S Ganapathy, RA Mashelkar. Unusual retardation and enhancement in polymer dissolution: Role of disengagement dynamics. Chem Eng Sci 50: 2557–2569, 1995. 80. AA Ouano. Solvent-property relationships in polymers. In: RB Seymour, GA Stahl. eds. Macromolecular Solutions. New York: Pergamon Press, 1992, pp. 208–219. 81. E Steiner, H Divjak, W Steiner, RM Lafferty, H Esterbauer. Rheological properties of solutions of a colloid-disperse homoglucan from Schizohyicum commune. Progr Coll Polym Sci 77: 217–220, 1988.

82. H Mark. Der Feste Korper. Leipzig, Germany: Hirzel, 1938. 83. R Houwink. Relation between the polymerization degree determined by osmotic and viscometric methods. J Prakt Chem 157: 15–18, 1940. 84. Q Wang, PR Ellis, SB Ross-Murphy, W Burchard. Solution characteristics of the xyloglucan extracted from Detarium senegalense Gmelin. Carbohydr Polym 33: 115–124, 1997. 85. SB Ross-Murphy. Rheological methods. In: HS Chan. ed. Biophysical Methods in Food Research. Critical Reports on Applied Chemistry, Vol. 5. Oxford: Blackwell Scientific Publications, 1984, pp. 138–199. 86. ER Morris, AN Cutler, SB Ross-Murphy, DA Rees, J Price. Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions. Carbohydr Polym 1: 5–21, 1981. 87. VJ Morris. Gelation of polysaccharides. In: SE Hill, DA Ledward, JR Mitchell. eds. Functional Properties of Food Macromolecules. Gaithersburg, MD: Aspen Publishers, 1998, pp. 143–226. 88. PA Williams, GO Phillips. Introduction to food hydrocolloids. In: PA Williams, GO Phillips. ed. Handbook of Hydrocolloids. Boca Raton, FL: CRC Press, 2000, pp. 1–20.

3

Carbohydrates: Starch

Lorraine L. Niba

Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University

CONTENTS I.

Starch Composition and Structure ........................................................................................................................3-2 A. Amylose ........................................................................................................................................................3-2 B. Amylopectin ..................................................................................................................................................3-3 C. The Starch Granule ......................................................................................................................................3-3 D. Non-Starch Components ..............................................................................................................................3-3 II. Starch Sources ......................................................................................................................................................3-4 A. Grain Starches ..............................................................................................................................................3-4 B. Root and Tuber Starches ..............................................................................................................................3-5 C. Other Sources of Starch ................................................................................................................................3-5 III. Starch Physicochemical Properties and Functionality..........................................................................................3-6 A. Starch Gelatinization ....................................................................................................................................3-6 B. Starch Retrogradation....................................................................................................................................3-7 C. Starch Damage ..............................................................................................................................................3-7 D. Interactions with Acids, Sugar, and Salts......................................................................................................3-7 IV. Starch Hydrolysis..................................................................................................................................................3-8 A. Enzyme Hydrolysis ......................................................................................................................................3-8 B. Acid Hydrolysis ............................................................................................................................................3-9 C. Alkaline Hydrolysis ......................................................................................................................................3-9 D. Heat-Induced Hydrolysis ..............................................................................................................................3-9 V. Starch Modification ..............................................................................................................................................3-9 VI. Starch in Food Applications................................................................................................................................3-10 A. Functional Properties ..................................................................................................................................3-10 B. Value-Added Food Applications ................................................................................................................3-10 VII. Starch Nutritional Quality ..................................................................................................................................3-11 A. Starch and Glycemic Index ........................................................................................................................3-11 B. Resistant Starch ..........................................................................................................................................3-12 VIII. New Starch Technologies....................................................................................................................................3-13 A. Genetic Modification ..................................................................................................................................3-13 B. Resistant Starch Production by Autoclaving ..............................................................................................3-14 C. Other Procedures ........................................................................................................................................3-14 References ....................................................................................................................................................................3-14 Starch is the major source of calories and dietary energy in most human food systems. As the primary human metabolic substrate, starch is preferentially digested, absorbed and metabolized. Most diets worldwide have a substantial starchy component as a main or side item. For instance, potatoes are a major item in most northern European diets, rice is popular in Asian diets, maize-based foods are common in Latin America, and starchy root and tuber crops constitute a significant part of the diet in most tropical areas.

Starch occurs naturally in plants and is the storage polysaccharide of plants. It is heterogeneous, consisting of two glucose polymers: amylose and amylopectin. It is a polymer of glucose and a complex carbohydrate, which finds multiple applications in various industries such as pharmaceuticals, textiles, paper, and the food industry. Starch performs various functions in food systems. It is used as a carrier in various products, as a texture modifier, as a thickener, and as a raw material for the production 3-1

3-2


of other valuable food ingredients and products. Physiologically, it is a source of energy. In addition, starches that are resistant to amylase digestion have properties similar to soluble fiber.

I. STARCH COMPOSITION AND STRUCTURE Starch is composed of two basic molecular components: amylose and amylopectin. These are identical in their constituent basic units (glucose), but differ in their structural organization (linkages). These variations in the linkages in turn affect their functionality in food applications. Amylose is a straight chain molecule, while amylopectin is a branched molecule. In addition, each is hydrolyzed, digested, and absorbed differently. Amylose is hydrolyzed mainly by amylases, while amylopectin requires debranching enzymes such as pullulanase for complete hydrolysis. As a result of their structure, the nature and products of hydrolysis of amylose and amylopectin differ. The proportions of amylose and amylopectin in foods therefore influence the extent of digestibility of the starch. The ratios of amylose to amylopectin vary among starch sources and play a considerable role in determining reactions and physicochemical properties of starches in processing and food applications (1–6). Most tuber starches contain high levels of amylopectin, imparting a “waxy” texture. Amylose and amylopectin are the polymers which constitute the starch granule (Table 3.1).

A. AMYLOSE Amylose is composed of D-glucose molecules, which are linked in an α-1→4 conformation. The glucose monomers therefore form a linear straight chain polymer. Amylose is less predominant (about 20%) and typically constitutes about 20–40% in proportion (7). Amylose contains α-1→4 glycosidic bonds and is slightly soluble in water. Amylose molecules are arranged in a helical conformation. This facilitates formation of complexes with iodine, lipids, and other polar substances (2,8,9). The iodide ions are sequestered in the central tunnel of the helix. Amylose forms a blue complex with iodine, which can be read at about 650 nm. The starch iodine test is often used to determine amylose content of various starches and starch types (4,10). Amylose is more suitable for the starch-iodine test. The affinity of pure amylose for iodine is 19–20% compared to only about 1% for amylopectin (1,2,6). Amylose would adsorb 19–20.5 g of iodine per 100 g compared to only about 1.2 g for amylopectin. The starch iodine test is often considered to be a measure of apparent amylose content of the starch. Amylose is the key component involved in water absorption, swelling, and gelation of starch in food processing. High amylose starches are therefore most commonly applied in food products that require quick-setting gels such as candies and confectionery. Amylose is more susceptible to gelatinization and retrogradation, and hence is most commonly involved in resistant starch formation.

TABLE 3.1 Properties of Amylose and Amylopectin (1–6,9) Property

Amylose

Structure

Linear (branched chains isolated from some starches) Up to 1 000 000 α-1→4 Blue 19–20.5% 1.2–1.6 Complexes with polar agents Crystalline Phosphorus-free

Molecular weight Glycosidic linkage Iodine complex Iodine affinity Blue value Polar agents X-ray diffraction pattern Phosphorus Association with lipids α-Amylase hydrolysis products Hydrolysis to maltose Pullulanase Gel stability Susceptibility to retrogradation Common sources

High Glucose, maltose, maltotriose, mainly oligosaccharides 100% No effect Firm, translucent, quick-setting gels Retrogrades readily Typically higher in cereal starches (e.g., corn starch)

Amylopectin Branched chains (long segments of linear chains in some starches) Up to 5 000 000 α-1→4 and α-1→6 Purple 0–1.2% 0–0.05 Does not complex with polar agents Amorphous 0.06–0.9% phosphorus (mostly in root/tuber starches) Low Small amounts of reducing sugars, mainly oligosaccharides 55–60 (100% with limit dextrinase and β-amylase) Debranches α-1→6 linkages Clear viscous gels Mostly stable Typically high in tubers and root starches (e.g., tapioca starch)


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B. AMYLOPECTIN Amylopectin consists of D-glucose units which are linked in an α-1→4 conformation as is the case with amylose, as well as D-glucose units in an α-1→6 conformation. Amylopectin is therefore highly branched as the α-1→4 linear chains are punctuated with the α-1→6 linkages. The α-1→6 constitute about 5% of the structure of amylopectin and gives rise to the branching (11). The amylopectin molecule therefore is much larger than the amylose molecule. The larger molecular size of amylopectin from amylose facilitates separation of these two polymers by size exclusion chromatography (2,7). Negligible amounts of unbranched amylopectin (A chains) in some starches have also been reported. In addition, there are long unbranched portions of the glucose polymer in some amylopectin molecules. While the straight chain of amylose is readily hydrolyzed by β-amylases, de-branching enzymes have to be used to obtain full hydrolysis of amylopectin (2,3,8). Amylopectin has short branched chains and branch linkages, and thus cannot form the helical complex with iodine. The branched dextrin of amylopectin, however, gives a purple color with the iodine complex, identifiable at about 550 nm (3,9). The enzymes required for amylopectin hydrolysis vary from those required for hydrolysis of amylose. Pullulanase, an enzyme which is specific for the α-1→6 glycosidic linkage, and other debranching enzymes are needed to hydrolyze amylopectin. The properties of amylopectin in food applications differ considerably from those of amylose. Amylopectin gels are more flexible and resistant. Amylopectin also is much more resistant to retrogradation than amylose. High amylopectin starches (waxy starches) are therefore commonly used in noodle processing and in some baked products to extend shelf-life. They also are used to improve freezethaw stability due to their resistance to retrogradation.

C. THE STARCH GRANULE The basic components of starch, amylose and amylopectin, are located in granules. The size, shape, and characteristics of the granules are specific to the plant source. The growth and development of the granule originates at the center of the granule, which is known as the hilum. Under magnification and polarized light, native starch granules typically appear to have a cross-like structure, similar to a maltese cross, exhibiting birefringence. The size and shape of this cross-like shape varies among botanical starch sources. For instance, starch from pinto bean has elliptical shaped lobes, while some starches have more than four lobes (12). The ordered arrangement of amylopectin molecules intertwines to form three-dimensional double helices between adjacent branches of the same amylopectin molecule or between adjacent clusters. The double helices are

FIGURE 3.1 Tapioca starch granules.

stabilized by weak van der Waals and hydrogen bonds. The various arrangements of the helices result in the presence of crystalline regions on the granule (11,13,14). The nature of these regions becomes clear by their X-ray diffraction patterns. The crystalline patterns vary under X-ray diffraction patterns. These polymorphic arrangements occur in two patterns, which are classified as A or B, and an intermediate form or mixture of A and B forms, known as C type (11,14,15). Most cereal starches have A type patterns. Root and tuber starches such as potato starch contain mostly B type patterns, while legume starches have a combination of both polymorphic A and B forms, and hence are classified as C forms (11,14,15). The crystalline nature and diffraction of starch is greatly altered by processing (16). Natural starch granules are insoluble in water, which is why starch is separated by sedimentation. This shape is disfigured and lost as starch loses its structure with modification such as heat and moisture. Granules vary in shape and size and are characteristic of the starch sources. These shapes may be round, lenticular, or oval (11,17). Starch granule properties are used as diagnostic characteristics for identification and characterization of starches, based on structure and shape (Figure 3.1).

D. NON-STARCH COMPONENTS Various non-starch components are covalently linked to amylose or amylopectin in starch. Structural and functional proteins are present which surround the starch granule. The protein friabilin, responsible for hardness of the endosperm in most cereals, is located on the granule. In addition, the enzyme responsible for starch synthesis, granule-bound starch synthase (GBSS), is located on the granule (18,19). Wheat starch characteristics are especially influenced by the presence of proteins. The proportion of protein in starches could be up to 0.5%.

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Most starches contain glycolipids, complexed to amylose or amylopectin. Wheat starch, for instance, contains amylose-lipid complexes. The nature of lipids present in starches differs depending on the origin and nature of the starch. Lipids such as monoglycerides and lysophosphatidyl choline form complexes with amylose and amylopectin. Lipids may occur on the surface of the starch granule as well as in the interior of the granule. Lipids that occur within the starch granule are typically monoacyl lipids which could either be mostly free fatty acids or lysophospholipids (20). In addition, most of the lipids on the surface of the granules are monoacyl lipids. Some lipids are non-starch lipids and not associated with starch, but rather occur in the endosperm (21). Phosphorus is a common constituent of many starches, occurring primarily as phosphate monoesters on amylose and amylopectin. Rice starch, corn starch, wheat starch, and potato starch contain various proportions of organic phosphorus or phosphate groups (22,23). Banana starches are reported to contain potassium and magnesium (24). Non-starch components such as protein and lipids influence starch behavior in food applications. Functional properties such as water absorption, gelatinization, and starch hydrolysis are influenced by the presence of these components. The presence of lipids, for instance, affects water absorption and hence gelatinization properties. This in turn influences the formation of resistant starch and starch susceptibility to enzymatic digestion.

II. STARCH SOURCES Starch is obtained from various plant sources. The most common sources of dietary and industrial starch are grains, such as maize and wheat, and roots such as potato and cassava (tapioca). Roots and tubers are significant sources of dietary starch (25).

A. GRAIN STARCHES The grains primarily used as dietary and industrial starch sources include various cereal grains, mainly maize, wheat, and rice. Legumes and pulses also contribute considerably to dietary starch consumption. Corn starch, from maize (Zea mays), is the most commonly used source of industrial starch. Corn contains about 86% starch on a dry weight basis. As a high amylose starch, it forms heavy and easy setting gels, and therefore is commonly used for thickening. Corn starch is also used as a carrier, as an ingredient for various applications, and as raw material for other industrial products. For instance, it is hydrolyzed in various ways to obtain sweeteners and glucose. Starch from wheat (Triticale aestivum) and rice (Oryza sativa) is also a predominant ingredient in food industry applications. Other cereal grains such as sorghum (Sorghum bicolor) and barley (Hordeum distichon) are sources of starch, less commonly used than maize or wheat starch (Figure 3.2).

Grain starches tend to have high levels of amylose. Furthermore, these starches typically contain amylopectin in the crystalline regions. The amylose of these starches meanwhile may form complexes with glycolipids (26). Most cereal starch sources such as maize and wheat starches are A-type starches (15). Various legumes contain up to 45% starch (12). Legumes commonly used as sources of starch include pinto bean, faba bean, moth bean, chickpea, and mung bean. As a result of their high amylopectin content, some legume starches such as mung bean starch have restricted swelling and increased overall stability during processing. They are therefore of high suitable quality for application in food products such as starch noodles (27). Most legumes contain B-type starches that are generally more resistant to digestion (28,29). In addition, other legume starches such as pea starch meanwhile contain Ctype starches. Legume starches have lower digestibility than other starches and hence result in a lower post-prandial glycemic and insulin response (30) (Figure 3.3).

B. ROOT AND TUBER STARCHES Among the root starches, potato (Solanum tuberosum) starch and tapioca (Manihot esculenta) or cassava starch are the most predominant industrial starch sources. Root starches have high amylopectin content and therefore have greater clarity, minimal flavor, and acceptable water absorption, and subsequently swelling capacity. Tapioca (cassava) starch is a major ingredient in dietary and industrial starch application. Also known as yucca or manioc, this root crop is the primary source of dietary energy in various tropical regions of the world. Tapioca starch has unique attributes that make it particularly desirable in food applications. Potato is a dietary staple of most European and Scandinavian diets. Potato starch has high water-binding capacity and a bland taste, and is commonly used in the food industry in many applications for thickening and texture modification. Other dietary and industrially important root and tuber starch sources include banana and plantain (Musa spp.), taro (Colocasia esculenta), cocoyams (Xanthosoma spp.), and various yams (Dioscorea spp.), sweet potato (Ipomea batatas) (31,32). Even though there are multiple sources of dietary starch in the tropics, including grains and legumes, roots and tubers constitute dietary staples in most areas as their cultivation is suited to the hot humid tropics. When freshly harvested, they are high in moisture, containing about 70–80% moisture and between 16–24% starch (32). Starchy foods are generally processed in some manner prior to utilization in food preparation. In addition, they are processed into raw material for secondary products. These therefore satisfy needs for calories, food preferences, and convenience foods (Figure 3.4). Some of these find limited use in industrial applications such as is the case with yam starches (32). Root


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starches contain amylopectin in the crystalline regions, while amylose is more common in the amorphous regions of the starch granule (26).

C. OTHER SOURCES

OF

STARCH

In addition to the major sources of dietary and industrial starch such as maize and tapioca starch, other starchcontaining plants find considerable application as dietary and commercial sources of starch. These include lesser known sources of starch such as sago (Metroxylon sagu),

arrow root (Maranta arundinacea), and edible canna (Canna edulis). Sago starch is obtained from the trunk of the plant Metroxylon sagu. The starch is used in various food products as it has high storage stability. Refined sago starch finds application in noodles, as well as raw material in industry for monosodium glutamate (MSG), glucose, and caramel (33). It is susceptible to enzymatic hydrolysis to glucose, which can then be fermented to produce fermentation products. Arrowroot starch contains up to 23% amylose and is used in dietary applications as a thickener in various

FIGURE 3.2 Some common cereal grain starch sources: (a) wheat, (b) barley, (c) maize.

FIGURE 3.3 Some legume starch sources: (a) pinto bean, (b) black-eye pea.

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FIGURE 3.4 (a) Brazil yam, (b) cassava (tapioca) root, (c) plantain.

sauces. Another lesser known root crop commonly used in starch production for food application is edible canna, obtained from Canna edulis (34). The root contains up to 16% high amylose starch. The separated starch is used in production of noodles in various parts of Asia, particularly Vietnam. The properties of the canna starch are desirable for noodle production.

III. STARCH PHYSICOCHEMICAL PROPERTIES AND FUNCTIONALITY The application of starch in food products is greatly influenced by its physicochemical properties and interactions with various components. The reaction of starch molecules in foods is essential for the multiple properties that they contribute to the quality of food products. For instance, water absorption and gel formation are extremely essential for the thickening properties of starch. In addition, hydrolysis and digestion of starch, for instance, are not feasible if starch is not gelatinized as the amylases and starch hydrolysis enzymes do not interact with intact, ungelatinized granules. Characteristics such as gelatinization temperature, granule size, and shape are specific to the type of starch. They are diagnostic properties characteristic of native

starches and can therefore be used for identification. The quality of products formulated with starch, such as carriers and thickeners, is largely affected by its functional and pasting properties (35,36). Water-holding capacity, solubility, and paste viscosity are important parameters that influence the quality of products such as carbohydrate-based fat substitutes (37). These in turn influence gelling ability, water- and fatbinding ability, slicing ability, and hence textural quality of food products. Functionality and physicochemical properties vary among starches as they are influenced by the ratios of amylose to amylopectin. High amylopectin starches for instance are preferred for high viscosity products. In addition, the presence of phosphate esters in some starches such as potato starch may influence starch waterbinding capacity by weakening the bonds between starch molecules due to ionic repulsion.

A. STARCH GELATINIZATION Gelatinization occurs when the ordered structure of the starch granule is disrupted and reorganized in the presence of heat and sufficient moisture. The granules are disrupted with absorption of water, losing their organized molecular structure, to facilitate swelling (29,38).


Starch gelatinization is critical in the utilization of starch in food applications. Native starch granules are insoluble in cold water and gelatinization is essential to facilitate water absorption and enhances the chemical and physical reactivity of inert starch granules in food processing (11). Granular characteristics of starches are characteristic of the plant source. The structure of the granule in turn influences the structure of the gels or pastes formed on heating. Gelatinization results in starch swelling, and formation of a viscous paste that may be opaque or translucent depending on the nature of the starch (12). Gelatinization is followed by gelation, a process in which the swollen granules are disrupted and amylose is released into the starch-water medium. The leaching of amylose from gelatinized granules contributes to the thickening characteristics of starch and gel formation, a colloidal dispersion of starch in water. The leached amylose in the starchwater system associates to form a structural network to entrap the granules, resulting in the formation of a gel. Viscosity of starches such as maize and tapioca starch are greatly influenced by ratios of amylose to amylopectin (10). Genetically modified high amylose starches form highly resistant and firmer gels (39). Increasing amylose content also increases early onset of gelation. Starches with low levels of amylose such as waxy maize — less than 1% amylose — do not form gels effectively. Instead, they form clear pastes that are generally resistant to syneresis (11). The strength of the starch gel is influenced by the presence of ionic components which may interact with the negatively charged starch molecules. Water absorption and swelling of starch is limited by the presence of amylose-lipid complexes (20).

B. STARCH RETROGRADATION Cooling of gelatinized starch results in the re-association of the leached amylose from gelatinized granules. This is the process of retrogradation. Retrogradation is also referred to as setback, and occurs with re-crystallization of amylose. Amylose is much more susceptible to retrogradation and amylopectin is only minimally involved in starch retrogradation even though amylopectin has been shown to influence retrogradation and syneresis in corn starch gels (5). This re-association and re-crystallization of amylose causes release of the water absorbed and bound during gelatinization, leading to the phenomenon known as syneresis. Retrogradation of starch in food products is a concern as it affects product quality. The stability of starch-containing products during cold storage in particular is greatly affected by the extent of retrogradation. Freeze-thaw cycles result in extensive retrogradation and syneresis. Retrogradation of starch in some instances enhances quality as such starches are resistant to enzyme hydrolysis

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and hence more stable. Cooling retrograded starches at room temperature prior to freezing at ⫺20°C results in the formation of resistant starch as the retrograded starch is no longer susceptible to enzyme hydrolysis (29). This procedure is used in the production of industrial resistant starch.

C. STARCH DAMAGE Starch damage is the modification or destruction of starch granule structure to the extent that it affects physicochemical properties such as water absorption. This in turn influences functionality of damaged starch in food applications, and subsequently, the quality of the final product. Starch damage results from various processes such as milling of grains. Starch damage affects the susceptibility of starch to hydrolysis and reactions as enzymes do not properly interact with the restructured granules. Starch damage by processing or mechanical action causes a cracked appearance to granules. Extensive starch damage causes disruptions in the molecular structure of the starch. Modification to the starch granule therefore results in increased swelling ability and is more susceptible to enzymatic hydrolysis (19). In addition, cold water solubility of starch is enhanced. This affects the applicability in baking and food applications.

D. INTERACTIONS WITH ACIDS, SUGAR, AND SALTS The presence of chemical components such as sugar and salts has a great effect on the characteristics of starch in food systems. The granule surface structure is affected and restructured in the presence of acid, as there is de-polymerization and hydrolysis of amylose and amylopectin (40). This results in lower viscosities of starch pastes. Solubility of starch is enhanced by acid. These effects are due to the disintegration of the component amylose and amylopectin at the low pHs typical of highly acidic solutions. Starch competes with sugars such as glucose, fructose, and sucrose for water absorption. Gelling and swelling of a starch is therefore modified in the presence of sugars. This is because sugars contain hydrophilic hydroxyl groups identical to the glucose monomers of starch. As a result they decrease the water activity of the starch-water system. There is an overall increase in the free volume of water, reducing its effectiveness as a desirable plasticizer required to facilitate starch gelatinization (41,42). Slade and Levine (1988) report that sugar has an anti-plasticization effect on starch. The sugars bind the water, reducing its availability for starch gelatinization (43). Consequently, sugars elevate the temperature at which the gelatinization of various starches occurs. The ionic nature of salts is responsible for their interaction with starch and the subsequent effects on starch physicochemical properties. Starch molecules possess a

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weakly charged ionic structure. In the presence of cations, the granules are stabilized and protected, whereas in the presence of anions, the hydrogen bonds are ruptured. This destabilizes the granules, enhancing and facilitating gelatinization (44,45). Various salts such as phosphates form complexes with the amylose and amylopectin, a property exploited for use in industrial in starch modification. Salts overall delay loss of birefringence and depress overall extent of gelatinization. Sodium chloride has great influence on starch physicochemical properties. Sodium chloride increases the gelatinization temperature of various starches. At concentrations of 6–9%, sodium chloride solution inhibits starch gelatinization (46). Various procedures for starch pre-treatment are commonly used in food processing. A common procedure is alkalization, in which alkalizing agents are added to maize, wheat, or rice in the preparation of tortillas, Chinese wheat noodles or rice dumplings, respectively. Common alkalizing agents include sodium hydroxide, or sodium and potassium carbonate (47). Addition of alkali contributes to improving starch swelling capacity. The presence of lime (calcium hydroxide) has been shown to decrease starch crystallinity in corn (48). Gelatinization temperature of corn starch is also increased by the presence of lime, attributed to cross linking of calcium with the starch, as well as due to ionic interactions with hydroxyl groups on the starches (47). It is expected that these would in turn lead to variations in gelatinized starch quality characteristics — color, gelation, and retrogradation tendency.

IV. STARCH HYDROLYSIS Starch hydrolysis is the cleaving of the starch polymer to short chain fragments such as dextrins and maltose, or to the glucose monomers. Starch hydrolysis is essential in many aspects of the application of starch. For instance, starch is hydrolyzed by various means for the production of sweeteners. Hydrolysis products of starch are multifold and include products such as dextrins and simple sugars. Starch hydrolysis is carried out primarily by the use of enzymes or chemicals, or in combination.

A. ENZYME HYDROLYSIS Enzymatic hydrolysis of starch is carried out for various purposes, but most notably for the industrial production of maltose syrup (49). The key enzymes used for starch hydrolysis are ␤-glucosidases, which hydrolyze the amylose and amylopectin in starch. These include α-amylase, amyloglucosidase, and pullulanase. The extent of starch hydrolysis is quantified by various parameters. This could be the hydrolysis index (HI), or by the dextrose equivalent (DE). The hydrolysis index

quantifies the proportion of starch hydrolyzed. Dextrose equivalent describes the potential for starch conversion to dextrose (glucose) and is defined as the sum of reducing sugars expressed as dextrose. This is because starch in its native form has few reducing sugar ends. The number of reducing ends is influenced by the proportion of amylopectin. Degree of polymerization is indicative of the number of glucose residues. Amylose from starches such as maize or wheat have DP of 200–1200, while amylose from potato or tapioca starch have DP of about 1000–6000. Hydrolysis of starch to maltodextrins is achieved by use of α-amylase enzymes. These enzymes are categorized either as endoamylases, exoamylases, debranching enzymes, or transferases (26). The endoamylases, the most common being α-amylase (EC 3.2.1.1), are specific for the α-1→4 linkage in amylose and amylopectin. Their hydrolysis products from starch hydrolysis are mainly oligosaccharides and dextrins (26). Exoamylases, on the other hand, have the ability to hydrolyze both the α-1→4 and α-1→6 bonds of amylose and amylopectin. A common example is amyloglucosidase (EC 3.2.1.20). β-Amylase is an exoamylase that has the ability to hydrolyze the α-1→4 bond of amylose. Debranching enzymes used in starch hydrolysis are targeted at hydrolyzing the α-1→6 bonds in amylopectin. These include pullulanases. Hydrolysis products of these are mainly maltose and maltotriose. The transferases have low activity with regard to starch hydrolysis but are involved in formation of new glycosidic linkages (26). Enzymatic hydrolysis of starch is influenced by the presence of non-starch components such as lipids, particularly lipids bound to amylose. This is because the presence of these complexes renders the amylose less susceptible to hydrolysis enzymes (20). Additional enzymes such as lysophospholipase are therefore sometimes required for complete hydrolysis of starch in the production of glucose from starch. Amylase enzymes produced by lactic acid bacteria — Lactobacillus plantarum, Lactobacillus amylophilus, and Lactobacillus delbruecki, in particular — are used for industrial hydrolysis of starch for conversation of starch to glucose. This is a process known as saccharification (50). Yeasts such as Saccharomyces cerevisiae, which produce α-amylase, are also used in bioreactors for enzymatic hydrolysis of starch and subsequent fermentation of the hydrolysis product (glucose) by the yeast strains (33). These micro-organisms produce heat-stable amylase which can survive the high bioreactor process temperatures required for gelatinization and hydrolysis of the starch. The lactobacilli produce enzymes that hydrolyze the starch to glucose, and then the bacteria ferment the starch of the industrial production of lactic acid. Immobilized enzymes also are used in industrial hydrolysis of starch. The enzymes are extracted from an


industrial source, usually microorganisms such as Aspergillus, and then immobilized on inert particles such as silica (51). This ensures that the enzyme has optimum activity and access for starch hydrolysis. Co-enzymes and ionic particles such as calcium are required for starch hydrolysis. While traditionally acids (mainly hydrochloric acid) have been used for hydrolysis of starch, there has been an increase in use of industrial enzymes for starch hydrolysis. Most of these convert starches for the production of maltodextrin, modified starches, glucose syrup, or fructose syrup. Hydrolysis of starch in foods is increased by processing. Enzymatic hydrolysis of starch in various legumes for instance is enhanced by soaking and sprouting. Gelatinization of starch is required prior to enzymatic hydrolysis.

B. ACID HYDROLYSIS Acids are used to facilitate the hydrolysis of starch. The α-1→4 linkages in amylose and amylopectin are susceptible to hydrolysis at the low pH typical of acids. Hydrochloric acid at low concentrations (0.36% w/v) hydrolyzes starch (52). The use of acids in combination of alcohols has been suggested for starch hydrolysis. Formation of limitdextrins with varying degrees of polymerization occurs in the presence of various alcohols such as methanol, ethanol, and propanol. These alcohols are possibly involved in disrupting the hydrophobic and hydrogen bonds of the starch helical structure in the granule. Increase in temperature further increases the susceptibility of starch to acid hydrolysis in alcohol (52).

C. ALKALINE HYDROLYSIS Alkaline hydrolysis of starch is enhanced and influenced in the presence of heat and inorganic salts. There is complete hydrolysis of starch with microwave heating in the presence of metal chlorides (53). The theoretical yield of glucose (111%) is obtained in the presence of chloride salts such as lithium chloride, barium chloride, and iron trichloride. On the other hand, acid hydrolysis of starch is greatly limited in the presence of sulfate salts. In the presence of sulfate salts — sodium sulfate, magnesium sulfate and or zinc sulfate — acid hydrolysis is actually greatly impeded (53).

D. HEAT-INDUCED HYDROLYSIS Extrusion of starch is used in combination with enzymes for effective starch hydrolysis. The starch is treated under conditions of high temperature, high pressure, shear, and moisture (54). Heat stable amylase is used for starch hydrolysis. Extrusion cooking facilitates disruption of the granule structure and the crystallinity. This renders the

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amylose and amylopectin susceptible to gelatinization (55). Application of extrusion in starch hydrolysis has the advantage in that the process conditions can be modified such that the extent of hydrolysis is controlled for desired end products and dextrins (55).

V. STARCH MODIFICATION Native starches such as tapioca starch often require considerable modification to enhance quality and versatility in food applications, and for storage stability. The components of starch — amylose and amylopectin — are highly sensitive to shear, stress, acidity, and high temperatures, and are typically altered by heat-moisture conditions of processing (15). Most native starches such as tapioca starch have limited swelling power and solubility. Modification is essential to improve paste clarity, paste stability, resistance to degradation, and freeze-thaw stability. Modification of starch is important to improve the reactivity of glucose, as well as introduce reactive side chains (56). The integrity and structure of the granule is also enhanced by modification. Additional side chains interfere with potentially deleterious post-process starch properties such as retrogradation. Most starches used in food applications are modified starches. Modification of starches is by physical and chemical procedures. Modification procedures include acetylation, hydroxypropylation, and a combination of hydroxypropylation and cross-linking (57). Hydroxypropylated starches are most commonly used in the food industry (57). Stabilization of starch is facilitated by use of acetates and hydroxypropyl esters (58). These modification procedures greatly increase freeze-thaw stabilization and increase resistance to process conditions such as heat and shear. Cross-linking is commonly carried out with various chemical agents such as phosphorus oxychloride, sodium trimetaphosphate, and anhydrides (58). Cross-linked starches are more resistant to process conditions such as temperature and acidity as a result of the fact that the hydrogen bonds have been reinforced and act as bridges. These are useful in preventing re-crystallization of amylose and the subsequent retrogradation in processed starchy foods. Some procedures that have been shown to be effective in modification of banana starch include cross-linking with sodium trimetaphosphate, formation of starch phosphate with sodium tripolyphosphate, and hydroxypropylation using a combination of sodium hydroxide and sodium sulphate (24). These procedures result in starches with enhanced water-binding capacity, and in most cases, increased solubility. Starch phosphates in particular have increased freeze-thaw stability. Acid-thinned starches are obtained by reducing the concentration of concentrated starch slurry with a mineral

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acid at 40–60°C, to obtain a desirable viscosity. The starch is recovered after the acid is neutralized (59). The granule structure of the starch is not destroyed in the process, but various changes to the properties of the starches occur. Starch solubility and gel strength for instance are increased, while starch viscosity is decreased (60). Rate of starch hydrolysis is increased with increasing concentration of acid (59). Physical modification procedures that have been used include pre-gelatinization. Pre-gelatinization increases swelling power and paste clarity of banana starch (24). Extrusion cooking of starch is used to improve quality and characteristics of starch.

VI. STARCH IN FOOD APPLICATIONS Starch is a functional ingredient in many food products. There are multiple functions of starch in food products. Most commonly, starch is used as a bulking agent, binder, carrier, in fat-replacers, as a texture-modifier, and as raw material for other starch-related products. It is a basic ingredient in products such as breads, puddings, marinades, and sauces, and also serves a considerable function in other products such as powdered spices and beverages. The applicability and utility of a starch in food products is enhanced by factors such as its composition and functionality. Starch is a substrate for lactic acid bacteria in fermentation to produce lactic acid (61). Starch-based foods play a major role in the diet in various areas because of their bulking quality, and ability to contribute to satiety. Fermentation of cassava for instance imparts a sour taste that is sometimes highly desirable.

A. FUNCTIONAL PROPERTIES Starch is used as to facilitate thickening and gel formation in various food products such as fruit preparations (62). The consistency of products such as tapioca pudding and many custards would not be attainable without the thickening and stabilizing properties of starch. High amylose starches have high viscosity, and form thick gels. This enhances their properties as thickeners in food products. Starches with lower amylose content are better suited for use in certain types of noodles, such as Japanese noodles (23). High amylose starches are desirable for application in fried products as they have minimal fat absorption. High amylose starches are also applicable as thickeners and for use as gelling agents in foods such as jellies. High amylose starch gels set rapidly hence are desirable in production of confectionary and candies (56). Other desirable properties of high amylose starches include their flexibility, water resistance, and tensile strength (63). Starches with high swelling ability and high viscosity are desirable for various types of Asian noodles (18).

Starches high in amylopectin and low in amylose (waxy starches) such as waxy wheat are produced for use in such products. The higher levels of amylopectin further contribute to extending shelf-life, by reducing retrogradation and staling in baked products. High amylopectin starches are less susceptible to retrogradation, and hence very applicable in improving freeze-thaw stability (63). Modified starches are highly effective as stabilizers in products such as yogurt (57). The presence of side groups such as acetyl and hydroxyl groups in modified starches, however, results in interactions with amylose and amylopectin, improving overall stabilizing ability. Fermented, sun-dried cassava starch is commonly used in baked products in various parts of South America and Brazil. This is unique in that the fermentation facilitates expansion which is desirable in the baked products (64). Viscosity of sour starch pastes is lower than for nonfermented starch, attributable to the solubilization of amylopectin. Starch is used to improve quality of extruded food products. Addition of cassava starch to cassava flour prior to extrusion increases water solubility, but decreases water absorption and bulk density properties (65). Shear thinning of starch is an important characteristic with regards to stability of starch pastes during processing, particular in food products that require extensive stirring and agitation. Removal of lipids (defatting) in sorghum starch has been associated with increased shearthinning characteristics (66). Starches that are resistant to shear thinning are generally highly desirable to ensure product stability and suitable consistency. Products of starch hydrolysis find considerable application in food products. Maltodextrins, for instance, are commonly used in heat-stable gels (67).

B. VALUE-ADDED FOOD APPLICATIONS Starch is used as a basic ingredient in starch-based fat substitutes. These simulate the functional properties of fats, particularly texture modification, but with less caloric value. Various starch-based fat substitutes are commonly used in industry. Some examples of these include TrimChoice (Specialty Grain products, NE) made from hydrolyzed oat starch, Amalean (American Maize Products, IN) made from modified high-amylose corn starch, and SlenderLean (National Starch, NJ) made from tapioca starch (37). Starch-based fat substitutes are especially applicable in baked products and value-added foods. Resistant and minimally digestible starches are used in value-added food products. Most of these products are targeted at the management of diet-related diseases such as obesity and type II diabetes. An example of such a product is Extend, a snack bar formulated with resistant starch (corn and rice starch), which has been formulated for the


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Native starch

Starch processing techniques Hydrolysis (enzyme / acid / other)

Chemical modification

Maltodextrins, glucose

Cross-linked, esterified starches

Heat-moisture (microwaving / other)

Extrusion

Multi-cycle autoclaving

Genetic modification

Starch products Solubilized starch

Extrudates

Resistant starches (RS 3)

High amylose or amylopectin

Functional food ingredients

Gel, functional properties

Applicable properties in foods Heat stable gels, ingredients

Freeze-thaw stability; functionality

Functional, gelatinization properties

Swelling, hydrolysis

FIGURE 3.5 Outline of starch processing and applications in foods.

management of type II diabetes. The product ensures a slow and sustained digestion of the resistant starch, and minimal absorption of glucose, mitigating the problems of high post-prandial blood glucose levels (Figure 3.5).

VII. STARCH NUTRITIONAL QUALITY Starch is the primary nutrient involved in energy intake and regulation. Typically, most adults require about 200 g carbohydrate daily to facilitate brain and muscle function. The digestibility and absorption of starch has significant nutritional and physiological implications. As the primary source of energy, starch is rapidly metabolized and absorbed. The extent of starch digestibility is influenced by the nature of the starch, food processing, and physiological status. There is a dichotomy of starch functionality as a nutrient. On the one hand, it is a source of glucose, the primary substrate for cell metabolism. On the other hand, starch resistant to digestion (resistant starch) is minimally digestible and only minimally absorbed, and therefore is not physiologically available.

A. STARCH AND GLYCEMIC INDEX Starch is the primary source of metabolizable energy, and therefore its availability and digestion are important. High

starch foods are rapidly digested and metabolized. Glycemic index (GI), the post-prandial blood glucose response to a particular food, has been used to differentiate the metabolic response to dietary carbohydrates (68). Glycemic index is indicative of the relationship between a food and the implications of starch digestibility, absorption, and metabolism. Foods that are high in readily digestible starch result in high levels of glucose in the blood. These are classified as high GI foods. Typically, most tropical root starchy staples such as cassava and yams have high levels of readily digestible waxy starches (high amylopectin). These are rapidly and readily absorbed, resulting in elevated levels of glucose in the blood. This is, however, modified to a large extent by other factors associated with the nature of the starch, its processing, preparation, and consumption. Starch is hydrolyzed by salivary and pancreatic amylases to yield monosaccharides such as glucose and fructose, and maltodextrins. These are transported via the hepatic portal vein and available for metabolism. Starch digestion and metabolism has been classified into three categories: rapidly digestible starch, slowly digestible starch, and resistant starch (69). Rapidly digestible starch, which typically is completely digested, is associated with post-prandial glucose response, and hence has effect on insulin levels. Rapidly available glucose meanwhile describes glucose and sucrose obtained as hydrolysis

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products of rapidly digestible starch. Rapidly digestible starch occurs most commonly in highly processed foods such as puffed wheat cereal, while slowly digestible starch occurs in foods such as legumes and pasta (69). Starch digestion and glycemic index also have been associated with satiety. Rapidly digestible starches are quickly absorbed and metabolized, whereas slower digesting starches are only slowly absorbed and therefore improve satiety (70). These have also been shown to improve exercise endurance (71). There are differences in the metabolic response to dietary carbohydrates. Post-prandial blood glucose and insulin responses vary depending on the nature of carbohydrates, particularly starch. Physiological conditions such as type II diabetes and obesity have been associated with starch metabolism. Other conditions such as coronary heart disease are linked to metabolism of glucose derived from dietary starch. The physiological consequence of starch consumption is influenced by the extent of its digestibility and metabolism. Digestibility of starch is determined by its availability and susceptibility to digestive enzymes. Susceptibility of starch to digestive enzymes is in turn influenced by the chemical nature of the starch and the changes that result from processing. Starch digestibility is influenced by various factors such as processing, storage, amylose content, and presence of dietary fiber (72). The ratios of amylose to amylopectin are important in starch digestion and metabolism. Consumption of modified high amylopectin starches have been shown to result in an increase in serum free fatty acids and serum glucose levels. This is probably as a result of gluconeogenesis. Conversely, modified amylose cornstarch is highly digestible, and results in lower insulin levels. Starch that is digested and absorbed, however, has physiological effects, some of which have been linked to disease conditions. Researchers have demonstrated that the consumption of high starch diets in human test subjects apparently leads to an overall decrease in overall energy intake, compared to a high-sucrose (simple sugar) or high fat meals (70,73). This indicates that high starch may have potential for a high satiety value, but with low caloric density, and hence the lowered energy intake. Type II diabetes, a condition that results from inadequate production of insulin to facilitate glucose uptake, is exacerbated by the presence of glucose in the blood. Clinical manifestations of Type II diabetes include fainting and dizzy spells as a result of low brain glucose levels. Starch and glucose metabolism have been also associated with obesity and accumulation of fat, as glucose is involved in fat metabolism. The nature of starch and the level of amylose in the starch play a considerable role in the diabetic process and insulin response. Long-term consumption of a high amylose corn starch (70% amylose) by hyper-insulinemic

subjects results in a normal insulin response (74). High amylose starch in the diet reduces insulin response (75). Meanwhile legume starches such as pure pea starch have been shown to be even more effective than corn starch in reducing hyperglycemia, as has been demonstrated with purified pea starch (76). The conversion of sugars, which are starch hydrolysis products, into fat has been implicated in diabetes and cardiovascular disease and obesity. The consumption of simple sugars and refined grain foods has been linked to higher rates of cardiovascular disease and Type II diabetes, particularly in instances of insulin resistance (68,77).

B. RESISTANT STARCH Resistant starch is non-digestible starch which occurs in foods in various forms. Resistant starch is described in various ways, including as starch and starch degradation products not absorbed in the gut (78,79). Resistant starch occurs in four categories, primarily dependent on their mode of origin. These are described as: type 1 (RS1): physically entrapped starch in the cell matrix of whole or partially milled grains, hence is inaccessible; type 2 (RS2): native granular starch, mostly B-type legume starches which are may be ungelatinized during processing; type 3 (RS3): retrograded starch, particularly from food processing; and type 4 (RS4): chemically modified starch (29,79–82). Resistant starch levels in food products are influenced by various factors including the nature of the starch, the mode of food processing and preparation, and storage conditions (83). Physical inaccessibility such as cell wall structure and the presence of dietary fiber influences levels of resistant starch, particularly in legumes (72). Type 3 resistant starch (RS3), which is retrograded starch, is the most commonly occurring form of resistant starch in processed foods. Starch in cooked then cooled foods such as pasta, rice, and lentils exhibits considerably reduced susceptibility to enzymatic digestion, indicating the formation of resistant starch (72). This is attributable to the retrogradation which occurs following cooling of gelatinized starch. Amylose is more susceptible to retrogradation than amylopectin. Resistant starch formation is therefore influenced by ratios of amylose to amylopectin. Processing of starchy foods, in addition to factors such as starch amylose: amylopectin ratios and chemical modification, influences their digestibility. This is as a consequence of the disruption of the physical and chemical structure of the starch (84,85). Retrogradation of amylose with processing is mainly thought to be responsible for this alteration in susceptibility to digestive enzymes. In some cases, however, partial damage to starch molecules which would otherwise be physically entrapped


by the cell wall and inaccessible to digestive enzymes, may improve their susceptibility to digestion (86). Consumption of resistant starch yields physiological effects similar to soluble fiber (82). Fermentation products include short chain fatty acids such as acetate, propionate, and butyrate, which facilitate absorption of minerals, excretion of bile acids, and consequently protect against colorectal cancer. Resistant starch-containing foods such as legumes, and retrograded starches, however, have been associated with disease prevention. Fermented corn porridge, commonly consumed among some indigenous populations, has been shown to contain considerable amounts of starch that is resistant to digestion and subsequently is protective against various colon conditions (87). Experimental evidence using high resistant starch breakfast cereals in humans shows an improved glucose tolerance (88). In rural South Africa, however, consumption of cold maize porridge, which is high in retrograded starch and has rather low starch digestibility and a low glycemic index, has been associated with low levels of diabetes mellitus (87,89). High resistant starch foods which have low glycemic index are effective in lowering the concentrations of highdensity-lipoprotein (HDL) cholesterol and in improving glucose tolerance in incidence of diabetes and insulin resistance (77,88,90). Root and tuber starches, unlike grain starches, are high in amylopectin and do not have the same restricting nature of the cell wall. They are, therefore, generally more digestible. Processing techniques such as autoclaving to reduce starch digestibility by increasing resistant starch levels have been suggested in foods (91). Digestibility of legume starch is increased by processes such as soaking and sprouting (30).

VIII. NEW STARCH TECHNOLOGIES The functionality and applicability of starch in so many food applications and its importance as a food ingredient have led to continuous efforts to improve and optimize properties and versatility of starch. Some techniques currently used include genetic modification to modify starch yields and quality, multi-cycle autoclaving for production of resistant starch, and new processes such as microwave hydrolysis of starch.

A. GENETIC MODIFICATION Genetic modification of starch most commonly targets the enzymes of the starch biosynthetic pathway. The activities of these enzymes dictate and determine the quantities of starch synthesized as well as specific characteristics such as ratios of amylose to amylopectin. Their activity therefore influences starch properties: its reactivity, functionality, and applicability in food processing and in food applications.

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Genetic modification of cereal starch is commonly employed to modify ratios of amylose to amylopectin, and hence improve functionality and nutritional quality of starch. The primary enzymes involved in starch synthesis include the starch synthases, starch branching enzymes and adenosine diphosphate-glucose pyrophosphorylase (ADP-glucose-phosphorylase). The starch synthases occur both as a granule-bound synthase (GBSS) or located in the soluble phase, and catalyze the formation of the α-1→4 glucan chains by adding ADP-glucose to the non-reducing end of the primer. The starch branching enzyme catalyzes formation of the α-1→6 branches of amylopectin molecules. The ADP-glucose pyrophosphorylase catalyzes the formation of ADP-glucose (56). Other important enzymes are starch debranching enzymes and phosphorylases. A major contribution of genetic modification is the modification of various cereal starches to reduce amylose content and produce high amylopectin starch. These waxy starches are desirable for various characteristics. They are desirable in various noodles, for modification of amylose characteristics in extrudates, and to extend the shelf-life of baked goods (18). Waxy starches are produced by modification of the enzyme involved in amylose synthesis, granule-bound starch synthase (GBSS). While naturally occurring mutations in various wheats have resulted in waxy wheat starch, biotechnology to modify the expression of the GBSS genes is used to produce waxy starches, including rice, maize and wheat. Modification by decreasing the levels of enzymes such as starch synthase and starch branching enzyme is employed to increase amylose levels. Modification by decreasing levels of GBSS results in increased amylopectin levels. Regular cereal starches (up to 27% amylose) typically form opaque pastes and firm gels. Genetic modification techniques are applied to either decrease or increase the amylose to amylopectin ratios. Low amylose cereal starches (waxy maize, waxy rice, waxy wheat) lack GBSS, and therefore contain less than 1% amylose. These therefore do not effectively form gels but instead clear pastes. Genetically modified high amylose starches form highly resistant and firmer gels (41). Increasing amylose content also increases early onset of gelation. High amylose maize starch — amylomaize — is modified to have high levels of amylose, 50–70%. The granules of amylomaize are more resistant to swelling and therefore form much firmer and more rigid gels (11,92). Genetically modified potato starch has been shown to be suitable in processing and preparation of starch noodles, as these have greater transparency and higher flexibility (93). This may be due to higher amylose content. Modification of starch synthesis to increase overall yields of starch in food products is carried out by modifying

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levels of adenosine triphosphatase (ATPase) and starch branching enzymes (56). Genetic modification of reactive groups such as phosphates is used to change the composition of starch by decrease of the starch branching enzyme (56).

B. RESISTANT STARCH PRODUCTION AUTOCLAVING

BY

Autoclaving and steam processing are used in the production of resistant starch. Resistant starch produced by this technique is retrograded starch (RS3), as it involves gelatinization and subsequent retrogradation of starch, rendering it resistant to digestive amylases. Autoclaving has been shown to modify resistant starch content in grain sorghum (94). High amylose starches which are most susceptible to retrogradation are therefore preferred for this process. High pressure autoclaving has been standardized for the production of resistant starch (91,95). Starch with a high volume of water is gelatinized in a high pressure autoclave with stirring until a homogenous gel is obtained. The mixture is then cooled and frozen to facilitate retrogradation.

C. OTHER PROCEDURES Other procedures employed in starch modification include microwave solubilization (96,97). Corn starch modified by microwave heating for a short period of time (32–90 seconds) at 900 W has decreased swelling ability (96). Microwave pre-solubilization of starch at 180 W for 10 minutes is employed in food analysis (97). In the presence of dilute hydrochloric acid, there is complete hydrolysis of starch in 5 minutes of microwave processing. This is attributable to the superheating produced by the presence of the ions. These procedures are proposed to substitute for the more expensive and time-consuming enzyme hydrolysis procedures commonly used.

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4

Functional Properties of Carbohydrates: Polysaccharide Gums

Steve W. Cui and Qi Wang

Food Research Program, Agriculture and Agri-Food Canada

CONTENTS I. Introduction ............................................................................................................................................................4-2 II. Functional Properties of Polysaccharide Gums ......................................................................................................4-2 A. Viscosity Enhancing or Thickening Properties ................................................................................................4-2 B. Gelling Properties ............................................................................................................................................4-2 C. Surface Activity and Emulsifying Properties ..................................................................................................4-3 III. Chemistry, Functional Properties, and Applications of Polysaccharide Gums in Food and Other Industries ......4-3 A. Gums from Exudates ......................................................................................................................................4-3 1. Gum Arabic ................................................................................................................................................4-3 2. Tragacanth Gum ........................................................................................................................................4-4 3. Gum Karaya ................................................................................................................................................4-4 4. Gum Ghatti ................................................................................................................................................4-5 B. Gums from Plants ............................................................................................................................................4-5 1. Galactomannans (Locust Bean, Tara, Guar, and Fenugreek Gums) ..........................................................4-5 2. Pectins ........................................................................................................................................................4-6 3. Konjac Glucomannan ................................................................................................................................4-7 4. Soluble Soybean Polysaccharides ..............................................................................................................4-7 5. Flaxseed Gum ............................................................................................................................................4-8 6. Yellow Mustard Gum ..................................................................................................................................4-8 7. Cereal β -Glucan ........................................................................................................................................4-8 8. Psyllium Gum ............................................................................................................................................4-9 9. Pentosans/Arabinoxylans ..........................................................................................................................4-10 C. Gums from Seaweeds ....................................................................................................................................4-10 1. Agar ..........................................................................................................................................................4-10 2. Algin (Alginates) ....................................................................................................................................4-11 3. Carrageenans ............................................................................................................................................4-11 D. Gums from Microbial Fermentation ............................................................................................................4-12 1. Xanthan Gum ............................................................................................................................................4-12 2. Gellan Gum ..............................................................................................................................................4-13 3. Curdlan Gum ............................................................................................................................................4-13 4. Dextran ....................................................................................................................................................4-14 E. Chemically Modified Gums ..........................................................................................................................4-14 1. Microcrystalline Cellulose (MCC) ..........................................................................................................4-14 2. Carboxymethylcellulose (CMC) ..............................................................................................................4-14 3. Methylcellulose ........................................................................................................................................4-15 4. Hydroxypropylcellulose and Hydroxyethylcellulose ..............................................................................4-15 5. Chitin and Chitosan ..................................................................................................................................4-16 IV. Future Prospects and New Development ..............................................................................................................4-16 References ....................................................................................................................................................................4-16

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I. INTRODUCTION Gums are long chain polysaccharides widely used in the food and many other industries as thickeners, stabilizers, and texture modifiers. Gums and related polysaccharides are produced in nature as storage materials, cell wall components, exudates, extracellular substances from plants or microorganisms, and in some cases from exoskeletons of shellfish such as lobsters, shrimps and crabs (e.g., chitosan). Some polysaccharides are simple in sugar composition, such as cellulose and β-D-glucans, which contain only one type of monosaccharide (e.g., β-D-glucose), while others are rather complex and may contain up to six types of monosaccharides plus one or two types of uronic acids. Common monosaccharides and uronic acids present in natural polysaccharides include D-glucose, D-galactose, D-mannose, D-xylose, L-arabinose, L-rhamnose, L-fucose, D-galacturonic acid, D-gulucuronic acid, D-mannuronic acid, and L-guluronic acid. The primary structure of a polysaccharide, i.e., monosaccharide composition, linkage patterns, and molecular weight, defines the solubility and conformation of the polymer chains in aqueous solutions, which in turn dictate the functional properties of the gums exhibited in food and other systems. Polysaccharides can be linear or branched polymers. With the same molecular weight, linear polysaccharides generally have poorer solubility and higher viscosity than branched counterparts due to their extended conformation in solutions (if soluble or dispersible). Perfectly linear homoglycans such as cellulose are either difficult to dissolve or insoluble in aqueous medium due to excessive intra- and intermolecular interactions (mainly through hydrogen bonding), which make them less useful as hydrocolloidal gums. Irregularity introduced by substitution or branching to the linear chain increases solubility. Highly branched polysaccharides are usually very soluble but exhibit lower viscosity in solutions because of their smaller hydrodynamic volumes compared to liner molecules with the same molecular weight. The variations in monosaccharide composition, linkage patterns, molecular weight, and molecular weight distribution of gums contribute to the unique functional properties exhibited by each gum. The goal of the present chapter is to provide information on the basic structural and functional properties and major applications of all commercial gums and some emerging gums in food and other industries. For detailed descriptions of chemical structure, molecular characterization, physicochemical properties, and applications of these gums, readers are referred to several comprehensive books and chapters (1–5).

II. FUNCTIONAL PROPERTIES OF POLYSACCHARIDE GUMS A. VISCOSITY ENHANCING OR THICKENING PROPERTIES When polysaccharide gums are dissolved into solution, one remarkable phenomenon is the considerable increase in solution viscosity; gums restrict the movement of water molecules and in extreme cases gels are formed. The ability of polysaccharide gums to increase viscosity or to thicken the aqueous system is the most important property of such polymers. The shape and conformation of polysaccharides are determined by their primary sequence structure. Once the structure is determined, the shape and/or conformation of a polysaccharide are more or less fixed, and the molecular weight (size) and number of polysaccharide molecules in a given volume (concentration) become important in determining their functional properties. In addition, environmental factors such as solution pH, temperature, presence of certain ions, and ionic strength of the system have significant influences on the conformation of polysaccharide chains, and hence their functional properties. Solution viscosity of a gum almost always increases with concentration, but not necessarily in a linear manner. At low concentrations, dilute gum solutions normally exhibit Newtonian flow behavior (independent of shear rate) in which polymer molecules are free to move independently without intermolecular entanglements. For most random coil polysaccharides, the relationship between zero-shear specific viscosity (ηsp) and concentration (c) follows ηsp ∝ c1.1–1.3. When the polymer concentration is increased to a critical point (critical concentration C*), the viscosity of the solution increases sharply due to entanglement of polymer molecules. This is called the semi-dilute region within which polysaccharide gums usually exhibit shear thinning flow behavior where viscosity decreases with increase in shear rate. The viscosity of most gum solutions decreases with increased temperature, although some gums are more resistant to temperature changes. For example, the viscosity of xanthan gum solution is relatively unchanged over a wide range of temperatures (⫺4°C to 93°C) (4). There are other extremes, such as methyl cellulose, where the viscosity increases as the temperature increases, and eventually gels are formed at higher temperature. Other factors influencing viscosity include pH, ionic strength, and presence of co-solutes with effects differing in individual gums.

B. GELLING PROPERTIES All hydrocolloids have viscosity enhancing or thickening properties, but only a few are able to gel. Gelation of polysaccharides is caused by the cross-linking (covalently


and/or non-covalently) of long polymer chains to form a continuous three-dimensional network which traps and immobilizes water and forms a firm and rigid structure resistant to flow under force. Gelation of polysaccharides usually involves three stages: 1) Polysaccharide gums have to be dissolved/dispersed at temperature above the melting point. At this stage, polymer chains exist in a coiled conformation. Upon cooling, polymer chains start to form ordered structures such as helices. 2) Formation of a gel network with further cooling. At this stage, helices begin to aggregate by forming cross-links or super-junctions, and a continuous network is eventually developed. 3) Aging stage where existing helices or aggregations are further enhanced and some new helices are formed. Contraction of gel networks may occur with the liberation of free water (“weeping” or syneresis). Most of the polysaccharide gels are thermally reversible below 100°C with defined setting and melting temperature ranges. There is a minimum concentration for each polysaccharide, below which gel cannot be obtained. Some gels exhibit thermal hysteresis where the melting temperature is significantly higher than the setting temperature, e.g., agarose gels (6). However, there are a few gelling gums which do not follow the above rules. Some gums form gels upon heating while others can form gels by changing ionic strengths and pH or introducing specific ions. A wide range of gels with different textures, such as soft, elastic, very firm, and brittle, can be prepared by selecting different types of polysaccharides and by varying gelation conditions.

C. SURFACE ACTIVITY AND EMULSIFYING PROPERTIES Although polysaccharides are hydrophilic compounds not conventionally perceived to be surface active, many polysaccharide gums are used to stabilize emulsions that already contain an emulsifier (proteins or surfactants). The universal role of gums in emulsion systems is to thicken the continuous phase, thereby inhibiting or slowing droplet flocculation and/or creaming. There are a few exceptions of gums that actually exhibit surface activity; these gums play double roles in emulsion systems: as an emulsifier and a thickener. In most cases, surface activity of these gums is attributed to the protein component associated with the polysaccharides, while in other situations, the surface activity is due to the presence of hydrophobic functional groups, such as in the cases of methyl cellulose and propylene glycol alginate. Although it is still controversial regarding what is responsible for the surface activity, fenugreek gum does exhibit excellent emulsifying and emulsion stabilizing properties even at very low protein content (e.g., ⬍0.5%) (7). Detailed applications of these gums as emulsifiers are described in the following sections.

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III. CHEMISTRY, FUNCTIONAL PROPERTIES, AND APPLICATIONS OF POLYSACCHARIDE GUMS IN FOOD AND OTHER INDUSTRIES A. GUMS

FROM

EXUDATES

The earliest gum known to humans is from plant exudates. Many plants exude a viscous, gummy liquid when wounded and the liquid will dry to form hard, glassy, tear-drop-like balls or other shapes of masses. The exudates are hand collected, sorted/graded, and further processed to meet the application needs. Gum arabic, tragacanth, karaya, and ghatti are exuded gums that are commercially significant. 1. Gum Arabic

a. Source and structure Gum arabic, or acacia gum, is prepared from the exudate of Acacia trees, mostly from senegal species, and sometimes mixed with seyal species. Natural gum is in the form of spherical balls resembling tear drops, collected by hand and processed before use. Almost all commercial gum arabic is produced from the Sahelian regions of Africa. Gum arabic consists of a mixture of a relatively lowmolecular-weight polysaccharide (~0.25 ⫻ 106 daltons, a major component) and a high-molecular-weight hydroxyproline-rich glycoprotein (~2.5 ⫻ 106 daltons, a minor component) (8). It is a heavily branched polysaccharide; the main chain consists of (1→3)-linked β-D-galactopyranosyl residues. The side chains are two to five units in length made of (1→3)-linked β-D-galactopyranosyl units, joined to the main chain by (1→6)-linkage. Both main and side chains are substituted by α-L-arabinofuranosyl, α-L-rhamnopyranosyl, β-D-glucuronopyranosyl, and 4-O-methyl-β-D-glucuronopyranosyl units. The monosaccharide composition of gum arabic varies with gum sources, e.g., gum from Acacia senegal contains about 44% galactose, 27% arabinose, 13% rhamnose, and 16% glucuronic acid of which only 1.5% are 4-O-methylated. In contrast, gum arabic from Acacia seyal contains 38% galactose, 46% arabinose, 4% rhamnose, and 12% total glucuronic acid (of which 5.5% are 4-O-methylated) (8). These compositional and structural differences affect their functionalities, e.g., gum arabic from Acacia senegal is a much better emulsifier than gum from Acacia seyal. b. Functional properties and applications Gum arabic is readily dissolved in water to give clear solutions with light colors ranging from very pale yellow to orange brown. It is a typical low viscosity gum and the solutions exhibit Newtonian flow behavior even at concentrations as high as 40%. Higher concentration solutions can be

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prepared up to 55%. A major functional property of gum arabic is its ability to stabilize oil-in-water emulsions. The protein-rich high-molecular-weight species are preferentially adsorbed onto the surface of oil droplets while the carbohydrate portion inhibits flocculation and coalescence by electrostatic repulsions and steric forces (8). The major application of gum arabic is in the confectionary and beverage industries for stabilizing emulsions and flavor encapsulation. In the confectionary industry, gum arabic is used to prevent sugar crystallization and to emulsify the fatty components. Examples of such products include pastilles, caramel, and toffee. Gum arabic has also been used in chewing gums, cough drops, and candy lozenges. Good stability under acidic conditions makes gum arabic useful in beverages, e.g., it is used as an emulsifier in the production of concentrated citrus juices and cola flavor oils of soft drinks (3). Gum arabic stabilized flavor oils can be spray-dried to form microencapsulated powders that can be easily incorporated into dry food products such as soup and dessert mixes.

The viscosity of the suspension reaches a maximum after 24 hours at room temperature, and hydration can be accelerated by an increase in temperature. The suspension typically exhibits shear thinning behavior. The ability to swell in water, forming thick, viscous dispersions or pastes, makes it an important gum in the food, pharmaceutical, and other industries. It is the most viscous of the natural water-soluble gums and is an excellent emulsifying agent with good stability to heat, acidity, and aging. Food applications of tragacanth gum include salad dressings, oil and flavor emulsions, ice creams, bakery fillings, icings, and confectionary. In the pharmaceutical and cosmetic industries, tragacanth gum is used as an emulsifier and stabilizer in medicinal emulsions, jellies, syrups, ointments, lotions, and creams. Gum tragacanth is also a good surface design thickener since it is a good medium for mixing with natural dyes and conveying controlled design onto fabric. It allows easy painting, stamping, and stenciling, and ensures a good control over color placement.

2. Tragacanth Gum

3. Gum Karaya

a. Source and structure Tragacanth gum is dried exudates from branches and trunks of Astragalus gummifer Labillardiere or other species of Astragalus grown in West Asia (mostly in Iran, some in Turkey). After hand collection, the exudates are graded, milled, and sifted to remove impurities. Tragacanth gum is composed of a water-soluble fraction and a water-insoluble fraction. The water-soluble fraction, accounting for 30–40% of total gum, is a highly branched neutral polysaccharide consisting of L-arabinose side chains attached to D-galactosyl backbones (9, 10). The D-galactosyl residues in the core chains are mostly 1→6-linked, sometimes 1→3-linked, whereas the branching L-arabinosyl residues are mutually joined by 1→2-, 1→3-, and/or 1→5-linkages. The waterinsoluble fraction, the major fraction (60–70%), is an acidic polysaccharide consisting of D-galacturonic acid, D-galactose, L-fucose, D-xylose, L-arabinose, and L-rhamnose, and is called tragacanthic acid or bassorin. It has a (1→4)-linked α-D-galacturonopyranosyl backbone chain with randomly substituted xylosyl branches linked at the 3 position of the galacturonic acid residues. Some of the xylosyl residues are attached by an α-L-fucosyl or a β-D-galactosyl residue at the 2 positions (3, 11).

a. Source and structure Gum karaya is from the exudates of Sterculia urens, trees of the Sterculiaceae family grown in India. It is a branched acidic polysaccharide with high molecular weight. Gum karaya contains 37% uronic acid and 8% acetyl groups. The backbone chain consists of (1→4)linked α-D-galacturonic acid and (1→2)-linked α-Lrhamnosyl residues with side chains of (1→3)-linked β-D-glucuronic acid, or (1→2)-linked β-D-galactose on the galacturonic acid unit where one half of the rhamnose is substituted by (1→4)-linked β-D-galactose (12, 13). The quality of the gum varies significantly depending on the season of collection: summer usually gives high yields and high viscosity gum. During storage, the viscosity of gum karaya can be lost when exposed to high temperature and high humidity. The decrease in viscosity is more significant when the particle size is small. Preservatives may be added to prevent viscosity loss.

b. Functional properties and applications Tragacanth gum swells rapidly in both cold and hot water to form a viscous colloidal suspension rather than a true solution. When added to water, the soluble tragacanthin fraction dissolves to form a viscous solution while the insoluble tragacanthic acid fraction swells to a gel-like state, which is soft and adhesive. When more water is added, the gum first forms a uniform mixture; after 1 or 2 days, the suspension will separate into two layers with dissolved tragacanthin in the upper layer and insoluble bassorin in the lower layer.

b. Functional properties and applications Similar to gum tragacanth, gum karaya does not dissolve in water to give a clear solution but swells to many times its own weight to give a dispersion. The type of dispersion is influenced by the particle size of the product. For example, coarse granulated gum karaya produces a discontinuous, grainy dispersion whereas a fine powdered product gives a homogenous dispersion. Dispersion of gum karaya exhibits Newtonian flow behavior at low concentration (⬍0.5%) and shear thinning behavior at semi-dilute concentrations (0.5% ⬍ c ⬍ 2%) (13). Further increase in gum concentration produces a paste resembling spreadable gels. An increase in temperature improves solubility in water, but excessive heat will cause degradation of the polysaccharides, resulting in non-recoverable loss of viscosity. At


extreme pHs and in the presence of sodium, calcium, and aluminium salts, the viscosity of gum karaya dispersion decreases. Gum karaya is used to stabilize packaged whipped cream products, spread cheeses and other dairy products, frozen desserts and salad dressings, and as acid-resistant stabilizers in acidified products. It is also used as a water binder in bread, processed meats, and low-calorie dough-based products such as pasta (11). Other applications of karaya gum include dental adhesives, bulk laxatives, and adhesives for ostomy rings. Gum karaya is also used in the manufacture of long-fibered, lightweight papers in the paper industry and as a thickening agent in the textile industry to help print the dye onto cotton fabrics. 4. Gum Ghatti

a. Source and structure Gum ghatti is an amorphous translucent exudate of Anogeissus latifolia, a tree of the Combretaceae family grown in India. It contains L-arabinose, D-galactose, D-mannose, D-xylose, and D-glucuronic acid in the ratio of 10:6:2:1:2, plus traces of a 6-deoxyhexose. The detailed structure of gum ghatti has not been clearly established. Its main chain consists of β-D-galactopyranosyl residues connected by (1→6)-linkages and D-glucopyranosyluronic acid units connected by (1→4)-linkages (9). b. Functional properties and applications Similar to gum karaya and tragacanth, gum ghatti does not dissolve in water to give clear solutions, but can be dispersed to form a colloidal dispersion. The dispersion exhibits non-Newtonian flow behavior and its viscosity is between those of gum arabic and gum karaya dispersions at the same concentration. Gum ghatti is an excellent emulsifier and can be used to replace gum arabic in more complex systems (13). The pH of gum ghatti dispersions is 4.8, and the viscosity increases with increase in pH, reaching a maximum at pH 8 (3). The viscosity of gum ghatti dispersions increases with time regardless of solution pH; however, addition of sodium salts, such as sodium carbonate and sodium chloride, results in decrease in viscosity. Loss in viscosity also occurs when the gum dispersions are not protected by preservatives against bacterial attack. Gum ghatti is used as an emulsifier and stabilizer in beverages and butter-containing table syrups, and as a flavor fixative for specific applications. Gum ghatti is also used to prepare powdered, stable, oil-soluble vitamins, and as a binder in making long-fibered, lightweight papers.

B. GUMS

FROM

PLANTS

Gums of plant origin other than exudates are also important for food use. These include storage polysaccharides from seeds and tubers, mucilages from seed coats, and cell wall materials from fruits and cereals.

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1. Galactomannans (Locust Bean, Tara, Guar, and Fenugreek Gums)

a. Source and structure Galactomannans are a group of storage polysaccharides from various plant seeds. There are four major sources of seed galactomannans: guar (Cyamopsis tetragonoloba), locust bean (Ceratonia siliqua), tara (Caesalpinia spinosa Kuntze) and fenugreek (Trigonella foenum-graecum L.). Among these, only guar and locust bean gums are of considerable industrial importance and the use of tara and fenugreek is limited due to availability and price. Most of the guar crop produced worldwide is grown in India and Pakistan. The plant has also been cultivated in tropical areas such as South and Central America, Africa, Brazil, Australia, and the semi-arid regions of the southwest United States. Locust bean is produced mostly in Spain, Italy, Cyprus, and other Mediterranean countries. Fenugreek is grown in northern Africa, the Mediterranean, western Asia, and northern India, and has been recently cultivated in Canada. The production of commercial guar, locust bean, and tara gums is similar, involving separation of endosperms from the seed hull and germ, grinding and sifting of the endosperm to a flour of fine particle size and sometimes purifying by repeated alcohol washings. The final product is a white to cream-colored powder. The amount and molecular weight of galactomannans found in the endosperm extract can vary significantly depending on the source of seed and growing conditions. Most commercial gums contain >80% galactomannan. Low-molecular-weight grades are produced from acid, alkaline, or enzyme hydrolysis of native gums. Fenugreek gum is extracted from the endosperm or ground whole seed with water or dilute alkali, and yields vary from 13.6% to 38%, depending on the variety/cultivar and extraction methods (14). Commercial fenugreek gum products, such as Fenu-pure and Fenu-life, contain over 80% galactomannas with about 5% proteins. Laboratory-prepared material involves pronase treatment of the gum samples, which produces a product of much higher purity with less than 0.6% protein contaminates (15). Seed galactomannans consist essentially of a linear (1→4)-β-D-mannopyranose backbone with side groups of single (1→ 6)-linked α-D-galactopyranosyl units. The molar ratio of galactose to mannose varies with origins, but are typically in the range 1.0:1.0~1.1, 1.0:1.6–1.8, 1.0:3.0, and 1.0:3.9~4.0 for fenugreek, guar, tara, and locust bean gums, respectively. The distribution of D-galactosyl residues along the backbone chain is considered irregular, where there are longer runs of unsubstituted mannosyl units and block condensation of galactosyl units (16, 17). b. Functional properties and applications The solubility of galactomannan gums increases with the degree of galactose substitution. Guar and fenugreek gums are readily dissolved in cold water whereas locust bean gum is only slightly soluble in cold water but can be dissolved in

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hot water. The hydration rate and solution viscosity depend on factors such as particle size, pH, temperature, etc. Guar gum solutions are reported to be stable over the pH range 4.0–10.5, and the highest hydration rate is reported at ~ pH 8.0 (18). Hydration rates are reduced in the presence of salts and other water-binding agents such as sucrose. Like many polysaccharides found in nature, these galactomannans are polydisperse, high-molecular-weight polymers. Average molecular weight varies, typically from 1.0 to 2.5 million Daltons. The galactomannan molecules exist as an extended ribbon-like structure at solid state and adopt a flexible coil-like conformation in solution. All four types are highly efficient thickening agents. Given the same molecular weight and polymer concentration, the thickening powder decreases in the order of the increase of galactose contents, i.e., locust bean > tara > guar > fenugreek. The rheological properties of some galactomannan solutions show a considerable departure from classical random coil-like behavior (19). In particular, there is a lower coil overlap parameter C*[η] ~2.5 in comparison with C*[η] ~4 for most other disordered coils, and a stronger dependence of specific viscosity on concentration (ηsp∝ c~4.5 in contrast to ηsp ∝ c~3.3). This is attributed to intermolecular associations at high concentrations. Galactomannan gums are compatible with most hydrocolloids. There is a useful synergistic increase in viscosity and/or gel strength by blending galactomannan gums with certain linear polysaccharides including xanthan, κ-carrageenan, and agarose. The synergistic interactions are more pronounced with galactomannans of lower galactose contents. Fenugreek and guar gums are non-gelling polysaccharides whereas locust bean and tara gum solutions may form weak gels upon freeze-thaw treatment, or by adding large amounts of ethylene glycol or sucrose. Gelation of galactomannans can also be induced by the addition of cross-linking agents such as borax and transition metal ions. The synergistic interactions of locust bean and tara gums with some gelling polysaccharides, such as κ-carrageenan and agarose, may enhance gelation, impart a desirable elastic character, and retard syneresis in these gels. These mixed gels have been used to form sheeted, fruit-flavored snack products and to produce hair gels. Polysaccharides are generally considered non-surface active agents and the apparent surface activity is frequently attributed to the presence of small amounts of proteins. However, purified fenugreek gum (with less than 0.6% proteins and used in less than 1%) appears to be more efficient than guar and locust bean gums in lowering interfacial free energy. Fenugreek gum is also more concentration-efficient than gum arabic and xanthan gum in stabilizing oil/water emulsions. Guar and locust bean gums are the most extensively used gums in the world. In the food industry they are widely used as thickening and stabilizing agents, usually in

amounts of beef) (14). Therefore, the reactivity of different hemoglobins in various muscle foods should be considered as a causative factor in addition to fatty acid unsaturation and endogenous antioxidant capacity. Compartmentation of cellular and extracellular reactants should be critical in controlling rates of lipid oxidation. Takama et al. (15) suggested that minced flesh of trout was susceptible to rancidity due to the dispersed blood pigments in the flesh caused by the mechanical destruction of the tissue. Crushing plant tissue brings formerly segregated reactants together to stimulate various reactions including oxidation of lipid (16). Critical cellular components and additional factors that control rates of lipid oxidation are discussed below. In any discussion pertaining to lipid oxidation, it is important to realize that any component that accelerates lipid oxidation under one set of conditions can be inhibitory under different conditions.

O2 ⫹ 2H+ → H2O2

⫺•

[reaction 2]

Ferrous iron (Fe2+) can then react with H2O2 or preformed lipid hydroperoxides to produce hydroxyl or alkoxyl and hydroxyl radicals, respectively (reactions 3 and 4). Hydroxyl and alkoxyl radicals are capable of abstracting a hydrogen atom from a polyunsaturated fatty acid and hence initiate/propagate lipid oxidation (17). Fe2+ ⫹ H2O2 → Fe3+ ⫹ −OH ⫹ ⫺•OH

[reaction 3]

Fe2+ ⫹ LOOH → Fe3+ ⫹ ⫺•OH ⫹ LO⫺• [reaction 4] Reaction 3 is termed the Fenton reaction. Hydroxyl radical can also be produced via the Haber-Weiss reaction (reaction 5). A “ferryl ion” is produced from Fenton reagents and relevant as an initiator of lipid oxidation (reaction 6); even in the absence of H2O2, ferryl ion can be produced from the reaction of Fe2+ and the “perferryl ion” complex (Fe2+O2) (18). O2 ⫹ H2O2 → O2 ⫹ OH− ⫹ •OH

[reaction 5]

Fe2+ ⫹ H2O2 → Fe2+O ⫹ H2O

[reaction 6]

⫺•

Ferryl ion Chelators such as ethylenediaminetetraacetic acid (EDTA) and adenosine diphospate (ADP) are widely used to enhance the ability of iron to promote lipid peroxidation (19). Ascorbate increased the ability of iron to stimulate lipid oxidation by reducing ferric iron (Fe3+) to ferrous iron (Fe2+) (20). Antioxidant properties of ascorbate and metal chelators are discussed later (Section V.F and V.G). Ferric iron can also be reduced enzymatically (e.g., membrane bound reductase, ADP and NADH). Although Fe2+ did eventually stimulate lipid oxidation in sarcoplasmic reticulum, increasing concentrations of Fe2+ increased the lag phase prior to lipid oxidation; this suggested that Fe2+ initially was an antioxidant by reducing membrane antioxidant radicals to their active form (21). Fe2+ could then stimulate lipid oxidation after the antioxidant capacity was exhausted. Lipolysis, cooking temperatures, ascorbate, peroxides and extended storage times have the ability to increase iron concentrations in biological systems by stimulating the release of iron from proteins including ferritin, transferrin, hemoglobin, and myoglobin (22–26).

A. METALS Low-molecular-weight metals are potent catalysts of lipid oxidation. Copper and iron are two of the more potent metal catalysts in biological systems. Only ferrous ions and oxygen are needed to produce hydrogen peroxide (H2O2) as seen in reactions 1 and 2: Fe2+ ⫹ O2 → Fe3+ ⫹ ⫺•O2

[reaction 1]

B. HEME PROTEINS Hemoglobin and myoglobin are the predominant heme proteins (HP) in muscle foods. The blood protein hemoglobin is a tetrameric protein while myoglobin from the interior of muscle cells is a monomer (single polypeptide chain). Hemoglobin tetramers dissociate into monomers and dimers upon dilution and with decreasing pH (27, 28).

Lipid Chemistry and Biochemistry

8-5

Certain aquatic and land animals possess multiple hemoglobins with different chromatography characteristics (29, 30). These factors can cause erroneous determination of hemoglobin and myoglobin content in tissue extracts. Heme proteins consist of a globin chain(s) and a heme ring(s), the latter containing an iron atom. The iron is primarily in the ferrous state (HP-Fe2⫹) in vivo. Met heme protein (HP-Fe3⫹) accumulates post mortem via a proton or deoxygenated HP mechanism (reactions 7 and 8) (31). A general term for the formation of metHP from ferrousHP is heme protein autoxidation. oxy(+2)HP + H+ → met(+3)HP + •OOH

[reaction 7]

deoxy(+2)HP + O2 → met(+3)HP + ⫺•O2

[reaction 8]

Met heme protein formation is likely critical to the onset of lipid oxidation since metHP reacts with either H2O2 or lipid hydroperoxides to form the ferryl HP radical that is capable of initiating lipid oxidation (reactions 9 and 10) (32). MetHP is much more likely to unfold and release its heme group compared to ferrous HP (33, 34). Released or displaced heme can react with lipid peroxides to form various lipid radical species that have the ability to propagate lipid oxidation processes (reaction 11) (35–37). Bohr effects occur in certain fish hemoglobins (38). This decreases oxygen affinity of the heme protein at post mortem pH values and hence increases met heme protein formation (reaction 8). It is still unclear if ferrous forms of heme proteins can react with lipid hydroperoxides to stimulate lipid oxidation processes although some potential pathways have been suggested involving oxyHP and deoxyHP (39, 40). met(⫹3)HP ⫹ H2O2 → HP+•ferryl(⫹4) ⫽ O + H2O [reaction 9] met(⫹3)HP ⫹ LOOH → HP+•ferryl(⫹4) ⫽ O + LOH [reaction 10] heme(⫹3) ⫹ LOOH → Heme(+3)-O• ⫹ LO• [reaction 11]

C. PEROXIDES

D. ROLE

OXYGEN

OF

Oxygen not only peroxidizes alkyl radicals to propagate lipid oxidation (Figure 8.4) but also is a source of activated oxygen species (Figure 8.5). Unlike ⫺•O2, •OOH can cross membranes, which may increase its pro-oxidative character (51). The oxygen concentration in marine oils is fairly constant between 20°C and 60°C but rapidly decreases between 60°C and 80°C (52). The O2 concentrations in these oils at 20°C (0.44 to 1.25 mM) exceed the O2 concentration found in water at the same temperature (around 0.30 mM). In 80% oxygen and 20% carbon dioxide packaging, oxygen penetrated 1.7 to 11 mm into different muscle foods (beef ⬎ pork ⬎ lamb) (53). At high ratios of [O2]/[H2O2], the ferryl ion initiation (reaction 6) is believed to dominate while Fenton reagents (reaction 3) are more prevalent at lower ratios (18). In CCl4-induced lipid peroxidation of hepatocytes, a distinct maximum was obtained at 7 mm Hg oxygen while iron-mediated lipid oxidation in microsomes differed in oxygen dependence depending on whether initiation or propagation phases were considered (54). Metmyoglobin formation in beef occurred most rapidly at around 11 mm Hg oxygen (55). Non-destructive oxygen sensors are available to measure the oxygen content in headspace of different packaging systems (56). Carotenoids are believed to be more effective antioxidants at low oxygen concentrations compared to higher oxygen concentrations (57).

O2

e–

– + –•O e /2H 2

H2O2

e –/H +

HO•

↔

There are numerous sources of hydrogen peroxide in biological systems. Equations 1 and 2 describe an iron, oxygen, and proton-mediated mechanism of formation. NADPH-cytochrome P450 reductase produces ⫺•O2 that dismutates to H2O2 (19). Production of H2O2 in mitochondrial and peroxisomal fractions has been described (41). H2O2 production in erythrocytes was mainly attributed to hemoglobin autoxidation (42). H2O2 was produced at a rate of 14 nmol/g of fresh weight/30 min in turkey muscle at 37°C (43). High concentrations of H2O2 will cause release of iron from the heme ring of heme proteins (25).

Like H2O2, lipid hydroperoxides (LHP) react with metals or heme proteins to produce free radical species that propagate lipid oxidation. Further, the collection of volatiles that produce rancid odor result from LHP breakdown. Trace amounts of LHP are required for lipoxygenase activity, converting iron in the active site from the ferrous to ferric form (44). Reduction of lipid hydroperoxides to alcohols with compounds such as ebselen and triphenylphosphine often abolishes any lipid oxidation that was observed prior to reduction (45, 46). Tocopherolmediated lipid peroxidation was found to require Cu2+ and low levels of lipid hydroperoxides (47). Fe2+ reacts with lipid hydroperoxides around 20 times faster than with hydrogen peroxide (48). Protein hydroperoxides may also exacerbate lipid oxidation processes (49). Non-lipid surfactant hydroperoxides increased rates of lipid oxidation in oil-in-water emulsions (50).

+

HOO•

H2O

+

H

pKa 4.8

e–/H +

FIGURE 8.5 One-electron reductions of oxygen.

H 2O

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E. LIPOXYGENASES AND MYELOPEROXIDASES Lipoxygenases are responsible for flavor deterioration in beans during frozen storage (58). Lipoxygenases “initiate” lipid oxidation processes by hydrogen abstraction from a polyunsaturated fatty acid. The off-flavor is due to the volatiles that are produced from breakdown of the lipoxygenase-derived lipid hydroperoxides. Fresh fish aromas are believed to result in part from lipoxygenases that enzymically peroxidize fatty acid substrates (59). These enzymes may also be responsible for formation of rancid odors by providing critical amounts of lipid hydroperoxides that can be broken down by metals or heme proteins to produce rancid odor. Some of the confusion surrounding the presence or absence of lipoxygenases in animal tissues may be due to the quasilipoxygenase activities of myoglobin and hemoglobin (60, 61). Esculetin has been utilized as a specific lipoxygenase inhibitor; however, esculetin is a phenolic compound that has general free radical scavenging ability and should not be considered a specific inhibitor of lipoxygenase. Myeloperoxidases are found in white blood cell neutrophils. Myeloperoxidase catalyzes the reaction of chloride and hydrogen peroxide that produces hypochlorous acid which in turn reacts with •−O2 and yields hydroxyl radical (62). This reaction was found to be six orders of magnitude faster than the Haber-Weiss reaction (reaction 5) and does not require iron.

F.

LIPOLYSIS

Lipolysis results in the formation of free fatty acids. Lipolysis occurs due to enzyme action or heat and moisture. Free fatty acids are responsible for both undesirable and desirable flavors (e.g., milk rancidity or positive flavors in cheese, bread, and yogurt). Cabbage phospholipase D decreased the formation of lipid oxidation products in beef homogenates and egg yolk phosphatidylcholine liposomes (63). Adding free fatty acids to fresh salmon flesh, at levels of free fatty acids that accumulated during 6 months at –10°C storage, increased taste deterioration in fresh minced salmon (64). The amount of taste deterioration from each fatty acid was 22:6n-3 > 16:1n-7 > 18:2n-6 > 20:5n-3). This suggested that hydrolysis of triacylglycerols negatively impacted sensory quality. A review on lipolysis effects in fish muscle indicated that triacylglycerol hydrolysis results in increased lipid oxidation while phospholipid hydrolysis was inhibitory (65).

G. PHOTOACTIVATED SENSITIZERS (SINGLET OXYGEN) Oxygen can exist in the triplet (3O2) or singlet state (1O2). Triplet oxygen is the normal state of oxygen while singlet oxygen is generated via photosensitization by natural pigments in food (e.g., riboflavin or chlorophyll). The two

electrons in the antibonding 2p orbitals of 3O2 have the same spin and are in different orbitals. This creates a small repulsive electronic state. In 1O2, the two electrons are in a single antibonding orbital and have opposite spins; therefore, electrostatic repulsion will be great. 1O2 is thus at a higher energy state than 3O2, and 1O2 is more electrophillic than 3O2. This causes 1O2 to react readily with moieties of high electron density such as double bonds in unsaturated fatty acids (8). This direct addition of 1O2 to unsaturated fatty acids initiates lipid oxidation without the need for hydrogen abstraction as is the case with free radical-mediated initiation. Nine or more conjugated double bonds (e.g., carotenoids) are required for physical quenching of singlet oxygen (66). Other compounds such as tocopherols and amines can quench singlet oxygen by a charge transfer mechanism (67).

H. FAT CONTENT Release of c-9 aldehydes into headspace decreased with increasing oil content in oil-in-water emulsions (68). This suggested that the impact of certain odor compounds is decreased by elevated levels of fat via solubilization of the component into the oil phase. A study was conducted that examined the effect of added triacylglycerols on rates of hemoglobin-catalyzed oxidation of washed cod muscle lipids. No difference in rate or extent of lipid oxidation catalyzed by hemoglobin was obtained when washed cod muscle (around 0.7% phospholipids) was compared to the washed cod muscle containing up to 15% added triacylglycerols (69). This indicated that triacylglycerols did not accelerate rates of lipid oxidation during storage. Similar non-effects of added triacylglycerols were obtained in cooked lipid-extracted muscle fibers (70). Increasing fat contents did not increase oxidized oil odor in frozen stored catfish (71).

I. EFFECT

OF

COOKING

Consumers are finding less time to prepare meals. The food industry is responding to this by increasing the availability of pre-cooked meats. A major problem with precooked meats is the development of an objectionable warmed-over flavor via lipid oxidation (72). This warmed-over flavor occurs more rapidly during refrigerated compared to frozen storage temperatures. It has been suggested that released iron from heme proteins promotes warmed-over flavor in pre-cooked beef (23). The evidence for this was that the low-molecular-weight fraction in an aqueous extract of beef muscle stimulated lipid oxidation of washed muscle fibers much better than the high-molecular-weight fraction (73). On the other hand, in precooked fish, heme proteins were believed to be the active catalysts due to higher pro-oxidative activity in the highmolecular-weight fraction of the fish muscle (74).


Polyphosphates inhibited lipid oxidation in precooked beef, which may be due to iron chelating properties of the phosphates (73). Inhibitors of warmed-over flavor were produced in meat during retorting but could not be extracted from raw beef. This suggests that the high temperature processing caused formation of products that inhibit lipid oxidation (75). Browning reactions that involve carbohydrates and amino acids were believed to impart this antioxidant effect. Lipid oxidation is much less of a problem in precooked meats that are cured. Cured meats contain nitrite in the formulation. The primary way that nitrite is believed to exert its antioxidant effect is by conversion of nitrite to nitric oxide (NO) that binds to the iron atom in the heme ring of heme proteins. The NO-ligand may be antioxidative by preventing release of heme or iron during cooking and storage or by decreasing heme protein reactivity. Nitrite can also act as an antioxidant by chelating metals and scavenging free radicals. Nitrite may be toxic at elevated levels and therefore it is critical to control the residual nitrite content in the product.

IV. MEASURING RATES OF LIPID OXIDATION IN FOOD SYSTEMS Lipid hydroperoxides are primary lipid oxidation products that are precursors to rancidity. Lipid hydroperoxides need to be broken down to form the low-molecular-weight volatile compounds (secondary products) that impart rancidity. It is imperative to measure primary and secondary lipid oxidation products. To accentuate this point, tocopherol enriched lipoproteins had higher levels of conjugated dienes (primary product) than lipoproteins containing little tocopherol (76). Standing alone, this errantly suggests that tocopherol was a pro-oxidant. Fortunately, these researchers also measured thiobarbituric reactive substances (TBARS) which indicated less formation of the secondary products in the tocopherol enriched samples. Apparently, tocopherol stabilized the hydroperoxides. Thus, a more complete picture is realized when measuring both primary and secondary lipid oxidation products. Sensory analysis should be done whenever possible since human subjects can determine the point at which the product becomes undesirable which ultimately determines shelf life. Degree of rancidity or quality perception is harder to pinpoint using chemical indicators of lipid oxidation. Single time point measurements are also discouraged. Primary and secondary lipid oxidation products commonly increase, reach a maximum, and then decrease substantially. This can create a situation where one sample is perceived to be minimally oxidized but in fact had undergone extensive oxidation well before the measurement. Thus, measuring lipid oxidation products at multiple time points during storage is suggested so that a kinetic curve can be obtained which demonstrates

8-7

a lag phase, exponential phase and plateau, or decrease phase. Common lipid oxidation indicators that are measured during storage of lipid-containing foods include lipid peroxides, conjugated dienes, headspace volatiles, thiobarbituric acid reactive substances (TBARS), anisidine value, oxygen consumption, and carotene bleaching. A description of these and other methods including those used in fried products is available (8). Very good correlations between TBARS and headspace volatiles (e.g., hexanal, pentenal) were determined in cooked turkey during 4°C storage (77). TBARS are unlikely to provide useful results if the starting material has already undergone considerable oxidation. Rancidity can develop before any detectable change in fatty acid composition occurs. For example, no difference in fatty acid composition was found when fresh mackerel muscle was compared to extensively rancid mackerel muscle (78). This should not be a surprise considering that extremely small amounts of fatty acid precursors are required to produce the amount of volatiles needed for sensory impact (79). Numerous pitfalls exist when measuring rates of lipid oxidation. Thermogravimetric methods entail weighing the sample until a rapid increase in weight occurs due to oxygen adding to the lipid. This can be done under isothermal conditions or programming from ambient to elevated temperatures. The drawback is that by the time a spike in weight occurs, detection of rancidity had previously occurred. Bulk oils are sometimes heated to 90°C to shorten the storage period needed to produce quantifiable levels of lipid oxidation. The amount of oxygen that is soluble in oil decreases substantially at elevated temperatures. This causes the mechanism of oxidation to be different from that which would occur at lower temperatures. Both the AOM and Rancimat method have been considered unreliable due to the high temperatures that are used (80). More reasonable methods to accelerate the rate of lipid oxidation in oils and emulsions are to store samples at 50°C and add metals or hemin to the system. It is interesting to note that fish held at –10°C was more susceptible to lipid oxidation than muscle stored at around 0°C. The temperature deceleration effect was apparently less substantial than the effect of freeze concentration of reactants (81). The mechanism of lipid oxidation at –20°C (commercial storage) may also be different than –10°C considering that less tissue damage should occur at the lower temperature due to faster freezing rate and smaller sized ice crystals.

V. ANTIOXIDANTS Food antioxidants are used to inhibit lipid oxidation reactions that cause quality deterioration (e.g., flavor, color, texture, nutrient content). It is important to note that any compound that is antioxidative under one set of conditions

8-8


can become pro-oxidative under different conditions. As an example of this point, ascorbate has been found to both inhibit and accelerate lipid oxidation depending on the concentration of linoleate hydroperoxides in the system (82). The main antioxidant mechanisms are free radical scavenging, chelation of metals, removal of peroxides or reactive oxygen species, and quenching of secondary lipid oxidation products that produce rancid odors (83).

A. FREE RADICAL SCAVENGERS Some typical free radicals that can initiate/propagate lipid oxidation and hence be scavenged by antioxidants include hydroxyl (•OH), alkoxyl (LO•), peroxyl radicals (LOO•), and ferryl heme protein radicals (HP+•ferryl(+4)=O) (84). • OH is one of the strongest biological oxidants (Table 8.1) and therefore will react with nearly any molecule that it encounters. This might limit the amount of •OH that will react with fatty acids. Peroxyl radicals are likely prevalent since the reaction of oxygen with alkyl radicals that forms after hydrogen abstraction from a fatty acid is highly favored both thermodynamically and kinetically. Alkoxyl radicals will form due to breakdown of lipid hydroperoxides by heme or reduced metal complexes. Alkoxyl radicals can undergo β-scission reactions that produces a short chain alkyl radical (RCH2•) that reacts readily with O2 to form peroxyl radicals (17). There are numerous free radical scavengers (FRS) that are either endogenous to the food or incorporated during processing. The antioxidant effectiveness will depend on hydrogen bond energies (85). The donation of hydrogen from a generic phenolic antioxidant to an alkoxyl radical is depicted in Figure 8.6. The ability of a particular FRS to donate hydrogen to a free radical can be predicted from standard one-electron reduction potentials (17). Any compound that has a reduction potential lower than that of a free radical is capable of donating hydrogen to that free radical (Table 8.1). For example, catechol has a lesser reduction potentials than alkoxyl radical. Thus, catechol can donate hydrogen to the alkoxyl radical. This donating

TABLE 8.1 Standard One-Electron Reduction Potentials of Components Involved in Free Radical Reactions [Oxidized / Reduced] Couple HO•, H+ / H2O LO•, H+/ LOH LOO•, H+ / LOOH PUFA•, H+ / PUFA-H Catechol•, H+ / catechol α-Tocopheroxyl•, H+ / α-tocopherol Ascorbate• −, H+ / ascorbate− Adapted from Ref. 17.

E° ′ (mV) 2310 1600 1000 600 530 500 282

ability of catechol competes with the undesirable reaction of alkoxyl radicals with polyunsaturated fatty acids (PUFA-H) (Table 8.1) and hence inhibits lipid oxidation processes. It should be kept in mind that the reduction potential of a compound changes as a function of pH, temperature, and concentration of the compound(s) of interest. A potential drawback is that the FRS becomes a free radical itself after donating hydrogen to the alkoxyl radical (Figure 8.6) (Table 8.1). The most efficient FRS exist as low energy radicals after scavenging. The benefit of existing as a low energy radical is that the radical is unlikely to abstract hydrogen from polyunsaturated fatty acids. Low energy radicals result from resonance delocalization (Figure 8.6). The conjugated ring structure of the phenolic allows the phenolic radical to reside at multiple sites on the molecule. As the radical migrates from site to site, a low energy radical results that possesses low reactivity. Evidence of low reactivity can be gleaned from the one-electron reduction potentials. Any radical with reduction potential less than a polyunsaturated fatty acid (e.g., catechol radical) cannot abstract hydrogen from the fatty acid; hence the antioxidant radical cannot initiate/propagate lipid oxidation processes (Table 8.1). Efficient FRS in their radical form should also not react with oxygen. If reaction with oxygen occurs, a free radical peroxide forms (FR-OOH). The free radical peroxide cannot be regenerated by reducing equivalents as can occur when the FRS is in a resonance delocalized form (FR•). The net effect is depletion of the antioxidant upon reaction with oxygen. Further free radical peroxides can decompose to species capable of furthering oxidation. Note that ascorbate has a one-electron reduction potential that is less than tocopherol (Table 8.1) and thus ascorbate can regenerate tocopherol from tocopheroxyl radicals. Thus, phenolic compounds are efficient FRS due to their hydrogen donating properties and resonance delocalization of the phenoxyl radical. There is a multitude of phenolic free radical scavengers available to food scientists. The synthetic phenolics butylated hydroxy toluene (BHT), butylated hydroxy anisole (BHA), tertiary butyl hydroquinone (TBHQ), and propyl gallate (PG) (Figure 8.7) are commonly used in the food industry due to their low cost of production although consumers prefer natural FRS such as tocopherols and plant phenolics.

B. SYNTHETIC PHENOLICS Propyl gallate (PG) is poorly soluble in oils and sensitive to heat degradation (e.g., frying temperatures). Substitution of the propyl group with octyl or dodecyl groups provides more heat stability and lipid solubility. Gallates have been used to stabilize meat products, baked goods, fried products, confectionaries, nuts, and milk products (86). Butylated hydroxyanisole (BHA) volatilizes upon frying,


8-9

O•

OH LO•

O

O

O

LOH •

•

•

FIGURE 8.6 Free radical scavenging by a phenolic compound and resonance stabilization of the resulting phenoxyl radical. Adapted from Ref. 85.

OH OH

OH

OH OH

(H3C)3C

C(CH3)3

COOC3H7

CH3

PG

C(CH3)3

OH

BHT

TBHQ

OH

OH

C(CH3)3

C(CH3)3 OCH3

3-BHA

OCH3

2-BHA

FIGURE 8.7 Structures of various synthetic antioxidants.

but residual BHA does protect fried foods. BHA is a mixture of two isomers (Figure 8.7). The −C(CH3)3 group on the conjugated ring increases oil solubility and enhances resonance stabilization of the phenoxy radical. This alkyl group on the conjugated ring also enhances hydrogendonating properties. Butylated hydroxytoluene is highly soluble in oil due to its two −C(CH3)3 groups and single methyl group (Figure 8.7). TBHQ has two hydroxy groups and significant solubilities in a wide range of fats, oils, and solvents. The order of antioxidant efficacy in fish oil stored at 60°C was TBHQ> PG=BHA>BHT (87).

C. TOCOPHEROLS AND TOCOTRIENOLS Tocopherols are of plant origin and exist in four forms (α, β, γ, δ). The structures of the isomers are illustrated in Figure 8.8. Tocopherols are soluble in oils and ethanol. When tocopherol reacts with a peroxyl radical, at least five resonance structures of the tocopherol radical can form (83). BHA, BHT, and PG are considerably more stable to heat treatment than α-tocopherol (86). α-, β-, γ-, and δ-tocopherol inhibited formation of

cholesterol oxidation products to different degrees in metal-induced oxidation of unilamellar phospholipidcholeseterol liposomes (88). In beef muscle, tocopherolquinone and 2,3-epoxy-tocopherolquinone were the dominant tocopherol oxidation products and lower amounts of 5,6-epoxy-tocopherolquinone and tocopherolhydroquinone were detected (89). This was consistent with mainly a peroxyl radical scavenging function of tocopherol but also some scavenging of other free radicals. Predominant amounts of the 2,3- and 5,6-epoxy-tocopherolquinone products would suggest a nearly exclusive mechanism of peroxyl-radical scavenging. When examining Atlantic mackerel, a substantial amount of tocopherol was present in stored muscle that was highly rancid (90). This suggested that tocopherol was not an effective antioxidant in the mackerel muscle. Tocotrienols are similar in structure to tocopherols but contain three unsaturated units in the isoprenoid chain. γ- and δ-tocotrienols extended shelf life of coconut fat better or in a manner similar to their corresponding tocopherols during 60°C storage and exposure to frying temperatures (91).

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CH3 O

R2

CH3 CH3 (-CH2 --CH2 -(-CH2-(-CH2)3 -CH3

HO R1

Type

R1

R2

Alpha

-CH3

-CH3

Beta

-CH3

-H

Gamma

-H

-CH3

Delta

-H

-H

FIGURE 8.8 Structures of tocopherols.

D. PLANT PHENOLICS Simple plant phenolics contain a single conjugated ring with various substitutions. These compounds are usually water soluble and examples are gallic acid and hydroxycinnamic acid. Anthocyanidins are 3-ringed structures that exist as protonated cations at acidic pH values, are colorless open-ringed structures at intermediate pH values, and are anions at higher pH values. Glycosylated anthocyaninidins are termed anthocyanins and are the common red pigments in fruits. Flavan 3-ols are colorless compounds that are common in tea. Epicatechin is an example of a flavan-3-ol (Figure 8.9). Quercetin is a flavonol and is one of the most abundant flavonoids (Figure 8.9). Flavonoids is a general term that includes anthocyanins, flavonols, flavones, isoflavones, and chalcones. A quercetin metabolite was found to have antioxidant properties in a liposomal membrane (92). Linked flavan-3-ol repeated molecules are high molecular weight, generally poorly soluble in water, and referred to as proanthocyanidins, procyanidins, tannins, or heteropolyflavans (93). Extensive structural diversity exists

in different plant phenolics. In rosemary leaf extract, carnosol, carnosic acid, rosmarinic acid, and rosmaridiphenol have antioxidant potency (86). The rate of peroxyl radical scavenging by quercetin and epicatechin was greater in non-polar solvents compared to hydrogen bonding solvents (94). In a liposomal model system that generate free radicals during metalinduced peroxidation, 1) antioxidant activity increased with increasing hydroxy substitutions present on the B ring for anthocyanidins but the opposite was observed for the flavan-3-ol, catechin, 2) substitution by methoxyl groups decreased antioxidant activity of anthocyanidins, and 3) substitution of a galloyl group at position 3 of the flavonoid moiety decreased antioxidant activity of the catechin (95). Many phenolic antioxidants have been characterized in grapes, berries, teas and spices. Beet root pigments were found to have free radical scavenging properties (96). Betanidin 5-O-beta-glucoside in beet was found to inhibit lipid oxidation at low concentrations (97). In pineapple juice, phenolic compounds containing cysteine, glutamyl, and glutathione linkages were identified (98). Proteins (casein or albumin) decreased antioxidant efficacy of tea flavanols (99). Freezing and storage had negligible effects on antioxidant capacity of raspberry phenolics (100). Ferryl myoglobin, a possible pro-oxidant in muscle tissue, was reduced by epigallocatechin gallate from green tea (101). The antioxidant effects of tea catechins in raw chicken muscle were attributed to free radical scavenging ability and iron chelating effects (102).

E. CAROTENOIDS Carotenoids are fat-soluble pigments. Canthaxanthin and astaxanthin possess oxo groups at the 4 and 4⬘-positions in the β-ionone ring (Figure 8.10). β-carotene and zeaxanthin do not contain oxo groups and were found to be less inhibitory to methyl linoleate peroxidation than canthaxanthin or astaxanthin (103). β-carotene, however, can scavenge free radicals. Peroxyl radicals either add directly to the hydrocarbon portion of the molecule displacing an unsaturation site or add to the β-ionine ring forming a β-carotene cation radical; these oxidation products, however, are

OH

OH OH

HO

OH HO

O

O

OH OH

O

Quercetin

FIGURE 8.9 Structures of the flavonoids quercetin and epicatechin.

OH OH

Epicatechin


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OH

(a)

O (b) HO

O OH (c)

(d)

FIGURE 8.10

O

HO

O

Structures of various carotenoids. (a) β-carotene, (b) zeaxanthin, (c) canthaxanthin, (d) astaxanthin.

susceptible to breakdown that results in formation of alkoxyl radicals (83). There is evidence that carotenoids are effective antioxidants at low oxygen concentrations but not higher oxygen concentrations (57). Carotenoids including lycopene can inactivate singlet oxygen by physical quenching of the activated oxygen specie (104).

F. OTHER FREE RADICAL SCAVENGERS AND REDUCTANTS Uric acid is present in plasma and can inhibit lipid oxidation by scavenging free radicals or singlet oxygen (105). Ascorbate is believed to scavenge tocopheroxyl free radicals thereby regenerating tocophopherol (17). Ascorbate can also scavenge various free radicals such as •−O2, • OOH, and •OH (48). Like flavonoids, ascorbate reduces hypervalent forms of heme proteins to potentially inhibit lipid oxidation in muscle foods (106). Heme oxygenase converts heme into bilirubin. Bilirubin is believed to scavenge free radicals, which results in formation of biliverdin that is reduced back to bilirubin by NADH and biliverdin reductase. This redox cycle was used to explain the high antioxidant power of bilirubin in vivo (107). It is not known how effective bilirubin inhibits lipid oxidation in food systems. Ubiquinol is a phenolic compound that is conjugated to an isoprenoid chain and is associated with mitochondrial membranes. Oxidation of ubiquinol results in formation of semiubiquinone radical. Dietary

ubiquinone increased ubiquinol levels in lipoproteins and decreased lipid oxidation rates (108). Ubiquinol is considered a weak free radical scavenger due to internal hydrogen bonding that interferes with abstraction of its phenolic hydrogen by free radicals (109). A potent natural antioxidant from shrimp was tentatively identified as a water-soluble, polyhydroxylated derivative of an aromatic amino acid (110).

G. METAL INACTIVATORS Ethylenediamine tetraacetic acid (EDTA) can inhibit lipid oxidation by forming an inactive complex with metals. EDTA can either promote or inhibit lipid oxidation depending on the iron/EDTA ratio, which modulates the effective charge in the system (111). EDTA is approved for use in foods at low concentrations. It is poorly soluble in fats and oils but only small amounts are needed for maximum activity. EDTA protected lard better than a combination of BHT and citric acid (86). It should be noted that EDTA indirectly acts as a free radical scavenger. Jimenez and Speisky (112) showed that glutathione scavenged free radicals less effectively in the presence of copper than when EDTA was mixed with copper prior to addition of gluathione. The ability of EDTA to tie up copper or form a chelate with glutathione apparently increased the free radical scavenging ability of glutathione. The carboxylic acid groups of EDTA are protonated at low pH values

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(i.e., when pH is below the pKa for the acid groups of EDTA). This interferes with the ability of EDTA to complex metals or other cellular components. Desferrioxamine is often used as a “metal chelator” in research studies, but this can lead to errant results since desferrioxamine can also act as a free radical scavenger (113). EDTA, tartaric acid and citric acid are other commonly used metal chelators in the food industry. Citrate esters improve oil solubility but at least two free carboxyl groups are needed for effective metal inactivation. Propylene glycol increases solubility of citric acid in oils and fats (86). Sodium tripolyphosphate can act as an antioxidant via metal chelation (114). A disadvantage of using metal chelators in general is that iron bioavailability during digestion may be compromised. Ceruloplasmin inhibits metal-catalyzed oxidation via its ferroxidase activity. The ferroxidase converts Fe2+ to Fe3+, a less catalytic form of iron (115). Transferrin (plasma protein) and ferritin (muscle cell protein) can inactivate metals by chelation of iron but also can release iron causing a pro-oxidant effect; lipolysis and ascorbate, respectively are capable of triggering the iron release (22, 116). Carnosine is a B-alanylhistidine dipeptide found in skeletal muscle at high concentrations. It is capable of chelating copper, scavenging peroxyl radicals, and forming adducts with aldehydes (117). Histidine was found to inhibit nonenzymatic iron mediated lipid oxidation apparently due to formation of an inactive chelate but histidine was also found to activate enzymic pathways of lipid oxidation (118).

H. ENZYMES THAT INACTIVATE OXIDATION INTERMEDIATES Superoxide anion (⫺•O2) can be produced by heme protein autoxidation or by any process that causes addition of an electron to oxygen (119). Superoxide can reduce Fe3⫹ to Fe2⫹, the more pro-oxidative form of iron. In addition, the pKa of ⫺•O2 is around 4.5. Thus at pH values below 4.5, the conjugate acid •OOH is the predominant form which can directly initiate lipid oxidation (84). Superoxide dismutase is present in cells and extracellular fluids to remove ⫺•O2 resulting in formation of oxygen and hydrogen peroxide. Hydrogen peroxide (H2O2) can react with either lowmolecular-weight iron or heme proteins to form free radicals that initiate/propagate lipid oxidation processes. Biological systems are equipped with antioxidants to deal with this stress. Catalase, a heme-containing enzyme reacts with H2O2 to form water and oxygen (120). In plants and algae, ascorbate peroxidase removes H2O2 and forms monodehydroascorbate and water. Glutathione peroxidase removes H2O2 and forms water and oxidized glutathione. The reaction of glutathione peroxidase with lipid hydroperoxides results in formation of an alcohol, water, and oxidized glutathione. Compounds such as methionine and thiodipropionic acid can also decompose peroxides but at much slower rates than the enzymes.

I. SCAVENGING OF LIPID OXIDATION BREAKDOWN PRODUCTS Lipid oxidation breakdown products (e.g., aldehydes, ketones, hydrocarbons) form a mixture of volatiles that causes objectionable flavors and odors. Carnosine, anserine, histidine, lysine, albumin, and sulfur or amine containing compounds have the ability to bind aldehydes and therefore decrease rancidity in foods (83). These “scavengers” should be examined in relation to browning of beef considering that lipid oxidation derived aldehydes accelerated the conversion of oxyMb to metMb and hence have the capacity to accelerate browning in beef (121).

J. OTHER MECHANISMS

OF

ANTIOXIDANT ACTION

Spermine was found to inhibit lipid oxidation in hepatocytes of CCL4-treated rats; a possible mechanism was formation of polyamine-phospholipid complexes (122). Conjugated linoleic acid (CLA) has been shown to decrease rates of lipid oxidation in muscle tissue (123). The mechanism may be related to the ability of dietary CLA to decrease polyenoic fatty acid concentrations in the muscle (124). Organosulfur compounds such as diallyl sulfide and N-acetyl cysteine may exert their antioxidant protection by modulating antioxidant enzymes such as catalase and glutathione-s-transferase (125).

K. INTERFACIAL, CHARGE, AND LOCATION EFFECTS Deciding which antioxidant(s) to utilize in a particular food system is a formidable task. Having water and lipid soluble antioxidants was found to maximize extension in shelf life of mayonnaise prepared from fish oil (126). However, cost limitation is a factor that limits amounts of antioxidant addition. Most foods have a water phase, lipid phase and water-lipid interface. Location of different antioxidants should affect antioxidant potency. Membrane phospholipids are believed to be more prone to lipid oxidation than triacylglycerols in muscle foods so protecting membrane lipids is desired (127). δ-Tocopherol could be preferentially incorporated into isolated membranes compared to triacylglycerols by proper selection of antioxidant solvent (ethanol instead of corn oil) (128). In minced chicken muscle containing added triacylglycerols, δ-tocopherol could be preferentially incorporated into the membrane fraction if the antioxidant was added to the lean muscle before addition of TAG lipids (129). Hydrophilic antioxidants (trolox and ascorbic acid) were generally more effective than more hydrophobic compounds (tocopherol and ascorbyl palmitate) in bulk oils while the hydrophobic compounds were more effective in oil-inwater emulsions (130, 131). However, when comparing carnosic acid to the more hydrophobic methyl carnosate, the latter was a more effective antioxidant in both bulk oils and emulsions (132). Benzoic acid, a water-soluble phenolic, partitioned into the oil phase of a whey-protein


stabilized emulsion more than could be explained by oil/water partitioning alone (133). This suggested that benzoic acid bound to protein adsorbed at the interface. In oil-in-water emulsions, excess surfactant solubilized phenolic antioxidants into the aqueous phase but the removal of antioxidants from the oil or oil interface phases did not accelerate lipid oxidation (134). The ability of excess surfactant to cause lipid hydroperoxides and iron to partition into the aqueous phase (away from oil droplets) may explain the ability of excess surfactant to inhibit lipid oxidation in oil-in-water emulsions (50, 135). Positively charged protein emulsifiers inhibited lipid oxidation more effectively than negatively charged emulsifiers in oil-inwater emulsions (136). This was attributed to the ability of the positive charge of the protein interface to repel iron away from the oil phase. The ability of Trolox to inhibit lipid oxidation in liposomes was least when the membrane bilayer and trolox molecule were negatively charged and removing the repulsive forces by altering membrane type or pH increased antioxidant efficacy (137). More studies are needed to evaluate the distribution of antioxidants in different phases in conjunction with lipid oxidation kinetics during storage.

VI. PRODUCTION OF FATS AND OILS Production of fats and oils from plant, animal, fish, and dairy lipids can be broken into four classifications: recovery, refining, conversion, and stabilization. Pressing or solvent extraction are common processes to liberate oil from plant seeds. Care should be taken during transportation of seeds to prevent cell rupture prior to oil extraction. Lipases and lipoxygenases in the cytosol that mix with TAG prematurely due to decompartmentation will be detrimental to oil quality (i.e., formation of free fatty acids and peroxidized lipids prior to extraction will reduce TAG purity and hence yields). Heating during or prior to the pressing step (115°C for 60 min) inactivates lipases and lipoxygenases. Other benefits of heating are rupturing of cell wells, decrease in oil viscosity, and coagulation of proteins. Elevated moisture levels are discouraged due to the ability of excess water to facilitate hydrolysis of esterified lipids. Recovery of animal fat and marine oil is also a high temperature process called rendering. Trimmings, cannery waste, bones, offal, tallow and lard can be subjected to rendering to produce valued added oils and fats. Refining is the removal of non-TAG components including free fatty acids, phospholipids, pigments, protein, and wax. The degumming step is a water wash that removes phosphatides (e.g., lecithin, phospholipids). Hydration in the presence of heat makes phosphatides insoluble in the oil allowing removal by centrifugation. Heating of oil contaminated with phosphatides can result in foaming and even fire due to the surfactant properties of the phospholipids. The next step in refining is neutralization. Free fatty acids and phosphatides react with sodium

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hydroxide to form a soap (e.g., saponified material). Subsequent bleaching removes undesirable pigments typically by use of neutral clays. Waxes are then removed by cooling the oil to around 7°C for 4 hours and filtering at 18°C. The final step in refining is deodorizing, which removes hexane, pesticides, and peroxide decomposition products that can potentially impart off-odors and off-flavors. Deodorization is accomplished by steam distillation at high temperatures (180°C to 270°C) under vacuum. Freshly deodorized oils should have a peroxide value of zero and a free fatty acid content of less than 0.03% (138). Ideally fat-soluble antioxidants such as tocopherols are retained in the purified oil. Unfortunately, refining strips antioxidants from the TAG which often requires post-processing addition of antioxidants to pure oil. The conversion processes winterization and fractional crystallization are physical processes that alter the lipids and thus are out side the scope of this chapter. Various chemical processes of conversion (e.g., interesterification) and stabilization (e.g., hydrogenation) are described later in this chapter. Stabilization techniques for fats and oils are also discussed in the preceding section on antioxidants.

VII. MODIFICATION OF LIPIDS AND PRODUCTION OF SPECIALTY FATS This section describes the numerous chemical processes that are available to modify the functional properties of food lipids. Functional properties include 1) oxidative stability, 2) plastic range, 3) flavor properties, 4) nutrient content, 5) health promoting effects, and 6) caloric value. Increasing fatty acid saturation or redistributing fatty acids on the glycerol backbone to improve functionality can be accomplished in bulk oils by treatment with lowmolecular-weight catalysts. In other cases, more specific alteration of lipids is accomplished through the use of enzymes to improve functionality. Endogenous enzymes in yeasts, molds, and bacteria utilize nonlipid or lipid containing carbon sources to produce a wide array of different specialty lipids (e.g., cocoa-butter substitutes, triacylglycerols rich in omega-3 fatty acids, biosurfactants, polyunsaturated fatty acids, wax esters, and hydroxy fatty acids). A thorough description of the emerging fields of “lipid biotechnology” and “structured lipids” is available (139, 140). Some specific examples that utilize lipases to produce specialty lipids are cited in Section VII.C of this chapter. The opportunity to modify lipids “pre-harvest” is addressed in Section VII.D.

A. HYDROGENATION Hydrogenation is done for two important reasons: 1) provides a semi-solid fat at room temperature from an oil source and, 2) increase oxidative stability during storage. Hydrogenation involves mixing oil with a catalyst such as nickel at elevated temperatures (140°C to 225°C).

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Hydrogen gas is then introduced with agitation. Once the desired saturation is obtained, the material is cooled and the catalyst is removed by filtration. Typical products that result include shortenings and margarine. A disadvantage of this process is the formation of trans fatty acids that are considered unhealthy.

O O — C — CH2 — R OCH3

B. NON-ENZYMATIC INTERESTERIFICATION Factors that contribute to textural properties of fats include not only degree of fatty acid unsaturation and the chain length but also the location of fatty acids on the glycerol backbone. Chemical interesterification “randomizes” the location of the different fatty acids, thereby improving the utility of the fat. Spreadability, melting point, and solid-fat content temperature profile are modified by the randomization. This process typically involves the use of sodium methoxide (0.1%) as a catalyst. The catalyst should function at low temperatures (around 50°C) to avoid polymerization and decomposition of lipids during interesterification. Moisture inactivates the catalyst. Therefore, the water content must be below 0.01%. Free fatty acids and lipid peroxides must be below 0.1% and 1%, respectively. The catalyst must be soluble in the lipid. The mechanism of interesterification using alkaline bases involves nucleophillic attack by the catalyst towards the slightly positive carbonyl carbon. This attack liberates a fatty acid methyl ester and a resulting glycerate anion (Figure 8.11). The glycerate anion is the nucleophile for subsequent carbonyl attacks. This process continues until all the available fatty acids have exchanged positions. Sodium methoxide also removes an acidic hydrogen from the carbon alpha to the carbonyl carbon. The carbanion produced is a powerful nucleophile. On occasion randomness is not desirable. If the fat is maintained below its melting point, interesterification proceeds with the formation of more saturated triacylglycerols. This “directed interesterification” produces a product with a higher solids content at higher temperatures, which extends its plastic range. A practical application of interesterification involves the modification of lard. In its native form, lard has negative attributes including grainy texture, poor appearance, poor creaming capacity, and limited plastic range (141). The graininess is due to a preponderance of palmitic acid at the sn-2 position. Randomization decreases the amount of palmitic acid and the sn-2 position and hence decreases graininess. Directed interesterification resolves the plastic range problem. Improvement in plasticity and stability is due to alterations in the polymorphic behavior. The interesterified lard crystallizes into a β⬘-2 form that promotes the improved functionality (8). Fish oils are liquid at room temperature due to their high content of polyunsaturated fatty acids including

O :O:

C — CH2 — R OCH3

Glycerate monoanion

Fatty acid methyl ester

FIGURE 8.11 Proposed mechanism of chemical interesterification. Adapted from Ref. 141.

omega-3 fatty acids (e.g., 22:6 and 20:5). Ingestion of omega-3 fatty acids are noted for their ability to decrease incidences of various diseases but are also highly susceptible to lipid oxidation in foods, which causes off-flavors and off-odors. A possible route to increased consumption of these fatty acids with less quality loss during storage is chemical interesterification. Interesterification of a hydrogenated vegetable oil and the fish oil will produce a mixture of fatty acids on the glycerol backbone ranging from highly saturated to highly unsaturated. The saturated TAGs can be removed by low temperature fractional crystallization and centrifugation. The fraction obtained with intermediate unsaturation (moderately higher temperature crystallization) comprises TAGs containing both saturated fatty acids and the highly coveted omega-3 fatty acids. Compared to the starting fish oil, this results in triacylglycerols 1) with a greater plastic temperature range increasing product applications, 2) more resistance to lipid oxidation due to the incorporation of the saturated fatty acids, and 3) a relatively more stable source of omega-3 fatty acids for incorporation into foods. An area of concern would be the stability of the omega-3 fatty acids at the temperatures used during chemical interesterification. An alternative process that requires lower reaction temperatures is enzymatic interesterification.

C. ENZYMATIC MODIFICATION

OF

LIPIDS

Enzymatic interestification is accomplished using lipases from bacterial, yeast, and fungal sources. The regio- and stereospecificity obtained through the use of lipases is a marked advantage over chemical interesterification. Enzymatic interesterification requires less severe reaction conditions, products are more easily purified, and produces less waste than chemical interesterification. Enzymatic interesterification is more expensive at the present time although advances are expected to lower costs. In any event, certain processes can be accomplished


with lipases that are not achievable via chemical interesterification. For example, it is optimal to incorporate stearic acid (18:0) at the sn-1 or sn-3 position because stearic acid is least absorbed at these positions compared to the sn-2 position (142). This is advantageous since caloric value is decreased while maintaining a long chain saturated fatty acid that expands the plastic range. An sn-1,3 lipase facilitates the regioselectivity desired whereas chemical interesterification cannot. Since fatty acids at the sn-2 position are more efficiently absorbed than those at the sn-1,3 positions, the ideal location for essential fatty acids is at the sn-2 position. Fatty acids at sn-2 will be shuffled to other positions on the triacylglycerol in chemical interesterification, which is undesirable. The sn-1,3 lipases, however, allow those endogenous fatty acids to remain at the sn-2 site. The major triacylglycerols in cocoa butter all contain oleic acid at the sn-2 position (1-palmitoyl-2-oleoyl-3stearoyl-glycerol, 1,3-dipalmitoyl-2-oleoyl-glycerol, and 1,3-distearoyl-2-oleoyl-glycerol). Palm oil is rich in palmitic and oleic acid but lacks appreciable amounts of steric acid. Thus, palm oil has been reacted with stearic acid and an sn-1,3 specific lipase; replacement of palmitic acid with stearic acid at the sn-1 or sn-3 position produced an effective cocoa butter substitute. Chemical interesterification will randomize the location of all the fatty acids and produce a less effective substitute. Cocoa butter is an expensive material due to its limited quantities and unique melting properties (hard and brittle at room temperature but melts as it is warmed in the mouth). The reaction of fatty acids with esters such as those found in triacylglycerols is termed acidolysis. Another acidolysis reaction involves incorporating capric acid (10:0) and caproic acid (6:0) into an oil stock. This is beneficial since these fatty acids are readily oxidized in the liver and therefore are excellent sources of energy as opposed to normal storage fat for individuals having deficiencies in fat absorption. Transesterification is the exchange of acyl groups between two esters, specifically between two tricacylglycerols. Mixtures of hydrogenated cotton oil (rich in stearic and palmitic acid) and olive oil (rich in oleic) can be reacted in the presence of the proper lipase and minimal water to create a cocoa butter substitute. To separate the desired TAG from the undesired TAG, trisaturated TAG can be removed by crystallization in acetone or temperature differentials that crystallize out the more saturated triacylglycerols. This process can also be performed using sodium methoxide catalyst instead of lipases but again the randomization of oleic acid from the sn-2 position in the chemical interesterification should produce a less effective substitute than a sn-1,3 lipase-driven interesterification that regiospecifically alters the starting oil stocks, thereby maintaining oleic acid at the sn-2 position.

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Alcoholysis is the esterification reaction between an alcohol and an ester. The most common alcoholysis is the production of mono- and diacylglycerol surfactants (e.g., emulsifiers) by reacting glycerol with triacylglycerols. Specifically, this reaction is termed glycerolysis and is usually performed using nonspecific lipases. The newly formed mono- and diacylglycerols are isolated by temperature-induced crystallization. In glycerolysis, Tc is defined as the critical temperature below which monoacylglycerols crystallize out of the reaction mixture (143). This pushes the equilibrium of the reaction to produce more monoacylglycerols. Vegetable oils have low melting points and hence low Tc due to the abundance of polyunsaturated fatty acids compared to animal fats. By reducing the temperature below the Tc for vegetable oils (Tc = 5°C to 10°C), yields of monoacylglycerols can be increased.

D. MODIFICATION

OF

LIPIDS PRIOR TO HARVEST

Genetic manipulation of lipid biosynthesis is a possible route to improve functionality of TAG and phospholipids (144). For example, the overexpression of cis-9 desaturase in transgenic tomato results in increases in 16:1(cis 9), 16:2(cis 9,12), and 18:2(cis 9,12) fatty acids, which enhance positive flavor attributes in the fruit mediated by a lipoxygenase/hydroperoxidelyase/isomerase/reductase enzyme system (145, 146). Apparently the enhanced fatty acids are precursors for the desirable flavor compounds in tomato. In cell membranes, phosphatidyl glycerol containing two saturated fatty acids is correlated with decreased chilling injury in plants. Incorporating plastidic sn-glycerol-3-phosphate acyltransferase (GPAT) from a chillinginsensitive species (spinach) into a moderately chilling sensitive species (tobacco) increased disaturated phosphatidyl glycerol and was successful at decreasing chilling injury in the tobacco (147). Oxidative stability of transgenic canola oil was improved by decreasing the activity of a cis 15-endogenous desaturase using antisense technology (148). This lowered the 18:3 (cis 9,12,15) content from 6.9% to 1.4% in the oil. Although lipid stability was improved by this technique, 18:3 is an essential fatty acid so functionality is improved at the expense of nutritional quality. A thorough review of genetic engineering of crops that produce modified vegetable oils is available (149).

E. FAT REPLACERS Fat replacers can be primarily carbohydrate, protein, or lipid based. Protein or carbohydrates replacers are called mimetics and tend to absorb water readily but cannot carry lipid-soluble flavor compounds. The other category of fat replacers, called substitutes, will typically contain fatty acids esterified to a carbohydrate. The fatty acids provide desirable physical properties of fats but are not readily cleaved by lipases during digestion. In other words, the lipids are not metabolized and therefore caloric intake is

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reduced per gram or fat ingested. An example of a fat substitute is raffinose polyester. Raffinose is made up of galactose-glucose-fructose units. The eleven available hydroxy groups can potentially be esterified with fatty acids. As the degree of substitution increases the susceptibility to hydrolysis and absorption will decrease (150). Sucrose fatty acid esters act as emulsifiers, texturizers, and protective coatings in various foods products. Benefat consists of short chain fatty acids (e.g., 2:0, 3:0, 4:0) and a long chain saturated fatty acid (stearic acid, 18:0). Short chain fatty acids have low caloric value because they are easily hydrolyzed by digestive lipases and readily converted to carbon dioxide (151). Stearic acid is only partially absorbed, especially if located at the sn-1 or sn-3 position. Benefat is around 5 kcal/g while typical fats are 9 kcal/gram. Currently, Benefat is produced by base-catalyzed interesterification of hydrogenated vegetable oils with TAGs of acetic, propionic, and/or butyric acids (152). The ratios of the short chain fatty acids and the long chain saturated fatty acid can be varied not only providing a low caloric intake but also the physical properties required for specific food applications. Benefat can be used in cookies, baked goods, dairy products, dressings, dips, and sauces (150).

VIII. CHEMISTRY OF FRYING Frying of oils results in distinctive fried flavors and undesirable off-flavors if the oil is overly deteriorated. Off-flavors are manifested via 1) hydrolysis, 2) oxidation, and 3) polymerization reactions. The interaction of steam, water, and oil will hydrolyze TAG into mono-acylglycerols, di-acylglycerols, and free fatty acids. With increased time even glycerol will be produced due to complete hydrolysis of an individual triacylglycerol. Little glycerol can be detected in frying oils since glycerol volatilizes around 150°C and frying temperatures are typically higher. Factors that control hydrolysis are oil temperature, interface area between oil and aqueous phases, water level, and steam level (153). Metals that contaminate the oil interact with lipid hydroperoxides to form free radical species that initiate and propagate oxidation reactions in the presence of oxygen. Frying temperatures will greatly increase the rate of these fundamental lipid oxidation processes and stimulate reactions that may not occur at lower temperatures resulting in an array of oxidation products including aldehydes, ketones, alcohols, esters, hydrocarbons, and lactones. These low-molecular-weight compounds that form due to degradation of the frying oil are considered “volatile,” contributing desirable and undesirable flavors. Polymerization is common in frying where molecules cross-link often as a free radical-free radical reaction. As polymerization increases so too does viscosity of the oil. Most polymerized products are nonvolatile (e.g., dimeric fatty acids, TAG-trimers) and hence

do not produce flavor. However, with further heating these non-volatile compounds can be degraded to off-flavor and toxic products. Degradation products negatively affect not only flavor and safety but also color and texture of the fried products. Antioxidants and antifoam are added to frying oil to extend frying life. Other measures of delaying degradation of oil quality include utilizing fresh oil, using an oil low in polyunsaturated fatty acids and contaminating metals, filtration of oil with adsorbents, turnover of oil, and decreasing exposure of oil to oxygen. Antifoam will aid in reducing exposure of oil to oxygen. Continuous heating is better than discontinuous heating in extending frying life of the oil (153). Not all oxidation that occurs with frying is negative. For instance, 2-4-decadienal is considered a positive flavor compound. Often a preliminary batch of fried foods is prepared and discarded so that the subsequent batches have a desired flavor profile. More unsaturated oils oxidize faster than less saturated oils which decreases the amount of time needed to obtain a proper frying flavor in the food. Free fatty acid content is an unreliable measure of frying oil quality. There still is not a fully appropriate single test to assess frying oil quality. The FoodOil sensor (FOS) (Northern Instruments Corp., Lino Lakes, MN) measures dielectric constant of frying oil compared to fresh oil and has had some success. The dielectric constant increases with increasing polarity so that once a certain value is reached the oil needs change.

IX. FOOD IRRADIATION The purpose of food irradiation is to destroy microorganisms and hence extend shelf life. Lipids can be adversely affected. Typical dosages range from 1 to 10 kGy. Sterilization is achieved at doses of 10–50 kGy. When ionization radiation is absorbed by matter, ions, and excited molecules are produced. These ions, and excited molecules can dissociate to form free radicals. Reactions induced by irradiation prefer to react near the oxygen portion of TAG (154). Reaction occurs preferentially near the oxygen due to the high localization of electron deficiency on the oxygen atom. This explains the preponderance of aldehydes with the same chain length as the most abundant parent fatty acid (cleavage at location b) (Figure 8.12). Cleavage at locations c and d results in hydrocarbons that have one and two carbons less, respectively, than the parent fatty acid, which also is more common than a random assortment of hydrocarbons. Alternatively, free radicals can combine. For instance, two alkyl radicals react to form a dimeric hydrocarbon; acyl and alkyl radicals result in a ketone; acyloxy and alkyl radicals produce an ester; alkyl radicals can react with various glyceryl residue radicals to form alkyl glyceryl diesters and glyceryl ether diesters.


a

b

CH2 O

O

c

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d

C CH2 CH2

CH2

CH2

CH2

O CH

O

C

R

O CH2

O

C

R

FIGURE 8.12 Cleavage sites on a triacylglycerol due to radiolysis. Adapted from Ref. 8.

Irradiation was found to accelerate lipid oxidation in raw pork patties and raw turkey breast that was aerobically packed (155, 156). Lipid oxidation was accelerated by irradiation (3 kGy) in aerobically packed, pre-cooked chicken (157). Irradiation caused formation of a brown pigment in raw beef and pork, but not turkey (158). Carbon monoxide was implicated as the cause of pinking in irradiated raw turkey breast muscle (159).

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9

Fats: Physical Properties

Francisco J. Hidalgo and Rosario Zamora Instituto de la Grasa

CONTENTS I. II.

Introduction..............................................................................................................................................................9-2 Crystallization and Polymorphism ..........................................................................................................................9-2 A. Crystalline Structure of Triacylglycerols ........................................................................................................9-2 B. Polymorphism and Phase Behavior of Natural Fats........................................................................................9-3 1. Milk Fat ....................................................................................................................................................9-3 2. Palm Oil ....................................................................................................................................................9-4 3. Lauric Fats ................................................................................................................................................9-4 4. Liquid Oils ................................................................................................................................................9-4 5. Hydrogenated Fats ....................................................................................................................................9-4 6. Cocoa Butter ............................................................................................................................................9-4 7. Confectionery Fats ....................................................................................................................................9-4 C. Techniques to Determine Crystallization and Polymorphism ........................................................................9-5 1. Infrared and Raman Spectroscopy............................................................................................................9-5 2. X-Ray Diffraction ....................................................................................................................................9-5 3. Microscopic Techniques ..........................................................................................................................9-6 III. Thermal and Rheological Properties, and Other Physical Constants......................................................................9-6 A. Melting ............................................................................................................................................................9-6 1. Melting Points ..........................................................................................................................................9-6 2. Specific Heat and Heat of Fusion ............................................................................................................9-8 B. Plasticity ..........................................................................................................................................................9-8 C. Viscosity ........................................................................................................................................................9-10 D. Vapor Pressure ..............................................................................................................................................9-10 E. Smoke, Flash, and Fire Points ......................................................................................................................9-10 F. Heat of Combustion ......................................................................................................................................9-11 G. Thermal Conductivity ....................................................................................................................................9-11 H. Thermal Diffusivity........................................................................................................................................9-12 I. Thermal Expansion ........................................................................................................................................9-12 J. Dielectric Constant ........................................................................................................................................9-12 K. Density ..........................................................................................................................................................9-12 L. Solubility........................................................................................................................................................9-13 M. Surface Tension, Interfacial Tension, and Emulsification ............................................................................9-13 N. Ultrasonic Properties......................................................................................................................................9-15 IV. Optical and Spectroscopic Properties....................................................................................................................9-15 A. Color ..............................................................................................................................................................9-15 B. Refractive Index ............................................................................................................................................9-17 C. Ultraviolet Spectroscopy................................................................................................................................9-17 D. Infrared (IR) Spectroscopy ............................................................................................................................9-17 E. Raman Spectroscopy......................................................................................................................................9-19 F. Nuclear Magnetic Resonance (NMR) Spectroscopy ....................................................................................9-19 1. Low-Resolution NMR ............................................................................................................................9-19 2. High-Resolution 1H NMR ......................................................................................................................9-20 3. High-Resolution 13C NMR ....................................................................................................................9-21 References ......................................................................................................................................................................9-21 9-1

9-2


I. INTRODUCTION The physical properties of fats and oils are of great practical importance so it is necessary to understand the makeup of these materials and how they should be used (1–9). Thus, many technical applications of fatty materials, including their uses in edible products, depend on the oiliness, surface activity, solubility, melting behavior, or other physical properties peculiar to long-chain compounds (10). Because fats and oils are mainly composed of mixtures of triacylglycerols, the physical properties of these molecules are going to determine the physical characteristics of the oil or fat. Thus, these characteristics are dependent on such factors as seed or plant source, degree of unsaturation, length of carbon chains, isomeric forms of the constituent fatty acids, molecular structure of the triacylglycerols, and processing. This chapter will review the most important physical properties of triacylglycerol molecules as well as of the most common edible fats and oils.

Long spacing

Short spacings

Angle of tilt

FIGURE 9.1 The triclinic unit cell for long-chain compounds.

II. CRYSTALLIZATION AND POLYMORPHISM TABLE 9.1 The Seven Crystal Systems

Crystallization from solution is usually a slow process that first requires supercooling and then leads to nucleation and crystal growth. A high degree of supercooling will be conductive to nucleation, and very small crystals will be formed. At temperatures closer to the crystallization point, crystal growth will be favored and large crystals will be formed (2). Once formed, crystals, which may be stable or metastable, are able either to modify their habit or undergo phase transitions, respectively. Both processes result in polymorphic behavior, a behavior common to fats and other lipids (11–22).

Tetragonal

A.

Monoclinic

CRYSTALLINE STRUCTURE OF TRIACYLGLYCEROLS

In the solid state, molecules adopt the ideal conformation and arrangement in relation to their neighbors in order to optimize intra- as well as intermolecular interactions and achieve close-packing. The smallest building unit of a crystal, the repeating unit of the whole structure, is called the unit cell (Figure 9.1). The crystal structure is obtained by repetition of this unit in the three axial directions (5). Only seven different cells are necessary to include all possible point lattices. These correspond to the seven crystal systems into which all crystals can be classified (Table 9.1). Of these seven crystal systems, it is now accepted that three predominate in crystalline triacylglycerols (23). Usually, the most stable form of triacylglycerols has a triclinic subcell with parallel hydrocarbon–chain planes (T||). A second common subcell is orthorhombic with perpendicular chain phases (O⊥). The third common subcell type is hexagonal (H) with no specific chain plane conformation (24). This hexagonal form exhibits the lowest stability.

System Cubic

Rhombohedral Hexagonal Orthorhombic

Triclinic

Angles and Axial Lengths All axes equal and all at right angles a = b = c and α = β = γ and = 90º Two of three axes equal and all at right angles a = b ≠ c and α = β = γ and = 90º All axes equal and none at right angles a = b = c and α = β = γ and ≠ 90º Two axes = 120º and the third at 90º relative to them a = b ≠ c and α = β = 90º and γ = 120º All axes unequal and all at right angles a ≠ b ≠ c and α = β = γ and = 90º Three unequal axes having one pair not equal to 90º a ≠ b ≠ c and α = γ = 90º ≠ β All axes unequal and none at right angles a ≠ b ≠ c and α ≠ β ≠ γ and ≠ 90º

Source: Ref. 11.

Interpretation of X-ray crystallography data from trilaurin and tricaprin resulted in representation of triacylglycerols in a tuning fork conformation when crystalline (Figure 9.2). The fatty acids esterified at the sn-1 and sn-2 positions of glycerol are extended and almost straight. The sn-3 ester projects 90º from sn-1 and sn-2, folds over at the carboxyl carbon, and aligns parallel to the sn-1 acyl ester. Molecules are packed in pairs, in a single layer arrangement, with the methyl groups and glycerol backbones in separate regions. The main cell is triclinic centered and contains two molecules; the subcell is also triclinic. In addition to these bilayer structures, triacylglycerols may also be arranged in trilayers (Figure 9.2) (25–27).


9-3

TABLE 9.2 Characteristics of the Polymorphic Forms of Monoacid Triacylglycerols α Form

β’ Form

β Form

H 4.15 720 Least dense Lowest Amorphous-like

O⬜ 4.2, 3.8 727, 719 Intermediate Medium Rectangular

T|| 4.6, 3.9, 3.7 717 Most dense Highest Needle shaped

Characteristic Chain packing Short spacing (Å) IR bands (cm-1) Density Melting point Morphology

1 3 2

Source: Refs. 10, 12, 15.

Bilayer

Trilayer

FIGURE 9.2 Double and triple chair arrangements of β form.

Thus, a trilayer structure occurs when the sn-2 position of the triacylglycerol contains a fatty acid that is either cisunsaturated or of a chain length different by four or more carbons from those on the sn-1 and sn-2 positions (28). Also, a trilayer structure has been predicted to arise if the sn-2 position contained a saturated acyl ester with unsaturated moieties occupying the sn-1 and sn-3 positions (29). When unsaturation results in a trans configuration around the carbon–carbon double bond, the crystal structure exhibits the normal bilayer appearance (12).

B. POLYMORPHISM AND PHASE BEHAVIOR NATURAL FATS

OF

Polymorphism is the ability of fat crystals to exist in more than one crystal form or modification. In the case of natural fats, these crystal forms are α, β, and β, in order of increasing stability (Table 9.2). The changes among these phases are monotropic, and, therefore, proceeds in the solid phase from lower to higher stability. The forms differ in crystalline structure and in melting points, and correspond to the crystal structures described for natural fats in Section II.A. Thus, the most stable and with highest melting point T|| is the β polyform. Another polyform, with variable stability and a melting point lower than β, is β, which has orthorhombic subcell packing (O⊥). Finally, phases with the hexagonal subcell have the lowest melting point and represent the α polymorph. In addition to these three basic polymorphs, other polymorphs showing subtle differences may be observed. Within groups having the same subcell, lower melting polymorphs are designated with a progressively higher subscript. In addition, the bilayer or trilayer structure of the triacylglycerol is designated with 2 or 3 following the

polymorph description. Thus, β2-2 designates a bilayer of a β polymorph with the second highest melting point. The fatty acid makeup and position in the glycerides of the fat solids and temperature history are the two main factors in determining polymorphic behavior (1). Other factors include kind and quantity of impurities, nature of possible solvent, and degree of supercooling. A high level of fatty acids of identical chain length results in a slow conversion rate of β to β and a coarsening of crystal structure. The more heterogeneous of fatty acid makeup, the more likely it will be β and fine-grained or needlelike crystals. Thus, mixed fatty acid triacylglycerols, such as those in lauric fats, tend to be β-stable. If a fat is cooled rapidly, the tendency is to form the small, α-crystals. These generally do not last long and convert rapidly to the β needlelike crystals. These β crystals are considered highly stiffening and, hence, are the form of choice for plastic shortenings (1). Depending on the glyceride composition and the temperature history, the β-form may convert to the most stable β-form. This form has large, coarse, platelike crystals. These are not stiffening; hence, those hydrogenated fats exhibiting this behavior are the choice for the solids in fluid shortenings (1). Generally speaking, β-forms melt about 5–10°C higher than the α-forms, and the β-forms also melt about 5–10°C higher than the β-forms. Fats that tend to crystallize in β-forms include soybean, corn, olive, sunflower, and safflower oils, as well as cocoa butter and lard. On the other hand, cottonseed, palm, and rapeseed oils, milk fat, tallow, and modified lard tend to produce β crystals that tend to persist for long periods. 1. Milk Fat As with many natural fats, the temperature at which crystallization occurs influences milk fat firmness, crystalline conformation, and percentage of solid fat. Hardness variability in milk results from different thermal treatments and may be better understood considering the presence of three milk fat fractions, which are observed by using differential scanning calorimetry. These fractions are defined as high-, middle-, and low-melting fractions (HMF, MMF,

9-4


and LMF, respectively). LMF is liquid at ambient temperature. Stable polymorphs of MMF and HMF were found to be a mixture of β-2 + β-3, and β-2, respectively (30,31). 2. Palm Oil Palm oil is expressed from the pulp of the oil palm (Elaeis guineensis) fruit and is unique among vegetable oils because of the large percentage (10–15%) of saturated acyl esters at the sn-2 position of the triacylglycerols. In addition, it has almost 5 % of free fatty acids that play a role in the hardness. At room temperature, the oil appears as a slurry of crystals in oil. Three polymorphs have been determined: β2, α-2, and the stable β1 form (32). The β stability has resulted in the addition of palm oil to oils destined for shortening or margarine, since β-tending fats can result in gritty textures. 3. Lauric Fats Lauric fats are those fats rich in laury acyl esters in the triacylglycerol molecule, mainly coconut oil and palm kernel oil. Two polymorphs have been identified in these fats: α and β-2. The α form is fleeting and can be recognized only after rapid cooling, as it quickly transforms into the β-2 polymorph (33–35). The melting point of these fats is sharp at 22°C for coconut and 25°C for palm kernel oil (36). 4. Liquid Oils Evaluations of polymorphism in fats that are liquid at room temperature are limited. Cottonseed and peanut oils crystallize in a β2 form that is transformed into a stable β1-2 form. Four other oils (corn, safflower, sunflower, and soybean) show polymorphism similar to that of peanut and cottonseed, but these four fats developed a stable β-2 form (12). 5. Hydrogenated Fats Complete hydrogenation eliminates the asymmetry, often leading to β stable polymorphs. Thus, soybean, peanut, sunflower, corn, and sesame oils, are converted to hydrogenated fats having stearoyl esters and consequently show the stable β-2 form. For oils rich in palmitic, hydrogenation leads to fats containing a high proportion of 1,3dipalmitoyl-2-stearoyl-sn-glycerol (PStP). Because the rearrangement of PStP into a stable β form is hindered by misalignment of the methyl end plane of the β unit cell (23), a fat rich in this triacylglycerol will stay in the β form. On the other hand, a hydrogenated fat rich in StPSt can transform into a stable β form. The high PStSt fats have equally stable β and β forms, and any transformation to the β form occurs over a long period of time (12).

6. Cocoa Butter Cocoa butter occupies a special place among natural fats because of its unusual and highly value physical properties. Products containing cocoa butter, such as chocolate, are solid at room temperature; have a desirable “snap”; and melt smoothly and rapidly in the mouth, giving a cooling effect with no greasy impression on the palate. The main characteristic of cocoa butter is the presence of a high content of symmetrical monounsaturated triacylglycerols (1-palmitoyl-2-oleyl-3-stearoyl-sn-glycerol (POSt), 1,3-distearoyl-2-oleyl-sn-glycerol (StOSt), and 1,3-dipalmitoyl-2-oleyl-sn-glycerol (POP) account for about 80% of the total). The polymorphic behavior of cocoa butter is more complex than that of its component glycerides, and a specific system for cocoa butter is often used. This was introduced by Wille and Lutton (37) and recognizes six different polymorphs –I, for the lowest melting form, through VI, for the highest melting form (Table 9.3). Another system in use recognizes only five polymorphs, designated γ, α, β, β2, and β1, in order of increasing stability and melting point (42–45). The desirable physical properties of cocoa butter and chocolate – snap, gloss, melting in the mouth, and flavor release – depend on the formation of polymorph V or β2, which has to be obtained under controlled temperature conditions (41,46). After a long storage or unfavorable storage conditions such as extreme temperatures, chocolate may show “bloom.” This is a grayish covering of the surface caused by crystals of the most stable β phase (phase VI) (41). Eventually the change progresses to the interior of the chocolate and the resulting change in crystal structure and melting point makes the product unsuitable for consumption. 7. Confectionery Fats Cocoa butter is the primary fat used in chocolate. Its expense has led to the development of other fats, used alone or in combination, to replace some or all cocoa butter in cocoa-containing confections. These confectionery TABLE 9.3 Nomenclature and Melting Point (°C) of Cocoa Butter Polymorphs Form I II III IV V VI a

Melting Pointa

Form

Melting Pointa

17.3–17.9 23.3–24.4 25.5–27.7 27.3–28.4 33.0–33.8 34.6–36.3

γ α

16–18 21–24

β β2 β1

27–29 34–35 36–37

Values correspond to the range of the different values described in the literature. Source: Refs. 12, 37–45.


or specialty fats can be classified into cocoa butter equivalents (CBE) and cocoa butter substitutes (CBS) (47). Essentially, a CBE is a mixed fat that provides a fatty acid and triacylglycerol composition similar to those of cocoa butter. A CBS is a fat that provides some of the desired physical characteristics to a confection independent of its dissimilar chemical composition to that of cocoa butter. Miscibility is an important characteristic of confectionery fats. When fats of different composition are mixed, the melting point or the solid fat content of the blend may be lower than that of the individual components (eutectic effects). This happens when cocoa butter is mixed with a CBS that may lead to unacceptable softening. Mixing of cocoa butter and CBE gives no eutectic effect, and this type of fat can be used in any proportion with cocoa butter, analogously to milk fat (33). However, it has been reported that minor components of milk fats exert a significant influence on the crystallization behavior when milk fat is mixed with cocoa butter and other confectionery fats, having, for instance, a softening effect and antibloom properties (8,48,49). CBEs are generally based on three raw materials – shea oil, illipe butter, and palm – oil and processed by fractionation. CBEs also require the same tempering procedures as cocoa butter, since they will exhibit polymorphism similar to that of cocoa butter. It is also possible to tailor make CBEs to higher solid content and melting point than some of the softer types of cocoa butter. These fats are described as cocoa butter improvers (CBIs). CBSs are available in two types, lauric and nonlauric. Lauric CBSs are based on palm kernel oil or coconut oil and are not compatible with cocoa butter. They do not need tempering, and the crystals formed are stable. Nonlauric CBSs are produced by hydrogenation of liquid oils, frequently followed by fractionation and/or blending. These products, especially those made from palm olein, are very stable in the β form. Nonfractionated CBSs are used in compound-coating fats for cookies. The fractionated, hydrogenated CBSs have better eating quality and can tolerate up to 25% cocoa butter when used in coatings.

C. TECHNIQUES TO DETERMINE CRYSTALLIZATION AND POLYMORPHISM The techniques used to elucidate crystal structures are either spectroscopic or microscopic. Spectroscopic techniques include X-ray diffraction and Raman and infrared spectroscopy. Microscopic techniques include polarized light and electron microscopy (50). 1. Infrared and Raman Spectroscopy The region of major interest in an IR spectrum for the study of fat polymorphism is the methylene rocking vibration mode which appears between 670 and 770 cm1. The

9-5

spectra vary according to the polymorphic form. The nomenclature for polymorphs is as follows: a doublet at 728 and 718 cm1 indicates the presence of the β form, a singlet at 720 cm1 the α form, and a singlet a 717 cm1 the β modification (51). In addition, vibrational (infrared and Raman) spectra have given fruitful information on molecular conformations of aliphatic chains and olefinic groups, and methyl end packings (52–54). Raman spectroscopy is also used to identify the different states of order of lipids (55). Two regions are of interest: the C-C stretching vibration region at about 1100 cm1, and the C-H stretching vibration region at about 2850 cm1. Lipids with crystalline chains show two sharp peaks near 1065 and 1130 cm1, whereas these peaks are shifted towards a broad band near 1090 cm1 when the chain melts. The ratio 1080/1130 can be taken as a measure of the degree of a liquid-type order (56). In addition, a peak at 2850 cm1 corresponds to symmetric vibrations of methylene groups characteristic for the liquid state, whereas a peak at 2890 cm1 is caused by antisymmetric vibrations of methylene groups and dominates when hydrocarbon chains are crystalline (57). 2. X-Ray Diffraction The principle of X-ray diffraction is to excite an anticathode which will emit X-rays being diffracted by the crystal structure at a specific angle. The angle depends on the distance between two crystal planes, d, and d is different for each crystal structure. The chain packing of the triacylglycerol molecules determines the spacing between adjoining molecules. The cross-sectional structures determine the short spacings (Figure 9.1). Each of the chainpacking subcells is characterized by an unique set of X-ray diffraction lines in the wide-angle region between 3.5 and 5.5 Å. The nomenclature used to identify lipid crystal forms was proposed by Larsson (40) and is based on the following criteria: a form that gives only one strong short-spacing line near 4.15 Å is termed α; a form that gives two strong short-spacing lines near 3.80 and 4.20 Å and also shows a doublet in the 720 cm1 region of the infrared absorption spectra is termed β; a form that does not satisfy criterion two is termed β (Table 9.2). X-ray diffraction is a powerful analytical technique to identify polymorphic phases unambiguously in both pure triacylglycerol systems and edible fats (43,58–60). In addition, recent developments in high-energy accelerators and X-ray detectors have reduced the exposure times of the sample to the order of milliseconds. Thus, with synchrotron radiation X-ray diffraction, the kinetics of rapid triacylglycerol polymorphic transformations has been elucidated under both isothermal and nonisothermal conditions in pure and mixed triacylglycerol systems (18,61–63). The complex structure of the crystal network is determined by the fractal dimension, D, which describes the

9-6


relation between the number of crystals in a crystal aggregate and its radius, R (64,65). In general, the higher the D value, the more compact the crystal dispersion and, in the case of triacylglycerols, its value changes with ageing of the system after crystallization. In turn, the magnitude of D affects the value of the elastic modulus and, therefore, the texture of the crystal dispersion at a given temperature (e.g., mouthfeel, spreadability). Although the rheology of fat crystal dispersions determines important properties in vegetable oil system (i.e., texture, sedimentation), still more research is needed to understand the structure-property relationship (65). 3. Microscopic Techniques Polarized light spectroscopy allows under certain circumstances to differentiate the α (platelets), β (small needles), and β (larger and growing in clump) forms. This technique makes it possible to view crystals in the range of 0.5–100 µm (66). In contrast to polarized light microscopy, electron microscopy resolves details in areas of 0.1 µm. However, since fine structures of fats are temperature sensitive, special techniques such as freeze-fracture or freeze-etching are required (66). Structures of liquid and crystallized fat in systems such as butter and margarine can be characterized in micrographs due to the amorphous appearance of the originally liquid fat (67).

III. THERMAL AND RHEOLOGICAL PROPERTIES, AND OTHER PHYSICAL CONSTANTS A.

TABLE 9.4 Melting Points (°C) of Common Triacylglycerols Form a

TAG

LaLaLa MMM PPP StStSt AAA PStP PPSt PStSt StPSt POP PPO POSt StPO PStO StOSt StStO StESt StRSt PLP OPO POO StOO OOO EEE LLL LnLnLn

α 15.0 33.0 45.0 54.9 62.0 47.0 47.4 50.6 51.8 18.1 18.5 18.2 25.3 25.5 23.5 30.4 46.0 25.8 – – 4.0 1.5 32.0 15.5 33.7 44.6

β’ 34.5 46.0 56.6 64.1 69.0 68.9 59.9 60.8 64.0 30.5 35.4 33.2 38.7 37.4 36.6 42.2 58.0 48.0 18.6 – 2.5 8.6 11.8 37.0 21.0 –

β 46.5 58.0 66.1 73.4 78.0 65.5 62.9 65.0 68.5 35.3 40.4 38.2 40.5 – 41.2 42.1 61.0 – – 18.7 19.2 23.0 5.1 42.0 10.0 24.2

a

Abbreviations: TAG, triacylglycerol. Fatty acids in TAG: A, arachidic; E, elaidic; L, linoleic; La, lauric; Ln, linolenic; M, miristic; P, palmitic; R, ricinoleic; and St, stearic. Source: Refs. 7, 8, 41, 68.

MELTING

1. Melting Points The melting point is the temperature at which a solid fat becomes a liquid oil. Thus, an individual fatty acid or triacylglycerol has a specific complete melting point for each polymorphic form (Table 9.4 collects the melting points of common triacylglycerols in their three polymorphic forms). Complications arise in fats and oils because they are essentially mixtures of mixed triacylglycerols which crystallize in several crystal forms (Table 9.5 collects triacylglycerol composition of some common fats and oils). These molecules, although of the same chemical structure, differ in chain length, unsaturation, and isomerism. Each component in these products has its own melting point. Fats, therefore, do not have sharp melting points, but a melting range. What is commonly known as the melting point of a fat is in reality the end of the melting range. Table 9.6 collects melting and solidification points of some common fats and oils. In addition, softening points (74) and congeal points (75) are sometimes reported.

The more complex and diversified the mixture of triacylglycerols in the fat, the greater the melting range. If the melting range is less than 5°C, the fat is considered to be non-plastic (cocoa butter, for example). If the melting range is significant (in certain cases it may exceed 40°C) the fat is called plastic. This happens for the majority of natural and processed fats. The temperature at which a fat or oil is completely melted depends on various factors (12), including the average chain length of the fatty acids (in general, the longer the average chain length, the higher the melting point); the positioning of the fatty acids on the glycerol molecule (as an example, safflower oil, which has a long average chain length, will melt like a medium chain length triacylglycerol); the relative proportion of saturated to unsaturated fatty acids (the higher the proportion of unsaturated fatty acids, the lower the melting point); and the processing techniques employed, for example the degree and selectivity of hydrogenation and winterization.

6.1

11.1

5.2

PStSt

2.3 film >min

Compute effective permeability PO , PCO 2

2

Calculate EMA END

FIGURE 119.7 Description of steps involved in MAP design.

of both gases in the package from which the equilibrium time can be calculated as follows: Vf e teq O2 24 3600 PO2 A

冢y

RO2 M e yOe 2 PO2 A

冢y

RO2 M e yOe 2 PO2 A

eq O2

ln

i O2

冢y

RCO2 M e e yCO 2 PCO2 A

冢y

RCO2 M e e yCO 2 PCO2 A

i CO2

ln

冣

冣

Vf e eq t CO 2 24 3600 PCO2 A

eq CO2

(119.35)

冣

冣

(119.36)

Equations 119.35 and 119.36 show that the variables that affect the time required to reach the

119-12


Optimum window Average composition Fi

0.21

lm lin

CO 2(atm)

e

0.14 Best CO 2

N

or

m

al

Best O 2

lin

e

0.07

(0,0.0003)

0.07

0.14

0.21

O 2 (atm)

FIGURE 119.8 Determination of equilibrium gas composition to be achieved by the selected film.

equilibrium are O2 and CO2 permeability of film, free volume inside the package, the concentrations of O2 and CO2 at the beginning and at equilibrium and the product weight, A and e. The highest time of tO2 and tCO2 is the one considered for simulation of gas composition inside the package. Usually the initial concentrations are those in atmospheric air and the concentrations at equilibrium are the ones for optimal preservation of the produce. But in passive MAP the gas composition changes with time due to which respiration rate also changes and achieves constant rate at the once equilibrium gas composition reaches. Hence, it is necessary to consider the effect of change in gas composition on the respiration rate and thereby on the equilibrium time. ix. The last step consists of the calculation of the changes in package gas composition with time. The program uses an optimisation routine named fsolve to solve the two ordinary differential Equations 119.1 and 119.2 for O2 and CO2 concentrations, respectively. The program calculates the O2 and CO2 concentrations at every minute. The respiration rate is re-calculated at new gas composition, i.e., composition after one minute and is used to calculate the next O2 and CO2. In this way gas composition is predicted until the eq equilibrium time teq (t eq O2 or t CO2, whichever is higher) is reached.

VI. PERFORATION-MEDIATED MAP A major challenge is to develop films that have greater permeability and have a wider range of β values than existing

types. Films of enhanced permeability are necessary for packaging high respiration rate products and for preventing the development of anaerobiosis. A wider range of β values, especially those below 3, is necessary to better match the respiration behaviour of many products. The use of either perforation systems or microporous films is a possible solution to meet these two requirements. These systems and films have permeabilities many orders of magnitude higher than those of non-perforated polymeric films, as well as β values between 0.8 and 1. The use of perforations in MAP has been recently reported by Fonseca et al. (36), Silva (37) and Emond (38). The gas transfer coefficients through perforations can be described by Equations 119.37 and 119.38 characteristics (36). This model has a functional form similar to the one that would be expected by dimensionless analysis and has the advantage of having less number of parameters than those suggested by Emond (38) and Silva (37). Γ is the tortuosity of the path through which the pores pass and is equal to 1.14. The other parameters are shown in Table 119.5. ε PO2 NP 2 a Dbp Lpc Γ ε PCO2 NP β 2 a Dbp Lpc Γ

(119.37) (119.38)

VII. MACROSCOPIC HOLES IN COMBINATION WITH POLYMERIC FILM Since β value of polymeric films is much larger than the respiratory quotient, RQ, there will be more CO2 leaving the package than O2 entering. Nitrogen cannot make up the volumetric difference, so the package free volume tends to shrink during storage. This volume shrinkage can be eliminated by introducing a very small hole that allows a convective influx of air to balance the total pressure inside the bag with the air outside. This, of course, alters the composition of the gas in the package relative to what it would have been in the absence of the hole. For some products, it is necessary to have a lower CO2/O2 permeability ratio than available polymer choices permit in TABLE 119.5 Parameters of the Mathematical Model Shown in Equations 119.37 and 119.38 Parameter a b c DO2 DCO2

Value 6.42 106 1.45 0.598 1.14 16.4 106 m2.sec1 20.6 106 m2.sec1

An Interactive Design of MA-Packaging for Fresh Produce

order to achieve the CO2/O2 composition for optimal extension of shelf life. This can be solved by making the area of the holes large enough to provide non-selective permeation in parallel with the film; thus reducing the net CO2/O2 permeability ratio. The macroscopic perforation in the polymeric film represents an alternate route for gas transport, which is in parallel to the barrier formed by the plastic material. The total flow through polymeric film having NH number of holes is Total permeation

冧冦

冧

(119.39)

The macroscopic perforations in polymeric films have diameters of the order of 104 m or greater, whereas the mean-free path of gas molecules at atmospheric pressure is much less, being about (1 or 2) 107 m (25). Therefore, transport through the perforation may be treated as macroscopic diffusion in a cylindrical pathway filled with air. If the distance between perforations is much greater than their radius, the diffusive pass length becomes the length of cylindrical pore plus the radius of the hole. Diffusive flux in this case obeys Fick’s law: π RH2 DO2 PO2 Pfilm O2 NH (e RH)

冤

(119.40)

π RH2 DCO2 film NH PCO2 PCO 2 (e RH)

(119.41)

冥

Product Characteristics Product selected Varity/cultivar Product weight (M), kg Respiration rate modela True density (ρ), kg.m3 Optimum MA conditionsb O2 minimum (ymin O2 ), atm O2 maximum (y max O2 ), atm CO2 minimum (ymin CO2), atm CO2 maximum (ymax CO2), atm Temperature (T), °C Package Characteristics Top Width (WT), m Bottom width (WB), m Length (LT), m Height (HT), m Total volume (Vt), m3 Free volume (Vf), m3 2 Film area (ATray F ), m

Mango Nam dok mai 1.0 MMUc 1044 0.03 0.07 0.05 0.08 10 0.16 0.12 0.14 0.09 1.792 103 0.834 103 0.0224

a

Charoenchaitawornchit et al. (23). Kader (39). c Michaelis-Menten type equation with uncompetitive inhibition of CO2. b

0.24

冥

冤

TABLE 119.6 Product and Package Characteristics for the Case Study

0.21 0.18

VIII. DEVELOPMENT OF SOFTWARE

y CO 2, atm

冦

Permeation Permeation through through film one hole NH

119-13

0.15 0.12

1

0.09 0.06

The graphical user interface of the software was built in Matlab programming language. The same tool was used to solve the design steps as explained in Figure 119.7.

0.03

11 12

0.00 0

IX. CASE STUDY A case study is presented to illustrate the use of the software to design MAP for whole mango. In this case study, the design procedure is explained for two kinds of package: permeable polymeric film and microscopic perforation (hole) with permeable polymeric film. A tray type package as shown in Figure 119.5 is selected for the present case study. The product and package characteristics are shown in Table 119.6. For the selected commodity and at given optimum conditions βmin and βmax were found to be 1.71 and 3.84, respectively. As in Figure 119.9, the thick rectangle indicates the limits, inside which lines must run so that the corresponding films are eligible for selection. As can been

0.03 0.06 0.09 0.12 0.15 0.18 0.21 0.24 y O2, atm

FIGURE 119.9 Optimum window and lines established from the β relation of selected films for mango (the number given to each line corresponds to the serial number of films in Table 119.7).

seen, there are 11 films selected from the database of 27 films. These films will modify the package atmosphere leading to the optimum level for the selected commodity provided that the area of film is kept as shown in Table 119.7. The equilibrium modified atmosphere parameters such as eq yOeq2, yCO , AF and teq are calculated and are shown in 2 Table 119.7. All the 11 films were found to give the optimum atmosphere, but the area of film required is different for all of them. Moreover, the film area required to achieve equilibrium is different from the possible area

119-14


TABLE 119.7 List of Selected Polymeric Films for Mango Equilibrium Parameters of MAP Sr. Selected Polymeric No. Films*

Film ␤

AF, m2

y eq O2, atm

y eq CO2, atm

teq, day

1 2 3 4 5 6 7 8 9 10 11 12

2.09 2.64 3.33 3.71 2.85 3.09 2.64 2.27 3.13 3.39 3.53 9.40

0.021 0.093 0.363 0.458 0.745 1.170 1.350 1.670 1.970 22.20 23.70 0.022

0.055 0.049 0.045 0.045 0.047 0.046 0.049 0.052 0.046 0.045 0.045 0.065

0.076 0.062 0.050 0.045 0.058 0.054 0.062 0.070 0.053 0.049 0.047 0.037

3.47 3.97 2.84 2.55 3.46 3.10 3.96 4.64 3.05 2.79 2.67 3.33

Ethyl cellulose Polyvinyl chloride (RMF) Low density polyethylene Methyl rubber Cast polypropylene Oriented polypropylene Polyvinyl chloride (VF) Rubber hydrochloride Polyvinyl chloride (AF) Polyethylene terephthalate Nylon multilayer Polybutadiene ¢

* Thickness 1 mil; ¢ Not suitable for mango.

(ATray 0.16 0.14 0.0224 m2) for the selected tray F type of package. For the present case, ethyl cellulose film is selected because it presents the minimum difference between ATray and AF. If the area of any other chosen film F is kept 0.0224 m2, the package will not achieve the optimum levels. The change in O2 and CO2 in MAP with ethyl cellulose film is shown in Figure 119.10 (a). The rest of the films are not suitable due to their high β values. Nevertheless, these films could be used for MAP of mango after making certain number of holes to increase the gas transfer across the film (Figure 119.10(b)). Table 119.8 shows the results of microperforations with polymeric films having β value more than βmax. It is necessary to decrease the β value of the film in order to produce the atmosphere within the optimum range of mango, by

Gas composition, atm

0.25 0.20

(a) Ethyl cellulose film

yO

(b) Polybutadiene with holes

yO

2

2

Butyl rubber Cellulose acetate Ceramic-filled LDPE Ceramic filled polystyrene Ethylene vinyl acetate Linear LDPE Natural rubber Neoprene Polybutadiene Polybutadiene styrene Polyethylene (Irradiated) Polyvinyl chloride (VA) High density polyethylene * Thickness of film 1 mil.

Film ␤

0.05 0.00 0

10

20

30

40

50

60

70

4.31 8.14 4.73 5.79 5.93 3.90 6.93 7.50 9.40 8.72 5.04 6.49 4.85

80

9

Time, h

FIGURE 119.10 Steady-state establishment of O2 and CO2 in MAP with (a) Ethyl cellulose film having thickness 1 mil, area 0.0146 m2; (b) Polybutadiene film having thickness 1 mil, area 0.0224 m2, number of holes 2, diameter of each hole 0.0001 m.

making tiny holes in polymeric film. The number of holes and diameter depends on the permeability and area of the film as shown in Table 119.8. Although the polymeric films are the most useful packaging material, they are unable to produce the optimum atmosphere for the high respiration and transpiration rates and higher CO2 tolerance commodities. The perforation-mediated package is one such alternative, where the regulation of the gas exchange is achieved by single or multiple perforations or tubes that perforate an otherwise impermeable covering. This perforation-mediated MAP has special interest in fresh-cut products, e.g., cut Galega kale (21). Table 119.9 shows the design output for the perforation-mediated MAP for cut Galega kale at 20 and 5°C. The resultant steady state establishment of O2 and CO2 in perforation-mediated MAP is shown in Figure 119.11.

2

DH, m

NH

Net ␤

0.0224 0.0224 0.0224 0.0224 0.0224 0.0224 0.0224 0.0224 0.0224 0.0224 0.0224 0.0224 0.0224

0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

14 13 14 14 14 14 03 14 03 03 14 14 09

0.84 0.88 0.94 0.90 0.96 0.95 4.05 1.04 4.97 4.52 0.91 1.20 0.80

AF, m

2

0.10

Equilibrium Parameters of MAP Polymeric Film

2

y CO

0.15

TABLE 119.8 EMA Parameters Achieved by Microperforated Polymeric Film *

y CO

yeq O2,

atm

0.141 0.137 0.140 0.140 0.139 0.140 0.059 0.138 0.052 0.050 0.140 0.136 0.147

yeq CO2, atm

teq, day

0.075 0.076 0.067 0.070 0.066 0.066 0.035 0.062 0.030 0.034 0.070 0.054 0.072

1.0 1.0 1.0 1.0 0.9 0.9 2.4 1.0 2.0 2.0 1.0 1.0 2.0

An Interactive Design of MA-Packaging for Fresh Produce

TABLE 119.9 Design Output of Perforation-Mediated MAP System for Cut Galega Kale Equilibrium Parameters of MAP Temperature, °C

DP, m

LP, m

NP

␧

yeq O2, atm

yeq CO2, atm

teq, day

20 5

0.015 0.009

0.019 0.015

1 1

0.43 0.43

0.041 0.022

0.194 0.200

0.5 1.2

0.25 Gas composition, atm

y O2

y CO

2

0.20 0.15 0.10 0.05 0.00 0

10

20

30

40 50 Time, h

60

70

80

90

FIGURE 119.11 Steady state establishment of O2 and CO2 in perforation-mediated MAP at 5°C, diameter of perforation 0.009 m, length 0.015 m, Np 1, ε 0.43, weight of cut Galega kale 0.4 kg.

ACKNOWLEDGEMENTS The authors acknowledge financial support from the Irish Government, under National Plan 2000–2006. Dr. Luís M. Cunha acknowledges financial support from Fundação para a Ciência e a Tecnologia (FCT) through “Financiamento Plurianual — POCTI.”

REFERENCES 1. D Zagory, AA Kader. Modified atmosphere packaging of fresh produce. Food Technology 42(9): 70–77, 1988. 2. JP Emond, F Castaigne, CJ Toupin, D Desilets. Mathematical modelling of gas exchange in modified atmosphere packaging. Transactions of the American Society of Agricultural Engineers 34: 239–245, 1991. 3. NH Banks, DJ Cleland, AC Cameron, RM Beaudry, AA Kader. Proposal for a rationalized system of units for postharvest research in gas exchange. HortScience 30: 1129–1131, 1995. 4. HW Peppelenbos, J Van’t Leven. Evaluation of four types of inhibition for modeling the influence of carbon dioxide on oxygen consumption of fruits and vegetables. Postharvest Biology and Technology 7: 27–40, 1996.

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5. PV Mahajan, TK Goswami. Enzyme kinetics based modeling of respiration rate for apple. J. of Agricultural Engineering Research 79:399–406, 2001. 6. R Lakakul, RM Beaudry, RJ Hernandez. Modeling respiration of apple slices in modified-atmosphere packages. Journal of Food Science 64:105–110, 1999. 7. M Prasad. Development of modified atmosphere packaging system with selective polymeric films for storage of red delicious apples. Unpublished Ph.D. Thesis, Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur, India, 1995. 8. G Gunes, CB Watkins, JH Hotchkiss. Physiological responses of fresh-cut apple slices under high CO2 and low O2 partial pressures, Postharvest Biology and Technology 22: 197–204, 2001. 9. HW Peppelenbos, J van’t Leven, BH van Zwol, LMM Tijskens. The influence of O2 and CO2 on the quality of fresh mushrooms. In: GD Blanpied, JA Barstch, JR Hicks, eds. Proceedings of the 6th International Controlled Atmosphere Research Conference Ithaca, NY, USA, 1, 1993, pp. 746–758. 10. C Maneerat, A Tongta, S Kanlayanarat, C Wongs-Aree. A transient model to predict O2 and CO2 concentrations in modified atmosphere packaging of bananas at various temperatures. In: JR Gorny, ed. Proceedings of the 7th International Controlled Atmosphere Research Conference Davis, CA, USA, 5, 1997, pp. 191–197. 11. MLATM Hertog, HW Peppelenbos, RG Evelo, LMM Tijskens. A dynamic and generic model of gas exchange of respiring produce: the effects of oxygen, carbon dioxide and temperature. Postharvest Biology and Technology, 14: 335–349, 1998. 12. Y Song, HK Kim, KL Yam. Respiration rate of blueberry in modified atmosphere at various temperatures. Journal of the American Society for Horticultural Science 117: 925–929, 1992. 13. DS Lee, Y Song, KL Yam. Application of an enzyme kinetics based respiration model to permeable system experiment of fresh produce. Journal of Food Engineering 27: 297–310, 1996. 14. JP Emond, KV Chau, JK Brecht. Modelling respiration rates of blueberry in a perforation-generated modified atmosphere package. In: GD Blanpied, JA Barstch, JR Hicks, eds. Proceedings of the 6th International Controlled Atmosphere Research Conference, Ithaca, NY, USA, 1993, pp. 134–144. 15. Y Makino, K Iwasaki, T Hirata. Oxygen consumption model for fresh produce on the basis of adsorption theory. Trans. of the ASAE 39: 1076–1073, 1996. 16. C Ratti, GSV Raghavan, Y Gariépy. Respiration rate model and modified atmosphere packaging of fresh cauliflower. Journal of Food Engineering 28: 297–306, 1996. 17. P Jaime, R Oria, J Burgos, ML Salvador. Influencia de la variedad, temperatura y composición de la atmósfera en la tasa respiratoria de cerezas “Burlat,” “Sunburst” y “Sweetheart.” VIII Congreso Nacional de Ciencias Hortícolas, Murcia, 248–254, 1999. 18. F Devlieghere, L Jacxsens, J Debevere. Modified atmosphere packaging: state of the art, 2000.

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19. AB Smyth, J Song, AC Cameron. Modified atmosphere packaged cut iceberg lettuce: effect of temperature and O2 partial pressure on respiration and quality. J. Agric. Food Chem. 46: 4556–4562, 1998. 20. CP McLaughlin, D O’Beirne. Respiration rate of a dry coleslaw mix as affected by storage temperature and respiratory gas concentrations. Journal of Food Science 64: 116–119, 1999. 21. SC Fonseca, FAR Oliveira, JM Frias, JK Brecht, KV Chau. Modeling respiration rate of shredded Galega kale for development of modified atmosphere packaging. J. Food Eng. 54: 299–307, 2002. 22. PV Mahajan, TK Goswami. Prediction of respiration rate of litchi (Litchi chinensis Sonn.) fruit by the principles of enzyme kinetics. Journal of Interacademicia 6(2): 156–165, 2002. 23. A Charoenchaitawornchit, S Kanlayanarat, A Tongta. Modelling of respiration and modified atmosphere packaging of mango “Nam dok mai.” Acta Hort. (ISHS) 599:489–494, 2003. 24. M Rusmono, AM Syarief, HK Purwadaria. Modelling Respiration of Edible-coated, Minimally Processed Mango in Modified Atmosphere Packaging. Quality assurance in agricultural produce, ACIAR Proceedings edited by G.I. Johnson, Le Van To, Nguyen Duy Duc and M.C. Webb, 2000. 25. S Fishman, V Rodov, S Ben-Yehoshua. Mathematical model for perforation effect on oxygen and water vapor dynamics in modified-atmosphere packages. Journal of Food Science 61: 956–961, 1996. 26. JC Guevara, EM Yahia, E Brito de la Fuente. Determinacion de un modelo matematico para predecir la concentracion de gases en nopales empacados en atmosferas modificadas pasivas y semiactivas. IX Congreso Nacional de Biotecnologia y Bioengenieria. Septiembre 10–14. Veracruz, Mexico, 2001. 27. DW Joles, AC Cameron, A Shirazi, PD Petracek, RM Beaudry. Modified atmosphere packaging of ‘Heritage’ red raspberry fruit: respiratory response to reduced oxygen, enhanced carbon dioxide and temperature. Journal of the American Society for Horticultural Science 119: 540–545, 1994. 28. M Zhu, CL Chu, SL Wang, RW Lencki. Influence of O2, CO2 and degree of cutting on the respiration rate of rutabaga. Journal of Food Science 66: 33–37, 2001. 29. MA Rao, TA Siebert, YD Hang, DL Dowing. Respiration rates and microbial growth in snap beans at 12 and 25°C, and different O2 and CO2 concentrations.

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120

Ohmic and Inductive Heating

Rhonda Bengtson, Emily Birdsall, Stuart Feilden, Sireesha Bhattiprolu, Sandeep Bhale, and Marybeth Lima

The Department of Biological & Agricultural Engineering, Louisiana State University

CONTENTS I. II.

Overview ............................................................................................................................................................120-1 General Information on Ohmic Heating ............................................................................................................120-2 A. Advantages ................................................................................................................................................120-2 B. Applications ..............................................................................................................................................120-2 C. Design ........................................................................................................................................................120-2 D. Cost ............................................................................................................................................................120-3 III. Parameters of Importance in Ohmic Heating ....................................................................................................120-3 A. Product Properties ......................................................................................................................................120-3 B. Texture Analysis ........................................................................................................................................120-4 C. Gelatinization ............................................................................................................................................120-4 IV. Modeling of Ohmic Heating Processes ............................................................................................................120-4 A. Basic Equations ..........................................................................................................................................120-4 B. Microbial Death Kinetics ..........................................................................................................................120-5 C. Vitamin Degradation Kinetics ....................................................................................................................120-5 V. Novel Uses of Ohmic Heating ..........................................................................................................................120-5 A. Background ................................................................................................................................................120-5 B. Blanching ..................................................................................................................................................120-6 C. Evaporation ................................................................................................................................................120-6 D. Dehydration ................................................................................................................................................120-6 E. Fermentation ..............................................................................................................................................120-6 F. Extraction ..................................................................................................................................................120-6 G. Summary of Novel Processes ....................................................................................................................120-6 VI. Future Research Directions ................................................................................................................................120-7 References ....................................................................................................................................................................120-7

I. OVERVIEW Ohmic and inductive heating are alternative methods of heating to conventional heating techniques. Ohmic heating, also known as Joule heating, electric resistance heating, direct electric resistance heating, electro heating, and electro conductive heating, is a process in which alternating electric current is passed through food material; heat is internally generated within the material due to its resistance to the applied electrical current. Inductive heating is a technique that uses electric coils to generate oscillating electric fields that send currents through the food. Ohmic heating is not a new technology; it was used as a commercial process in the early 20th century for the

pasteurization of milk (1). However, the “Electropure Process” was discontinued between the late 1930s and 1960s, ostensibly due to the prohibitive cost of electricity. Interest in ohmic heating was rekindled in the 1980s, when investigators were searching for viable methods to effectively sterilize liquid-large particle mixtures, a scenario for which aseptic processing alone was unsatisfactory. Very little information exists on inductive heating because its development in food processing is so recent. The purpose of this chapter is to present general information regarding ohmic heating, and to identify areas of study which will add to the knowledge base of this subject. This chapter is separated into several sections: (1) general information on ohmic heating, (2) modeling of 120-1

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ohmic heating processes, (3) novel uses of ohmic heating, and (4) future research directions.

II. GENERAL INFORMATION ON OHMIC HEATING A. ADVANTAGES Ohmic heating exhibits several advantages with respect to conventional food processing technologies, as follows: ●

●

●

●

● ●

Particulate foods up to 1 in3 are suitable for ohmic heating; the flow of a liquid-particle mixture approaches plug flow when the solids content is considerable (20–70%). Liquid-particle mixtures can heat uniformly under some circumstances (for example, if liquids and particles possess similar electrical conductivities, or if properties such as solids concentration, viscosity, conductivity, specific heat, and flow rate are manipulated appropriately). Temperatures sufficient for UHT processing can be rapidly achieved. There are no hot surfaces for heat transfer, resulting in a low risk of product damage from burning or overprocessing. Energy conversion efficiencies are very high. Relatively low capital cost.

B. APPLICATIONS Ohmic heating can be applied to a wide variety of foods, including liquids, solids, and fluid-solid mixtures. Ohmic heating is being used commercially to produce liquid egg product in the United States. It is also being used in the United Kingdom and Japan for the processing of whole fruits such as strawberries. Additionally, ohmic heating has been successfully applied to a wide variety of foods in the laboratory, including fruits and vegetables, juices, sauces, stews, meats, seafood, pasta, and soups. Widespread commercial adoption of ohmic heating in the United States is dependent on regulatory approval by the FDA, a scenario that requires full understanding of the ohmic heating process with regard to heat transfer (temperature distributions), mass transfer (concentration distributions, which are influenced by electricity), momentum transfer (fluid flow), and kinetic phenomena (thermal and possibly electrothermal death kinetics, and nutrient degradation). Larkin and Spinak (2) examined safety considerations for ohmically heated, aseptically processing, multiphase low acid food products, and discussed the need for providing information on equipment design, product specification, process design, and process validation for regulators. Full knowledge of these areas is critical to ensure that the food

FIGURE 120.1 Graphic of a static ohmic heating apparatus. (Courtesy of Barbara Corns, LSU Ag Center Communications.)

FIGURE 120.2 APV ohmic heater used for industrial applications. (Courtsey of Dr. Sudhir Sastry, Ohio State University.)

product receives adequate thermal treatment. Significant research strides toward widespread commercial use have been made, though more work remains to be done.

C. DESIGN Ohmic heating devices consist of electrodes, a power source, and a means of confining the food sample (for example, a tube or vessel). Appropriate instrumentation, safety features, and connections to other process unit operations (pumps, heat exchangers, holding tubes, etc.) may also be important. Ohmic heaters can be static (batch) or continuous. Figure 120.1 contains a graphic of a static ohmic heating apparatus, while Figures 120.2 and 120.3 represent an early1 continuous ohmic heater used by APV Baker, a U.K. company using ohmic heating for food processing, and a more recent continuous ohmic heating system developed by S. Sastry, respectively. Important design considerations include electrode configuration (current flows across product flow path or parallel to product flow path), the distance between electrodes, electrolysis (metal dissolution of electrodes, particularly at low frequencies), heater geometry, frequency of alternating 1

1980s and 1990s.


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mixture. Substantial research was conducted on this property in the early 1990s because of the importance of electrical conductivity with regard to heat transfer rate and temperature distribution. The electrical conductivity is determined using the following Equation (120.1): L σ⫽ ᎏ AR where ␴ ⫽ specific electrical conductivity (Siemens/m) A ⫽ area of cross section of the sample (m2) L ⫽ length of the sample (m) R ⫽ resistance of the sample (ohm) FIGURE 120.3 Ohmic heater developed by Dr. Sudhir Sastry. (Courtsey of Dr. Sudhir Sastry, Ohio State University.)

current, power requirements, current density, applied voltage, and product velocity and velocity profile. Additional factors regarding the food system used in an ohmic heater include the type of product and its properties, especially electrical conductivity and heating rate; others include percent solids, acidity, product viscosity, specific heat, and density, and solid particle size, shape, and orientation to the electric field. Substantial literature has been devoted to these topics; see, for example, references (3–10). Coated electrodes can minimize or eliminate electrolytic reactions; temperature measurement remains an area of concern, as many measurement methods influence the electric field during ohmic heating. Some success has been seen with thermocouples that are coated with material such as Teflon, however, non-invasive temperature measurements that do not interfere with the electric field remain a challenge, particularly with regard to temperature measurement inside particles.

General2 findings of numerous electrical conductivity studies are as follows: ●

Electrical conductivity is linearly correlated with temperature when the electrical field is sufficiently high (at least 60 V/cm). Non-linearities (sigmoidal curves) are observed with lower electrical field strength (3, 12). ●

●

●

D. COST Investigators (11) conducted an economic engineering analysis of ohmic food processing for low and high acid foods. They found that ohmic heating is an economically viable technology for processing low acid foods. Though ohmic heating was found to be more costly than conventional methods for processing high acid foods, the authors believed that ohmic heating was still viable in these cases because of its potential to produce superior product quality.

III. PARAMETERS OF IMPORTANCE IN OHMIC HEATING

The electrical conductivity is a function of food components; ionic components (salt), acid, and moisture mobility increase electrical conductivity, while fats, lipids, and alcohol decrease it.

●

Electrical conductivity increases as temperature and applied voltage increases, and decreases as solids content increases. Lowering the frequency of alternating current during ohmic heating increases the electrical conductivity. The waveform can influence the electrical conductivity; though alternating current is usually delivered in sine waves, sawtooth waves increased the electrical conductivity in some cases, while square waves decreased it (12). Electrical conductivity increases by heating cycle; preheated samples showed increased electrical conductivity as opposed to raw samples when both were subsequently subjected to ohmic heating (13).

The electrical conductivity of solids and liquids during ohmic heating of multiphase mixtures is also critically important. In an ideal situation, liquid and solid phases possess essentially equal electrical conductivities, and would thus (generally) heat at the same rate. When there are differences in the electrical conductivity between

A. PRODUCT PROPERTIES The most important parameter of interest in ohmic heating is the electrical conductivity of the food and/or food

2

These findings are true in general; some exceptions may exist depending on the situation.

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a fluid and solid particles, the particles heat faster than the fluid when their conductivities are lower than the fluid. Also, solid particulates heat more slowly than a fluid when the electrical conductivity of the solid is higher than that of the fluid. Fluid motion (convective heat transfer) is also an important consideration when there are electrical conductivity differences between fluids and particles. Other product properties that may affect temperature distribution include the density and specific heat of the food product. When solid particles and a fluid medium have similar electrical conductivities, the component with the lower heat capacity will tend to heat faster. High densities and specific heats are conducive to slower heating. Fluid viscosity also influences ohmic heating; higher viscosity fluids tend to result in faster ohmic heating than lower viscosity fluids.

B. TEXTURE ANALYSIS Sensory evaluation is critically important to any viable food process. Numerous publications have cited the superior product quality that can be obtained through decreased process time, though few published studies specifically quantify sensory and texture issues. Six stew formulations sterilized using ohmic heating before and after three years of storage were analyzed; the color, appearance, flavor, texture, and overall food quality ratings were excellent, “indicating that ohmic heating technology has the potential to provide shelf-stable foods closely equivalent to those prepared from scratch” (14).

C. GELATINIZATION Starch gelatinization is an important parameter in food processing, and can be either advantageous or disadvantageous depending on the desired product formulation. The electrical conductivity of a food product is influenced significantly by starch gelatinization (13). These investigators found that electrical conductivity decreased with the degree of gelatinization, and suggest that ohmic heating can be used in the development of a sensor to detect starch gelatinization. Ohmic heating was used to maximize the gel functionality of a seafood product (15). The ohmic heating process was superior to the conventional heating process due to rapid heating that deactivated enzymes, which in turn enabled strong gel formation.

IV. MODELING OF OHMIC HEATING PROCESSES A. BASIC EQUATIONS Considerable effort has been expended to model the heat transfer mechanisms and microbial death kinetics involved during ohmic heating. Models are of interest in the analysis

and design of ohmic heating processes to provide information about the temperature distribution throughout the process, especially “the cold spot,” and to provide accurate predictions of the minimum lethal processing time. Complexities in modeling heat transfer processes during ohmic heating arise when the liquid and particle possess different electrical conductivities, and because electrical conductivity is a (sometimes non-linear) function of temperature and frequency of alternating current. Basic equations regarding ohmic heating are included below. The temperature distribution in a fluid during ohmic heating is based on an energy balance as follows: ⭸Tf ⫽ ⵜ ⭈ (kf ⵜTf) ⫺ np Ap hfp(Tf ⫺ Tps) ⫹ u. f ␳Cpvz ᎏ ⭸z where ␳ ⫽ density Cp ⫽ specific heat T ⫽ temperature z ⫽ distance vz ⫽ fluid velocity f ⫽ fluid p ⫽ particle ps ⫽ particle surface k ⫽ thermal conductivity np ⫽ number of particles A ⫽ surface area of particles hfp ⫽ fluid to particle heat transfer coefficient . u f ⫽ internal energy generation rate of the fluid The temperature distribution in a particle during ohmic heating can be predicted with the conduction heat transfer equation with internal energy generation: ⭸T . ⵜ ⭈ (kⵜT) ⫹ u ⫽ ␳Cp ᎏ ⭸t where k ⫽ thermal conductivity The internal energy generation is: . u ⫽ |ⵜV|2 ␴ where V ⫽ voltage ␴ ⫽ electrical conductivity . u ⫽ energy generation rate per unit volume The voltage field is determined by solving: ⵜ ⭈ (␴ⵜV) ⫽ 0 Numerous models have been developed based on numerical solution of these equations with appropriate boundary conditions and assumptions, and also from


dimensional groupings. Though these models have contributed significantly to the understanding of heat transfer in ohmic heating, none have completely described the ohmic heating process to date. The voltage field (Laplace) equation for a single solid particle in a static heater has been solved (16). Numerical solutions and experimental simulations to more complex ohmic heating situations have been developed (17–22) using magnetic resonance imaging to rapidly map the temperature of food particles during ohmic heating.

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vitamin C degradation kinetics (27). This study was conducted at one electrical field strength (E ⫽ 23.9 V/cm). Others found that the ascorbic acid degradation rate in buffer solution during ohmic heating was a function of power, temperature, NaCl concentration, and products of electrolysis (10). Further research in this area could include the influence of electrical field strength, endpoint temperature, and frequency of alternating current on the degradation of food components during ohmic heating. The characterization of electrolysis is also a critical need in this area.

B. MICROBIAL DEATH KINETICS In terms of microbial death kinetics, considerable attention has been paid to the following question: does electricity result in microbial death, or is microbial death due only to heat treatment? The challenge in modeling microbial death kinetics is precise matching of time-temperature histories between ohmic processes and conventional processes. The FDA has published a comprehensive review of microbial death kinetics data regarding ohmic heating (23). Initial studies in this area showed mixed results, though the experimental details were judged insufficient to draw meaningful conclusions (1). Researchers compared death kinetics of yeast cells under time-temperature histories as identical as possible and found no difference between conventional and ohmic heating (24). More recent work in this area has indicated that decimal reduction times of Bacillus subtilis spores were significantly reduced when using ohmic heating at identical temperatures (25). These investigators also used a two step treatment process involving ohmic heating, followed by holding, followed by heat treatment, which accelerated microbial death kinetics; they hypothesized that electroporation may positively influence microbial death kinetics. The inactivation of yeast cells in phosphate buffer by low-amperage direct current electrical treatment and conventional heating at isothermal temperatures was examined (26). These researchers concluded that a synergistic effect of temperature and electrolysis was observed when the temperature became lethal for yeast. Further research regarding microbial death kinetics, survivor counts subsequent to treatment, and the influence of electricity on cell death kinetics are necessary to address regulatory issues. At the present time, assuming that microbial death is only a function of temperature (heat) results in an appropriately conservative design assumption.

C. VITAMIN DEGRADATION KINETICS Limited information exists regarding product degradation kinetics during ohmic heating. Researchers measured vitamin C degradation in orange juice during ohmic and conventional heating under nearly identical time-temperature histories, and concluded that electricity did not influence

V. NOVEL USES OF OHMIC HEATING A. BACKGROUND Early research on ohmic heating was conducted on heat transfer and sterilization of liquid-particle mixtures. In executing such studies, investigators observed unanticipated phenomena. For example, ohmically heating beetroot resulted in enhanced diffusion of betanin from the beetroot tissue when compared to beetroot tissue heated conventionally (28). These investigators hypothesized that the enhanced mass transfer could be due to electroosmosis. Investigators expanded on the aforementioned work and found that diffusion of beet dye from beetroot into a carrier solution from was enhanced as much as 40% during heating from 20°C to 80°C, and that the concentration of diffused dye was proportional to particle surface area, and a linear function of electric field strength (29). Other researchers ohmically heated Japanese white radish and found that the ohmic heating rate was influenced by frequency; as the frequency of alternating current decreased, the heating rate increased (30). These investigators used H-NMR analysis and hypothesized that at low frequency (50 Hz), rapid heating is due to electroporation of radish tissue membrane, which resulted in a decrease of electrical impedance. Subsequent studies (9, 31) have concluded that electroporation is the most likely mechanism for enhanced mass transfer effects during ohmic heating. Electroporation is defined as the formation of holes in a cell membrane resulting from local pressure of ions, which cannot initially permeate the cell membrane, but are forced against it by the electric field (32). The relatively low alternating frequencies employed during ohmic heating enable this charge build up to occur on the cell wall, resulting in the formation of pores. This also suggests that the lower the frequency of ohmic heating, the more pronounced the mass transfer effect; this concept has been demonstrated in the literature (31, 33–34). It was found that direct current resulted in less mass transfer enhancement than low AC frequency ohmic heating (at 15 V/cm, 250 Hz ⬍ DC ⬍ 50 Hz). Kulshrestha and

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Sastry postulated that a monopolar electric charge (DC) is not as effective as a bipolar electric charge at creating stress on the cell membrane, thus yielding less of an effect than low frequency alternating current (31). Electrically heating foods influences their mass transfer properties. This phenomenon has important implications for food processing operations that involve mass transfer. In 2001, the FDA reported that “A large number of potential future applications exist for ohmic heating, including its use in blanching, evaporation, dehydration, fermentation, and extraction” (23). In this section, we will report on some of the research regarding these novel uses.

when drying to initial or intermediate moisture contents. Zhong and Lima (40) showed that ohmic pretreatment accelerated the vacuum drying rate of sweet potato as much as 24%; these investigators also demonstrated that minimal ohmic treatment (electrical field strength of 50 V/cm and an endpoint temperature of 40°C) resulted in the maximal or near maximal acceleration of drying rate. These investigators suggested that because ohmic heating enhances drying rates and enhances extraction yields, the process could be ideal for the recovery of high value, heat labile components from biological materials using unit operations such as supercritical fluid extraction.

B. BLANCHING

E. FERMENTATION

Because blanching requires large volumes of water during processing, and often requires dicing vegetables, studies to increase the efficiency of blanching using ohmic heating are important. Wigerstrom (35) found that electric fields enhanced moisture loss during the blanching of potato slices. Mizrahi (36) determined that ohmic heating was an effective method for blanching because the rapid, uniform heating exhibited by ohmic heating eliminated the need for dicing vegetables. The quick process time and reduction in surface area (no dicing) reduced solute losses by an order of magnitude during blanching. Sensoy, Sastry, and Beelman (37) found that using ohmic heating during the blanching of mushrooms resulted in the shrinking of mushrooms at a lower temperature and with less water use as compared to conventional blanching. Lakkakula, Lima, and Walker (34) showed significant lipase deactivation in rice bran during ohmic heating, with and without a corresponding temperature increase. Taken collectively, these studies show that ohmic heating can increase process efficiency in blanching.

Cho, Sastry, and Yousef (41) found that mild electrical treatment significantly decreased the lag time of Lactobacillus acidophilus, possibly due to electroporation, which could enhance the transport of substrates across the cell membrane. These investigators also found that electricity applied later in the microbial growth cycle proved detrimental, possibly due to the enhanced transport of inhibitory substances across the cell membrane.

C. EVAPORATION Wang and Chu (38) studied the effect of ohmic heating on the vacuum evaporation of orange juice, and found that the evaporation rate could be increased as much as three times using ohmic heating, and resulted in enhanced product quality. The authors conclude that ohmic heating has potential as a fast evaporation method and recommend further development in this area.

D. DEHYDRATION Ohmic heating has also been used to enhance the drying rate of vegetable tissue. Wang and Sastry (39) showed that ohmically treating sweet potato prior to dehydration accelerated the hot-air drying rate significantly compared to raw, conventionally treated, and microwaved samples. Lima and Sastry (33) found that the lower the frequency of alternating current used in ohmic heating, the faster the hot-air drying rate. Maximum drying benefits were seen

F.

EXTRACTION

Ohmic heating has been used to enhance the extraction of components from foods. Katrokha, Matvienko, Vorona, Kupchik, and Zaets (42) used an electric field to extract sugar from sugar beets. Kim and Pyun (43) extracted soymilk from soybeans. Lima and Sastry (33) and Wang and Sastry (44) found that ohmically heating apple tissue prior to mechanical juice extraction significantly increased apple juice yields with respect to non-treated apple tissue, and that the lower the frequency of alternating current, the greater the extraction yield. Several studies have examined the diffusion of beet dye from beetroot. In addition to the pioneering work mentioned above, Lima, Heskitt, and Sastry (45) found that the diffusion enhancement beet dye due to ohmic heating was especially pronounced at lower temperatures (42°C vs. 58°C and 72°C), and could be related to the difference in electrical conductivity of beet tissue between conventional and ohmic cases at the same temperature. Kulshrestha and Sastry (46) showed that significant leaching of beet dye occurs with temperature increases of 1–2°C in ohmic heating. Lakkakula, Lima, and Walker (34) used ohmic heating to significantly increase the extraction of rice bran oil from rice bran (with moisture addition), especially at low (1 Hz) frequency.

G. SUMMARY

OF

NOVEL PROCESSES

There exists a strong potential to enhance mass transfer operations using ohmic heating, particularly because mild ohmic treatment has been shown to significantly increase


dehydration and extraction efficiencies. Future work in these areas includes establishing a more complete body of knowledge regarding the mechanisms for mass transfer effects, and process design to establish industrial processes that take advantage of this technology.

VI. FUTURE RESEARCH DIRECTIONS Though there has been a proliferation of published research on ohmic heating during the past fifteen years, there exist many opportunities for contributing to the body of knowledge regarding ohmic and inductive heating. In terms of inductive heating, so little information exists that substantial contributions can be made in all areas of research, including equipment design and instrumentation, process characterization, properties of importance in inductive heating, and modeling process transport phenomena and kinetics (microbial death and food degradation). In terms of ohmic heating, areas of future work include the following: ●

●

●

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Developing temperature measurement methods that are (preferably) non-invasive and that do not interfere with the electrical field for the internal monitoring of solid particles during ohmic heating. Developing models that correlate process parameters and process design with properties of the product (physical, electrical, chemical, biological, microbial) in order to standardize ohmic heating design and analysis, and to accurately quantify changes in process or product. Determining the influence of temperature and electrical field on the degradation kinetics of key pathogenic microorganisms. Developing the knowledge necessary to quantify the effects of electrical field on mass transfer properties in order to optimize promising applications of ohmic heating, including drying, extraction, blanching, fermentation, evaporation, and gelatinization. Quantifying electrolytic effects during ohmic heating, particularly the minimization of electrolysis at low frequencies, where several novel process options exist.

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9. 10.

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REFERENCES 1. Palaniappan, S., Richter, E. R. and S. K. Sastry. 1990. Effects of electricity on microorganisms: A review. Journal of Food Process Preservation 14: 393–414. 2. Larkin, J. and S. Spinak. 1996. Safety considerations for ohmically heating, aseptically processes, multiphase

19.

20.

low acid food products. Food Technology, May 1996, pp. 242–245. Palaniappan, S. and S. Sastry. 1991. Electrical conductivities of selected solid foods during ohmic heating. Journal of Food Process Engineering 14: 221–236. de Alwis, and P. Fryer. 1992. Operability of the ohmic heating process: Electrical conductivity effects. Journal of Food Engineering 15: 21–48. Reznick, D. 1996. Ohmic heating of fluid foods. Food Technology, May 1996, pp. 250–251. Kim, H., Choi, Y., Yang, T., Taub, I., Tempest, P., Skudder, P., Tucker, G. and D. Parrott. 1996. Validation of ohmic heating for quality enhancement of food products. Food Technology, May 1996, pp. 253–261. Sastry, S. 1996. Ohmic heating. McGraw-Hill Yearbook of Science and Technology, 1996. McGraw-Hill Book Company, pp. 127–130. Zoltai and Swearingen. 1996. Product development considerations for ohmic processing. Food Technology, May 1996, 263–266. Sastry, S. and J. Barach. 2000. Ohmic and inductive heating. Journal of Food Science 65(4): 42–46. Assiry, A., Sastry, S. and C. Samaranayake. 2003. Degradation kinetics of ascorbic acid during ohmic heating with stainless steel electrodes. Journal of Applied Electrochemistry 33: 187–196. Allen, K., Eidman, V. and J. Kinsey. 1996. An economic-engineering study of ohmic food processing. Food Technology, May 1996, pp. 269–273. Lima, M., Heskitt, B. and S. Sastry. 1999b. The effect of frequency and wave form on the electrical conductivitytemperature profiles of turnip tissue. Journal of Food Process Engineering 22: 41–54. Wang, W. and S. Sastry. 1997. Starch Gelatinization in Ohmic Heating. Journal of Food Engineering 34: 225–242. Yang, Cohen, Kluter, Tempest, Manvell, Blackmore, Adams. 1997. Microbiological and sensory evaluation of six ohmically heated stew type foods. Journal of Food Quality 20: 303–313. Yongsawatdigul, J., Park, J. and E. Kolbe. 1995. Electrical conductivity of Pacific whiting surimi paste during ohmic heating. Journal of Food Science 60(5): 922–925, 935. de Alwis, A. and P. Fryer. 1990. A finite element analysis of heat generation and transfer during ohmic heating of food. Chem. Eng. Sci. 45(6): 1547–1559. Sastry, S. and Q. Li. 1993. Models for ohmic heating of solid-liquid mixtures. In Heat Transfer in Food Processing, HTD, Vol. 254, Eds. M. Karwe, T. Bergman, S. Paolucci, pp. 25–33. American Society of Mechanical Engineers, New York. Sastry, S. and Q. Li. 1996. Modeling the ohmic heating of foods. Food Technology, May 1996, pp. 246–248. Fu, W. and C. Hsieh. 1999. Simulation and verification of two-dimensional ohmic heating in static system. Journal of Food Science 64(6): 946–949. Quarini, G. 1995. Thermalhydraulic aspcts of the ohmic heating process. Journal of Food Engineering 24: 561–574.

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21. Davies, L., Kemp, M. and P. Fryer. 1999. The geometry of shadows: effects of inhomogeneities in electrical field processing. Journal of Food Engineering 40: 245–258. 22. Ruan, R., Chen, P., Chang, K., Kim, H. and I. Taub. 1999. Rapid food particle temperature mapping during ohmic heating using FLASH MRI. Journal of Food Science 64(6): 1024–1026. 23. FDA, 2001. Kinetics of Microbial Inactivation for Alternative Food Processing Technologies: Ohmic and Inductive Heating. Available from http://www.cfsan.fda.gov/⬃comm/ift-ohm.html 24. Palaniappan, S., Sastry, S. and E. Richter. 1992. Effects of electroconductive heat treatment and electrical pretreatment on thermal death kinetics of selected microorganisms. Biotechnol. Bioeng. 39(2): 225–232. 25. Cho, H.-Y., Sastry, S. K. and A. E. Yousef. 1999. Kinetics of inactivation of Bacillus subtilis spores by continuous or intermittent ohmic and conventional heating. Biotechnol. Bioeng. 62(3): 368–372. 26. Guillou, S. and N. Murr. 2002. Inactivation of Saccharomyces cerevisiae in solution by low-amperage electric treatment. Journal of Applied Microbiology 92: 860–865. 27. Lima, M., Heskitt, B., Burianek, L., Nokes, S., and S. Sastry. 1999a. Ascorbic acid degradation kinetics during conventional and ohmic heating. Journal of Food Processing and Preservation 23(5): 421–434. 28. Halden, K., de Alwis, A. and Fryer, P. 1990. Changes in the electrical conductivity of foods during ohmic heating. International Journal of Food Sci. Tech. 25: 9–25. 29. Schreier, P., Reid, D. and P. Fryer. 1993. Enhanced diffusion during the electrical heating of foods. International Journal of Food Sci. Tech. 28: 249–260. 30. Imai, T., Uemura, K., Ishida, N., Yoshizaki, S. and Noguchi, A. 1995. Ohmic heating of Japanese white radish Rhaphanus sativus L. Int. J. Food Sci. Tech. 30, 461–472. 31. Kulshrestha, S. and S. Sastry. 2003. Frequency and voltage effects on enhanced diffusion during moderate electric field (MEF) treatment. Innovative Food Science & Emerging Technologies 4: 189–194. 32. Weaver, J. 1987. Transient aqueous pores: a mechanism for coupling electric fields to bilayer and cell membranes. In Blank, M. and E. Findl, Mechanistic Approaches to Interactions of Electric and Electromagnetic Fields with Living Systems (pp. 249–270). New York: Plenum Press.

33. Lima, M. and S. Sastry. 1999. The effects of ohmic heating frequency on hot-air drying rate and juice yield. Journal of Food Engineering 41: 115–119. 34. Lakkakula, N., Lima, M. and T. Walker. 2004. Rice bran stabilization and rice bran oil extraction using ohmic heating. Bioresource Technology 92: 157–161. 35. Wigerstrom, K. 1976. Passing an electric current of 50–60 Hz through potato pieces during blanching. U.S. Patent No. 3,997,678. 36. Mizrahi, S. 1996. Leaching of soluble solids during blanching of vegetables by ohmic heating. Journal of Food Engineering 29: 153–166. 37. Sensoy, I., Sastry, S., and R. Beelman. 1999. Ohmic blanching of mushrooms. Abstract No. 79 B-1, 1999 IFT Annual Meeting, Chicago, IL, July 24–28, 1999. 38. Wang, W. and C. Chu. 2003. Study of vacuum evaporation by using ohmic heating. Abstract No. 92 B-59, 1999 IFT Annual Meeting, Chicago, IL, July 12–16, 2003. 39. Wang, W. and S. Sastry. 2000. Effects of thermal and electrothermal pretreatments on hot air drying rate of vegetable tissue. Journal of Food Process Engineering 23(4): 299–319. 40. Zhong, T. and M. Lima. 2003. The effect of ohmic heating on vacuum drying rate of sweet potato tissue. Bioresource Technology 87: 215–220. 41. Cho, H.-Y., Sastry, S. K. and A. E. Yousef. 1996. Growth kinetics of Lactobacillus acidophilus under ohmic heating. Biotechnol. Bioeng. 49(3): 334–340. 42. Katrokha, I., Matvienko, A., Vorona, L., Kupchik, M. and V. Zaets. 1984. Intensification of sugar extraction from sweet sugar beet cossettes in an electric field. Sakharnaya Promyshlennost 7: 28–31. 43. Kim, J. and Y. Pyun. 1995. Extraction of soy milk using ohmic heating. Abstract, 9th Congress of Food Sci. and Tech., Budapest, Hungary. 44. Wang, W. and S. Sastry. 2002. Effects of moderate electrothermal treatments on juice yield from cellular tissue. Innovative Food Science & Emerging Technologies 3: 371–377. 45. Lima, M., Heskitt, B., and S. Sastry. 2001. Diffusion of beet dye during electrical and conventional heating at steady-state temperature. Journal of Food Process Engineering 24(5): 331–340. 46. Kulshrestha, S. and S. Sastry. 1999. Low frequency dielectric changes in vegetable tissue from ohmic heating. Abstract No. 79 B-3, 1999 IFT Annual Meeting, Chicago, IL, July 24–28, 1999.

121

Power Ultrasound

Hao Feng

Food Science and Human Nutrition Department, University of Illinois

Wade Yang

School of Agricultural and Environmental Sciences, Alabama A&M University

CONTENTS I. Introduction ........................................................................................................................................................121-1 II. Generation of Ultrasound and Ultrasound Systems ..........................................................................................121-2 A. Ultrasound Generation ................................................................................................................................121-2 1. Magnetostrictive Transducers ..............................................................................................................121-2 2. Piezoelectric Transducers ....................................................................................................................121-2 3. Comparison between Magnetostrictive and Piezoelectric Transducers ..............................................121-2 B. Power Ultrasound Systems ........................................................................................................................121-2 1. Conventional Power Ultrasound Systems ............................................................................................121-3 2. Variable Frequency Systems ................................................................................................................121-3 C. Cavitation ....................................................................................................................................................121-3 III. Selected Power Ultrasound Applications ..........................................................................................................121-4 A. Emulsification ............................................................................................................................................121-4 B. Cutting ........................................................................................................................................................121-5 C. Inactivation of Microorganisms ..................................................................................................................121-5 D. Enzyme Activity Control ............................................................................................................................121-5 E. Modification of Biopolymers ....................................................................................................................121-6 F. Separation of Bio-Polymers and Bio-Components ....................................................................................121-6 G. Case Study –– Power Ultrasound Enhanced Corn Pericarp Separation......................................................121-6 References ..................................................................................................................................................................121-7

I. INTRODUCTION Sound waves are mechanical vibrations that travel through solids in the form of transverse waves, and through liquids and gases in the form of longitudinal waves. Ultrasound refers to sound waves having a frequency higher than the range audible to humans. The lowest ultrasonic frequency is commonly taken as 20 kHz (1 Hertz ⫽ 1 cycle per second). The upper limit of ultrasound frequencies is not clearly defined but is usually taken to be 5 MHz for gases and 500 MHz for liquids and solids (1). Applications of ultrasound can be divided broadly into two categories: lowand high-power ultrasound (Figure 121.1).

The first category involves low amplitude sound waves and is also referred to as “low intensity,” “diagnostic,” or “high frequency” ultrasound. Low power ultrasound uses very high frequencies of 2 MHz to 20 MHz with low sound intensities of 100 mW/cm2 to 1 W/cm2. It measures the velocity and attenuation of the wave in a medium and utilizes such information in medical imaging (e.g., scanning an unborn fetus), chemical analysis, food quality assessment, and non-destructive testing (e.g., regular crack testing for aircraft structures). A low-power ultrasound measurement system is composed of a transducer, a signal generator, a digitizer, and a measurement cell. Possible applications of low-power ultrasound in food 121-1

121-2


0

10

102

103

104

105

106

107

Frequency

Human hearing

20 Hz – 20 KHz

Conventional power ultrasound

20 KHz – 100 KHz

Extended power ultrasound

20 KHz – 2 MHz

Diagnostic ultrasound

5 MHz – 10 MHz

FIGURE 121.1 Sound wave spectrum (from ref. (1)).

processing and quality determination include measurement of temperature and flowrate, determination of composition, determination of particle size, determination of creaming and sedimentation profiles, monitoring of phase transitions, study of gelation, ultrasonic imaging, fouling detection, and the study of molecular properties. The second category in often known as “power ultrasound” or “high intensity ultrasound.” Power ultrasound uses lower frequencies (typically 20 kHz to 100 kHz) and produces sound intensities of 10 to 1,000 W/cm2 with amplitudes ranging from about 5 to 50 microns. It finds applications in food processing operations such as emulsion generation, dispersion of aggregated materials, drying, inactivation of microbes and enzymes, heat and mass transfer enhancement, biological components separation, and modification and control of crystallization process (2). The purpose of this chapter is to provide an insight into the general principles of power ultrasound, as well as to review recent progress in the research and development of power ultrasound and its applications in the food processing industry. Readers who are interested in low-power ultrasound applications can refer to the comprehensive review articles of McClements (3, 4) for detailed information.

edge of each laminate attached to the bottom of a process tank or other surfaces to be vibrated. A coil of wire is placed around the magnetostrictive material. When a flow of electrical current is supplied through the coil of wire, a magnetic field is created. This magnetic field causes the magnetostrictive material to contract or elongate, thereby introducing a sound wave into a sonicating fluid. 2. Piezoelectric Transducers The heart of a piezoelectric transducer is a single or double thick disc of piezoelectric ceramic material, such as barium titanate, lead metaniobate, or lead zirconate titanate (PZT), sandwiched between electrodes that provide the attachment points for electrical contact. The ceramic assembly is compressed between metal blocks (one aluminum and one steel) to a known compression with a high strength bolt. When a voltage is applied across the ceramic through the electrodes, the ceramic expands or contracts, depending on polarity, due to changes in its lattice structure. This physical displacement causes a sound wave to propagate into a treatment solution. 3. Comparison between Magnetostrictive and Piezoelectric Transducers

II.

GENERATION OF ULTRASOUND AND ULTRASOUND SYSTEMS

A.

ULTRASOUND GENERATION

Ultrasound is generated via an ultrasonic transducer –– a device by which mechanical or electrical energy can be converted into sound energy. There are two fundamental transducer designs used for power ultrasonic applications today, magnetostrictive and piezoelectric, both powered by electricity. 1.

Magnetostrictive Transducers

Magnetostrictive transducers consist of a large number of nickel plates or laminations arranged in parallel with one

Piezoelectric transducers utilize the piezoelectric property of a material to convert electrical energy directly into mechanical energy. Magnetostrictive transducers utilize the magnetostrictive property of a material to convert the energy in a magnetic field into mechanical energy. Both types of transducers have advantages and disadvantages. A comparison between two types of transducers is given in Table 121.1.

B.

POWER ULTRASOUND SYSTEMS

A typical ultrasonic system comprises three essential parts (2): a generator that converts electricity into required high frequency alternating current, a transducer that converts the high-frequency alternating current into

Power Ultrasound

121-3

TABLE 121.1 A Comparison between Piezoelectric Transducers and Magnetostrictive Transducers Transducer

Frequency Range (kHz)

Piezoelectric Magnetostrictive

Wide range 18–30

Noise Less noisy Noisy

mechanical vibrations, and a delivery system to convey the vibration into a food system, such as the tank of the ultrasonic bath and the horn of the ultrasonic probe system. 1. Conventional Power Ultrasound Systems (1) In practice, ultrasound can be introduced into a food system to perform various applications in two ways: direct and indirect contacting. With the direct contacting method, a food is in direct contact with an ultrasonic element, which can be a thin metal blade in the case of a liquid whistle apparatus or a sonic horn for probe type designs. Transducer arrays can also be arranged with sonic horns attached to them inserting into a treatment chamber or flow cell of different geometry to facilitate various sonication treatments. The advantage of this arrangement is that ultrasound can be directly transmitted into a food system with less energy loss. It can be used to design a high surface power density (W/cm2) system or a high volumetric acoustic energy (W/cm3) system by controlling the treatment chamber volume. The disadvantages may include difficulty of temperature control, and the possibility of generating free radicals on the contacting surfaces when surface power density is high. Another direct contacting design is a sonic vibrating bar developed by a Canadian company (5). The metallic bar is driven by three magnets and vibrates at the audible frequencies. It can operate at power as high as 75 kW and is effective as a mixer and grinder. In an indirect contacting system, the ultrasound transducer is usually mounted onto a large surface to perform sonication treatments. The widely used ultrasonic baths are a good example of this design. In this category, ultrasound has to be transferred through a wall to reach the food system under treatment and thereby both the ultrasound power intensity (W/cm2) and the volumetric acoustic energy density (W/cm3) are low. It also has the problems of poor temperature control and difficulty in quantifying power delivered into the food system during sonication. Systems developed based on this design are widely used owing to their simplicity and ease of operation. The most successful application of the indirect contacting system is surface cleaning of jewelry and gun parts. In recent years, various food surface decontamination applications have been explored.

Reliability Improving reliability Reliable

Sweeping Not easy Easy

Aging Noticeable NA

Energy Efficiency ⬎70% 35–40%

2. Variable Frequency Systems Due to the ultrasound generation mechanism, traditional ultrasound systems are inherently single frequency units. In recent years, the concept of using more than one transducer each with different frequencies to enhance cavitation has been explored. Researchers have demonstrated several-fold increases in cavitation activities in lab testing in a dual or multi-frequency ultrasound system (6). Ultrasound units using this concept have to use more than one transducer, each with its own generator. This will complicate the design and operation, and will increase the cost of the unit. A new concept, the multi-frequency, multimode, modulated (MMM) technology was recently developed to facilitate variable frequency sonication applications. In a MMM unit (Figure 121.2), the ultrasonic power supply is able to produce variable frequencysweeping oscillations around a central operating frequency, and has an amplitude-modulated output signal (where the frequency of amplitude modulation follows sub-harmonic low frequency vibrating modes of the mechanical system). The MMM technology can utilize the coupled vibrating modes in a mechanical system by applying advanced digital signal processing to create driving wave forms that synchronously excite many vibrating modes (harmonics and sub-harmonics) of an acoustic load. It will help to produce uniform distribution of highintensity acoustical activity to make the entire available vibrating domain acoustically active while eliminating the creation of potentially harmful and problematic stationary and standing wave structures (7).

C.

CAVITATION

Most power ultrasound applications are based on the activity of cavitation, which refers to the formation, growth, and implosion of gas- or vapor-filled cavities in liquids when large acoustic pressure differences are applied. When sound waves travel through a liquid in the form of longitudinal waves comprising a series of compression and rarefaction portions, negative pressures are generated at the rarefaction portions. It is believed that nucleation is initiated at sites where the tensile strength of the liquid is dramatically lowered. One generally accepted nucleation mechanism states that gas entrapped in small-angle crevices, when subjected to negative

121-4


(A) Multifrequency ultrasonic power supply

(B) Ultrasonic converter

(C) Acoustical wave-guide

(D) Acoustical Load, Ultrasonic reactor, Oscillating mechanical system

Amplitude

Power

MMM ultrasonic generator /power supply

(Feedback loop)

(E) Acoustic activity sensor (item B ultrasonic converter, may replace this external sensor)

FIGURE 121.2 A multifrequency, multimode, modulated (MMM) ultrasound system (courtesy of MP Interconsulting).

acoustic pressure, undergoes expansion and forms small free bubbles (8). The behavior of the bubbles in a sonicating liquid determines the cavitation dynamics. Transient cavitation bubbles are generated when sound intensity is greater than 10 W/cm2. These bubbles, with effective residence time of ⬍100 nano-seconds, will experience large expansion in size in a few acoustic cycles and terminate in a violent collapse. The collapse of transient micro-bubbles can create extreme physical conditions, such as temperatures and pressures as high as 5,000 K and 1,000 atmospheres (8). It is believed that the localized high temperatures and pressures are responsible for most of the sonochemical and bactericidal effects. The release of the high pressure results in the formation of shock waves that often provide mechanical cleavage of large biopolymers. Stable cavitation is produced at fairly low sound intensities (1–3 W/cm2). Stable bubbles have a much longer residence time so that the mass transfer of gas into them will result in growth of bubble sizes. As bubble sizes increase, stable bubbles can transform into transient bubbles and undergo collapse. They can also float to the surface and be expelled. Stable bubbles oscillating in resonance with the applied acoustic field can generate intense local strains in the bubbles’ vicinity, which are the cause of many of the disruptive mechanical effects of sound (1). There are several parameters affecting cavitation activity in a sonicating liquid. Increasing frequency decreases the intensity of cavitation because, at high frequencies, the rarefaction cycle is too small to permit a bubble to grow to a size sufficient to cause implosion. Since the prerequisite for micro-bubble formation in a liquid is that the negative pressure must overcome the cohesive forces, it is more difficult to generate cavitation in viscous and high surface tension liquids. Cavitation intensity increases with temperature

when the acoustic power density is a constant. Increased vapor pressure makes it easier to surpass the crushing force, the difference between the hydrostatic pressure and the acoustic pressure, to generate cavitation. The effect of applied external pressure is two-fold. Higher external pressure will result in a higher cavitation threshold, as well as an increase in the intensity of bubble collapse.

III. SELECTED POWER ULTRASOUND APPLICATIONS A.

EMULSIFICATION

Emulsification is one of the earliest applications of power ultrasound. When a bubble collapses in the vicinity of the phase boundary of two immiscible fluids, the resulting shock wave can provide a very efficient mixing of layers (9, 10). Stable emulsions generated with ultrasound have been used in the textile, cosmetic, pharmaceutical, and food industries. The emulsions obtained by mechanical oscillations at ultrasound frequencies are known to have a number of advantages, including stable emulsions even without the addition of surfactant, and narrow mean droplet size distribution compared to other methods (11). Mongenot et al. (11) reported that encapsulations spray-dried from ultrasound-generated emulsions of maltodextrin better retained cheese aroma. In milk homogenization tests with ultrasound, shorter fermentation time of yogurt was found. Ultrasound treatment also altered the physico-chemical properties of the milk (12). The mechanical device used to acoustically generate emulsification can be a sonicator with a piezoelectric or magnetostrictive transducer, or a “liquid whistle” that is

Power Ultrasound

121-5

widely used for homogenization and emulsification applications in the manufacture of fruit juices, tomato ketchup, and mayonnaise (9).

B.

CUTTING

Ultrasonic knives made of titanium have been used in slicing or slitting of different food products. An ultrasonic cutting machine consists of a specially designed horn, used as a knife, driven by an ultrasound transducer, usually through a booster, and a precision positioning mechanism. The reciprocating vibration of the blades at ultrasonic frequency greatly reduces friction between the knife and the product, which ensures straight and clean cuts and results in products with uniform size, shape, and density. Ultrasonic knives can be used to cut sticky and brittle products. Nuts, raisins, and other hard fruits are cut cleanly without plowing or displacement, and peanuts are cut with minimum waste. Production costs and downtime associated with conventional cutting methods are minimized. With greatly reduced friction, there is minimal knife abrasion and blades stay sharper longer, which reduces annual maintenance costs. An ultrasonic cutting unit can be easily incorporated into an existing production line. It can also be a complete custom-built combined ultrasonic slitting and guillotine cutting station.

C.

INACTIVATION

OF

Several theories have been proposed to describe the inactivation mechanism of ultrasound. When ultrasonic waves pass through a liquid, bubbles or cavities can be formed if the amplitude of the waves is high enough. This phenomenon is known as cavitation. Cavitation can affect a biological system by virtue of a highly localized temperature rise and mechanical stress (22), which cause doublestrand DNA breaks, enzyme inactivation, and damage to liposomes. Application of ultrasound to a liquid also leads to the formation of OH⫺ and H⫹ species and hydrogen peroxide (23). These species also have important bactericidal properties. When ultrasound is combined with heat and pressure, the synergistic effect was attributed to the disruption of the bacterial spore cortex, which resulted in protoplast rehydration and loss of heat resistance (24). In the case of sonication assisted by elevated pressures, the increase in inactivation rate was probably due to an increase in bubble implosion intensity, as postulated by Pagán et al. (19). For certain microorganisms, such as L. monocytogenes, S. enteridis, and A. hydrophila, only additive effects were observed under the conditions tested by Pagán et al. (19). Ultrasound has also been tested for its efficacy on surface decontamination of poultry (25) and fresh produce (26), as well as on removal of biofilm (27). The use of ultrasound in microbial inactivation for a food system is still in the stage of laboratory testing. No commercial applications of food microbial reduction have been documented.

MICROORGANISMS

The bactericidal effect of ultrasonic waves has long been observed (13). At that time, the effect was attributed to the compression that ultrasound would generate in a liquid. However, the relatively low inactivation rate of sound waves compared with other methods prevented ultrasound from being used as a food preservation method. In the early years, the low inactivation capacity of ultrasound was related to the low power density used. A few studies have examined the use of ultrasound in conjunction with other preservation methods for the destruction of microorganisms. Neppiras and Hughes (14) reported that static pressure helped to increase the inactivation capacity of sound waves. Burgos et al. (15) showed that the heat resistance of bacteria spores decreased under sonication. Ultrasound has been combined with ozone (16) and H2O2 (17) to inactivate bacteria and spores and a synergistic effect was observed. When combining heat with power ultrasound (20 kHz) (thermosonication), the microbial inactivation rate was greater than the addition of the inactivating effect of heat to that of ultrasound when acting independently (18). Pagán et al. (19,20) and Mañas et al. (21) documented in their studies inactivation of L. monocytogenes with ultrasonic waves under pressure at sublethal (manosonication) and lethal (manothermosonication) temperatures and reported a significant increase in inactivation due to pressurization.

D.

ENZYME ACTIVITY CONTROL

Ultrasonic energy has been used either to increase or inhibit enzyme activity, depending upon the ultrasound intensity. At low ultrasound intensities, Ishimori et al. (28) achieved a two-fold increase in α-chymotrypsin activity in a casein substrate. Enzyme activity inhibition at high ultrasound intensity levels has long been observed. Inactivating enzymes with ultrasound, however, usually requires long treatment times and the presence of oxygen (29). To increase the effectiveness of ultrasound treatment, different combined processes have been used to inactivate food enzymes. The most recent development includes treatments using a combination of heat, low pressure and ultrasound to increase the inactivation rate (manothermosonication). Lopez et al. (30) studied tomato pectinmethylesterase (PME) and polygalacturonase (PG) inactivation kinetics using manothermosonication (MTS) in a buffer and reported D values of 0.85 min for PME, and 1.46 and 0.24 min for PG1 and PG2, respectively. Vercet et al. (31) applied MTS to inactivate enzymes in a tomato paste at 200 MPa and 70°C for 1 min. They found that PME residual activity in treated samples was undetectable and PG residual activity was 38%. As observed with microorganisms, enzyme inactivation by a combination of heat and ultrasound under pressure exhibited a synergistic effect. The enzyme inactivation efficacy of

121-6


heat is increased by a factor that is dependent on the nature of the enzyme and the working conditions (29). In laboratory tests, MTS has been proven to be an effective means to inhibit enzyme activity. An advantage of using MTS is that both microbial inactivation and enzyme activity inhibition can be achieved in one processing step.

TABLE 121.2 Starch Yields from Corn Flour and Hominy Feed Treated with Different Methods Fractions (%) Product Corn flour

E. MODIFICATION

OF

BIOPOLYMERS

Jackson et al. (32) used ultrasound to dissolve corn and sorghum starch granules after heating. They reported that ultrasonic vibrations disrupted swollen granules, thereby releasing amylose and amylopectin from the granules, which resulted in an increase in water solubility of the starch. Chung et al. (33) used power ultrasound to treat mung bean, potato, and rice starches with heating followed by ultrasound and reported that the average degree of polymerization did not change after sonication. They postulated that the changes in starch properties were induced by the disruption of swollen granules rather than the breakage of glucosidic linkages. In a study to examine the effect of ultrasound treatment on starch at different pH (2.0, 4.5, 7.0, 9.5, and 12.0), Zhang et al. (34) found that starches treated with power ultrasound had lower peak viscosity (PV) and higher pasting stability than did native starch. Ultrasound treated starches exhibited a decrease in the gelatinization enthalpy (⌬H) and an increase in the gelatinization onset (To). Ultrasound also significantly increased the in vitro digestibility of starches in the earlier stage.

F.

SEPARATION OF BIO-POLYMERS AND BIO-COMPONENTS

Studies utilizing the mechanical action of sonication have been performed in recent years to realize various bioseparation operations. Mason (35) reported a study using ultrasound to treat rice grains in which surface erosion and particle size reduction resulted in shorter cooking and gel times. In a study on rice starch isolation, Wang et al. (36) found that high-intensity ultrasound treatment resulted in a high starch recovery but a slightly higher residual protein content. Zhang et al. (37) tested power ultrasound as a means to recover starch from degermed corn flour and hominy feed, which are reasonably high in starch and may be a source of non-sulfate treated starch. They used five treatments to produce starch from degermed corn flour and hominy feed slurries (10% solid): control, ultrasound only, ultrasound followed by fine grinding, fine grinding followed by ultrasound, and fine grinding only. The total starch recovery data are listed in Table 121.2. The starch yield from the corn flour was 37.1% for the control, and those by ultrasound treatments were 65.5 to 67.0%, a 28.4 to 29.9% increase compared to the control. Similarly, starch yields from the hominy feed by ultrasound treatments were 45.4 to 45.8%, a 16.1 to

Hominy feed

Treatment

Starch

Gluten

Ultrasound Ultrasound-Grind* Grind-Ultrasound Grind Control Ultrasound Ultrasound-Grind Grind-Ultrasound Grind Control

66.8 65.5 67.0 34.7 37.1 45.6 45.4 45.8 34.6 29.3

15.7 16.3 15.9 8.4 5.9 22.5 26.7 27.5 21.2 12.3

Starch Recovery Fiber (%) 16.7 17.5 15.8 55.2 56.0 29.0 24.5 23.1 39.9 54.6

99.1 97.2 99.4 51.5 55.0 98.3 97.8 98.7 74.6 63.1

*Grind ⫽ fine grounding.

16.5% increase compared to the control (Table 121.2). Comparing with the total starch contents in the two products, ultrasound treatments recovered 97.3 to 99.5% starch from the degermed corn flour and 97.8 to 98.9% from the hominy feed. Obviously, ultrasound treatment is a very effective method to recover starch from low value degermed corn flour and hominy feed. Power ultrasound was also used to increase starch yield in a novel corn processing method, the quickgerm/quick-fiber process (38). In the experiments, yellow dent corn soaked in deionized water at 52°C for 24 hrs without addition of SO2 was wet-milled using a 100-g laboratory procedure with some modifications. Ultrasound treatments were performed at different process steps: first grind followed by ultrasound, ultrasound followed by second grind, second grind followed by ultrasound, fine fiber slurry treatment with ultrasound, and milling only (no ultrasound). A conventional wet milling treatment was used for comparison. Starch yield resulting from no ultrasound treatment was 61.7%, ultrasound treatments were 66.9–68.7%, and conventional wet milling was 68.9%. The characteristics of the starches produced with ultrasound treatments are similar to that from a conventional wet milling method as shown by color measurements and RVA curves.

G.

CASE STUDY –– POWER ULTRASOUND ENHANCED CORN PERICARP SEPARATION (39, 40)

Corn pericarp is a main source of dietary fiber. Refined corn pericarp has at least 92% dietary fiber, which places it among the most concentrated sources of edible fiber. It can be used as a supplement in dietary beverages, extruded breakfast cereals and snack foods, and breads and other bakery products. Currently, there are no rapid and effective pericarp separation methods available. It is imperative for

121-7

Average affinity (Pa)

Power Ultrasound

15000 14500 14000 13500 13000 12500 12000 11500 11000 10500 10000

13700±700 12700±700 11800±500

40&80 KHz

40 KHz Frequency

80 KHz

FIGURE 121.3 Pericarp affinity at three sonication frequencies. The samples were sonicated for 1 min at room temperature using the Zenith sonicator with a rating power of 925 W. 30000 Average affinity (Pa)

28000 26000

25400±2100

24000 22000

21100±1600

20000

20000±1500

18000 16000

15100±1200

14000 12000 10000 Level 2 (240 W, 0.17 W/cm2)

Level 4 (480 W, Level 6 (720 W, 0.33 W/cm2) 0.50 W/cm2) Power Level

Level 8 (960 W, 0.66 W/cm2)

FIGURE 121.4 Pericarp affinity at four ultrasonic power levels. The samples were sonicated for 1 min at room temperature using a VWR sonicator at 40 kHz. The wattage for power levels of 2, 4, 6, and 8 was 240, 480, 720, and 960 W, respectively, and the corresponding power intensities were 0.17, 0.33, 0.50 and 0.66 W/cm2, respectively.

such methods to be developed to facilitate the lab use for transgenetic research of corn as well as for industrial applications of the corn pericarp and other components. Yellow dent corn at about 13% moisture content was treated with two ultrasonic baths at different frequencies, treatment times, temperatures, and power levels. Corn sample (50 g) was sealed in a plastic bag containing 200 ml of water and placed in the ultrasonic baths for sonication tests. After sonication, the pericarp can be easily separated by mechanical friction or abrasion. To quantify the separation effect of ultrasound, the affinity of the pericarp (Pa) was measured with an Instron Testing Machine (Instron Corporation, Canton, MA). Figure 121.3 shows the average pericarp affinity for each of the three frequency conditions tested in the experiments: 40 kHz, 80 kHz, and a combination of 40 and 80 kHz. From Figure 121.3 it can be seen that pericarp sonicated at 40 kHz had a lower affinity (i.e., was easier to separate) than that sonicated at 80 kHz. Compared to the pericarp sonicated at 40 kHz or 80 kHz alone, the pericarp sonicated with a combination of 40 & 80 kHz resulted in

the lowest pericarp affinity. Figure 121.4 shows the average pericarp affinity at four power levels. One can see that the pericarp affinity showed a decreasing trend with the power level. The power level had a significant effect (α ⫽ 0.05) on the pericarp affinity. However, changes in sonication duration and sonication temperature did not show a marked effect on pericarp affinity.

REFERENCES 1. TJ Mason, JP Lorimer. Applied Sonochemistry: The Uses of Power Ultrasound in Chemistry and Processing. Weinheim: Wiley-VCH, Verlag GmbH, 2002. 2. M Povey, TJ Mason. Ultrasound in Food Processing. New York: Blackie Academic and Professional, 1998. 3. DJ Mcclements. Principles and instrumentation of ultrasonic analysis. Seminars in Food Analysis 4(2):73–93, 1999. 4. DJ McClements. Ultrasonic characterization of foods and drinks: principles, methods, and applications.

121-8

5.

6.

7. 8. 9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.


CRC-Critical-Reviews-in-Food-Science-and-Nutrition 37:1–46, 1997. JP Russell, M Smith. Sonic energy in processing: use of a large-scale, low-frequency sonic reactor. Advances in Sonochemistry 5:279–302, 1999. R Feng, Y Zhao, C Zhu, TJ Mason. Enhancement of ultrasonic cavitation yield by multi-frequency sonication. Ultrasonics Sonochem 9:231–236, 2002. Anno. MMM Basics. Active Ultrasonics, 2003. KS Suslick. Organometallic sonochemistry. Advances in Organometallic Chemistry 25:73–119, 1985. TJ Mason, L Paniwnyk, JP Lorimer. The uses of ultrasound in food technology. Ultrasonics Sonochem 3:S253–S260, 1996. Behrend, K Ax, H Schubert. Influence of continuous phase viscosity on emulsification by ultrasound. Ultrasonics Sonochem 7:77–85, 2000. N Mongenot, S Charrier, P Chalier. Effect of ultrasound emulsification on cheese aroma encapsulation by carbohydrates. J Agric Food Chem 48:861–867, 2000. H Wu, GJ Hulbert, JR Mount. Effects of ultrasound on milk homogenization and fermentation with yogurt starter. Innovative Food Sci & Emerging Technol 1:211–218, 2001. E Harvey, A Loomis. The destruction of luminous bacteria by high frequency sound waves. J Bacteriol 17:373–379, 1929. EA Neppiras, DE Hughes. Some experiments on the disintegration of yeast by high intensity ultrasound. Biotechnol Bioeng 4:247–270, 1964. J Burgos, JA Ordoñez, F Sala. Effect of ultrasonic waves on the heat resistance of Bacillus cerus and Bacillus licheniforms spores. Appli Microbiol 24:497–498, 1972. GR Burleson, TM Murray, M Pollard. Inactivation of viruses and bacteria by ozone, with and without sonication. Appli Microbiol 29:340–344, 1975. FIK Ahmed, C Russell. Synergism between ultrasonic waves and hydrogen peroxide in the killing of microorganisms. J Appl Bacteriol 39:31–40. 1975. JA Ordóñez, MA Aguilera, ML García, B Sanz. Effect of combined ultrasounic and heat treatment (thermoultrasonication) on the survival of a strain of Staphylococcus aureus. J Dairy Sci 54:61–67, 1987. R Pagán, P Mañas, A Palop, FJ Sala. Resistance of heatshocked cells of Listeria monocytogenes to manosonication and mano-thermo-sonication. Letters Appl Microbiol 28:71–75, 1999a. R Pagán, P Mañas, I Alvarez, S Condón. Resistance of Listeria monocytogenes to ultrasonic waves under pressure at sublethal (manosonication) and lethal (manothermosonication) temperatures. Food Microbiol 16:139–48, 1999b. P Mañas, R Pagán, J Raso, FJ Sala, S Condon. Inactivation of Salmonella enteritidis, Salmonella

22.

23.

24.

25. 26.

27.

28. 29.

30.

31.

32.

33.

34.

35.

36.

typhimurium, and Salmonella senftenberg by ultrasonic waves under pressure. J Food Prot 63:451–456, 2000. P Riesz, TK Kondo. Free radical formation induced by ultrasound and its biological implications. Free Rad Biol and Med 13:247–70, 1992. KS Suslick, Homogenous Sonochemistry. In: KS Suslick, ed., Ultrasound. Its Chemical, Physical and Biological Effects. New York: VCH, 1988, pp. 123–164. J Raso, A Palop, R Pagan, S Condon. Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment. Appl Environ Microbiol 85:849–854, 1998. HS Lillard. Decontamination of poultry skin by sonication. Food Technol, Dec. 72–73, 1994. IJ Seymour, D Burfoot, RL Smith, LA Cox, A Lockwood. Ultrasound decontamination of minimally processed fruits and vegetables. Int J Food Sci Technol 37:547–57, 2002. AM Rediske, BL Roeder, MK Brown, JL Nelson, RL Robinson, DA Draper, GB Schaalje, RA Robinson, WG Pitt. Ultrasonic enhancement of antibiotic action on Escherichia coli biofilms: An in vivo model. Antimicro Agnts Chemo 43:1211–1214, 1999. Y Ishimori, I Karube, S Suzuki. J Molec Catal 12:253, 1981. FJ Sala, J Burgos, S Condón, P Lopez, J Raso. Effect of heat and ultrasound on microorganisms and enzymes. In: New Methods of Food Preservation. GW Gould, ed. Gaithersburg, MD: Aspen Publishers, Inc., 1999, pp. 176–204. P Lopez, A Vercet, AC Sanchez, J Burgos. Inactivation of tomato pectin enzymes by manothermosonication. Z Lebensm Unters Forsch A 207:249–252, 1998. A Vercet, C Sanchez, J Burtino, L Montanes, PL Buesa. The effects of manothermosonication on tomato pectin enzymes and tomato paste rheological properties. J Food Eng 53:273–278, 2002. DS Jackson, C Choto-Owen, RD Waniska, LW Rooney. Characterization of starch cooked in alkali by aqueous high-performance size-exclusion chromatography. Cereal Chem 65:493–496, 1988. KM Chung, TW Moon, H Kim, JK Chun. Physicochemical properties of sonicated mung bean, potato, and rice starches. Cereal Chem 79:631–633, 2002. Z Zhang, H Feng, SR Eckhoff. Physical properties and enzymatic digestibility of power ultrasound treated cornstarch as affected by pH, Institute of Food Technologist, 2003 Annual Meeting, paper No. 60A-21, Chicago, IL, 2003. TJ Mason. Power ultrasound in food processing — the way forward. In: MJW Povey and TJ Mason, eds. Ultrasound in Food Processing. New York: Blackie Academic Professional, 1998, pp. 105–127. L Wang, YJ Wang. Neutral protease and high-intensity ultrasound treatment in improving rice starch isolation.

Power Ultrasound

Abstract of 2002 IFT Annual Meeting, Chicago, IL, July 12–16, 2003. 37. Z Zhang, H Feng, Y Niu, SR Eckhoff. Starch recovery from degermed corn flour and hominy feed using power ultrasound, Cereal Chem, 2005a, in print. 38. Z Zhang, H Feng, Y Niu, SR Eckhoff. Sonication enhanced cornstarch separation, Starch, 2005b, accepted.

121-9

39. W Yang, Z Liu, TJ Siebenmorgen. Effect of power ultrasound on the affinity between corn bran and endosperm. Abstract of the 2002 AACC Annual Meeting, Montreal, Canada, Oct. 13–17, 2002. 40. W Yang, Z Liu, TJ Siebenmorgen. Ultrasound processing of foods: A case study of corn component separation. ASAE paper No. 026022. St. Joseph, MI: ASAE, 2002.

122

Ultraviolet Light

Gilbert Shama

Department of Chemical Engineering, Loughborough University

CONTENTS I. II. III. IV. V.

The Ultraviolet Portion of the Electromagnetic Spectrum ..............................................................................122-1 The Effect of UV on Microorganisms..............................................................................................................122-1 UV Dose Lethality............................................................................................................................................122-3 Inactivation of Microorganisms on Foods........................................................................................................122-3 Combined Treatments Incorporating UV ........................................................................................................122-7 A. UV and Hydrogen Peroxide......................................................................................................................122-7 B. UV and Ozone ..........................................................................................................................................122-8 C. Other ........................................................................................................................................................122-8 VI. UV Hormesis ....................................................................................................................................................122-8 VII. Deleterious Effects of UV on Foods ................................................................................................................122-9 VIII. UV Technology ................................................................................................................................................122-9 A. UV Sources ..............................................................................................................................................122-9 B. UV Irradiation Equipment ......................................................................................................................122-12 References ..................................................................................................................................................................122-12

I. THE ULTRAVIOLET PORTION OF THE ELECTROMAGNETIC SPECTRUM

II. THE EFFECT OF UV ON MICROORGANISMS

Ultraviolet light forms part of the electromagnetic spectrum. The ultraviolet wavelength range is from about 10 to 400 nm, placing it between X rays and the visible part of the spectrum (Figure 122.1). Ultraviolet is frequently referred to as ‘non-ionising’ radiation, however the shortest ultraviolet wavelengths do bring about some ionisation. The ultraviolet portion of the spectrum has been sub-divided on a more or less arbitrary basis primarily for convenience. The term ‘vacuum ultraviolet’ is reserved for wavelengths below 200 nm, because in this region ultraviolet is strongly attenuated by air. It is usual to refer to the region between 200 and 300 nm as ‘far ultraviolet’ and that between 300 and 400 nm as ‘near ultraviolet.’ Alternative sub-divisions are often quoted in the scientific literature: thus UV-C is used for wavelengths in the range 100 to 280 nm, UV-B for 280 to 315 nm and UV-A for 315 to 400 nm. In what follows here, the abbreviation ‘UV’ will be used to denote UV-C.

The fate of a microbial cell, or a population of such cells, following exposure to UV will depend on a number of factors. The range of wavelengths used to irradiate the cells will be one such factor. All UV sources used for commercial and industrial disinfection are polychromatic but the spectral range emitted will depend on the type of source used. The most directly lethal wavelengths will be those that are maximally absorbed by the bases of DNA. The precise wavelength for maximum lethal effect varies from species to species because the DNA composition of species differs and because each DNA base has its individual peak absorptivity. Giese and Darby (2) compared the lethality of two UV wavelengths (280 and 301 nm) using a variety of bacteria and a bacteriophage (Table 122.1). There were greater differences in UV susceptibility between species at the highest wavelength (301 nm) but as the lethal effect of wavelengths in this part of the UV spectrum is low, the 122-1

122-2


log10λ(m)

−13 −12 −11 −10 −9 −8 −7 −6 −5 −4 −3 −2 −1

γ-rays

Ultra violet

X-rays

Infra red

Micro waves

1

0

2

3

Radio waves

Visible 100

200

Vacuum UV

400

300 Far UV

Near UV

UVC U V B

UVA

FIGURE 122.1 The electromagnetic spectrum.

TABLE 122.1 Germicidal Efficiencies at 280 and 301 nm

Citrobacter diversus Citrobacter freundii Klebsiella pneumoniae Bacteriophage ␾X-174

Mean Germicidal Efficiency* at 280 nm

Mean Germicidal Efficiency* at 301 nm

0.894 0.834 0.787 0.899

0.025 0.017 0.043 0.054

* Relative to 254 nm.

Germicidal effectiveness

Micro-organism

1 0.75 0.5 0.25 0 230

250

270 290 Wavelength (nm)

310

Data from (2).

FIGURE 122.2 Relative effectiveness (with reference to 254 nm) of wavelengths of the UV spectrum. Data from ref. (3).

differences are of no real significance and for all practical purposes it appears justifiable to use data that is speciesindependent (see Figure 122.2). This figure shows that wavelengths in the vicinity of 265 nm are the most effective at inactivating microbial cells. As the UV penetrates the microbial cell it will be attenuated by the various cellular structures and components. Attenuation effects are wavelength-specific. For example, the cell membrane may absorb 25% of the UV light at 280 nm but only 10% of the UV light at 254 nm, thereby decreasing the germicidal efficiency at 280 nm (2). Some organisms have evolved strategies for surviving irradiation by synthesising various UV-screening compounds (4). However, the majority of these compounds provide protection against UV-A and UV-B. A recent comparison of the sensitivity of a pigmented and a nonpigmented mutant of Rhodobacter sphaeroides to short wave UV showed no significant difference between the two cultures (5). Although UV can bring about changes to a number of cellular components, the most significant reactions in determining cell survival are those that occur between UV and the nucleic acids. If the retroviruses, which contain only RNA, are omitted from further discussion, it becomes possible to concentrate solely on DNA. The

interaction between UV and DNA will result in the formation of so-called ‘photoproducts.’ The most important of these are pyrimidine (i.e., thymine and cytosine) dimers. These are formed between two pyrimidine bases adjacent to one another on the same strand of DNA. Thymine dimers tend to predominate because thymine has a greater absorbance than cytosine in the germicidal wavelength range (6). Thymine (T) occurs in DNA in equal amounts with adenine (A) and it might be thought that species that contained a high proportion of A ⫹ T in their DNA might be particularly sensitive to UV, but this is not borne out by existing experimental evidence (5). Another type of photoproduct, ‘pyrimidine adducts’ are also formed between adjacent pyrimidine bases but at reduced rates of formation compared to dimers. At sufficiently high UV doses DNA-protein cross-links are formed, whilst at higher doses still, DNA strand breakages may be induced. A unique photoproduct –– the so-called ‘spore photoproduct,’ another dipyrimidine –– has been found in bacterial spores. Nearly all living cells possess the ability to reverse the damage caused to their DNA by UV by using one or more repair mechanisms of which there are three principal types. In photoenzymic repair, dipyrimidine dimers are

Ultraviolet Light

enzymically monomerized in the presence of light. The second type of repair process, excision-resynthesis repair, involves removing sections of damaged DNA and resynthesising them using the intact strand as template. Whilst in postreplication repair, undamaged sections of DNA are replicated and combined in such a manner that an intact double stranded molecule, identical with the original, is formed. The physiological state of cells is another factor that determines survival. It has been shown that microorganisms harvested at different stages of growth show differences in susceptibility to UV (7). Moreover, the physical state of cells can also have an effect on cell survival: when cell aggregates are irradiated, the cells on the outside can effectively shield those towards the centre of aggregates. This phenomenon manifests itself as a ‘tail’ in the so-called ‘dose–response curve’ for that species of organism. The dose response curve is simply a plot of the reduction in cell viability against dose. It has generally been accepted that the effect of UV exposure on living cells is solely determined by the dose absorbed. The UV dose is defined as the product of the exposure time and UV intensity (or ‘fluence rate’). In other words, a short, high intensity exposure is equivalent to a protracted low intensity one. This is the Bunsen-Roscoe reciprocity law. However, it has been known for some time that experimental data exists which casts doubt on the veracity of this law. Sommer et al. (8) compared survival data for three different strains of E. coli irradiated at constant doses achieved by varying the UV intensity and time of exposure. In all cases the most lethal effects were achieved at the highest intensities. These findings can be interpreted in mechanistic terms: irradiation of microbial cells will lead to the formation of DNA lesions as explained above, however this process is counteracted by the cells’ repair capabilities. As the UV intensity is increased the rate of lesion formation will eventually exceed the capacity of the repair systems. A similar argument has been used to explain the presence of ‘shoulders’ on the UV dose– response curves of certain microbial species. The most commonly used method of assessing cell viability following UV irradiation is to plate the cells out onto an agar medium. However, recent work suggests that there may be risks in using this type of assay to definitively define viability. Physical and chemical stresses applied to microbial cells can induce them to enter, what has been termed a ‘viable but non-culturable’ (VBNC) state. Of particular relevance is work that shows that cells of E. coli ‘killed’ by UV were capable of performing a number of metabolic functions 48 hours after being irradiated. These functions included expression of esterase activity, uptake of glucose and cell elongation (9). In the context of food processing knowledge of whether VBNC cells retain their pathogenicity could be of crucial importance. The evidence accumulated to date is that cells of

122-3

Salmonella typhimurium do not retain their pathogenicity (10) whereas E. coli cells do (11).

III. UV DOSE LETHALITY Compilations of data have been published which show the UV doses necessary to bring about specific reductions in the populations of a variety of microbial species (3). These data are often given in the form of the doses necessary to reduce the population size by one tenth (the ‘decimal reduction dose,’ D10) or by 1/e, i.e., 37% (D37). The very existence of this data seems to have perpetuated the notion that a single value of D10 can be ascribed to a particular species in much the same way as can the density of a solution of sugar of known concentration. Closer comparisons of data published for individual species reveal some quite large discrepancies. To quote just one example for the radioresistant bacterium Deinococcus radiodurans, estimates for the D37 dose in kJ/m2 include 0.40 ⫾ 0.13 (12), 0.34 ⫾ 0.05 (5) and 0.55 to 0.60 (13). Moreover, it is generally assumed that taxonomically closely related species have similar UV susceptibilities, whereas some quite large variations have been shown to exist (14). Decimal reduction doses, in common with all other measures of the effects UV on microbial populations, are subject to influence by all of the factors discussed above, i.e., the physiological and physical state of the cells as well as irradiation conditions. Van Gerwen et al. (15) adopted an interesting approach to the treatment of D10 doses for ionising radiation which might profitably be applied to existing UV data. These workers analysed over 500 estimates of D10 doses from the literature. After eliminating data clusters for unusually tolerant species such as D. radiodurans as well as data for particularly sensitive and highly resistant spores, they were able to specify an average D10 value for bacterial spores as well as one for vegetative cells. Until such an analysis becomes available of the UV data, the existing D10 compilations should be viewed upon as simply providing general guidance.

IV. INACTIVATION OF MICROORGANISMS ON FOODS Downes is credited in 1886 with discovering that the ultraviolet portion of the solar emission is lethal towards microorganisms (16). The first artificial UV source was patented in 1903 and soon after this UV sources were being used to disinfect water. The first recorded use of an artificial UV source for food disinfection was in 1906, for milk (17). Subsequent applications of UV in the early stages of its development as a method of disinfection are covered in the reviews of Moldovan (18) and Proctor and Goldblith (19).

122-4


Decimal reduction doses (D10) are typically obtained by irradiating dilute suspensions microorganisms in water or buffer. The difficulties in interpreting data obtained under these relatively well-defined conditions, as discussed above, are greatly compounded when such data is required for the surfaces of foods. There are a number of considerations that will influence the nature of the data obtained. Firstly, researchers have a choice of either working with the microflora naturally present on a particular food, or of artificially culturing organisms of interest and then applying them to the food in some way. Whilst the former approach might hold obvious attractions, it presents certain experimental difficulties. Samples of foods, even those taken from a single source, may harbour both different numbers, and different species of microorganisms, a problem made worse if more than a single source is used. This makes replication and statistical evaluation of results difficult. In addition to this, it has been accepted for some time that not all microbial species present on environmental samples — this includes foods — can be cultured under laboratory conditions. Whilst such difficulties may be greatly reduced by working with artificially cultured microorganisms, other complications arise. The association of microorganisms with foods is complex and can take more than one form. Microbial cells coming into contact with the food may adsorb to the food and then go on to multiply at its surface. This may simply lead to greater cell assemblages, however certain species may produce polysaccharides that serve not only to attach the cells more strongly to the food, but which can also protect them from external stresses. Moreover, the physiology of cells embedded in polysaccharide matrices, or ‘biofilms,’ is known to differ from those of planktonic cells (20). In addition, recent work has shown that certain elements of the natural microflora may offer protection against pathogens (22). In many cases the true state of the association between a particular food and its natural microflora is unknown, and the methods chosen by researchers to apply microorganisms to food surfaces may result in highly artificial associations. Often there are no alternatives to such methods. However, if these inherent experimental limitations are taken into account then the existing experimental data available for a number of different foods can provide a useful guide to assessing whether UV treatment of a particular food is feasible. Table 122.2 shows a compilation of recent data. The range of foods is quite diverse as are both the targeted microbial species and the dosages applied. Relatively few evaluations have been made of the effect of UV on the organoleptic characteristics of the foods. Such considerations are far from trivial and deleterious changes could lead to rejection by consumers despite the fact that treatment may have produced a safer product. When treating solid materials, such as foods, with UV, it is characteristic of the surfaces that are of primary

importance, as UV radiation will not penetrate very far beyond the surface layers of the material. On this basis, these surface properties can be expected to influence strongly the chances of microorganisms surviving irradiation. Evidence of the importance of macroscopic surface features is provided by work done on the irradiation of fish fillets by Huang and Toledo (33). These researchers found that UV was more effective in inactivating microorganisms on the surfaces of smooth-fleshed fish, such as mackerel, than on rough-fleshed fish such as mullet. This was because the surface of the mullet provided ridges around which microorganisms were shielded from incident UV light. Microscopic surface characteristics and irregularities may similarly be expected to affect microbial survival: surfaces which might appear smooth to the human eye, may at scales comparable to the dimensions of microorganisms (i.e., of the order of 1 to 10 µm) resemble a topography as rugged as that of the Himalayan foothills. One approach to account for surface topography and surface–organism interactions is to extend the concept of the decimal reduction dose (D10). A comparison of the D10 value for a particular microorganism in conditions where the nature of the medium surrounding the organism does not unduly influence microbial survival with that obtained on the surface of a food can provide some sort of measure of surface-related protective effects. When in dilute aqueous suspensions, microorganisms are essentially fully exposed to incident UV but, presumably for reasons of convenience, many workers have opted instead to irradiate colonies on the surface of agar to obtain ‘basal’ D10 values. Relatively few published studies contain sufficient data to enable this to be done but the data available is presented in Table 122.3. Implicit in this approach is the assumption that the inactivation data is approximately linear in form. This is the case for most of the data shown in the table, but there are instances where the data deviates significantly from linearity. A case in point is the data of Wong et al. (35) for both E. coli and Salmonella senftenberg on the surface of agar (i.e., the basal D10). In order to obtain D10 values under these conditions these workers effectively imposed linearity on their data to enable comparisons to be made with data for the surface of pork which was linear. Gardner and Shama (40) suggested an alternative approach to the use of decimal reduction factors. Working with model materials (cellulose filter papers), they proposed that the surfaces of foods could be considered as constituting a discrete number of zones each of which contained a certain fraction of the microbial surface population. Each zone was typified by a so-called ‘exposure factor.’ This quantified the degree of attenuation to incident UV. Thus factors near 1.0 indicated that the zone offered little attenuation, whilst zones with low factors offered high levels of attenuation, and therefore high UV doses were required in order to inactivate the organisms

Microbial Flora

Cultivated psychrophilic Pseudomonas sp. Thamnidium sp. Candida scotii

Natural microflora

Cultivated Pseudomonas spp. Micrococcus spp. Staphylococcus spp.

Natural microflora

Natural microflora

Cultivated S. typhimurium and natural psychrotrophic microflora

Cultivated S. typhimurium

Cultivated S. typhimurium, L. monocytogenes, E. coli O157:H7

Cultivated salmonellae

Cultivated E. coli O157:H7

Food

Beef (slices)

Beef (muscle and adipose tissue)

Beef

Bread (Baguette)

Chicken (whole)

Chicken (halves)

Chicken (skin)

Chicken (breasts)

Chocolate

Cider

TABLE 122.2 UV Disinfection of Foods

0.1–0.6

1.7

0.015

0.16–0.97

0.8

0.1

0.14–0.54

0.66

3.81 (mean reduction for all treatments).

Effects obtained by continuous long exposure (c. 3.5 days) at low UV intensities. UV irradiation resulted in increased growth lags and decreased growth rates for Penicillium sp., Pseudomonas sp. and Thamnidium sp. but not for C. scotii.

Other Comments

None evaluated.

No significant effects on colour or rancidity.

No adverse effect on organoleptic quality.

None evaluated.

No significant differences in odour or appearance between irradiated samples and controls.

None evaluated.

Treatment was affected by the original background microflora. Unable to consistently achieve the 5 log reduction required for the product by the regulatory authorities.

Photoreactivation was not observed.

Number of log reductions for all three bacterial species were decreased when the chicken was treated with the skin left on.

Although an immediate effect on surface contamination of treated, as opposed to untreated, chickens was observed, the shelf life was not significantly increased.

Shelf life extended by approx. 9 days.

Consumer desirability ratings based Type of UV source not stated but peak emissivity on colour were measured. Best results was at 366 nm with some contribution at were obtained for samples wrapped 253.7 nm. Shelf life increased by 1.5 to 2 days. immediately after irradiation.

None evaluated.

Adverse Effects

None evaluated. 5.3 for S. eastbourne in a 0.1 mm film of chocolate. No reduction in a 0.5 mm film.

0.48 for L. monocytogenes; 1.02 for S. typhimurium; 1.28 for E. coli. For chicken with the skin left on.

80.5% reduction (mean value for all doses).

0.5 (circa) for S. typhimurium no significant effect on psychrotrophs.

Not stated.

Not stated.

2.0 for the mixed bacterial culture.

Not stated.

Results highly variable: 0.1 to 2.2 for a dose of 77 kJ/m2 for Pseudomonas sp.

77.0

Not measured.

No. of Log Reductions

UV Dose kJ/m2

(Continued)

31

30

29

28

27

26

25

24

23

21

Reference

Ultraviolet Light 122-5

Not explicitly stated.

0.25 and 1.0

Botrytis cinerea

Strawberries

None evaluated.

1.1 at a dose of 0.08 kJ/m2. 2.5–4.0 at a dose of 0.5 kJ/m2.

Up to 0.5

Cultivated S. enteritidis

Shell Eggs

None evaluated.

2 at a dose of 10 kJ/m2. 3 at a dose of 30 kJ/m2.

Shelf life extended by 4–5 days. Extensive tests performed. At a dose of 2.5 kJ/m2 fruit had a higher anthocyanin content and were firmer than controls. Suggestion of damage to fruit at the higher dose (10 kJ/m2) on the basis of electrical conductivity measurements.

Type of source not stated. Eggs were rotated during treatment.

High intensity source. Sterilisation was not as effective at the ends of the sausages as towards the centre due to shadowing effects.

Up to 39

Natural microflora

Shell Eggs

2

1.92

None evaluated.

Both low pressure and high intensity sources used. Poor effect on mullet attributed to the rough surface of the fish. Best results obtained by combining UV irradiation with chlorinated water wash. Shelf life extended by at least 7 days for mackerel.

5 for E. coli 4 for B. subtitlis.

Cultivated E. coli B. subtilis

Sausages

Not determined.

None evaluated.

Oocyst viability was determined using mice.

D values At 1000 uW/cm2 S. senftenberg: Skin 490 s Muscle 1064 s E. coli: Skin 592 s Muscle 1205 s

Cultivated E. coli S. senftenberg

Pork (skin and pork muscle)

Not stated.

2.5 for mackerel 0.3 for mullet For low pressure source at a dose of 3.0 kJ/m2. 2.7 for mackerel 0.4 for mullet For high intensity source at a dose of 60 kJ/m2

None evaluated.

Other Comments

None evaluated.

Natural microflora

Maple syrup

3.0–75

5

Adverse Effects

3.8 on skin 4.6 on muscle for S. senftenberg. 1.6 on skin 1.5 on muscle for E. coli.

Natural microflora

Fish (fillets)

0.14

No. of Log Reductions

Maple sap was stored in tanks and continuously irradiated. Under these conditions the bacterial count was maintained below 4.0 ⫻ 105 per ml for 11 days.

Harvested Cryptosporidium parvum oocysts

Cider

UV Dose kJ/m2

The treated sap retained its flavour and colour.

Microbial Flora

Food

TABLE 122.2 (Continued )

39

38

37

36

35

34

33

32

Reference

122-6 Handbook of Food Science, Technology, and Engineering, Volume 3

Ultraviolet Light

122-7

TABLE 122.3 Decimal Reduction Doses (D10) for Various Foods Food

Bacteria

Chicken

Chocolate Pork

a

UV Intensity W/m2

D10 Values kJ/m2

Listeria monocytogenes Salmonella typhimurium E. coli O157:H7 Salmonella eastbourne

Peptone water 0.079 Peptone water 0.13 Peptone water 0.061a Peptone water 0.18a

Chicken (with skin) 1.948 Chicken (with skin) 0.888 Chicken (with skin) 0.677 Chocolate (0.1 mm thickness) 0.797

Chicken (without skin) 1.911 Chicken (without skin) 2.439 Chicken (without skin) 1.06 Chocolate (0.3 mm thickness) 16.28

E. coli Salmonella senftenberg

Agar 1.77 Agar 0.21

Pork skin 5.92 Pork skin 4.90

Pork muscle 12.05 Pork muscle 10.64

Reference

5 5

29

5 19

30

10 10

35

Estimate based on only two data points.

TABLE 122.4 Enhancement of UV Disinfection by Hydrogen Peroxide

1.0 0.9 Exposure factor.

0.8 0.7 0.6

Medium

0.5

Coarse cellulose filter paper Fine cellulose filter paper Distilled water

0.4 0.3 0.2

a

0.1

Ratio of First Order Inactivation Rate Constants (UV ⴙ H2O2a/UV)

Reference

3.2 5.3 4.8

43 43 44

H2O2 concentration 1% (w/v).

0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Population fraction.

FIGURE 122.3 Zone model for UV inactivation of microorganisms on a solid surface (40).

present. Figure 122.3 shows a typical food surface simulation. In this instance the surface of the food comprises three zones, containing 22, 53 and 25% of the microbial population and with exposure factors of, respectively, 0.85, 0.29 and 0.08. An extension of this approach to actual food surfaces might go some way to providing data that would enable the UV doses necessary to bring about given reductions in the viability of microbial populations to be predicted.

V.

COMBINED TREATMENTS INCORPORATING UV

A. UV AND HYDROGEN PEROXIDE The germicidal effects of UV can be enhanced by hydrogen peroxide. Hydrogen peroxide possesses antimicrobial properties in its own right, but when combined with UV produces synergistic disinfection effects. This has been

demonstrated both for bacterial spores (41) and for vegetative bacteria (42). Hydrogen peroxide undergoes photolysis in the presence of UV to yield hydroxy radicals (OH°): H2O2 → 2OH° These short-lived radicals are highly reactive and will attack microorganisms indiscriminately. The germicidal effect of hydrogen peroxide in aqueous solutions increases with concentration. However, as hydrogen peroxide absorbs UV quite strongly, concentrated solutions of hydrogen peroxide will attenuate incident UV resulting in reduced activity against microorganisms. Therefore, an optimum concentration exists at which the synergistic germicidal effect is at its greatest. This concentration has been shown to be approximately 1% (w/v) for Bacillus subtilis spores either in suspension (41) or on solid surfaces (43). Irradiation of hydrogen peroxide solutions at this concentration results in enhanced rates of spore inactivation compared to UV alone as Table 122.4 shows. Perhaps the most successful commercial application of the combined UV–hydrogen peroxide treatment has

122-8


been in the decontamination of beverage cartons (45). Although hydrogen peroxide treatment is currently being evaluated for decontaminating a wide variety of foods, it is somewhat surprising that not more attention has been paid to the combined treatment, particularly as UV can reduce residual hydrogen peroxide levels to those considered acceptable.

B. UV AND OZONE The classification of ozone by the Food and Drugs Administration in the USA as being generally recognised as safe (GRAS) has led to growing interest in its use as a germicide in the food industry (46). In common with hydrogen peroxide, ozone is known to exhibit synergistic disinfection effects under conditions of UV irradiation. The mechanism by which free radicals and other oxidising species are liberated during UV irradiation are quite complex but well established (47). Despite the advantages that ozone residuals are shortlived, very little work has been published on the combination of ozone and UV of direct relevance to foods. One early study (22) did show that a synergistic inhibitory effect against two species of moulds, (Thamnidium sp. and Penicillium sp.) was obtained when beef slices were irradiated with UV in the presence of gaseous ozone.

C. OTHER An interesting combination involves the use of long wave UV with a class of compounds known as the furanocoumarins. These tricylcic compounds exert their lethality by intercalation within the double helical structure of DNA and cross-linking with DNA bases. One particular attraction in the context of food processing is that furanocoumarins occur naturally in a number of food plant species including lemons, celery and parsley. Bintsis et al. (48) showed that the combination of UV and relatively high concentrations of furanocoumarins inactivated both pathogenic bacteria such as E. coli O157:H7 and Listeria monocytogenes, and the natural brine microflora. Further work in this field, possibly in relation to minimal processing, might yield more positive outcomes. The anatase crystalline form of titanium dioxide when irradiated by UV light in the presence of water generates free hydroxy radicals. This phenomenon has been widely exploited in environmental applications and some of these are of interest in food processing. One such application is the destruction of ethylene by UV-irradiated titanium dioxide (49, 50). Ethylene is known as the ‘ripening hormone’ and its accumulation in storage depots of certain horticultural products, i.e., fruit, vegetables and flowers, leads to considerable losses through accelerated senescence resulting in unmarketable produce.

Immobilized titanium dioxide was recently shown to be active in treating the water used in bean sprout cultivation (51).

VI. UV HORMESIS The uses of UV described so far have related to its direct effects against microorganisms. However, there seems to be a growing interest in the treatment of fruit and vegetables with UV in order to bring about indirect effects in plant tissues that confer resistance to a variety of fungal pathogens. To date this interest appears to be restricted to that of the research community with little evidence of its adoption by growers or processors. To some extent this is due to the fact that the magnitude of the effects obtained is due to the particular cultivar used or the precise conditions under which it was grown. For example, it is known that there are seasonal variations in susceptibilities of fruits to stresses (52) and such factors would need to be taken into account in commercialising any process based on irradiation with UV. However, as the changes that UV induces in plant materials become better defined and understood, irradiation of agricultural produce might well provide a viable method of reducing postharvest losses in the future. It has been claimed that postharvest losses of agricultural produce are significant in developed countries, but that in developing countries they can be catastrophic. A number of measures are applied in developed countries to reduce such losses. Principal amongst these is postharvest storage at low temperature. Fewer losses are sustained if in addition to cold storage fungicides are used. These are typically applied to the crops some short while before they are harvested. Interest in the potential use of UV stems from pressures from the regulatory authorities to reduce the dependence on chemical fungicides as primary disease control agents. To these concerns must be added those of consumers who are increasingly demanding food free from chemical additives and produced by methods that cause minimal environmental impact. The application of UV to the treatment of agricultural produce is termed ‘hormesis.’ Hormesis may be defined as a beneficial plant response resulting from the application of a low dose of a so-called ‘stressor.’ A variety of physical treatments, including UV irradiation, may serve as stressors. Although the phenomenon of hormesis was described in the 1940s, it is really in the last 20 years that it has excited interest as a practical method of reducing postharvest losses of crops (53). Irradiation of fruits and vegetables with UV will result in at least some direct inactivation of microorganisms present at the surface, but hormesis is quite distinct from purely surface disinfection and may even be considered as additive to it. The evidence for hormetic effects is that fungi deliberately inoculated into fruit some distance from

Ultraviolet Light

the surface are inhibited following low level irradiation (54). Moreover, hormetic effects have been shown to be reversible by illuminating the plant tissue with visible light following UV irradiation. This would seem to implicate genetic involvement in the response of the plant to UV. Indeed, the isolation of a gene in grapefruit that is activated in response to UV and produces an isoflavone reductase-like protein has recently been announced (55). In addition, it seems that there is a delay after irradiation before the maximum protective effect is achieved; this delay can be as long as 96 hours (56). Table 122.5 reveals that hormesis can be induced in a number of different crops. The only exception appears to be cactus pears where at best UV treatment resulted in no positive benefits (58). The optimal doses for achieving hormetic effects range from 0.12 to 9.0 kJ/m2, this is a much narrower range of doses than those used in irradiating foods for obtaining germicidal effects (see Table 122.3). For certain crops the nature of at least one ‘hormetin,’ the substance produced in response to the application of UV, has been identified. However, it seems likely that the formation of a number of quite different classes of compounds is probably elicited by hormesis. In many cases the exact identity of these compounds have yet to be described. Typically researchers have confined themselves to assaying only one or two previously identified compounds. The enzyme phenylalanine ammonia-lyase (PAL) has been found in a number of different fruits including sweet potato (54). This enzyme is associated with lignin biosynthesis which is a common plant response to fungal attack. There is mounting evidence to suggest that phytoalexins are produced following irradiation. Phytoalexins are low molecular weight compounds that are produced by plants in response to microbial infection or physical damage. The phytoalexin scoparone has been detected in a number of citrus fruits (52, 62), whilst 6-methoxymellein has been found in carrots (59) and resveratrol in grapes (72). The effects of irradiation with low doses are not restricted to the inhibition of fungal pathogens. A number of workers have shown that ripening of fruit can also be delayed (70). Premature ripening, as may occur for example in storage, is also the cause of postharvest losses as over-ripe produce is not marketable. There is compelling evidence that even more subtle effects than hormetic ones may be at work and that if these are better understood even greater protection may be given to agricultural produce without resorting to fungicides. The natural epiphytic (i.e., surface-associated) microflora found on certain fruits may inhibit certain fungal pathogens but is itself not affected by low UV doses. This has been demonstrated for the epiphytic yeast Debaromyces hansenii, which survives UV doses on the surfaces of peaches which would normally inactivate it on artificial surfaces. This strongly implicates one or more

122-9

control factors which are imposed by the fruit. D. hansenii has been shown to be inhibitory towards the soft rot fungus Monilinia fructicola without forming antibiotics. A synergistic protection of peaches against this particular fungus has been demonstrated by the application of the yeast with low UV doses (21). Of related application to the subject discussed above is the possibility of producing functional foods through UV treatment. Mau et al. (73) showed that the vitamin D2 content of a number of different mushroom species could be increased by irradiating them with UV-B. Cantos et al. (74) proposed subjecting grapes to UV-C pulses to increase their resveratrol content.

VII. DELETERIOUS EFFECTS OF UV ON FOODS Most of the data available on the damaging effects of UV on foods relates to the work done on applying hermetic UV doses to fruits and vegetables. These effects are summarised in Table 122.4. In general, UV doses much above the levels that bring about beneficial hermetic effects, approximately 0.12 to 9.0 kJ/m2, may result in surface discoloration, accelerated senescence or sprouting. Relatively few adverse effects following UV irradiation were reported in the range of foods listed in Table 122.2, this was mainly because such evaluations were often outside the scope of the work reported. In fact, very few studies have specifically focussed on this aspect of the UV treatment of foods. The extended irradiation of cold liver oil resulted in the formation of toxic aldehydes (75). Studies with artificial food colouring showed that prolonged UV irradiation led to the formation of breakdown products that were DNA damaging (76). However, the UV doses necessary to bring about these effects were not specified.

VIII. UV TECHNOLOGY A. UV SOURCES Despite being published 20 years ago, a very useful source of fundamental information on UV sources remains that of Phillips (1). UV sources are in the main gas discharge sources containing xenon and mercury or xenon and argon. The low vapour pressure mercury source is perhaps the most commonly used method of achieving disinfection. It operates optimally at a temperature of approximately 40°C and is often referred to as ‘monochromatic’ but actually 90% of its output is emitted at 253.7 nm which is fortuitously close to the maximum absorptivity of DNA. Increasing use is being made of medium and high pressure UV sources in water disinfection and this trend may extend to the food industry. These are not as efficient as the low pressure sources in emitting in the germicidal range but their higher power ratings

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TABLE 122.5 UV Hormesis of Horticultural Produce UV Dose Range Investigated kJ m⫺2

Crop (Variety)

Targeted Pathogen

Apple (Red Delicious)

Penicillium expansum

7.5

Not determined

Treated fruit stored at 24°C for 14 days. The earliest application of UV treatment (96 hours) before inoculating with P. expansum provided the best defence against disease. Combining UV irradiation with other disease prevention measures, harpin, chitosan and yeast antagonists Candida saitoana and C. oleophilia offered no advantages.

56

Cabbage seeds (Acc 16 Hybrid)

Xanthomonas campestris

1.3–7.5

3.6

Irradiation performed on cabbage seeds which were stored for up to 8 months. Improvements in quality and growth response observed at optimum dose. Disease resistance of treated seeds decreased with storage time.

57

Cactus Pear (Gialla)

Not specified

0.75

Not determined

UV treatment did not reduce the incidence of decay. Skin damage observed following irradiation.

58

Carrot (Caropak)

Botrytis cinerea

0.11–0.88

0.44–0.88

Both fresh and aged carrots were studied. Aged carrots had been stored at 1°C for 4 months. After irradiation carrots were stored at 1°C for 25 days. Only carrots that had been surface-wounded responded to UV treatment at 1°C but intact roots responded to treatment at 20°C. Treatment of fresh carrots gave higher resistance to storage rots. Exposure to UV induced 6-methoxymellein production.

59

Grape (Italia)

Botrytis cinerea

0.125–4.0

0.125–0.5

Irradiated grapes were stored at either 3°or 21°C. Grapes irradiated 24–48 hours before inoculating with B. cinerea showed a lower disease incidence than those inoculated immediately before irradiation. Doses above 1.0 kJ m⫺2 resulted in skin discolouration. Treatment within the optimum range did not significantly reduce the numbers of yeasts antagonistic towards pathogenic moulds.

60

Grapefruit (Star Ruby)

Penicillium digitatum

0.5–3.0

0.5

Quality and disease resistance determined after storage at 7°C for 4 weeks followed by 1 week at 20°C. Scoparone and scopoletin levels were increased at all UV doses. Rind browning and tissue necrosis occurred at UV doses ⬎1.5 kJ m⫺2.

61

Kumquat (Nagami)


0.2–15

1.5

Scoparone levels increased following irradiation at all UV exposures. After 2 weeks of storage at 17°C UV-treated fruit showed signs of damage, however at lower temperatures UV damage was practically absent even at the highest dose used.

62

Lemon (Eureka)


0–15

5

Irradiated fruit was stored in the dark at 17°C. UV was only effective in suppressing decay in fruit that had been irradiated at least 24 h before inoculation with P. digitatum. Increased levels of scoparone were found in irradiated fruits.

63

Mango (Tommys Atkins)

Not specified

4.9 and 9.9

4.9

Quality and disease resistance determined after storage at 5°C for 14 days followed by 7 days at 20°C. Treatment at 4.9 kJ m⫺2 resulted in improved appearance and texture of fruit. Irradiation induced spermidine and putrescine. The higher dose induced senescence.

64

7.33

Quality and disease resistance determined after storage at 20–25°C for four weeks. UV exposure was generally better than gamma or electron beam irradiation at

65

Onion Aspergillus spp., 0.44–19.10 (Walla Walla) Penicillium spp., Erwinia spp.

Optimal UV Dose kJ m⫺2

Additional Details

Reference

(Continued)

Ultraviolet Light

122-11

TABLE 122.5 (Continued ) Crop (Variety)

Targeted Pathogen

UV Dose Range Investigated kJ m⫺2

Optimal UV Dose kJ m⫺2

Additional Details

Reference

reducing the incidence of disease and improvement in marketability and storage life. UV irradiation did not affect texture or flavour. Some UV doses induced sprouting. Orange Not specified (Biondo Comune, Washington Navel, Tarocco, Valencia Late)

0.5–3.0

Not determined

Quality and disease resistance determined after storage at 7°C for 4 weeks followed by 1 week at 20°C. Peel quality was affected in all cultivars with the exception of Valencia L. Percentage of damaged fruit at the higher dosages decreased as the season progressed. UV irradiation at 0.5 kJ m⫺2 was effective in reducing decay development. The higher dose of 1.5 kJ m⫺2 was more effective but only in early harvested fruit. Concentrations of scoparone and scopoletin increased in all varieties with increasing dose.

52

Oranges (Shamouti, Valencia)


0.2–15

9.0

After 2 weeks of storage at 17°C UV-treated fruit showed signs of damage, however at lower temperatures UV damage was practically absent even at the highest dose used. Scoparone levels increased following irradiation at all UV exposures.

62

Peach (Elberta)

Monilinia fructicola

0.84–40

7.5

Exposure to UV delayed ripening, suppressed ethylene production and increased phenylalanine ammonia-lyase activity. Doses of 40 kJ m⫺2 increased susceptibility to brown rot. Irradiation resulted in increased numbers of the antagonist yeast Debaryomyces hansenii on the surface of the fruit.

66

Pepper (Bell Boy, Delphin)

Natural infections and Botrytis cinerea

0.22–2.20

0.88 for Botrytis cinerea

Fruit were stored at either 13° or 20°C following irradiation. All doses tested provided protection against natural infection. UV provided protection against B. cinerea only when artificial inoculation occurred after irradiation but not before. Two successive exposures at 0.44 kJ m⫺2 were equivalent to a single exposure at 0.88 kJ m⫺2.

67

Potato (Superior)

Fusarium solani Erwinia carotovora

7.5–15

Not determined

Potatoes stored at 8°C for 3 months. Disease suppression increased with UV dose. Doses higher than 15 kJ m⫺2 associated with induction of sprouting and were not investigated.

68

Sweet Potato (Jewel)

Fusarium solani

1.3–20

3.6

Tubers stored for up to 8 weeks at 25⫺27°C. All UV exposures resulted in increased phenylalanine ammonia-lyase activity.

54

Tomato Alternaria 1.3–40 (Tuskegee alternata 80–130, FloraBotrytis cinerea dade, Better Boy) Rhizopus stolonifer

3.6–7.5

UV doses of 3.6 and 4.8 kJ m⫺2 delayed ripening whilst doses of 40 kJ m⫺2 resulted in skin discolourization.

69

Tomato (Capello)

-

3.7–24.4

3.7

Study aimed at delaying senescence only. Treated fruit were stored at 16°C for 35 days. High UV doses caused abnormal browning of the surface of fruits. Treatment with doses of 3.7 kJ m⫺2 delayed ripening for 7 days. This correlated with increased amounts of putrescine in the fruits.

70

Zucchini Squash (Tigress)

Not specified

0.49–9.86

Not determined

Fruit sliced prior to irradiation and stored at 5° and 10°C for up to 18 days. Doses above 4.9 kJ m⫺2 retarded microbial growth. Treatment at the higher doses resulted in slight discolouration for fruit stored at 10° but not 5°C.

71

122-12


mean that greater overall outputs can be obtained. ‘Doping’ with e.g., metal halides can modify the spectral output of these sources. Recent advances have led to the development of pulsed UV sources in which the duration of the pulse varies from nano- to milli-seconds. Comparisons with continuous i.e., conventional sources have shown that pulsed sources are more effective at decontaminating surfaces (77, 78). Similarly tunable excimer lasers have been used to disinfect food packaging (79). Plasmas, generated in vacuum or at atmospheric conditions also have potential for use in surface disinfection (80).

B. UV IRRADIATION EQUIPMENT The history of treating water precedes that of food disinfection and it is therefore not surprising therefore that most innovations relate to the treatment of liquids and water at that. Novel thin film devices have been proposed for disinfecting liquids, such as cider and syrups, that have low UV transmittance (81) but such systems have low liquid throughputs. Solids are generally conveyed on belts past UV sources. These may be mounted on the walls of enclosures to create ‘UV tunnels.’ Shama et al. (82) proposed the hydraulic conveying of particulate matter over UV sources. Some innovative designs have been reviewed in relation to UV curing of inks on curved surfaces which could well have applications to food treatment (83).

10.

11.

12.

13.

14.

15.

16.

17.

18.

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59. J Mercier, J Arul, C Julien. Effect of UV-C on phytoalexin accumulation and resistance to Botrytis cinerea in stored carrots. J Phytopath, 139:17–25, 1993. 60. F Nigro, A Ippolito, G Lima. Use of UV-C to reduce storage rot of table grape. Postharvest Biol Tech, 13: 171–181, 1998. 61. G Dhallewin, M Schirra, M Pala, S Ben-Yehoshua. Ultraviolet C irradiation at 0.5 kJ. M-2 reduces decay without causing damage or affecting postharvest quality of star ruby grapefruit (C. paradisi Macf.). J Agric Food Chem, 48:4571–4575, 2000. 62. V Rodov, S Ben-Yehoshua, JJ Kim, B Shapiro, Y Ittah. Ultraviolet illumination induces scoparone production in kumquat and orange fruit and improves decay resistance. J Am Soc Hort Sci, 117:788–792, 1992. 63. S Ben-Yehoshua, V Rodov, JJ Kim, S Carmeli. Preformed and induced antifungal materials of citrus fruits in relation to the enhancement of decay resistance by heat and ultraviolet treatments. J Agric Food Chem, 40:1217–1221, 1992. 64. GA Gonzalez-Aguilar, CY Wang, JG Buta, DT Krizek. Use of UV-C irradiation to prevent decay and maintain postharvest quality of ripe ‘Tommy Atkins’ mangoes. Int J Food Sci Tech, 36:767–773, 2001. 65. JY Lu, C Stevens, P Yakubu, PA Loretan. Gamma, electron beam and ultraviolet radiation on control of storage rots and quality of Walla Walla onions. J Food Proc Pres, 12:53–62, 1987. 66. C Stevens, VA Khan, JY Lu, CL Wilson, PL Pusey, MK Kabwe, ECK Igwegbe, E Chalutz, S Droby. The germicidal and hermetic effects of UV-C light on reducing brown rot disease and yeast microflora of peaches. Crop Prot, 17:75–84, 1998. 67. J Mercier, M Baka, B Reddy, R Corcuff, J Arul. Shortwave ultraviolet irradiation for control of decay caused by Botrytis cinerea in bell pepper: induced resistance and germicidal effects. J Amer Soc Hort Sci, 126:128–133, 2001. 68. B Ranganna, AC Kushalappa, GSV Raghavan. Ultraviolet irradiance to control dry rot and soft rot of potato in storage. Can J Plant Path, 19:30–35, 1997. 69. J Liu, C Stevens, VA Khan, JY Lu, CL Wilson, O Adeyeye, MK Kabwe, PL Pusey, E Chalutz, T Sultana, S Droby. Application of ultraviolet-C light on storage rots and ripening of tomatoes. J Food Prot, 56:868–872, 1993. 70. R Maharaj, J Arul, P Nadeau. Effect of photochemical treatment in the preservation of fresh tomato (Lycopersicon esculentum cv Capello) by delaying senescence. Postharvest Biol Tech, 15:13–23, 1999.

71. M Erkan, CY Wang, DT Krizek. UV-C irradiation reduces microbial populations and deterioration in Cucurbita pepo fruit tissue. Env Exp Bot, 45:1–9, 2001. 72. E Cantos, JC Espin, FA Tomas-Barberan. Postharvest stilbene enrichment of red and white table grape varieties using UVC irradiation pulses. J Agric Food Chem, 50:6322–6329, 2002. 73. JL Mau, PR Chen, JH Yang. Ultraviolet irradiation increased vitamin D2 content in edible mushrooms. J Agric Food Chem, 46:5269–5272, 1998. 74. E Cantos, JC Espin, FA Tomas-Barberan. Postharvest induction modeling method using UV irradiation pulses for obtaining resveratrol-enriched table grapes: a new “functional” fruit? J Agric Food Chem, 49:5052–5058, 2001. 75. F Niyati-Shirkhodaee, T Shibamoto. Formation of toxic aldehydes in cod liver oil after ultraviolet irradiation. J Amer Oil Chem Assoc, 69:1254–1256, 1992. 76. A Ozaki, M Kitano, N Itoh, K Kuroda, N Furusawa, T Masuda, H Yamaguchi. Mutagenicity and DNAdamaging activity of decomposed products of food colours under UV irradiation. Food Chem Toxicol, 36:811–817, 1998. 77. KF McDonald, RD Curry, TE Clevenger, K Unklesbay, A Eisenstark, J Golden, RD Morgan. A comparison of pulsed and continuous ultraviolet light sources for the decontamination of surfaces. IEEE Trans Plasma Sci, 28:1581–1587, 2000. 78. NJ Rowan, SJ MacGregor, JG Anderson, RA Fouracre, L McIlvaney, O Farish. Pulsed-light inactivation of food-related microorganisms. Appl Env Micro, 65: 1312–1315, 1999. 79. K Warriner, G Rysstad, A Murden, P Rumsby, D Thomas, WM Waites. Inactivation of Bacillus subtilis spores on packaging surfaces by u.v. excimer laser irradiation. J Appl Micro, 88:678–685, 2000. 80. M Moisan, J Barbeau, MC Crevier, J Pelletier, N Philip, B Saoudi. Plasma sterilization. Methods mechanisms. Pure Appl Chem, 74:349–358, 2002. 81. G Shama, C Peppiatt, M Biguzzi. A novel thin film photoreactor. J Chem Tech Biotech, 65:56–64, 1996. 82. G Shama, DWM Gardner, AP Martin, NL Mason. Disinfection of particles using ultraviolet light. Trans IChemE Part C, 72:197–200, 1994. 83. RW Stowe. UV curing on curved surfaces and complex (3D) objects. In: JP Fouassier, JF Rabek. eds. Radiation Curing in Polymer Science and Technology. Volume 4. New York: Elsevier, 1993, pp. 179–193.

123

Aseptic Processing: Basic Principles and Advantages

K.P. Sandeep and Josip Simunovic

Department of Food Science, North Carolina State University

CONTENTS I. Introduction ............................................................................................................................................................123-1 A. Components of an Aseptic Processing System ..............................................................................................123-1 1. Historical Perspective ..............................................................................................................................123-2 2. Advantages and Disadvantages................................................................................................................123-2 B. Important Facets of Aseptic Processing ........................................................................................................123-3 1. Fluid Mechanics ......................................................................................................................................123-3 2. Kinetics ....................................................................................................................................................123-4 3. Heat Transfer............................................................................................................................................123-6 C. Processing Details ..........................................................................................................................................123-6 1. Issues to be Dealt with for Liquid and Particulate Foods........................................................................123-6 2. Product Heating and Cooling ..................................................................................................................123-6 3. Sterilization of Equipment ......................................................................................................................123-7 4. Heat Exchange Equipment and their Suppliers ......................................................................................123-8 D. Packaging Details ..........................................................................................................................................123-8 1. Types of Packaging Systems....................................................................................................................123-8 2. Testing of Package Integrity ..................................................................................................................123-10 E. Issues related to Process Validation..............................................................................................................123-10 References ..................................................................................................................................................................123-12

I. INTRODUCTION A. COMPONENTS SYSTEM

OF AN

ASEPTIC PROCESSING

Aseptic processing consists of pumping, deaeration, heating of the product (also referred to as “pre-sterilization”), passage of the product through a holding tube wherein the product attains the required temperature for the required amount of time (achievement of commercial sterility), cooling of the product, possible holding of the product in an aseptic surge tank, and subsequent packaging of the product in a pre-sterilized container under aseptic conditions. This process results in a high quality shelf-stable product in a hermetically sealed container. Pumping of the product at a constant rate (also referred to as “timing”) is very important to ensure that all parts of the product receive uniform and the required amount of

heat treatment. The most common type of pump used for this purpose is the piston type positive displacement pump wherein slippage (back flow of the product) is minimal. This type of pump delivers the product at a constant rate even if the pressure against which it is pumping fluctuates (for example, when fouling in the heat exchanger increases the pressure or when the opening and closing of valves causes changes in pressure). For certain fluid products, homogenization of the product may be required and this is achieved by using a homogenizer, which can also serve as the “timing” device. Deaeration of the product removes excess air in the product. It is accomplished in a vessel maintained at a certain degree of vacuum by means of a vacuum pump. The product is fed into the vessel at 55–70°C through a nozzle at the center of the vessel. Vacuum is controlled to obtain a product flash of about 5°C. An internal spiral 123-1

123-2


condenser condenses vapors and other condensable gases. The deaerated product is discharged through the bottom and pumped to the heating section. This serves the purposes of increasing the rate of heat transfer during heating and cooling, maintaining the specific volume of the product in the holding tube (to avoid reduction in holding time due to expansion of product), maintaining a constant fill rate and prevention of foaming during packaging, and minimizing oxidation reactions within the product during storage. The deaerator is placed before pre-heating in cases where loss of volatiles at elevated temperatures is of concern and after pre-heating in other instances since it is easier to remove air at higher temperatures due to its expansion. The high temperatures required for aseptic processing (125–145°C) are achieved by direct or indirect contact heat exchangers (usually with steam as the heating medium) and a back pressure valve (placed after the cooling unit) that maintains high enough pressure to prevent boiling (or flashing) of the product. The required back pressure for the process can be achieved by using a pistontype air actuated valve, diaphragm valve or pressurized tanks. Heating of the product can be accomplished by direct (steam injection of infusion) or indirect (tubular, shell and tube, plate, scraped surface, microwave, radio frequency, or ohmic) means. The choice of the type of heating and cooling equipment depends on a variety of factors such as type of product (acid or low-acid, viscous or non-viscous, fluid or particulate, heat sensitive or heat stable), potential for fouling, ease of cleaning, and as always the cost of the heat exchanger. An additional useful characteristic of the back pressure valve is that it also provides a dampening effect against the fluctuating or pulsating action of pumps used in aseptic processing. The holding tube is an important part of the aseptic processing system since this is where the product receives the required time-temperature treatment in order to render the product commercially sterile. The holding tube is always inclined upwards (requirement from regulatory agency) with a vertical rise of at least ¼ per foot length of the tube. This vertical rise ensures that the product completely fills the holding tube (with no air pockets) and that the product returns to the supply tank when the pump is shut off (thereby minimizing contamination issues). An aseptic surge tank provides the means for product to be continuously processed even if the packaging system is not operational due to any malfunction. It can also be used to package the sterilized product while the processing section is being re-sterilized. 1. Historical Perspective Historically, the first aseptic packaging of food (milk in metal cans) was done by Nielsen in Denmark in 1913. In 1917, Dunkley of the U.S. sterilized cans and lids by

saturated steam and filled pre-sterilized product in it. In 1923, aseptically packaged milk from S. Africa reached a trade fair in London in perfect condition. The work of Olin Ball and the American Can Research Department laid the foundation of aseptic processing in the U.S. as early as 1927 when the HCF (heat, cool, fill) process was developed. This was followed by the Avoset process in 1942 (steam injection of the product coupled with retort or hot air sterilization of packages such as cans and bottles) and the Dole-Martin aseptic process in 1948 (product sterilization in a tubular heat exchanger, metal container sterilization using superheated steam at temperatures as high as 450°F since dry heat requires higher temperature than wet heat, followed by aseptic filling and sealing of cooled product in a superheated steam environment). The early 1960s was marked with the advent of a form-fill-seal package — tetrahedron package. The late 1960s saw the advent of the Tetra Brick aseptic processing machine and the late 1970s saw the advent of the Combibloc (blank carton) aseptic system. Soon, aseptic filling in drums and bag-in-box fillers were established. One of the major landmarks in the history of aseptic processing is the approval of use of hydrogen peroxide for the sterilization of packaging surfaces by the FDA in 1981. In recent years, a major break-through for the aseptic processing industry was in 1997 when Tetra Pak received a no-objection letter from the FDA for aseptic processing of low-acid foods containing large particulates. 2. Advantages and Disadvantages Better product quality (nutrients, flavor, color, texture), less energy consumption, fewer operators, less space requirements, eliminating the need for refrigeration, easy adaptability to automation, use of any size package, use of flexible packages, and cheaper packaging costs are some of the advantages of aseptic processing over the conventional canning process. It also does not have the problem of texture changes associated with frozen products and increased permeability of packaging material such as ethylenevinylalcohol (to oxygen) due to the high temperature as in retortable pouches. Some of the reasons for the relatively low number of aseptically processed products include slower filler speeds and higher overall cost. Aseptic processing also requires better quality control of raw products, better trained personnel, and better control of process variables and equipments. It is also subjected to stringent and extensive validation procedures. Some of the disadvantages of aseptic processing include increased shear rates, degradation of some vitamins (some vitamins are stable at pasteurization temperatures but not at sterilization temperatures), separation of solids and fats, precipitation of salts, and change in flavor or texture of the product (steam injection followed by flash cooling may eliminate offflavors) relative to what consumers are accustomed to.


Though aseptic processing could potentially result in better product quality, one has to keep in mind that several chemical changes take place during temperatures encountered during aseptic processing which do not occur under normal processing conditions. Some of these chemical changes include age gelation, browning reactions, oxidation reactions, and changes in pigments and have been described in detail in references (1, 2). Due to some of the stringent regulatory requirements of aseptic processing, many processors adopt an aseptic process, but package it in non-aseptic containers. This results in products that are called “extended shelf-life products.” Such processes are easier to adopt, require less monitoring (since the resulting product-package combination does not need to be sterile), and are easier to file with regulatory agencies. One such process involves ultra-pasteurization of milk wherein extended shelf-life can be obtained. Notwithstanding the problems associated in producing aseptically processed foods, several companies have adopted this technology. Some of the products that are aseptically processed include fruit juices, milk, condensed milk, coffee creamers, puddings, soups, butter, gravies, and jelly. Some of the companies that deal with aseptic processing and packaging equipment are International Paper, Tetra Pak, Combibloc, Elopak, Cherry Burrell (tubular: Unitherm; plate: Thermaflex; SSHE: Thermutator; steam injection: Aseptic direct steam incorporation), Alfa Laval (Plate: Steritherm; SSHE: Contherm; steam followed by SSHE: Viscotherm; steam injection: VTIS — Vacu-therm instant sterilizer; corrugated tube: Spiraflo), ASTEC, VRC, APV (Plate: Juicematic; Plate for low-acid: Super ultramatic; steam injection: Uperizer), FranRica, Benco, Scholle, Bosch, and Metal Box.

B. IMPORTANT FACETS

OF

ASEPTIC PROCESSING

Some of the important facets of aseptic processing include fluid mechanics (residence time and residence time distribution of the fluid elements and particles in the product), kinetics (of microbial destruction, enzymatic inactivation, and nutrient destruction), and heat transfer (transfer of heat from the heating medium to the liquid and particulate portions of the product and accumulation of F0 or F-value at 121.1°C and z 10°C, where z is the temperature change required for an order of magnitude change in the decimal reduction time, D). 1. Fluid Mechanics The Food and Drug Administration (FDA) only credits heat treatment experienced in the holding tube, which makes its design critical. The velocity profile of the fluid in the holding tube is affected by the degree of its deviation from the behavior of a Newtonian fluid. The degree of deviation is characterized by the flow behavior index, n, for Ostwald-de-Waale fluids. For a Newtonian fluid

123-3

(n 1) flowing under laminar conditions in a straight tube of circular cross-section, the maximum velocity occurs at the center of the holding tube and its magnitude is twice the average velocity of the fluid. For pseudoplastic fluids (n 1), differences between the maximum and average velocity becomes smaller as n decreases. In other words, the velocity profile becomes flatter. For the extreme case (n 0), the plug flow profile is attained. However, for most cases (n 0), the maximum velocity occurs at the axis of the tube, which means that the minimum residence time corresponds to the residence time of particles located along the center-line of the tube. Consequently, these particles receive the least amount of heat treatment. Thus, the holding tube length required to achieve the required F0 value (time-temperature effect) can be calculated based on the knowledge of this minimum residence time, but this will result in an over-processed product. This is where the residence time distribution (RTD) of the particles comes into the picture. To understand RTD, we begin with the following equation, which describes the velocity profile for flow of a Newtonian fluid under laminar conditions in a pipe of circular cross-section: u 2 苶u [1 (r2/R2)]

(123.1)

Where u is fluid velocity in m/s, u苶 is average fluid velocity in m/s, r is the radial distance from the center of the tube in m, and R is the radius of the tube in m. Thus, it can be seen that different fluid elements (at different radial locations) spend different amounts of time in the tube. For instance, a fluid element traveling at the center of the tube will travel twice as fast as the average fluid element. The distribution of times spent by various fluid elements within the tube is referred to as the residence time distribution (RTD) of the fluid elements. Similarly, when different particles are flowing through the tube, they spend different times in the tube, and the distribution of these times is the RTD of the particles. The RTD of the particles depends a great deal on the RTD of the fluid. It also depends on flow rate and viscosity of the carrier medium, and also the size, density, and concentration of particles. Analysis of particle RTD is relatively simple when there is only one type of particle in a system. However, when different types of particles (especially particles of different densities) are present in a product, the flow behavior is quite different from the situation when they are each present as the only particle type in suspension. For instance, in a mixture of two types of particles, denser particles (which traveled slowly at the bottom of the tube when present alone) could be sped up by foreign particles due to collisions, and in turn, the foreign particles could get slowed down. Thus, an analysis has to be performed for each combination of particle types present in a system and direct inferences cannot be made from RTD of each particle type separately.

123-4


The existence of a RTD for the particles results in some particles receiving more heat treatment than others in the holding tube. From a safety standpoint, the fastest particle is what is of concern and the holding tube length is based on the fastest particle residence time. Thus, it can be seen that if the particle RTD is narrow, the quality of the product would be high since the difference between the fastest and slowest particle residence time is not very high. The wider the RTD of the particles in the holding tube section, the more non-uniform the process. One of the techniques that can be used to narrow the RTD of the fluid and particles is the use of helical tubes. When a non-Newtonian (power-law) fluid flows through a straight tube under laminar flow conditions, the velocity profile is given by the following equation. Non-Newtonian fluids: 3n 1 r u u苶 1 n 1 R

冤冢冣冥 n 1 n

(123.2)

Thus, for a pseudoplastic fluid (n 1), the maximum fluid velocity is given by: 3n 1 umax u苶 n 1

(123.3)

Hence it can be seen that the maximum velocity in the case of a pseudoplastic fluid is less than twice the average fluid velocity. Thus, the RTD of the fluid is narrower for a pseudoplastic fluid in comparison with that for a Newtonian fluid. Hence, the RTD of particles is also narrower when the carrier medium is a pseudoplastic fluid. Further details of RTD of fluid elements and particles have been presented by (3). The average fluid velocity can be calculated once the volumetric flow rate of the product is known. To determine the distribution of fluid residence times, salt injections, dye tracers, and fine particles are used. Magnetic resonance imaging can also be used under certain circumstances to obtain a fluid flow profile. Fluid flow profiles, though important, are usually not the target, since the species of concern are the slow-heating particles. Particle residence times, residence time distributions, and velocities can be determined by using a stop-watch, digital image analysis, LASER-Doppler velocimetry, and also with the aid of magnetically tagged particles. 2. Kinetics Sufficient heat has to be applied to a food product to inactivate microorganisms that cause food spoilage and food poisoning, and inactivate enzymes. However, the color, texture, flavor, and nutrients within the food must not be destroyed to an unacceptable level. This is where optimization of the process comes into play. Several combinations

of time and temperature could be used to destroy the microorganisms of concern. Out of these, the combination that results in the least nutrient destruction would be the desired combination. In order to arrive at this combination, a thorough understanding of the kinetics of microbial and nutrient destruction is essential. The heat resistance of microorganisms is affected by several factors. Some of these factors include water activity, pH, lipids and oily materials, dielectric constant, ionic species, ionic strength, oxygen level, organic acids, and antibiotics. Methods to measure heat resistance in the temperature range of 60–135°C are the end-point method (a number of replicate containers with a known number of spores are heated successively for longer periods of times until no survivors are obtained by culturing each container) and the multiple-point method (a batch of spores is heated continuously and samples are withdrawn at selected intervals, followed by determination of the number of survivors). The most general equation representing the kinetics of microbial inactivation, enzymatic inactivation, nutrient destruction, or other chemical reactions is dc kncn dt

(123.4)

with c being the concentration of the reacting species at time t, kn being the specific reaction rate, and n being the order of the reaction. To determine the order of the reaction, the logarithm of the equation is taken on both sides and a graph is plotted between ln(dc/dt) versus ln(c). The intercept of the graph is ln(kn) and the slope is the rate of the reaction. dc Zero order reaction (n 0): k0 dt c0 c k0t

Integrating, we get:

(123.5) (123.6)

Caramelization of sugar and degradation of vitamin C in model meat systems are examples of reactions that fall under this category. dc First order reaction (n 1): k1c dt Integrating, we get:

ln(c/c0)

This can also be written as:

(123.7)

k1t

c/c0 ekt

(123.8)

Most reactions, including microbial inactivation, enzymatic inactivation, and nutrient destruction, fall under this category. dc Second order reaction (n 2): k2c2 dt Integrating, we get: 1/c 1/c0 k2t

(123.9) (123.10)


Destruction of thiamin in milk falls under this category. Other orders of reaction (fractional): Reactions involving the color (n 1.31 0.18), texture (1.13 0.20) of peas and the texture (0.36 0.17), and appearance (0.44 0.16) of beans fall under this category. The more common way of representing the rate of microbial destruction is through the use of decimal reduction time (D). The relationship between the rate of a reaction and decimal reduction time is N/N0 ekt

(123.11)

123-5

Writing the above equation for two different temperatures, yields: E 1 1 k ( ) e R T Tref kref g

The activation energy is the minimum energy which molecules must have for the reaction to occur and the exponential term “eE/RgT” is the fraction of molecules that collectively have the minimum energy. The above equation can be simplified to:

log10 (N/N0) kt/2.303 with D 2.303/k

E 2.303 Rg(T)(Tref)/z

1. Thermal death time (TDT) method (D-z model): TDT is the time required for total destruction of a microbial population or the time required for destruction of microorganisms to an acceptable level. The plot of D (logarithmic scale) versus T is referred to as the “phantom” TDT curve (or the thermal resistance curve). The slope of this curve is “1/z.”

For unimolecular reactions A varies from 1014 to 10 s , for bimolecular reactions it varies from 104 to 1011 s1, and intermediate values for chain reactions. For heat resistant bacterial spores, A is extremely large, of the order of 1030–1060 s1 and E can be up to 500 kJ/mol. For Clostridium botulinum spores, A 2 1060 s1 and E 310.11 kJ/mol-K for the range of temperatures from 100°C to 150°C (4). It is assumed that A and E are independent of temperature, but this may not be the case always. There are several approaches available to correct this assumption or approximation — absolute reaction rate theory and quotient indicator method are two of them. Once the time-temperature profile within a product is determined, the degree of lethal treatment delivered is determined by determining the F-value of the process. The F-value of a 20 1

F

冕 10 t

TT ref z

dt

(123.17)

0

(123.13)

The points on this curve correspond to the combination of time and temperature that results in 90% reduction in microbial count. A similar curve with either 100% reduction in microbial count (based on experiments) or acceptable levels of reduction in microbial counts is referred to as the TDT curve and the time on the curve at a given temperature is the TDT at that temperature. Note that the slope of this curve is also “1/z.” 2. Arrhenius kinetics method (k-E model): The basic equation is as given below. k A eE/RgT

(123.16)

(123.12)

if k is represented in s1 and D in s. Thus, N/N0 10t/D where N0 and N are the initial and final number of microorganisms, respectively, and t is time. The D value determined at a reference temperature (Tref or Tr) is denoted by Dref or Dr. The ratio of Dref to D is referred to, as the lethal rate. The effect of temperature on rate of reaction can be described by one of the following two models:

log(D1/D2) (T2 T1)/z

(123.15)

(123.14)

where A is a pre-exponential factor, collision number, frequency factor (s1), E is the activation energy (J/kg-mol), and Rg is the universal gas constant ( 8314 J/kg-mol-K).

process is the time (in mins) at a reference temperature that would produce the same degree of microbial destruction as in the process under consideration. It is computed as follows. For a constant temperature process, the above equation reduces to F 10

TTref z

∆t

(123.18)

In aseptic processing, the reference temperature is usually chosen as 121.1°C (250°F) and the z value chosen is 10°C (or 18°F). The F-value computed with these values of Tref and z is referred to as the F0 value of the process. The use of a time-temperature integrator (TTI) as an alternative to temperature measurement or microbiological testing for process evaluation (determination of F-value) is becoming popular. A TTI can be an enzyme such as amylase or peroxidase that denatures (an unwinding of the structure) as it is heated. If the reaction kinetics of the

123-6


temperature-induced denaturation match those of the microbial death kinetics for the target species, it is possible to use such TTIs as non-biological markers of a process.

balance, fouling and enhancement of heat transfer, and techniques to estimate the temperature history of a product.

3. Heat Transfer

C. PROCESSING DETAILS

Heat is transferred from the heating medium (steam or hot water) to the product in the heat exchanger. For liquid foods, the rate of increase of product temperature depends on the overall heat transfer coefficient between the heating medium and the product. For particulate foods, there is an additional factor — heat from the liquid portion of the product has to be transferred to the particulates. This is affected by the convective heat transfer coefficient between the particles and the fluid and also thermal conduction within the particulates (governed by the thermal diffusivity of the particles). Efforts have been geared towards decreasing fouling (deposition of the food material on the heat exchanger) and increasing the rate of heat transfer between the heating medium and the product by not only minimizing fouling but also by new designs of heat exchangers and the change of processing parameters. During aseptic processing, the FDA does not credit lethality accumulation (accumulation of F-value) within a product in the cooling section. This is because particulates could possibly break up in the cooling section and thus, due to their smaller size, cool rapidly, thereby not accumulating the required F-value. Also, due to the uncertainties in the temperature distribution within the product in the heat exchanger, lethality credit is not given in this section. Lethality credit in the heat exchanger could possibly be included if the time-temperature history within the heat exchanger could be determined or modeled conservatively. Some of the techniques used to determine the temperature distribution within a product include the use of thermocouples, resistance temperature detectors, data tracers, infrared imaging, thermochromic dyes, magnetic resonance imaging, thermoluminescent markers, and magnetic particles. The non-invasive techniques among these are of more interest in aseptic processing. However, each of those techniques has their limitations in determining the center temperature of particles under unobstructed flow conditions. Thus, researchers are focusing on developing reliable techniques that can be used to non-invasively determine the internal temperature of flowing particles. Further details of the heat transfer aspects of aseptic processing have been described in detail by (5). This includes discussion of convective heat transfer coefficient, steam quality, dimensionless numbers governing heat transfer, natural (free) and forced convection, transient heat transfer within particles, hydrodynamic and thermal entrance lengths, heat transfer coefficient in straight tubes, heat transfer coefficient in helical tubes, heating media and equipment, co-current and counter-current heat exchangers, governing heat transfer equations and energy

Some of the important processing and packaging details to be considered during aseptic processing and packaging of liquid and particulate foods are discussed in this section. 1. Issues to be Dealt with for Liquid and Particulate Foods In aseptic processing, if we ensure that the slowest heating point (critical point) within the product is sufficiently processed, the entire product will be sufficiently processed. The critical point for a fluid product is generally at the center of the product. There are exceptions to this — for example, for flow in a helical tube, the fastest fluid element is located away from the center of the tube (towards the wall of the tube). The critical point with a particulate product is usually the center of the particle that receives the least heat treatment (critical particle). The critical particle in a system containing only one type and size of particle is the fastest particle in the holding tube. In a multi-particle product, the critical particle is the slowest heating particle, which is not necessarily the fastest particle since slower particles may potentially have a lower thermal diffusivity than the fastest particle. Mathematical modeling (with conservative assumptions) of the process would be one way to narrow down the choice of the critical particle. 2. Product Heating and Cooling Heating of the product is the first step towards delivering the required F-value to the product. For fluid products, a plate, tubular, scraped surface, steam injection/infusion, and volumetric heating mechanisms (ohmic, microwave, and radio frequency) can be used. It has been shown that the use of higher temperatures for shorter times is the generally preferred technique from the standpoint of nutritive value of the product. Thus, techniques to rapidly heat products are being sought. Product cooling is the final heat exchange operation prior to filling into aseptic packaging containers. Since the product temperatures achieved in aseptic processing are typically higher than in other thermal sterilization methods, and no creditable microbial lethality is accumulated in the cooling section (as opposed to the canning where creditable lethality can also be accumulated during the cooling stage), there is a need for rapid reduction of product temperature during cooling in order to minimize the negative effects of high temperature on various product quality characteristics such as nutrient, color, flavor, and texture retention. This reduction of temperature can be


performed, using appropriate coolant fluids, in the same types of heat exchangers listed for product heating (plate, tubular, and scraped surface heat exchangers), with the exception of steam injector equipment and volumetric heating equipment. For products where water is added through direct steam injection, flash or vacuum cooling can be employed to remove the added water as well as to effect very rapid cooling by boiling off the water under reduced pressure. The removal of the sensitive heat of evaporation causes the product material to cool to ambient or final filling very rapidly, typically within seconds. Indirect vacuum cooling equipment employs evaporating environment of cooling fluid to surround the flowthrough tubes of process material to effect rapid indirect, vacuum-driven cooling of product.

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Some of the techniques used for sterilization are listed below.

a. Radiation UV-C Radiation (250–280 nm)

Optimum effectiveness at 253.7 nm; applicable only to smooth, even surfaces. Infrared Radiation

Applicable only to smooth, even surfaces (Al lids coated with plastic laquer). Ionizing Radiation

Co-60 or Cs-139; 25 kGy (2.5 Mrad); 100 keV of electron beam (empty sealed containers such as bag-in-box).

b. Heat Saturated Steam

3. Sterilization of Equipment Sterilization of the processing, packaging, and the airflow system prior to processing are of utmost importance. This is what is referred to as pre-sterilization. Pre-sterilization of the air system is done by high efficiency particulate arresting (HEPA) filtering or incinerated air. For equipment, it is accomplished by steam, hydrogen peroxide, or other disinfectant solutions. For filling lines, pre-sterilization is done with steam or water at high pressure. The recommended heating effect for pre-sterilization (using hot water) of the processing equipment for low-acid foods is the equivalent of 250°F for 30 minutes. The corresponding combination for acid or acidified products is 220°F for 30 minutes. This often involves acidification of the water (to below a pH of 3.5 for acid products) used for sterilization. Pre-sterilization of an aseptic surge tank is usually done by saturated steam and not hot water due to the large volume associated with the surge tank. Once the product is processed, the system has to undergo a clean in place (CIP) operation. The CIP cycle for low-acid foods involves the use of hot water, alkali, hot water, acid, and hot water sequentially. The CIP cycle for high-acid foods is hot water, alkali, and hot water sequentially. A detailed description of fouling, cleaning, and disinfection has been presented by (6). Sterilization of the food contact surface of packaging material is the next point of consideration. For non-sterile acidic products (pH 4.5), a 4D process is required. For sterile, neutral, low acid products (pH 4.5), a 6D process is required. However, if there is possibility that C. botulinum is able to grow in the product, then a full 12D process is required. It has been suggested that only 3% of the total number of microorganisms on the package surface are spores. An upper value of 1,000 microorganisms per m2 (30 spores per m2) has been assumed for plastic films and paperboard laminates on reels, and 3,000 microorganisms per m2 (90 spores per m2) for prefabricated cups.

165°C and 600 kPa for 1.4 s (cups) and 1.8 s (lids); disadvantages include need for high pressure, removal of air (to promote heat transfer), and possible dilution of product as steam condenses. Superheated Steam

220–226°C for 36–45 s. Hot Air

315°C (surface temperature reaches 145°C for ⬃3 min); suitable only for acidic products. Hot Air and Steam

Hot air is blown through a nozzle in such a way that the base and walls are uniformly heated; used for cups and lids made of PP which is thermally stable up to 160°C. Extrusion

During extrusion of plastic granules prior to blow molding of plastic containers, temperatures of 180–230°C are reached for up to 3 min. However, because the temperature distribution inside the extruder is not uniform and the residence time of the plastic granules varies considerably, it is not possible to guarantee that all particles will achieve the minimum sterility. It has been suggested that extrusion results in a 3–4D process. Thus, aseptic filling into extruded containers should be used only for acidic products. For low-acid products, a hydrogen peroxide treatment is usually done.

c. Chemical Treatment Hydrogen Peroxide

Dipping, spraying, rinsing processes, or combined with UV-C or heat at least 80°C and 30% concentration is required; residual peroxide should be less than 100 ppb at time of filling and must decrease to 1 ppb within 24 hours. Since it is hard to detect peroxide in foods, containers filled with water are run through the machine initially. Peracetic Acid

Produced by oxidation of acetic acid by hydrogen peroxide; effective even at 20°C (1% solution will eliminate

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7–8 logs of the most resistant spores in 5 min at 20°C; maximum usable temperature is 40°C). Ethylene Oxide

It is a toxic gas and can penetrate porous materials; thus it is used for pre-sterilization of paperboard-based packaging materials (particularly preformed carton blanks which are to be assembled in an aseptic filler). Verification of sterilization is done by inoculation of the surface of the web, cup, or lid stock with the proper concentration of the test organism and allowing this to dry. The system is then run as in a commercial run and the finished containers are filled with an appropriate growth medium and observed for growth. Two of the most important factors affecting the success of the tests are the choice of the indicator organisms and the physical state of the microorganisms used. The indicator organisms used are: B. stearothermophillus strain 1518 (for superheated steam, peroxide steam, and extrusion), B. polymyxa PSO (for dry heat), B. subtilis strain A (for peroxide UV), C. sporogenes PA 3679 (for ethylene oxide), and B. pumilus (for gamma radiation). 4.

Heat Exchange Equipment and their Suppliers

The various types of heat exchangers used in heating and/or cooling products include plate, tubular, scraped surface, microwave, radio frequency, and ohmic heating devices. Each of these devices has their advantages, disadvantages, and range of applicability. Some of the systems that handle the liquid and solid portions of a particulate food separately are the Jupiter system, rotaholder, and the fluidized bed system (7).

a. Plate Heat Exchanger Numerous companies produce plate heat exchangers. Some of the well-known major suppliers of integrated aseptic processing lines including plate heat exchangers are Tetra Pak Inc (Vernon Hills, IL, USA), Alfa Laval, Inc. (Glen Allen, VA, USA), FMC FoodTech (FranRica, Madera, CA, USA), Waukesha Cherry-Burrell (Delawan, WI, USA), Invensys/APV (New York, NY, USA), and Stork Food and Dairy Systems (Gainesville, GA, USA). b. Tubular Heat Exchanger In addition to companies listed above, there are several companies producing Tube in tube and tube in shell heat exchangers. They are Feldmeier Equipment Inc. (Syracuse, NY, USA) and Rossi & Catelli (Parma, Italy). c. Helical Heat Exchanger VRC Co. Inc. (Cedar Rapids, IA) and GEA-AG (Bochum, Deutschland). d. Steam Injection and Steam Infusion Unit Most of the major heat exchanger producer companies listed under plate heat exchanger also offer steam injection direct heaters in various configurations.

e. Scraped Surface Heat Exchanger (SSHE) Major suppliers of scraped surface heat exchanger equipment are the same equipment companies providing the integrated aseptic processing systems. f. Continuous Flow Volumetric Heaters Radio Frequency Heater

Radio Frequency Co., Inc. Millis, MA. Continuous Flow Ohmic/Electro Heater

Invensys/APV (New York, NY, USA) and Raztek (Sunnyvale, CA, USA). Continuous Flow Microwave Heaters

Industrial Microwave Systems (Morrisville, NC, USA), Keam Holdem Associates (Auckland, NZ), and Armfield Limited (Ringwood, England).

D. PACKAGING DETAILS 1. Types of Packaging Systems Modern aseptic packaging units for foods cover an extensive range of materials, shapes, and sizes. For individual and family-size packages, traditional paperboard laminate package types are still the dominant form, with reclosable lids and pouring spouts introduced more recently. Some of the well known companies providing the packaging equipment and materials are Tetra, SIG Combibloc and Elopak. These types of packages — thermoformed polymer cups and pots with peel-off laminate layer tops and, depending on package size, re-closable lids are also well known and on the market for a considerable time — the packaging units and materials are provided by Robert Bosch Corporation. Individual serving size bottles with re-closable snap-on or screw-on lids made of high density polyethylene, polyethyleneterepthalate, or other polymers have also been introduced to the market by companies like Sidel, Krones AG, and Stork. Production of fluid products and beverages aseptically packaged into conventional packages such as glass bottles and aluminum cans has also been increasing during the last several years. Multi-layer laminate bags and pouches in forms from single serving mini-pouches, stand-up pouches and bags fitted with various pouring and dispensing closures and spouts are also on the market. Small 2–3 oz packages to 1–3 gallon bag and bag-in-box package types for institutional use to 50–60 gallon bags in boxes for industrial ingredient and raw material use are provided by LiquiBox, Rapak, Astepo, Scholle, etc. Aseptic packaging systems are generally classified into the following categories.

a. Can Systems Pioneered by Martin in the late 1940s, the first system was commissioned by Dole Corp. (CA) in 1950 for soups. It uses superheated steam at 225°C for up to 40 s to sterilize can and ends, with temperature not to exceed 232°C since


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tin flow underneath the enamel can occur, resulting in blister formation during seaming of the lid (the lining compound is still at ⬃220°C and is plastic; thus, the seamed can should be transported in vertical position for at least 15 s after seaming to allow compound to settle and hermetically seal the can). For composite cans consisting of a spirally wound body made from laminations of foil, plastics, and paper with metal ends, hot air at 143°C for 3 min is used to sterilize the packaging materials as steam would cause swelling of the paper layers. The Dole, Serac, and Remy systems are examples of systems that fall under this category.

Impaco, DuPont, Prodo-Pak, and Thimmonier systems fall under this category.

b. Bottle Systems

d. Cup Systems

Glass

Preformed Plastic Cups

Saturated steam or dry heat (when dry heat is used, extended cooling by sterile air is required to minimize thermal shock when cool product is filled in it; no commercial unit, yet).

The cups are fed onto a conveyor which is inside a sterile tunnel supplied with sterile air. The cups are sprayed with hydrogen peroxide and after about 3 s, the solution is removed with compressed hot air at ⬃400°C with the inside surface of the cups reaching ⬃70°C which completes the surface sterilization and reduces the peroxide residue to acceptable levels. The cups are then filled and sealed with aluminum foil (sterilized by peroxide with residue removed by heat) with a thin coating of a thermoplastic to provide heat sealability. The Metal Box, Gasti, Crosscheck, Hamba, Ampack, and Remy systems fall under this category.

Plastic

Non-sterile bottles: After blowing, the plastic bottles are conveyed into a sterile chamber which is kept at a slight over-pressure of sterile air. The bottles are inverted and sprayed inside with hydrogen peroxide and passed through a hot air tunnel to evaporate the residual peroxide. The bottles are rinsed with sterile water and then filled. A chemically sterilized, heat sealable closure such as a plastic film or cap is then applied. Sterile Blown Bottles

Bottles are extruded, blown with sterile air, and sealed under conditions that ensure internal sterility of the container. The sealed bottles are introduced into a sterile chamber (maintained at a slight positive pressure) where the outside surfaces are sterilized by hydrogen peroxide sprays. The closed top of the bottle is cut away, the neck trimmed, the bottle filled, and a foil cap or heat sealable sterile closure applied. Single Station Blowing, Filling, and Sealing

This is a complex system. The separate operations of parison extrusion, blow molding, bottle filling, and sealing all take place in sequence in a single mold. Sterility of the inside surface of the container is ensured by the high temperature (164 to 234°C) of the plastic material during extrusion of the parison, and the use of sterile air for blowing. After filling, the tube projecting from the bottle mold is vacuum-formed or sealed with jaws into a cap which closes the bottle. No special arrangements to ensure sterility are required since the filling and sealing are carried out within the closed mold.

c. Sachet and Pouch Systems Form-Fill-Seal Systems

A vertical form-fill-seal machine operates in a sterile chamber. The packaging material is passed through hydrogen peroxide and then drained and dried. The Asepak,

Layflat Tubing

This system uses a blown film polymer in the form of a layflat tubing so that only a transverse seal is required to form the bag. It is assumed that the inside of the tubing is sterile due to the temperature achieved during the extrusion process. The tubing is fed from the reel into a sterile chamber in which an over-pressure of air is maintained. The sachets are sealed at the bottom, cut, and moved into a filling station. After filling, they are sealed at the top and leave the chamber through a water seal.

Form-Fill-Seal Cups

The plastic material (usually polystyrene) in the form of a web is fed from a roll into a thermoformer. Sterilization of the web is done prior to forming using a hydrogen peroxide bath. It then passes through a tunnel where it is heated to 130–150°C to prepare it for thermoforming. Mechanical force and compressed air is used to form the container in a water-cooled mold below the web.

e. Carton Systems Form-Fill-Seal Cartons

The packaging material is supplied in rolls which have been printed and creased (for ease in the forming process). A polyethylene (PE) strip is sealed to one edge and the packaging material sterilized using a wetting system or a deep bath system. The sterilized packaging material is fed into a machine where it is formed into a tube and closed at the longitudinal seal by a heat sealing element. In this process, the PE which was added prior to sterilization is heat sealed across the inner surface of the longitudinal seal to provide protection of the aluminum and paperboard layers from the product which could corrode or swell the layers if such a strip were absent. Product is then filled into the tube and a transverse seal made below the level of product, thus ensuring that the package is completely filled. The TetraPak and International Paper systems fall under this category. Prefabricated Cartons

In this method, prefabricated carton blanks are used, the cartons being die-cut, creased, and the longitudinal seam

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completed at the factory of origin. The cartons are delivered to the processors in lay-flat form, ready to be finally shaped in the filler and the top and bottom seams formed and bonded. Stacks of blanks are loaded into a magazine from which they are individually removed by suction pads, opened up into a rectangle, and placed on a mandrel. PE at the bottom of the carton is softened by hot air. The bottom is then folded by transverse and longitudinal folders and sealed. The top is then pre-folded. All of this takes place in a non-sterile zone. The inside surface is then sterilized by peroxide in a sterile zone (over-pressure of sterile air). The carton is then filled, closed, and heat sealed.

f. Bulk Packaging Systems Metal Drum

Two major systems are in use and both use a 55 gallon metal drum constructed from steel with an electrolytically coated tin lining outside. The ends are double-seamed onto the body of the drum during manufacture and filling takes place through a threaded hole in which a cap is swaged after filling. The Scholle, FranRica, and CherryBurrell systems fall under this category. Bag-in-Box

In this system, the product is filled into a plastic bag which when full is put into an outer container such as a drum or a paperboard box. For large containers, filling occurs after the bag is placed in the box. The Scholle, FranRica, Liquibox, and ELPO systems fall under this category. Further details of aseptic packaging systems are described in reference (8). In addition, details of some of the “FDA-accepted” low-acid packaging systems have been presented in reference (9). 2. Testing of Package Integrity Testing of the integrity of aseptic packages is usually done by one of the following methods.

a. Destructive Methods Teardown

The flaps of the package are unfolded and pressure applied to the package to check the tightness of the transverse seals. The quality of the transverse and longitudinal seals is determined by carefully pulling apart the seals — if the seal is good, the PE layers will be removed and the aluminum foil laid bare in the sealing zone. Electrolytic Test

This test is based on the principle that a tight plastic container is an electrical insulator. By introducing an electric potential across a brine-filled package which is partially immersed in a brine solution, the existence of holes in the package can be determined. Positive tests are generally followed by a dye test for confirmation.

Dye Test

After rinsing with water and drying, a solution of 0.5% Rhodamine B in isopropanol is applied to the critical areas of the package including the longitudinal and transverse seals. The carton is then allowed to develop for 5 minutes and dried in a warm cabinet overnight. The flaps of the package are unfolded and the dye coated paper removed and examined for ink penetration. Any sign of the pink ink indicates the presence of holes in the PE layers.

b. Nondestructive Methods These include visual inspection, computer-aided video inspection, and automatic profile scanning. c. Biotest Methods The package is filled with a nutrient broth, sealed, and placed in contact with a medium infected with a test organism. After contact for a certain period of time, the package is placed in an incubator and microbial growth is assessed after an appropriate period of time.

E. ISSUES RELATED TO PROCESS VALIDATION Thermal process validation involves three stages: process establishment, lethality assurance, and record keeping. Process establishment involves considerations related to product formulation and properties, initial temperature, container size and shape, location of thermocouple, critical point, container stacking, retort controls, and steam and water controls. Lethality assurance involves comparison of the actual lethality delivered to that based on the scheduled process. Record keeping involves maintenance of full records of the process history of all production runs which contain the details of all critical factors related to the scheduled process. In aseptic processing, biological validation tests are performed at various stages of the process — just after start-up, during the middle of the run, and just before shut-down. These tests account for variations during the process and also for factors such as fouling. The validation tests are conducted at different temperatures to document a positive/negative result at the end of the process. This will aid in determining the minimum allowable process temperature that will result in a safe process. Microbiological validation tests are done using PA 3679 inoculated within alginate particles. Care should be taken to ensure that the spores do not leach out into the fluid. If the target for the process was a 5D process, and an initial load of 105 spores per particle is used, a final count of 1 would indicate a safe process. The decimal reduction time of the organisms used is determined by means of thermal death time studies. Based on all of these tests, a process is designed and finally verification of the established process has to be conducted. During this process of verification, comparisons are made between actual temperatures and lethalities to the predicted temperatures and lethalities in


order to ensure that the mathematical model developed (details of mathematical models have been provided by (3)) results in a conservative prediction of process lethality. Once verification is successful, all the process and system parameters are noted down and care should be taken to ensure that these parameters remain within an acceptable range. Some of the parameters include hydration time, mixing/batching time, temperatures at various locations, product flow rate, back pressure, and product properties. The final step in commercialization of the product involves process filing with the FDA using form 2541c. A comprehensive overview of the procedures and processes involved in process filing for a product such as the one discussed above has been given in a report prepared based on the workshops organized by the Center for Advanced Processing and Packaging Studies (CAPPS) and the National Center for Food Safety and Technology (NCFST) — (10) and the detailed discussion of the form has been presented in reference (3). At pH values below 4.6, processes are aimed at controlling the survival and growth of spore-forming organisms such as B. coagulans, B. polymyxa, B. macerans, and butyric anaerobes such as C. butyricum and C. pasteurianum, but not C. botulinum. An F0 0.7 min is generally regarded as adequate for this. NFPA suggests F 8.3 93.3 10 min when pH is between 4.3 and 4.5; F 8.3 5 min 93.3 when pH is between 4.0 and 4.3. Below a pH of 3.7, processors are concerned with the control of non-sporing bacteria, yeasts, and molds. They can be generally controlled by heat processes with temperatures below 100°C. Aseptic process validation requirements differ depending on the country of marketing and distribution and product type being marketed. In North American countries, requirements and procedures are regulated by government regulatory agencies like the Food and Drug Administration in the U.S. In most other countries, validation procedures and requirements are typically defined and implemented by the food producers. In either case, the producers need to design and implement a treatment that imparts the degree of sterility to the least treated food segment sufficient to inactivate all microorganisms of public health significance and sufficiently reduce the risk of product spoilage by the more resistant spoilage-related microorganisms. Unlike in European countries (11, 12) where regulations are based on spoilage tests, the FDA requires microbiological tests to prove the safety of a process with sufficient latitude for variability in process conditions. In the U.S., different regulatory agencies and rules apply to different products. For example, UHT milk processing is covered under title 21 (parts 108, 113, 114) of the code of federal regulations (CFR). The process should also adhere to the pasteurized milk ordinance (PMO). When meat is involved, the regulations are imposed by the USDA. In addition to these regulations, certain states have state regulations imposed on certain processes. During the past

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few years, HACCP has gained tremendous importance and its implementation has been extended by the FDA to various products after its initial application to certain acidified and low-acid canned foods. The details of the requirements of a HACCP program are described in detailed in 21 CFR 113, 114. In addition, details of the evaluation of a HACCP program for a multiphase food product aseptically produced has been presented in reference (3). Shelf-stable low-acid food products constitute a special case since the conditions of storage and chemical composition are conducive to the growth and toxin formation by various strains of Clostridium botulinum, microorganisms capable of producing one of the most potent toxic substances known, the causing agent of potentially fatal botulism poisoning. Processes for treating these types of products need to be designed and validated to consistently deliver a 12D reduction of spores of the most resistant proteolytic strains of C. botulinum. For homogeneous materials like beverages, dairy products, purees, homogenates, and clear and smooth soups, procedures and methodology to establish and validate these types of processes are well known and established. General FDA Requirements for Establishment of Registration, Thermal Process Filing, and Good Manufacturing Practices for Low-Acid Canned Foods and Acidified Foods are covered in 21 CFR 108, 21 CFR 110, 21 CFR 113, and 21 CFR 114. These and other listed regulations and forms are also accessible through contact with FDA directly or from their web site. Aseptically processed low-acid particulate products present a formidable challenge to the processor in terms of the ability to design, document, and validate a process that will deliver adequate treatment to the fastest moving, slowest heating particle within a continuously processed system. Three decades of intensive research and development by numerous researchers and engineers have been invested in the development of knowledge methodology and a technology database to perform these documentation and validation studies to meet the regulatory agency requirements. Typical thermal process design for aseptically processed liquid foods, aseptically processed fluid homogeneous foods, and aseptically processed low-acid heterogeneous foods containing discrete particulates have been presented in reference (9). A series of industry-university-government workshops on aseptic processing of multiphase foods in 1995 and 1996, sponsored by the National Center for Food Safety and Technology in Chicago and the Center for Advanced Processing and Packaging Studies at North Carolina State University in Raleigh, resulted in a publication of the “Case study for condensed cream of potato soup from the aseptic processing of multi-phase foods workshop” (10). As a result of these workshops, criteria for demonstrating a safe process for aseptic particle-containing foods were established (13). Within one year, Tetra Pak Inc., in

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conjunction with the NFPA, developed the necessary data required for a low-acid canned foods filing for cream of potato soup, which was accepted by the FDA in May 1997. After this first successful filing, no further filings are known that were submitted to FDA and no aseptically processed and packaged low-acid products are known to be on the North American market. This illustrates the still-prevailing hurdle of regulatory requirements and the lack of appropriate validation techniques as one of the major remaining obstacles to wider commercialization of multiphase aseptic products and aseptically processed foods in general. As a result, there is an abundance and wide variety of fluid and homogeneous semi-fluid aseptically processed products on the market, but low-acid products containing particles, in spite of the obvious advantages of aseptic processing technologies to their quality and distribution, remain limited world-wide and absent on the North American market. Further development of technologically more sophisticated, but easier to implement validation methods and tools is expected to reduce this last remaining obstacle to aseptic technology penetration and implementation into a wider range of processed food products and biomaterials.

REFERENCES 1. Nielsen, S., Marcy, J.E., Sadler, G.D. Chemistry of aseptically processed foods. In Principles of aseptic processing and packaging edited by Chambers, J.V., Nelson, P.E. pp. 87–114, 1993. 2. Van Eijk, A. Flavorings for UHT-treated and aseptically packed soups and sauces. In Aseptic processing of foods edited by Reuter, H. Technomic Publishing Company. pp. 145–152, 1993.

3. Sastry, S.K., Cornelius, B.D. Aseptic processing of foods containing solid particulates. Wiley-Interscience. pp. 4–67, 86–129, 194–219, 2002. 4. Simpson, S.G., Williams, M.C. An analysis of high temperature short time sterilization during laminar flow. Journal of Food Science. Vol. 39: 1047–1054, 1974. 5. Sandeep, K.P., Puri, V.M. Aseptic processing of liquid and particulate foods. In Food processing operations modeling: Design and analysis, edited by Irudayaraj, J. Marcel Dekker, Inc. pp. 55–68, 2001. 6. Lewis, M., Heppell, N. Fouling, cleaning, and disinfection. In Continuous thermal processing of foods: Pasteurization and UHT sterilization. Aspen Publishers, Inc. pp. 331–368, 2001. 7. Willhoft, E.M.A. Aseptic processing and packaging of particulate foods. Blackie Academic and Professional. pp. 6–7, 1993. 8. Reuter, H. Aseptic packaging of food. Technomic Publishing Company, 1989. 9. David, J.R.D., Graves, R.H., Carlson, V.R. Aseptic processing and packaging of food: A food industry perspective. CRC Press. pp. 224–246, 1996. 10. Anonymous. Case study for condensed cream of potato soup from the aseptic processing of multi-phase foods workshop. Published by the National Center for Food Safety and Technology, Chicago, IL and the Center for Advanced Processing and Packaging Studies, Raleigh, NC, 1996. 11. Rose, D. Guidelines for the processing and aseptic packaging of low acid foods. Part I: Principles of design, installation and commissioning. CCFRA Technical Manual 11, 1986. 12. Rose, D. Guidelines for the processing and aseptic packaging of low acid foods. Part II. CCFRA Technical Manual 11, 1987. 13. Damiano, D., Digeronimo, M., Garthright, W., Marcy, J., Larkin, J., Sastry, S.K. Workshop targets continuous multiphase aseptic processing of foods. Food Technology. 51(10): 43–62, 1997.

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Food Irradiation Using Electron-Beam Accelerators

Rosana G. Moreira

Department of Biological and Agricultural Engineering, Texas A&M University

CONTENTS I. Introduction ........................................................................................................................................................124-1 II. Source of Ionizing Radiation ..............................................................................................................................124-1 A. Cobalt-60 ....................................................................................................................................................124-2 B. Cesium-137 ..................................................................................................................................................124-3 C. Electron Beam Accelerators ........................................................................................................................124-3 1. Van de Graaff Accelerator ....................................................................................................................124-3 III. Effect of Ionizing Energy on Food ....................................................................................................................124-4 A. Basic Interaction of Photons and Electrons with Matter ............................................................................124-5 B. The Effect of Ionizing Radiation on Microorganisms ................................................................................124-7 C. Chemical and Nutritional Changes in Foods ..............................................................................................124-7 D. Low Dose Irradiation for Surface Pasteurization of Fresh Produce ..........................................................124-7 IV. Dose Distribution Determination in Food Products ............................................................................................124-8 V. Conclusions ........................................................................................................................................................124-8 References ....................................................................................................................................................................124-8

I. INTRODUCTION Food irradiation involves exposing the food (packaged or in bulk) to controlled amounts of ionizing radiation to achieve certain desirable objectives. The technology has recently been identified by the Food and Drug Administration (FDA) and World Health Organization (WHO) as having significant strategic importance for the future of food safety worldwide. Food irradiation has been around for over 60 years and can offer a number of potential benefits including inactivation of microorganisms, inhibition of many enzymatic processes (such as those that cause sprouting and ripening), in addition to the fact that it can be used as an alternative to chemical treatment for disinfestations. The recent progress in the development of electron beam accelerators, together with the increased number of illnesses associated with produce-related foodborne disease outbreaks in the last years, provide the incentive for the development of an efficient technique to ensure hygienic quality of food products, especially those to be consumed raw or undercooked, to protect consumer health.

It is estimated that up to 81 million people a year are infected by foodborne illness, and of that number, 10,000 die (1). With an estimated 25% of all food production lost after harvesting to insects, bacteria, molds, and premature germination and the potential for continued rise in food poisoning incidence, irradiation could be used more widely to improve the quality and safety of the food supply in the future.

II. SOURCE OF IONIZING RADIATION The type of radiation used in processing materials is limited to high energy gamma-rays, X-rays, and accelerated electrons. These radiations are also referred to as ionizing radiations because their energy is high enough to dislodge electrons from atoms and molecules and to convert them to electrically charged particles called ions. Gamma-rays and X-rays, like radiowaves, microwaves, ultraviolet, and visible light rays, form part of the electromagnetic spectrum, occurring in the short wave length, high energy region of the spectrum. Their properties and effects on materials are 124-1

124-2


the same, but their origins are different. In most cases, X-rays with varying energies are generated by machines. Gamma-rays with specific energies come from the spontaneous disintegration of radionuclides. Only certain radiation sources can be used in food irradiation. These are the radionuclides cobalt-60 (60Co) or caesium-137 (137Cs); X-ray machines having a maximum energy of 5 MeV, or electron machines having a maximum energy of 10 MeV. Energies from these radiation sources are too low to induce radioactivity in food. As shown in Figure 124.1, gamma-rays and X-rays radiation can penetrate distances of a meter or more into the product, depending on the product density, whereas electrons, even with energy as high as 10 MeV, can penetrate only several centimeters.

A. COBALT-60 Naturally occurring and man-made radionuclides, also called radioactive isotopes or radioisotopes, are unstable, and emit radiation as they spontaneously disintegrate, or decay, to a stable state. The Becquerel (Bq) is the unit of radioactivity and equals one disintegration per second. The radionuclide used almost exclusively for the irradiation of food by gamma rays is 60Co. Until 1993, food

irradiation in the United States occurred exclusively from the use of 60Co (2). It is produced by neutron bombardment, in a nuclear reactor, of the metal 59Co, and then doubly encapsulated in stainless steel “pencils” to prevent any leakage during its use in a radiation plant. 60Co has a half-life of 5.3 years. The emitted gamma rays are photons with very short wavelengths, similar to ultraviolet light and microwaves but with much higher energies. Because gamma radiation does not elicit neutrons (i.e., the subatomic particles that can make substances radioactive), irradiated foods and their packaging are not made radioactive (3). Since 1960, the worldwide use of 60Co has resulted in the accumulation of a vast and successful record of safety and reliability (4). One economic factor related to 60Co is the fact that its supply is limited and is practically a monopoly of Nordion Inc. (Ontario, Canada). Prices have increased and a sufficient future supply is questionable. A significant increase in demand would require the development of increased nuclear reactor capabilities. In a typical gamma radiation facility, the radioactive material (137Cs or 60Co) is placed at the top of an elevator that can be moved up for use or down under water when not in use. Materials that need to be irradiated are placed around the radioactive material at a suitable distance for

Gamma rays Source pencils

Electron

X-rays

FIGURE 124.1 Typical sources used for food irradiation.

Target


their desired dose. Among the drawbacks to the use of radioactive material is that the isotope source emits rays in all directions and cannot be turned “on” or “off ” (4).

B. CESIUM-137 137

Cs is the only other gamma-emitting radionuclide suitable for industrial processing of materials. It can be obtained by reprocessing spent, or used, nuclear fuel elements and has a half-life of 30 years (6). The proposed use of 137Cs as a radiation source dates from the early 1970s. It was based on the availability of vast quantities of unprocessed and encapsulated 137Cs from US Government’s stock of byproducts from nuclear energy and nuclear weapon production programs. By 1988, the Department of Energy canceled the program and the option of using 137Cs as an ionizing radiation source for food safety was eliminated (5).

C. ELECTRON BEAM ACCELERATORS Some machine sources of radiation are suitable for irradiating certain materials. High energy electron beams can be produced from machines capable of accelerating electrons (accelerators). Electrons cannot penetrate very far into food, compared with gamma radiation or X-rays. However, X-rays can be produced when a beam of accelerated electrons bombards a metallic target. Although X-rays have good penetrability into food, the efficiency of conversion from electrons to X-rays is generally less than 10% (7). Electron accelerators offer certain advantages over radioactive elements, which make them more attractive for industrialization: (1) The efficiency for direct deposition of energy, (2) the easy variability of electron-beam current and energy to provide flexibility in the choice of surface and depth treatments for a variety of food items, and (3) the ease with which an electron accelerator can be turned off or on. There are two main differences between gamma rays and accelerated electrons. First, gamma penetration is higher than accelerated electrons, but the penetration capacity of the latter increases with their energy. Electrons at 10 MeV are more penetrating than those at 4 MeV. Second, the gamma dose rate from a typical 60Co irradiator is 1–100 Gy/min, whereas electron beams from an accelerator can produce 103–106 Gy/sec (5). A particle accelerator delivers energy to a chargedparticle beam by the application of an electric field. Acceleration of charge particles can be divided in two categories: electrostatic and electromagnetic acceleration. An electrostatic accelerator consists basically of two conducting surfaces with a large voltage difference and a particle with charge gains kinetic energy. The peak energy of the beam is limited by the voltage that can be sustained without breakdown (8). The Van de Graaff accelerator is an electrostatic accelerator and will be described below since it is the electron beam source simulated in this work.

124-3

Electromagnetic fields are required in order to obtain energies above a few million electron volts. Electromagnetic accelerators can be resonant or nonresonant. Nonresonant are pulsed and are essentially step-up transformers, with the beam acting as a high voltage secondary. Resonant means that electromagnetic oscillations in resonant cavities or waveguides are used to transform input microwave power from low to high voltage. There is also a close coupling between properties of the particle orbits and time variations of the accelerating field. The category of resonance accelerators includes the linac, cyclotron, and synchrotron. Linac is an abbreviation for linear accelerator and the charged particles moves on a linear path and are accelerated by time dependent electromagnetic fields. Likewise, the particle orbit in a cyclotron is a spiral and a circle for a synchrotron (8). 1. Van de Graaff Accelerator Mechanical transport of electric charges to the inner surface of a hollow electrode underlies the operation of electrostatic generators (Figure 124.2). In this type of accelerator a corona discharge from an array of needles in gas is used as the source of electrons. The electrons drift toward the positive electrode and are deposited in a moving belt. The belt, which is composed of an insulating material with high dielectric strength, is immersed in insulating gas at high pressure. The attached charge is carried mechanically

HV terminal

Support insulator Belt

+ HV plate Corona needles FIGURE 124.2 Schematic of the Van de Graaff accelerator.

124-4


against the potential gradient into a high-voltage terminal. The terminal acts as a Faraday cage and there is no electric field inside the terminal other than that from the charge on the belt. The charge flows off the belt when it touches a metal brush and is deposited on the terminal. The energy to charge the high-voltage terminal is supplied by the belt motor. High-voltage terminals are usually constructed as large, smooth spheres to minimize peak electric field stress. The current available to drive a load (such as an accelerated beam) is controlled by either the corona discharge current or the belt speed. Typical currents are in the range of 10 µA. Van de Graaff accelerators are excellent research tools since they provide a steadystate beam with good energy regulation (8). Electron beam facilities, widely used to irradiate medical equipment, have been built for food treatment. A conveyor or cart system moves the product to be irradiated under the electron beam at a predetermined speed to obtain the desired dosage. The products move in and out the irradiation area continuously. Energy penetration is about 3.86 cm in food products, so the thickness of items to be treated is limited to about 7.62 cm with double-sided treatment (9). X-rays are electromagnetic radiation produced when energetic electrons hit a target. In an X-ray machine, the electrons are emitted by a heated cathode whose potential may be the order of 30 to 50 kV above the target (made of a material such as tungsten or molybdenum). For food irradiation electrons from a linear accelerator operating at 5 MeV hit a target to make X-rays and then treat the food. The early design of accelerator systems provided poor penetration (produced only low energies, ⫺2 MeV), were difficult to control, and were unreliable. So, the irradiation market was taken over by 60Co (gamma). During the 1970s, several companies, including Varian Associates, Phillips, and Siemens, took a new look at the application of X-ray technology for radiographic and oncology therapy equipment (10). Their involvement in the improvement of durability and reliability of accelerated electron technology raised performance parameters to a new level (11). Today, industrial e-beam accelerators are characterized by (11): ●

● ●

Higher energy (10 MeV) and thus better penetration High duty cycles (7,000–8,000 hours/year) Fully automated electronic control systems featuring programmable logic controllers (PLCs)

The applications of electron beam processing have then increased substantially in the last decades and are still growing. A comparison among the three basic industrial electron accelerators is shown in Table 124.1 (10): (1) Direct Current machines (DC); (2) Rhodotrons, and (3) Pulsed and Continuous Wave Linear Accelerators (CW LINAC).

TABLE 124.1 Industrial Electron Accelerator Parameters Energy Max beam power Efficiency Duty factor Dimensions Approximate cost 1

Direct Current1

Rhodotron2

CW LINAC3

10 MeV 50 kW

10 MeV 200 kW

10 MeV 500 kW

⬍30% 5% ⬃1.0 ⫻ 1.0 ⫻ 4.0 m3 ⬃$2 M

38% 100% ⬃2.9 ⫻ 2.2 m2

40% 100% ⬃0.8 ⫻ 0.8 ⫻ 9.0 m3 ⬃$1.5 M

⬃$4 M

(25) 2 (24) 3 (10).

A Direct Current machine is a linear accelerator that operates at 50 kW to produce up to 10 MeV beams. In a linear accelerator particles move in a linear path and are accelerated by time-dependent electromagnetic fields. The Rodotron technology utilizes a coaxial accelerating cavity of 2 meters in diameter. 10 mA of electrons are sent into the cavity and undergo a first acceleration of 0.5 MeV. Electrons pass through an opening and then emerge into the second part of the cavity, as the electrical field is reversed, they gain once more 0.5 MeV. Around the cavity, window-frame magnets are bending electrons back into the cavity for further acceleration steps. Ten successive crossings would be required to obtain 10 MeV beams (12). A LINAC may be operated continuously, which is called a continuous-wave (CW) operation or may also be pulsed operated. If the accelerated beam current is small, most of the power in CW operation is not delivered to the beam but is dissipated in the structure walls. If the accelerator is pulsed operated, a larger fractional power is delivered to the beam, and the efficiency is improved (13). Low-energy applications with electron beams up to ⬃1 MeV are adequately served by DC machines. These accelerators have high beam power and plug efficiency, but are bulky, thus being difficult and costly to handle. Above ⬃3 MeV, the CW LINACs have the lowest beam power cost of any commercial accelerator (10). Electron beam irradiator facilities require shielding and product handling equipment similar to 60Co facilities, although some shielding requirements may be reduced due to the directionality of radiation fields generated by machine sources. In addition, semi permanent facilities may also be developed, because electron beam accelerators can be made transportable (4).

III. EFFECT OF IONIZING ENERGY ON FOOD Regardless of the source, the effect of ionizing energy on food is identical. Energy penetrates the food and its packaging but with X-rays and gamma-rays most of the energy simply passes through the food, similar to the way


124-5

E

E

Rayleigh scattering

e

E

Photoelectric

E+

E′ E

E

Ee Compton

E− Pair production

FIGURE 124.3 Basic interaction of photons with matter.

microwaves pass through food, leaving no residue. Most of the energy that does not pass through the food is converted to heat, but some produces DNA strand breaks that inactivate bacteria. E-beam accelerators work on the same principle as a television tube. Electrons are emitted from a cathode and accelerated by an electric or magnetic field in a vacuum. Instead of being widely dispersed and hitting a phosphorescent screen at low energy levels, the electrons in the accelerator are concentrated and accelerate to higher energy, approximately 99% of the speed of the light. The electrons then pass through a thin metal foil and enter air at normal pressure. Irradiation efficiency depends on both the accelerator characteristics and irradiation technique, as well as on a number of factors including the type of material, its geometric dimensions, its shape, the packaging material, etc. Processing capacity (kg/h) is directly related to the beam power (kW) and system efficiency, and inversely related to the dose. The dose unit in the system is the Gray (Gy) — the dose at which 1 Joule of energy is absorbed in each kilogram of substance; the dose rate (Dr) is expressed most often in kGy/s; the total dose absorbed by the irradiated material is directly related to the dose rate and the irradiation time. The dose rate depends on the current (I, mA), the crosssection area (A, m2) of the irradiation field, over which the electron is scanned, and the energy gradient or the stopping power (T, MeV/g/m2) of the electrons. In general, the dose rate increases with energy and beam current and decreases as the distance between the accelerator and the irradiated material increases.

The electron range, the distance the electron will penetrate, is inversely proportional to the density of the material being irradiated. The useful range can be increased by 2.5 times by using two-sided irradiation rather than one-sided irradiation (14). In the case of food materials, if radiation is done from one side only a detector can be placed in the back of the material to monitor beam penetration and dose rate.

A. BASIC INTERACTION OF PHOTONS AND ELECTRONS WITH MATTER Photons (gamma rays and X-rays) are electrically neutral and do not steadily lose energy as they penetrate food materials. Instead, they can travel some distance before interacting with an atom. Penetration depth of a given photon depends on the specific medium traversed and on the photon energy. When photons interact with matter, they might be absorbed and disappear or be scattered, changing direction of travel, with or without energy loss (Figure 124.3). By contrast, a charged particle (electron), being surrounding by its Coulomb electric force field, interacts with one or more electrons or with the nucleus of practically every atom it passes (Figure 124.4). Thus, it is convenient to think of the particle as losing kinetic energy gradually in a friction-like process, often referred to as the “continuous slowing-down approximation” (CSDA). In general, electrons have much less penetration power than gamma and X-rays. Because of the small mass and single negative charge, each time an electron approaches a target, it is deflected from its path by the orbital electrons and the positive atom nuclei. For those reasons, electrons have a poorer penetrability compared to gamma and X-rays.

124-6


E

E−

E

E

Es = W-Ui Elastic

Inelastic

E+

W

E

+

E E−

E− Positron

Bremsstrahlung

FIGURE 124.4 Basic interaction of electrons with matter.

4.32 30 25

2.16

20 0.00

15 10

−2.20

5

(a)

−4.41 −5.04

−2.52

0.00

2.52

5.04

4.32 30 25

2.16

FIGURE 124.6 Source direction in an apple.

20 0.00

15 10

−2.20

5

(b)

−4.41 −5.04

−2.52

0.00

2.52

5.04

FIGURE 124.5 (a) 1 MeV photons incident in an apple; (b) 1 MeV electrons incident in an apple.

Figures 124.5a and 124.5b show the results of a simulation of the energy deposition of 1 MeV photons and electrons, respectively, in an apple with tilted source direction (Figure 124.6). The simulated results clearly show that the absorbed energy is distributed into the entire apple when 1 MeV photons is used as the energy source (Figure 124.5a). However, electrons with the same energy only penetrate about 5 mm of the apple (Figure 124.5b). For surface pasteurization of fruits and vegetables, electrons beams are the preferable source of treatment.


B.

THE EFFECT OF IONIZING RADIATION MICROORGANISMS

ON

According to the target theory (15), lethality due to ionizing radiation occurs when the irradiated microorganisms are destroyed by the passage of an ionizing particle or quantum of energy through, or in close proximity to, a sensitive portion of the cell. This direct “hit” on the target causes ionization in this sensitive region of the organism or cell and subsequent death. Bacterial spores are more resistant to ionizing radiation than are vegetative cells. Gram-positive bacteria are more resistant than gram-negative bacteria. The resistance of yeast and molds varies considerably, but some are more resistant than most bacteria. According to reference (16), the efficacy of a given dose of irradiation to destroy a microbial population depends on the following: ● ●

●

●

●

●

The kind and species of the organism. The numbers of organisms (or spores) originally present. The more organisms there are, the less effective a given dose will be. The composition of the food. Proteins, catalase, and reducing substances (nitrites, sulfites, and sulphydryl compounds) may be protective. Compounds that combine with the SH groups would be sensitizing. The presence or absence of oxygen. The effect of free oxygen varies with the organism, ranging from no effect to sensitization of the organism. Undesirable “side reactions” are likely to be intensified in the presence of oxygen and to be less frequent in a vacuum. The physical state of the food during irradiation. Both moisture content and temperature affect different organisms in different ways. The condition of the organisms. Age, temperature of growth and sporulation, and state (vegetative or spore) may affect the sensitivity of the organisms.

C. CHEMICAL AND NUTRITIONAL CHANGES IN FOODS In general, the irradiation process produces very little chemical change in food. None of the changes known to occur have been found to be harmful or dangerous. Some of the chemical changes produce so-called “radiolytic” products. These products have proven to be familiar ones, such as glucose, formic acid, acetaldehyde, and carbon dioxide that are naturally present in foods or are formed by heat processing. The United States Food and Drug Administration has estimated that the total amount of

124-7

undetected radiolytic products that might be formed when food is irradiated at a dose of 1 kGy would be less than 3 milligrams per kilogram of food — or less than 3 parts per million (17). Research has shown that protein, carbohydrates, and fat are relatively stable to radiation doses of up to 10 kGy. Different types of vitamins have varied sensitivity to irradiation and to some other food processing methods. For example, vitamins C and B-l (thiamine) are sensitive to irradiation as well as to heat processing. The evidence suggests that irradiation does not induce special nutritional problems in food (18). The change in nutritional value caused by irradiation depends on a number of factors including the radiation dose to which the food has been exposed, the type of food, packaging, and processing conditions (temperature during irradiation). Most of these factors are also true for other food preservation technologies.

D. LOW DOSE IRRADIATION FOR SURFACE PASTEURIZATION OF FRESH PRODUCE Ingestion of raw vegetables and fruits has been linked to outbreaks of food borne illness. Contaminated artichoke, beet leaves, cabbage, carrots, cauliflower, celery, eggplant, endive, fennel, onion, lettuce, mushrooms, potatoes, tomatoes, cantaloupe, watermelon, raspberries, strawberries, apples, etc. have been vehicles for transmission of pathogens (19). The viability of pathogenic organisms on the surface of fresh fruits and vegetables can be significantly reduced by electron beam irradiation. By limiting the irradiation to the surface, changes in the quality of the bulk of the product can be minimized. Electron beams (e-beams) are produced by small accelerators (which do not produce radiation when not in use) and electron energies, which penetrate only a short distance into the tissue, can easily be obtained. The most difficult technical challenge for surface irradiation is the need to achieve a uniform dose over the entire surface. This is particularly difficult if there are deep recesses such as the area of the stem of an apple, or convoluted surfaces such as some type of lettuces. Most of the investigations in the area of e-beam accelerators to irradiate food have dealt with the effect of this energy source on the inactivation of microorganisms and on the product characteristic changes. No information is available in the literature regarding the development of methods for ensuring reliable quality control of the irradiation process on materials with irregular shapes, for example. Methods to continuously monitor the e-beam characteristics, such as the electron energy, electron current, scanner width, scanner uniformity, penetration depth, and conveyor speed need to be evaluated for these types of product.

124-8


IV. DOSE DISTRIBUTION DETERMINATION IN FOOD PRODUCTS In food irradiation, one way to verify dose distribution in an irradiated product is by using photo luminescent dosimeters (20). This dosimeter is a small plastic wafer that is irradiated with the food. The dosimeter documents the dose received so that the process can be controlled to ensure that the product receives doses within regulatory requirements (dose range). Shining blue light on the irradiated dosimeter induces it to emit a red light (fluorescence). The intensity of the fluorescence is proportional to the dose that the food received. A chemical dosimeter using a ferrous sulfate (Frike) solution was used by (21) to calibrate e-beam irradiation dose to different foodstuffs. The dosimeter uses the change in ultraviolet absorption caused by dose-dependent oxidation of ferrous to ferric ions. The homogeneity of the dose delivered to the products was verified with an ionizing chamber on a motor-driven scanning system. The dose can also be predicted by various mathematical methods. Most of the research advances in dose distribution calculations has been in the area of radiation therapy. Although the application and practice of e-beam irradiation techniques vary from one industry to another, engineers tend to learn from each other. Thus, it is worth reviewing some of the radiation applications in the medical industry to learn how to improve the application of this technique to reduce pathogens in the US food chain. One of the most important steps in radiation treatment planning process is the determination of radiation dose distribution in the body. There are two main methods for calculating the dose in the patient: measurement-based empirical methods and model-based methods (22). The empirical approach relies on experiments with dose measurements in a ‘phantom’ (a box filled with water). The measured dose is then slightly corrected to account for actual beam shape, patient shape, and density and composition of the body tissue. Measured-based methods are accurate if the beam shape is a simple square, the surface of material to be irradiated is flat and perpendicular to the beam, and the beam travels through homogeneous soft tissue. This method would not be accurate for a more complex 3D structure of foods since it does not adequately account for their inhomogeneity and the lack of secondary electron equilibrium in and around the typical food object. The model-based methods are then generally used to estimate the dose in a body in radiation treatment planning. Monte Carlo technique is today the most accurate means of dose calculation. Monte Carlo transport simulates the behavior of irradiation particles as they interact with atoms in the body during a typical radiation treatment. Simulation of radiation transport (electrons and photons) by the Monte Carlo method has been used to

simulate radiation treatment machine heads, absorbed dose distribution, energy distributions, and electron-treatment planning (23). As an example, Figures 124.5a,b show Monte Carlo simulation used to determine the dose distribution at the surface of an apple irradiated with e-beam generated by a Van de Graaff accelerator (1–2 MeV). The dose distribution was used to develop the best irradiation angle while rotating an irregularly shaped food material (the apple) for uniform surface irradiation.

V. CONCLUSIONS We know that consumption of food contaminated with pathogens (Salmonella and E. coli, for example) causes serious foodborne illness and even death. Also, foods may be used as vehicles to deliver biological agents to cause disease. The security and safety of our food supply and agricultural production will continue to be topics of widespread international interest in the years to come. Consequently, efforts across the food industry to improve and implement measures to enhance assurances of food safety will always be needed. Progress in electron beam accelerators will continue to further the development of efficient methods to ensure quality and safety of food products, especially those to be consumed raw or undercooked, to protect consumer health. Satisfactory irradiation of foods will be a common goal for producers, processors, government agencies, and consumers around the world. Advances in dosimetry methods in heterogeneous and irregular shaped materials such as foods will provide accurate, precise, and wide range dosimetry data for effective treatment planning of a wide number of food products.

REFERENCES 1. R Tauxe, H Kruese, C Hedberg, M Potter, J Madden, K Wassmuth. Microbial hazards and emerging issues associated with produce: A preliminary report to the National Advisory Committee on Microbiologic Criteria for Foods. J Food Protection 60(11): 1400–1410, 1997. 2. A Chapple. Bye, bye bacteria. Nuclear Energy. 3rd. quarter: 9–12, 1993. 3. JF Diehl. Safety of Irradiated Foods. New York: Marcel Dekker, 1995. 4. M Lagunas-Solar. Radiation processing of foods: An overview of scientific principles and current status. J Food Protection 58(2): 186–192, 1995. 5. JM Jay. Modern Food Microbiology. 5th ed. Gaithersburg, MD: Aspen Publishers, 1998. 6. M Satin. Food Irradiation. Lancaster: Technomic Publishing, 1993. 7. S Throne. Food Irradiation. New York: Elsevier Science Publishers, 1991.


8. S Humphries. Principles of charged particle acceleration. New York: John Wiley & Sons, 1986. 9. ADA (American Diet Association). J American Dietary Association 100: 246–253, 2000. 10. AS Alimov, EA Knapp, VI Shvedunov, WP Trower. High-power CW LINAC for food irradiation. Applied Radiation and Isotopes 43: 815–820, 2000. 11. J Ungrin. Development of accelerators for radiation applications. JL Duggan, IL Morgan, eds. Proceedings of 12th International Conference of Applied Accelerators in Research and Industry, University of North Texas, Denton, TX, November, 1992. 12. D Defrise, M Abs, M Genin, Y Jongen. Technical status of the first industrial unit of the 10 MeV, 100 kW Rhodotron. Radiation Physics and Chemistry 46: 473–476, 1995. 13. TP Wangle. Principles of RF Linear Accelerators. Los Alamos National Laboratory. New York: John Wiley & Sons, 1998. 14. W Scharf. Particle Accelerators and Their Uses. Philadelphia: Harwood Academic Publishers, 1986. 15. AP Cassaret. Radiation Biology. Englewood Cliffs, NJ: Prentice Hall, 1968. 16. WC Frazier, DC Westhoff. Food Microbiology. 4th ed. New York: McGraw-Hill, 1988. 17. FAO. Irradiation in the production, processing and handling of food. Washington, D.C. US Food and Drug Administration, final rule, Federal Register: 55(85): 18538–18544, May 1989. 18. C Merritt. “Radiolitic products — Are they safe?” Safety factors influencing the acceptance of food irradiation technology, IAEA TECDOC, Vienna, 1989.

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19. LD Beuchat, MP Doyle, RE Brackett. Irradiation inactivation of bacterial pathogens in ground beef. Report to the American Meat Institute, University of Georgia, Athens. Center for Food Safety and Quality Enhancement, 1993. 20. RD Hagenmaier, RA Baker. Microbial population of shredded carrot in modified atmospheric packaging as related to irradiation treatment. J Food Science 65: 293–299, 1998. 21. SV Calenberg, G Vanhaelewyn, OV Cleemput, F Callens, W Mondelaers, A Huyghebaert. Comparison of the effect of X-ray and electron beam irradiation on Some Selected Spices. Lebenesmittelwsissenschaft und Technologie 31: 252–258, 1998. 22. CLH Siantar, FI Moses. The PEREGRINE program: using physics and computer simulation to improve radiation therapy for cancer. European Journal of Physics 19: 513–521, 1998. 23. O Andreo. Electron pencil-beam calculations. In: Jenkins, Nelson and Rindi, eds. Monte Carlo Transport of Electrons and Photons. New York: Plenum Press, 1987. 24. Y Jogen, T Delvigne, M Abs, A Herer, JM Capdevila, F Genin, A Nguyen. Rhodotron accelerators for industrial electron beam processing; a progress report. In: S Maier, A Pacheco, R Pascula, CH Petit-Jean-Genaz, J Pool, eds. Proceedings of the 1996 European Particle Accelerator Conference, Institute of Physics, Bristol, 1996, pp. 2687–2692. 25. A J Sterling. Electron beam processing: a new business and a new industry. In: S Maier, A Pacheco, R Pascula, CH Petit-Jean-Genaz, J Pool, eds. Proceedings of the 1996 European Particle Accelerator Conference, Institute of Physics, Bristol, 1996, pp. 272–275.

125

Microwave Heating in Food Processing

Yi-Chung Fu

Department of Food Science, National Chung Hsing University

CONTENTS I. II. III. IV.

Introduction ....................................................................................................................................................125-1 Microwave Heating ..........................................................................................................................................125-1 Definition of Terms and Propagation of Waves ..............................................................................................125-2 Microwave Power Distribution ........................................................................................................................125-3 A. Electric Field Intensity ............................................................................................................................125-3 B. Lambert’s Law ........................................................................................................................................125-3 V. Interaction of Microwave with Food ..............................................................................................................125-4 A. Dielectric Properties ................................................................................................................................125-4 B. Geometrical Heating Effects — Corner, Edge, and Focusing Effects ....................................................125-5 C. Microwave Bumping ..............................................................................................................................125-5 D. Evaporative Cooling and Steam Distillation ..........................................................................................125-5 E. Lack of Crispness (Texture) and Browning (Color, Flavor) of Microwave Foods ................................125-6 F. Food Ingredients ......................................................................................................................................125-6 VI. Microwave Processing ....................................................................................................................................125-7 A. Drying and Dehydration ..........................................................................................................................125-7 B. Pasteurization and Sterilization ..............................................................................................................125-8 C. Tempering and Thawing ..........................................................................................................................125-9 D. Baking ....................................................................................................................................................125-9 VII. Radio Frequency Processing ........................................................................................................................125-10 VIII. Conclusion ....................................................................................................................................................125-11 References ................................................................................................................................................................125-11

I. INTRODUCTION Microwave heating of food has existed since 1949. Growth in the number of homes with microwave ovens, combined with the industrial use of microwaves, has created a large market for microwave-processed foods and, consequently, has changed food preferences and preparation methods and increased the need for research on the behavior of various types of foods during microwave heating. Using microwaves as a source of heat in the processing (thawing, heating, drying, etc.) of food materials is advantageous because it offers a potential for rapid heat penetration, reduced processing times, and, hence, increased production rates, more uniform heating, and improved

nutrient retention. The use of microwaves represents the use of sophisticated technology in the food industry. Lack of sufficient and unified knowledge of this complex and radically different heating process has been the primary contributor to its unpredictability. Emphasis should be on basic research to better understand the interaction between the microwave energy and product. This chapter will provide fundamentals of microwave heating and description of microwave processes in the food industry.

II. MICROWAVE HEATING The temperature of a material can be increased either directly or indirectly. The indirect methods are those in 125-1

125-2


which heat is generated external to the product and is transferred to it by conduction, convection, or radiation. The direct methods are those in which heat is generated within material itself. Dielectric heating is used with electrically non-conducting materials. The material to be heated is placed between two electrodes and forms the dielectric component of a capacitor. Excitation is by means of a high frequency voltage (2 to 100 MHz) applied to the condenser plates. Radio frequency heating, which is at a much lower frequency, has thrived as an industry alongside microwaves over the decades. Radio frequency heating in the United States can be performed at any of three frequencies: 13.56, 27.12, and 40.68 MHz. Microwave heating is a special field of dielectric heating in which very high frequencies (300 MHz to 30 GHz) are applied. Domestic microwave ovens operate at 2450 MHz and industrial processing systems generally use either 2450 MHz or 915 MHz (896 MHz in the UK). A domestic microwave oven is a multimode cavity in which electromagnetic waves form a resonant pattern. As dielectric materials are poor heat conductors, heat applied from the outside by convection, radiation, or conduction is inefficient. In some cases the heat applied causes a skin or crust to form on the outside which is in itself a thermal barrier. The single most important thing about microwave heating is the unique opportunity to create heat within a material — the volumetric heating effect — not achievable by any other conventional means. No temperature differential is required to force heat into the center of the material. Generation of heat within food products by microwave energy is primarily caused by molecular friction attributed to the breaking of hydrogen bonds associated with water molecules and ionic migration of free salts in an electric field of rapidly changing polarity. Substances that respond to and, therefore, can be processed by microwave energy are composed of polar (e.g., water), ionic, or conductive (e.g., carbon black) compounds. Non-polar substances, e.g., polyethylene and paraffin, are unaffected.

III. DEFINITION OF TERMS AND PROPAGATION OF WAVES James Clerk Maxwell (1831–1879) developed the classical theory of electromagnetism and correctly predicted that an electromagnetic wave has associated electric field E and magnetic field H. A uniform plane wave characterized by E ⫽ ax Ex propagating in a lossy medium in the ⫹z-direction has associated with it a magnetic field H ⫽ ay Hy. The solution to be considered here is that of a plane wave, which for the electric field attains the form E(x) ⫽ Emaxejωt⫺γ z

(125.1)

A propagation constant, γ, is defined as

γ ⫽ jkc ⫽ jω兹µ 苶苶 εc

(125.2)

εc ⫽ ε⬘ ⫺ jε⬙ ⫽ ε0(ε⬘r ⫺ jε r⬙)

(125.3)

ε⬘r ⬅ ε⬘/ε0 ; ε⬙r ⬅ ε⬙/ε0

(125.4)

with

where ε0 is the permittivity of free space (8.8542E-12 Farad/m). Hence, all the previous equations for nonconducting media will apply to conducting media if ε is replaced by the complex permittivity εc. The material’s ability to store electrical energy is represented by ε⬘, and ε⬙ accounts for losses through energy dissipation. ε⬘r is often called “relative dielectric constant.” This is somewhat inappropriate, as the term “constant” should be used only for true constants. ε⬘r varies significantly both with temperature and frequency for many typical workload substances. ε⬙r is called the relative dielectric loss incorporating all of the energy losses due to dielectric relaxation and ionic conduction. The ratio ε⬙/ε⬘ is called a loss tangent because it is a measure of the power loss in the medium:

ε⬙ σ tan δc ⫽ ᎏ ≅ ᎏ ε⬘ ωε

(125.5)

The quantity δc may be called the loss angle. The propagation factor e⫺γ z can be written as a product of two factors: E(x) ⫽ Emaxe⫺αze j(ωt⫺βz)

(125.6)

where α and β are the real and imaginary parts of γ, respectively. Since γ is complex, we write, with the help of Equation (125.3),

冢

σ γ ⫽ α ⫹ jβ ⫽ jω兹µ 苶ε苶 1 ⫹ ᎏ jωε

冣

1/2

ε⬙ 1/2 ⫽ jω兹µ 苶ε苶⬘ 1 ⫺ j ᎏ ε⬘ (125.7)

冢

冣

兹苶2π f 2 α⫽ ᎏ ε⬘苶苶苶1苶苶 ⫹苶tan 苶苶苶 δ苶 ⫺ 苶苶1冣苶 r 冢兹 c 兹苶

(125.8)

兹苶2π f 2 β⫽ ᎏ ε⬘苶苶苶1苶苶 ⫹苶tan 苶苶苶 δ苶苶 ⫹苶1冣苶 r 冢兹 c 兹苶

(125.9)

As we shall see, both α and β are positive quantities. The first factor, e⫺α z, decreases as z increase and thus is an attenuation factor, and α is called an attenuation constant. The second factor, e⫺jβz, is a phase factor; β is called a phase constant which expresses the shift of phase of the propagating wave and is related to the wavelength of radiation in the medium (λm) by λm ⫽ 2π/β which, in free space, reduces to λ 0 ⫽ 2π/β ⫽ c0/f. From Equation (125.6), the first exponential term gives the attenuation of the electric field, and, therefore, the


125-3

distribution of the dissipated or absorbed power in the homogeneous lossy material follows the exponential law (Lambert’s Law): Pdiss ⫽ Ptranse⫺2αz

(125.10)

where Ptrans is the power through the surface in the z direction. Theoretically, the power penetration depth, Dp, is defined as the depth below a large plane surface of the substance where the power density of a perpendicularly impinging, forward propagating plane electromagnetic wave has decayed by 1/e from the surface value, 1/e ⬇ 37% [1]. The absorbed power in the top layer of this thickness in relation to the totally absorbed power (per surface area), is then 63%. 1 Dp ⫽ ᎏ 2α

(125.11)

Substitution of Equation (125.8) into Equation (125.11) yields the general expression for the penetration depth: c Dp ⫽ ᎏ 4π f

冪莦 2 ᎏᎏᎏ 2 ε⬘r 冢兹苶1苶 ⫹苶t苶 an苶苶δ ⫺ 1冣

λ0 ᎏᎏᎏᎏ 1/2 ⫽ 2π 兹苶2苶 ε⬘r苶冢兹苶1苶 ⫹苶(苶 ε⬙r苶/苶 ε⬘r苶)2苶 ⫺ 1冣

(125.12)

The skin depth Ds, where the electric field strength is reduced to 1/e (and the power density thus to (1/e)2), is twice the power penetration depth, Ds ⫽ 2Dp.

IV.

MICROWAVE POWER DISTRIBUTION

Most practical materials treated by microwave power are nonhomogeneous and very frequently anisotropic; the permittivity of these materials changes with temperature and moisture content (drying process). Thermal losses from the material surface and heat transfer in the bulk of material give additional complications. The generation of heat in food materials is also accompanied by significant moisture migration which, in turn, affects the energy absorption characteristics of food creating a coupling of heat and mass transport that complicates mathematical analysis. From the physical point of view, microwave heating is a combination of at least four different processes: distribution of power, absorption of power, heat transfer, and mass transfer. The magnitude and uniformity of temperature distribution are affected by both food and oven factors such as: 1. Magnitude and distribution of microwave power where the food is placed; 2. Reflection of waves from the food surface and penetration depth, as characterized by the food geometry and properties;

3. Distribution of absorbed power as well as power dissipated at a particular point (electric field intensity) as functions of the material parameters, temperature, and time (due to drying); and 4. Simultaneous heat and mass transfer.

A. ELECTRIC FIELD INTENSITY Electromagnetic waves transport energy through space. The amount of microwave energy absorbed is, in turn, determined by the electric field inside the microwave applicator. It offers an intangible link between the electromagnetic energy and the material to be treated. For microwave heating, the governing energy equation includes volumetric heat generation that results in a temperature rise in the material: ∂T Qabs ᎏ ⫽ α∇2T ⫹ ᎏ ∂t ρCp

(125.13)

In this equation Qabs (watts/cm3) corresponds to volumetric rate of internal energy generation due to dissipation of microwave energy. Basically, the apparatus is placed in the oven at the position of interest and the rate of temperature rise, ∂T/∂t, is measured. Cp (cal/g °C) is the heat capacity of the material, and ρ (g/cm3) is the density of the material. Assuming no temperature gradients in a small mass of dielectric medium, the energy balance can be obtained by simplifying Equation (125.13): Pabs ∂T Qabs ⫽ ᎏ ⫽ ρCp ᎏ V ∂t

(125.14)

where Pabs is the total power absorbed by the dielectric medium (watts). Its relationship to the E-field at the location can be derived from Maxwell’s equations of electromagnetic waves [2]. Qabs ⫽ 2πf ε0ε eff ⬙ E 2rms

(125.15)

where f is the microwave frequency (2450 MHz), ε⬙eff is the dielectric loss factor for the dielectric material being heated, and Erms is the root mean square value of the electric field intensity. By knowing the rate of temperature rise, the heat generation, Qabs can be determined and equated to the electric field, Erms, using Equation (125.15).

ρC ∂T ᎏ ᎏᎏ 冪莦ᎏ 2莦 π莦 f莦 ε ε莦莦⬙ 莦莦∂莦 t

Erms ⫽

p

(125.16)

0 eff

B. LAMBERT’S LAW In several computational studies of microwave heating, the heat generation has been modeled by Lambert’s law, according to which the microwave power is attenuated exponentially as a function of distance of penetration into the sample [3–9]. It must be emphasized that these penetration depth

125-4


calculations are valid only for materials undergoing plane wave incidence and for semi-infinite media only and henceforth will be referred to as Lambert’s law limit [5, 8]. Although Lambert’s law is valid for samples thick enough to be treated as infinitely thick, it is a poor approximation in many practical situations and often does not describe accurately the microwave heating of food in a cavity. To determine the conditions of the approximate applicability of Lambert’s law for finite slabs, Ayappa and others [8, 9] compared it with microwave heating predicted by Maxwell’s equation. The critical slab thickness Lcrit (in cm) above which the Lambert’s law limit is valid can be estimated from Lcrit ⫽ 2.7/Dp ⫺ 0.08. Fu and Metaxas [10] proposed a new definition for the power penetration depth ∆p, which is the depth at which the power absorbed by the material is reduced to (1 ⫺ 1/e) of the total power absorbed. This definition allows a unique value of ∆p to be found for all thickness and also gives an indication of the validity of assuming exponential decay within the slab. Another approach is used where a spherical dielectric load is assumed to absorb energy from a surrounding radiation field [11]. The power absorption inside of dielectric medium can be estimated in the following way. Assume that the power flux (power per unit area) entering through the surface of the dielectric medium is uniform, and all the waves are transmitted into the medium, i.e., no wave reflection. Then power decays exponentially, P(x) ⫽ P0 ⭈ exp(⫺x/Dp) where, P0 is the incident power at the surface. From the Poynting theorem [12], the field energy that dissipates as heat in the enclosed volume is equal to the total power flowing into a closed surface minus the total power flowing out of the same closed surface.

冕

P0e⫺x/Dp ∑ Peff ⫽ ᎏ dx Dp e d(a ⫺ r) 冤冕 ᎏ D a

⫺(a⫺r)/Dp

⫽ ⫺P0

p

0

e ᎏ d(a ⫺ r) 冕ᎏ 冥 D ⫺a

⫺

0

⫺(a⫺r)/Dp

(125.17)

p

Pabs ⫽ ∑ Peff ⫽ P0[1 ⫺ e⫺2a/Dp]

(125.18)

where a is the radius of the spherical dielectric load, Peff is the effective magnitude of the Poynting vector, and Pabs is the total power absorption by the dielectric medium. The use of Lambert’s law requires an estimate of the transmitted power intensity Ptrans (Equation (125.10)), which is obtained from calorimetric measurements [4, 7] or used as an adjustable parameter to match experimental temperature profiles with model predictions [6]. Thus Ptrans measured by the above methods represents the intensity of transmitted radiation, the accuracy of the estimate

depending on the method used. Alternately if Ptrans is the incident power flux then Lambert’s law must be modified to account for the decrease in power, due to reflection at the surface of the sample. Since Lambert’s law does not yield a comprehensive approach, a more accurate estimation of the heating rate based on predicting or measuring the fundamentally nonuniform electric field intensity in a cavity should be the most important subject of current research. How the shape and volume (relative to the microwave oven) of a food material change the rate of heating must be investigated further. The interior electric field, the moisture movement in solid foods, and changes in the dielectric and other properties combined to make designing microwave processes a difficult task.

V. INTERACTION OF MICROWAVE WITH FOOD Food shape, volume, surface area, and composition are critical factors in microwave heating. These factors can affect the amount and spatial pattern of absorbed energy, leading to effects such as corner and edge overheating, focusing, and resonance. Composition, in particular moisture and salt percentages, has a much greater influence on microwave processing than in conventional processing, due to its influence on dielectric properties. Interference from side effects like surface cooling, interior burning, steam distillation of volatiles, and short cook time alter the extent of interactions.

A. DIELECTRIC PROPERTIES The dielectric properties of foods are very important in describing the way foods are heated by microwaves. The most comprehensive effort on dielectric properties data to date being that of von Hippel [13]. The dielectric properties of foods vary considerably with composition, changing with variation in water, fat, carbohydrate, protein, and mineral content [14]. Dielectric properties also vary with temperature. As indicated earlier, the dielectric properties affect the depth to which microwave energy penetrates into the food to be dissipated as heat. The magnitude of the penetration depth, defined as the depth at which 63% of the energy is dissipated, can be used quantitatively to describe how microwave energy interacts with the food. A large penetration depth indicates that energy is poorly absorbed, whilst a small penetration depth indicates predominantly surface heating. Dielectric properties data for agricultural products, biological substances, and various materials for microwave processing are widely dispersed in the technical literature [15–18]. Those literature data can provide guidelines, but variability of composition of food products, and other specific conditions for particular applications, often require carefully conducted measurements.


B.

GEOMETRICAL HEATING EFFECTS — CORNER, EDGE, AND FOCUSING EFFECTS

With conventional cooking methods, heat is transferred from outside to the food product by conduction, convection, or infrared radiation. There is a temperature gradient from the outside to the inside. It is often said that with microwaving, heating takes place from the inside to the outside. This is not true; heating occurs throughout the whole food simultaneously, although it may not be evenly distributed. Probably this misapprehension is due to the fact that surface temperatures tend to be lower than temperatures inside the food (this is because of evaporative cooling and geometrical heating effect). For foods with a high loss factor, most of the microwave energy of a wave impinging on the food will be absorbed near the surface, and penetration and in-depth heating will be limited. In general, the surface will heat more rapidly than the interior, but there are exceptions. Refraction and reflection at interfaces will cause reinforcement of the field pattern near corners and edges of rectangularly shaped foods, resulting in overheating. Core heating effects of the same nature occur in foods of spherical or cylindrical shape at certain dimensions, causing energy concentration and overheating of the central part. The concentration heating effect means maximum heating occurs in the center for certain spherical and cylindrical geometries [19]. The well-known explosion of eggs during microwave heating is one of the most significant demonstrations of core heating effect. This occurs because center heating cause formation of steam which induces an energy impulse with such high power as to move the surrounding mass parts away from each other. This kind of thermal behavior has already been observed by many people [3, 6, 19, 20] for cylindrical and spherical shaped foods. The maximum heating regions also move slowly from the center towards the surface when the diameter increases. If the diameter is much greater than penetration depth, the temperature profile will be similar to that observed for a “semi-infinite” body. That is, the temperature decreases exponentially from the surface in accordance to Lambert’s law which governs the absorption of microwave power. If the diameter is much less than penetration depth, the heating profile will be flat. In between these extremes the focusing effect occurs. Moreover, Mudgett [3] pointed out the effect of salt on drying behavior. With addition of sodium chloride, penetration depth decreases significantly and, therefore, the heating profile could shift from that of focusing and center heating to one of surface heating. Another reason for uneven heating in lossy products can be traced to the electromagnetic boundary conditions at edges and corners [21]. This is the so called edge and corner effects. In an electric field, where the wavelength is larger than the dimensions of the heated object, field

125-5

bending will give rise to concentrations at some locations. The convergence of two or more waves at a corner results in a higher volumetric power density than on the flat surface. Higher heating rates will thus be obtained at the corners. If the electric field is strong enough, an arc may emanate from there when the air ionizes [22]. Square containers can cause burning in the corners of the product due to a greater surface area/volume ratio, resulting in more microwave energy absorption. Circular or oval containers help reduce the strong edge and corner effects as energy absorption occurs evenly around the edge but core heating effects may then originate.

C. MICROWAVE BUMPING Another phenomenon during microwave heating is the “bumping” which may occur in microwave cooking. The term, “microwave bumping,” also known as microwave popping or microwave splattering, is descriptive of the explosion phenomenon and is characterized by a jostling or shaking of the container, usually accompanied by an audible explosion. When microwave bumping occurs, the explosive sounds which can be heard some distance away are annoying and an unexpected surprise to consumers. Microwave bumping is due to the explosion of food particulates, not localized boiling of the liquid. Increasing the viscosity of the liquid did not result in a significant difference in intensity or frequency of bumping. Degree of microwave bumping is believed to be directly related to local superheating effects. The higher the electric field intensity, the greater the incidence of bumping. Due to edge, corner, and focus heating effects by microwave, container shape influences heating pattern of a food product and location of bumping in the container. Sterilizing vegetable particulates which causes excessive softening and salting food particulates which causes high microwave heating rate are two indispensable conditions to produce microwave bumping [23].

D. EVAPORATIVE COOLING AND STEAM DISTILLATION During the heating process of foods containing water, the resulting evaporation at the surface causes a depression of the temperature, known as evaporative cooling. The surface of food is seen to be cooler than the region just below the surface and warmer than the surrounding air. This phenomenon is readily seen during the cooking of a meat roast [6, 24]. At the same time, this surface evaporation can cause steam distillation of certain flavor components. Flavor release in microwave cooking is increased by steam distillation. In microwave heating, water vapor (steam) is one of the most important transport mechanisms contributing to movement of flavor compounds within a food matrix.

125-6


Individual compounds that make up a flavor which are particularly low molecular weight and water-soluble may be driven off or steam distilled out of the product during microwave heating. Fruit and other “sweet” flavorings are more of a problem. They evaporate easily in foods with high initial water content because they contain a great number of short-chain, volatile flavoring substances. Moreover, they are often of a more hydrophilic character and therefore a great part of the flavoring substances migrates to the aqueous phase of the food, which selectively absorbs the great part of microwave energy [25]. The percent loss may range from less than 10% for high boiling compounds to 95% for very volatile compounds [26]. The latter are the ones that create a strong aroma which is necessary when the flavor is designed to impart a balanced aroma profile in the room during microwave heating [27]. In this case the flavor was added solely for aroma generation and contributed very little to the flavor profile of the microwave product itself. However, this phenomenon, flash-off, often leads to imbalance of flavor concentrations in a finished product with a different character from the flavor that was added before cooking. Formulations that compensate for flash-off may require a highly imbalanced flavor character prior to microwaving. The specific loss is dependent on the types of flavor components used and the food system in which it is incorporated. As the outward migration of water vapor is the most important factor influencing flavor retention in the food product, the flavorings used for microwave application should have low water vapor volatility unless the flavorings are intended to create the “oven aroma” of conventional cooking methods, or to cover undesirable off-notes released during microwave cooking.

towards the surface instead of towards the center [28, 29]. Water vapor generated inside the food continuously migrates to the surface, drawing flavoring substances with it on the way out. As the evaporation rate of water is not high enough to dry out the surface, the evaporated water is continuously replaced by migration of water from the inside [25]. For foods which require a long heating time, e.g., meat joints, the effect can be significant and the resulting moisture loss from the surface of the product can be appreciable. An electromagnetic phenomenon creating “hot” and “cold” spots is inherent in all microwave ovens and is responsible for much of the uneven cooking associated with them. Liquid products quickly dissipate the microwave energy and result in a more uniform product. Solid food products, multiphase systems, or frozen products develop hot and cold spots during heating which further complicate flavor delivery in these systems [27]. During microwave heating the low surface temperature and its much higher water activity (approximately 1.0) and the lack of prolonged baking time have the following consequences: (1) no crust is formed because the necessary physical changes (protein denaturation, starch gelatinization, etc.) are inhibited, and (2) the formation of many flavor compounds and/or pigments (Maillard browning reactions) do not occur to the required extent. Thus, some flavors that typically develop in a conventionally cooked product will not necessarily work in a microwaved product. Van Eijk [25] stated that the differences in flavor generation and the performance of flavoring substances in microwave foods can be explained satisfactorily by the differences in heating pattern, the corresponding differences in water vapor migration, and the resulting physical changes, particularly at the surface of the food. No athermal effects have been observed.

E. LACK OF CRISPNESS (TEXTURE) AND BROWNING (COLOR, FLAVOR) OF MICROWAVE FOODS

F. FOOD INGREDIENTS

The texture of a microwaveable food may directly affect its acceptance. Toughness or lack of crispness in bread slightly overcooked in a microwave oven may not directly change its flavor, but does influence the consumer’s perception of the product. The lack of conventional-styled browning and crisping in microwave ovens is due to the microwave frequency used. At 2450 MHz, the wavelength, 12.2 cm, is too long to create the intense surface heat which occurs at the higher infrared frequencies, limiting the food item to a temperature of approximately 100°C. This is ideal for wet foods like vegetables and stews, but unacceptable for pastry, breaded or batter-coated items, and roast meat. In contrast to the convectively heated food, we have relatively low temperature ambient air (60–75°C) with a rather high relative humidity in most cases during microwave heating. The level of maximum temperature and consequently of maximum water vapor pressure generally lies further below the surface. The main driving force, therefore, is directed

Some of the ingredients in foods such as water, ionized salts, and fats and oils, in particular, and the distribution of these ingredients in the food product, exert a strong influence on temperature level and distribution. These ingredients interact physically and chemically to an extent dictated by numerous factors including mode of heating. The dielectric and thermal properties of foods can be modified by adjusting food ingredients and formulations and are manageable within certain limits. Frozen pure water has no microwave dipole relaxation and is therefore microwave transparent. Frozen foods, however, are not microwave transparent since some of the water is still in free liquid form. So when deep-frozen foods are defrosted by microwave energy, particularly difficult problems arise once both ice and water are present. Hot spots and runaway heating may be the consequence in this case. Fats have a low dielectric loss and consequently do not generate as much heat directly from the microwave field. Once


heat has been generated, conduction and convection become the main mechanisms of heat transfer. Fats reach very high temperatures due to their high boiling points, whereas water is limited to a maximum temperature of 100°C. However, since the heat capacity of fats is about half that of water, they heat more quickly in the microwave. Factors that affect dielectric properties of water, including the presence of other interactive constituents such as hydrogen bonding resulting due to the presence of glycerol and propylene glycol, and sugar and carbohydrate-like polyhydroxy materials will also impact microwave heating [30]. Salts and sugars can be used to modify the browning and crisping of food surface. Heating a sample with higher salt content can change the microwave heating pattern from center heating to surface heating [31]. In addition to direct microwave interactions, lipids, salts, sugar, and polyhydroxy alcohols can also raise the boiling point of water. This allows the food to reach a higher temperature needed for the development of reaction flavors, and Maillard browning reactions. To obtain useful and meaningful information on the contributions of rates of flavor migration and kinetics of degradation under various conditions, Fu and others [32] designed an apparatus for on-line measurement of flavor concentration, to formulate a thermally stable flavordough system and to accomplish isothermal heating. Photoionization detection method [33] and a cold-trap, on-line sampling method [34] were to investigate migration of flavor compounds in a solid food matrix subjected to microwave heating. As the moisture concentration decreased below 0.1 g water/g solid during microwave heating of gelatinized flour dough, a type of encapsulation occurred that prevented flavor from being released. The results of microwave reheating of limonene-formulated dough showed limonene is very stable and no significant limonene concentration profile in the sample and less than 1% overall change in total limonene concentration [35].

VI. MICROWAVE PROCESSING In the quest for better quality of shelf-stable, low-acid foods, a number of emerging technologies have been considered [36]. Food engineering will continue to evolve. Although alternative processes have been developed over the years, thermally processed food products maintain a clear dominance in the marketplace, primarily as a result of the wealth of theoretical and empirical knowledge that has been developed regarding thermal inactivation of pathogenic microorganisms and their spores [37]. Microwave sterilization is a nontraditional but solely a thermal process and so can be regarded by technologist and regulators as another terminal thermal sterilization technique. Microwave heating offers numerous advantages in productivity over conventional heating methods such as hot air, steam, etc. These advantages include high speed,

125-7

selective energy absorption, excellent energy penetration, instantaneous electronic control, high efficiency and speed, and environmentally clean processes [38]. Currently, microwave and radio-frequency both are laboratory or pilot scale and there are no known large operating microwave systems operating in the food industry, except for bacon precooking or tempering. It remains a very exciting processing tool, unmatched by any other technology if attention is paid to their selection.

A.

DRYING AND DEHYDRATION

Microwave drying is rapid, more uniform, and energy efficient compared to conventional hot air drying and sometimes it results in an improvement in product quality. But it is highly unlikely that an economic advantage will be demonstrated if only bulk water removal by microwave heating is desired, such as occurs in the constant-rate region [39]. During the falling-rate period because of the low thermal conductivity and evaporative cooling effect, high product temperatures are not easily obtained using convective drying. Surface hardening and thermal gradients again provide further resistances for moisture transfer. Actually, it has been suggested that microwave energy should be applied in the falling rate period or at low moisture content for finish drying [40–43]. Correspondingly sensory and nutritional damage caused by long drying times or high surface temperatures can be prevented. It is important to understand the dielectric properties of the material with different moisture content during microwave drying. The ability of dielectric heating to heat selectively areas with higher dielectric loss factors and the potential for automatic moisture leveling afford a major advantage for even drying of these types of materials [39]. Because internal microwave heating facilitated a more predominant vapor migration from the interior of the material as compared to that during conventional drying, microwave dried products have been reported to show a higher porosity because of the puffing effect caused by internal vapor generation [44–46]. Similar results are also found for pasta drying. Microwave drying produces a slightly puffed, porous noodle which rehydrates in half the time required for noodles dried by conventional methods [47]. Tong and others [44] investigated temperature and pressure distribution in a dough system with porosity ranging from 0.01 to 0.7 during microwave heating using miniature fiber optic temperature and pressure probes. Pressure build-up to approximately 14 kPa occurred during the initial stages of the heating process when the initial porosity was less than 0.15 and disappeared when the pressure exceeded the rupture strength of the dough. Volume expansion was observed up to the point where the dough sample ruptured, producing visible cracks in the structure. So microwaves produce a pressure gradient that pumps out the moisture [44]. This property can be used to

125-8


advantage to speed up the drying process. If the pressure build-up did not exceed the rupture strength of the structure it might be the result of such enhanced porous structure of the samples. So, it is a difficult task for reducing drying time and increasing quality at the same time. Careful studies need to be done by applying the right amount of microwave energy in the process. Nonuniformities in the microwave electric field and associated heating patterns can lead to high temperature in various regions dried earlier, causing product degradation [48]. Improvement can be achieved by using a fluidized bed dryer, or spouted bed dryers to average the uneven electric field [49, 50]. The combination of microwave and vacuum drying [51–54] or freeze drying [55–59] also has a certain potential. The vacuum process opens the cell structures (puffing) due to the fast evaporation and an open pore structure is generated. Reduced drying time is the primary advantage of using microwaves in the freezedrying process. But no commercial industrial application can be found, due to high costs and a small market for freeze dried food products. Pasta and potato chips have been dried successfully. Freeze drying and vacuum drying, in conjunction with microwave energy, have also shown promise and interest from an academic point of view but not meeting the economic criteria. A relative new and successful combination of microwave energy and frying process is used to produce fried goods, such as chips, noodles, and chickens, with 60% reduced time, 50% reduced fat content, and 33–60% energy saving [60].

B. PASTEURIZATION AND STERILIZATION Pasteurization provides a partial sterilization of substances by inactivating pathogenic microorganisms, notably vegetative cells of bacteria, yeast, or molds. Products have to be refrigerated. Sterilization processes are designed to inactivate microorganisms or their spores. Thermal sterilization is usually done at temperatures in excess of 100°C which means they are usually done under pressure. Industrial microwave pasteurization and sterilization systems have been reported on and off for over 30 years [61–68]. Studies with implications for commercial pasteurization and sterilization have also appeared for many years [69–77]. Early operational systems include batch processing of yogurt in cups [78] and continuous processing of milk [79]. A very significant body of knowledge has been developed related to these processes. As of this writing, two commercial systems worldwide could be located that currently perform microwave pasteurization and/or sterilization of foods [68, 80]. As a specific example, Tops Foods (Belgium) [68] produced over 13 million ready meals in 1998 and have installed a newly designed system in 1999.

Microwave pasteurization can reduce the come-up time, which can shorten to a small fraction of the time used by the conventional process. After this, the microwave heated meals pass into a nonmicrowave hot air tunnel for the hold-time period, and then to the cooler. Microwave is difficult to hold a constant temperature and should not be used. Especially in Europe, food pasteurization by microwave processing has been successfully accomplished for decades. The major advantage of the microwave process is that the product may be pasteurized within a package. A product goes through the line in wrapping continually, package by package, pallet by pallet. Shelf life can be extended from days to over a month without preservatives. Typical sterilization temperatures in the product may be 121–129°C (250–265°F) with hold times of 20–40 minutes. The come-up time may be significantly reduced by microwaves. This reduced come-up time would provide greater product quality. The enhanced quality retention is due to the fact that quality attributes normally have much lower activation energy (10–40 kcal/mol) than the microbial spores (50–95 kcal/mol). The heat-up time of the microwave process is much faster than that in a retort, so the product’s organoleptic (texture, color, and flavor) and nutritional qualities could be considerably improved. Microwave sterilization is more flexible than ohmic heating and aseptic processing. It can sterilize liquids, semi-solid, and solids and it can also sterilize pre-packed food products. There are several practical concerns and problems that have to be solved before it can be applied at the industrial level. The main issue has been the regulation of process parameters so that commercial sterility can be achieved. For the conventional retort process, by monitoring the time-temperature history at the cold point using a thermocouple thermometer, it is reasonably easy and accurate to determine the microbial lethality through mathematic calculations. But, determining the microbial lethality for a microwave sterilization process is not straightforward. The cold point during microwave sterilization is not always located on the central axis. The problem of providing a uniformly heated product makes it extremely timeconsuming and costly to adjust the microwave pattern to produce the quality advantage theoretically possible by using microwaves. Each product could require custom adjustment. The presence of uneven heating (hot and cold spots) makes it very difficult to ensure that all portions of a meal have reached a kill temperature. Microbiological safety is the major reason for the slow acceptance of microwave sterilization. In addition, the technical ability to accurately measure the temperature distribution throughout an entire microwave sterilized product has not been demonstrated. From the engineering point of view, no computer simulation models are available for investigating the feasibility of microwave sterilization. These computer simulation models are not only required by the Food and Drug Administration (FDA) for regulating and


approving microwave sterilization processes, but also highly demanded by the food industry for performing the cost/ benefit analyses. Without the reliable inputs of dielectric properties, thermophysical properties, and boundary conditions, a computer model is completely useless. Unfortunately, literature values on these properties are only available at room temperature to 60°C and not readily available at sterilization temperature. In Europe, microwave-sterilized foods, primarily pasta dishes such as lasagna and ravioli, are on many grocery shelves with no reported difficulties. Safety regulations are less stringent in Europe. For example, in one implementation [68] the process design consists of microwave tunnels with several launchers in relation to the number of products (ready meals). Microwave-transparent and heatresistant trays are used with shapes adapted for microwave heating. Exact positioning of the package is made within the tunnel and the package receives a pre-calculated, spatially varying microwave power profile optimized for that package. The process consists of heating, holding, and cooling in pressurized tunnels. The entire operation is highly automated. But, microwave sterilization has not been approved on the use of microwave processing for food sterilization by the Food and Drug Administration in the United States (US-FDA). Today, there continues to be a great deal of interest and some R&D activity in pasteurization and sterilization by microwaves [81–88].

C. TEMPERING AND THAWING Thawing and tempering of frozen food materials is an important part of some food processes, especially in the meat industry and food service. Reducing thawing time by higher temperatures results in a decrease in product quality such as more dip loss and surface drying in addition to increased risk of microbial growth. Frozen foods can be considered to be the mixture containing two components: fixed structure of ice and biological material surrounded by monomolecular layer of strongly bound water, and loose liquid water saturated with dissolved salts. Dielectric activity of this mixture is much higher than that of pure ice, but much less than that of the same material at temperatures above zero. The loss factor (ε⬙ ) of water is approximately 12; while that of ice is approximately 0.003. The penetration depth in water (1.4 cm) is much lower than in ice (1160 cm) [89]. If the thickness values is much greater than penetration depth, the temperature profile will be similar to that observed for a “semi-infinite” body. That is, the temperature decreases exponentially from the surface in accordance to Lambert’s law. Surface layers thus absorb more energy and heat up a little bit faster than the inside of the product. But for thickness values smaller than a certain value, resonance can still not be avoided and inside of a slab can be heated directly at high intensity, resulting in quick thawing. As

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the loss factor increases with the temperature, the surface heats up faster and faster and the penetration depth continually decreases. Spots of free water and spots that have reached temperature 0°C absorb more energy than crystals of ice, which leads to further acceleration of heating. Microwave energy penetrates a food material and produces heat internally. The main advantage consists of speed because tempering by microwave takes minutes instead of hours or even dozens of hours. For example, a 20 cm thick piece of beef, frozen to ⫺16°C, thaws more than 10 hours at the surrounding temperature of ⫹4°C. On the other hand, the whole cycle of MW tempering with following slicing, modification, and repeated freezing takes only 30 minutes [90]. There are at least 400 tempering systems operating in the United States alone. Food is heated to just under freezing temperatures, allowing easy chopping, cutting, processing, etc. In the United Kingdom there are several large systems, up to 200 kW, utilized for tempering of frozen beef, as well as butter. The lower frequencies, e.g., 915 MHz band, are used to advantage for MW thawing and tempering of larger blocks of food. For example, when tempering 18 cm thick blocks at 915 MHz frequency, temperature gradient is half of the gradient for 2450 MHz frequency [90]. 915 MHz tempering systems, batch and continuous, are sold worldwide. Microwave thawing remains a major problem. A main difficulty is formation of wide temperature gradients (runaway heating) within the product. The preferential absorption of microwaves by liquid water over ice is a major cause for run-away heating. Maximum homogeneity is achieved with temperatures slightly above zero. After that the inhomogeneity rises again. Therefore it is advantageous to reduce the thawing process to plain tempering, i.e., to stop the heating at the temperatures ⫺5 to ⫺2°C. Another reason why tempering is preferred is that the progress of energy consumption is dependent on the temperature. With most biological materials and water, the energy consumption starts to rise sharply at temperatures above ⫺5°C; the less fat they contain the higher is the consumption. Since the thawed material has a much higher dielectric loss, microwave penetration depth at the surface is significantly reduced, in effect developing a “shield.” Surface cooling helps to reduce the gradient in a frozen food, thus enabling the microwave power to remain on longer to decrease the thawing time. The temperature uniformity during microwave thawing can be improved when appropriate sample thickness, microwave power level, frequency, and/or surface cooling are applied [64, 91–93]. Today, there continues to be a great deal of interest and some R&D activity in thawing and tempering by microwaves [94–97].

D. BAKING Baking, in all cases except unleavened products, involves the creation, expansion, and setting of edible foams through

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the use of heat. Proofing is the step of causing the dough to rise and precedes the final baking or frying in the case of donuts. During the baking of raw bread dough significant volume change occurs, and the dough is converted from a viscoelastic material containing airtight gas cells with the ability to expand to a rigid structure which is highly permeable to gas flow. The cell walls are elastic but strong and the increasing gas pressure must cease while the cell walls set. Baking is a complex physicochemical reaction in which all the events must be carefully timed and must occur in a welldefined sequence. All baked products form some sort of crust which acts as a shield, making heat even harder to reach the inside. The heat transfer problems encountered by conventional means can be easily overcome by microwave heating. Pei [98] reviewed heat and mass transfer in the bread baking process and discussed the application of microwave energy. Goedeken [99] investigated microwave baking of bread dough with simultaneous heat and mass transfer. Highly porous products, such as bread, lend themselves well to the use of microwave energy because of greater penetration of microwave energy resulting in more uniform energy distribution within the product. But the microwave application must be carefully controlled or heating and expansion will occur too quickly, and while the cake may look fully expanded and baked, it will collapse to a pancake when the microwave energy is removed. Bread baking by means of microwave energy was first reported in the literature by Fetty [100]. Decareau [101] noted the possibility of combining microwave energy and hot air to produce typically brown and crusted loaves of bread in a shorter time than by conventional baking methods. One microwave baking process that was quite successful for several years was the microwave frying of doughnuts. Frying times of approximately two-thirds normal time are possible with 20% larger volumes, or 20% less doughnut mix required for standard volume. Fat absorption can be 25% lower than conventional. This proofing system was developed by DCA Food Industries, which operated 2450 MHz and varied in output from 2.5 to 10 kW for production rates of 400–1500 dozen doughnuts per hour [102–104]. One difficulty in the microwave baking process was to find a microwavable baking pan that is sufficiently heat resistant and not too expensive for commercial use. A patent by Schiffmann and others [105] describes microwave proofing and baking of bread in metal pans. This technique utilizes partial proofing in a conventional proofing followed by proofing in a microwave proofer utilizing warm, humidity-controlled air and reduces the proofing time by 30–40%. This was then followed by microwave baking in a separate oven. Four patents by Schiffmann and others [104–107] describe procedures for the baking of bread utilizing metal pans and, in some cases, also provided for partial proofing of the bread in the pans. In the procedure described in the aforementioned patents, the microwave baking process involved the

simultaneous application of microwave energy and hot air to both bake and brown the bread, producing thoroughly browned and crusted loaves of comparable volume, gain structure, and organoleptic properties. It was found that the use of either 915 MHz or combinations of 915 and 2450 MHz were quite effective in baking a loaf of bread. The system of microwave frying doughnuts was very successful for quite some time during the 1970s. These doughnuts have longer shelf life, better sugar stability, and excellent eating quality. The larger volume and lower fat absorption provided high profits for the bakery. To date, some very sophisticated packaging along with advanced susceptor technology has been the predominant solution to the lack of conventional-styled browning and crisping. Susceptors rapidly heat to temperatures where browning readily occurs and thus help produce flavor in the product. However, susceptors solve the flavor-related problems only on the surface. Another possible solution to the lack of browning during microwave cooking is the addition of compounds which give a roasted or toasted reaction flavor. Today, there continues to be a great deal of interest and some R&D activity in baking by microwaves [108–115].

VII. RADIO FREQUENCY PROCESSING Radio frequency and microwave heating refers to the use of electromagnetic waves of certain frequencies to generate heat in a material [2, 116, 117]. Radio frequency heating, which is at a much lower frequency, has thrived as an industry alongside microwaves over the decades. Radio frequency heating in the United States can be performed at any of three frequencies: 13.56, 27.12, and 40.68 MHz. The heating mechanism of radio frequencies is simply resistance heating which is similar to ohmic heating. This lossy dielectric arises from the electrical conductivity of the food and is different from the resonant dipolar rotation of microwave frequencies. Unlike microwave sources, one cannot purchase an RF high power source. Due to the high impedance nature of RF coupling, the RF source and applicator normally need to be designed and built together. Manufacturers of RF equipment develop the whole system, rather than only the power source. Therefore, developments in RF processing must involve the commercial RF manufacturers. RF equipment is available commercially at much higher power levels than microwave sources. While commercial microwave sources are available only below 75 kW, RF equipments at hundreds of kW are very common. At these high levels, the price per watt of RF equipment is much cheaper than microwaves. In addition to higher power and lower cost, another advantage of RF equipment over microwaves is in the control area. In high power RF systems, the source and the load are commonly locked together in a feedback circuit. Therefore, variations in the load can be followed by the source without external controls [118].


Microwave or radio frequency? For the same electric field, the higher the frequency, the higher the amount of power into the material. This is the reason why microwaves are a conceptually more effective means of heating. However, RF equipment has several advantages which workers in the field of microwave processing may find more suitable for scale-up of some processes. The microwave fields attenuate within the bulk of conductive materials and materials with high dielectric loss. The penetration depth of microwaves is much lower. This is particularly troublesome for larger scale processes. But, this type of nonuniformities are frequency dependent and become less severe as frequency is lowered. Because of much longer wavelengths of radio frequencies, they have better uniformity. Also, the depth of penetration is much higher. So, in cases where uniformity of heating is a critical issue, use of the radio frequencies and 915 MHz microwave frequency may have potential for the future [119, 120]. Working at radio frequencies allows it to process a large range of material types, from the thin wide webs of the paper industry to large three-dimensional objects like textile packages. In general terms, microwave is better for irregular shapes and small dimensions and RF is better for regular shapes and large dimensions. Microwave is more suitable for hard to heat dielectrics. Actually, many applications can be done by either, but RF is cheaper if it fits. RF equipment is easier to engineer into process lines, and can be made to match the physical dimensions of the up- and down-stream plant. In the case of microwaves, in a continuous process, complex arrangements may be necessary to allow the product to move in and out of the enclosure without giving rise to excessive leakage of energy [121]. This is because the wavelengths at microwave frequencies (e.g., 12.54 cm at 2450 MHz) are very much shorter than those at radio frequencies (e.g., 1100 cm at 27.12 MHz). An overview of food and chemical processing uses of radio frequency can be seen in Minett and Witt [122] and Kasevich [123]. The industrial applications using radio frequency include textiles (drying of yarn packages, webs, and fabrics), food (bulk-drying of grains; moisture removal and moisture leveling in finished food products), pharmaceutical (moisture removal in tablet and capsule production processes), and woodworking (adhesive curing for wood joinery). Radio frequency heating has been used in the food processing industry for many decades. The post-baking of biscuits, crackers, and snack foods is one of the most accepted and widely used applications of RF heating in the food processing industry. A relative small RF unit can be incorporated directly into a new or existing oven line (a hot air oven or conventional baking line) to increase the line’s productivity and its ability to process a greater range of products. The benefits of RF-assisted baking are precise moisture control, reduced checking, improved color control, and increased oven line throughput [124]. RF drying is intrinsically self-leveling, with more energy being dissipated

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in wetter regions than in drier ones [121]. This RF leveling leads to improvements in product quality and more consistent final products. Recently, RF cooking equipment for pumpable foods has been developed. These devices involve pumping a food through a plastic tube placed between two electrodes, shaped to give a uniform heating [125]. The primary advantage of improved uniformity of heating was also shown for in-package sterilization of foods in large packages using radio frequency at 27.12 MHz, although enhanced edge heating continued to be an issue [120]. Defrosting of frozen food using RF was a major application, but problems of uniformity with foods of mixed composition limited the actual use. The interest in RF defrosting has increased again in the last number of years [125]. Today, the use of a more recent 50 Ω RF heating equipment which allows the RF generator to be placed at a convenient location away from the RF applicator gives the possibility of an advanced process control [126]. Whether conventional or 50 Ω dielectric heating systems are used, the RF applicator has to be designed for the particular product being heated or dried. RF post-baking, RF-assisted baking, and RF meat and fish defrosting systems will continue to benefit both existing and emerging food applications and the availability of low cost RF power sources could lead to a major growth in the use of RF heating in the commercial food sectors. RF heating is well established in industry and, for many applications, it is the standard method. Its equipment is well proven and also reliable. It is an excellent choice where it fits.

VIII. CONCLUSION The fundamentals of microwave heating should be studied in depth before spending a great deal of effort and time on trial and errors. Microwave and radio frequency heating all provide a product that is potentially superior in quality to the product produced by conventional techniques. This point is key to almost all industrial processes. The potential synergistic effects of microwaves combined with steam, forced-air convection, and/or infrared will probably lead the future expansion of microwave processing technology. Microwaves are an extremely expensive way to evaporate water as compared to frying, high-velocity hot air, or infrared. They can be commercially successful if the products are of high intrinsic economic value and can carry the extra cost burden put on them.

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126

Pulsed Electric Field in Food Processing and Preservation

Paul Takhistov Rutgers University

CONTENTS I.

II.

III.

IV.

V.

VI. VII. VIII.

IX.

Treatment Chambers and Equipment ..............................................................................................................126-2 A. Batch Type Processing (“Static”) Chambers ..........................................................................................126-3 1. Parallel Plate Electrode Chambers ....................................................................................................126-3 2. Glass Coil Static Chambers ..............................................................................................................126-3 B. Continuous Flow PEF Chambers ............................................................................................................126-4 1. Parallel Plate Chambers ....................................................................................................................126-4 2. Co-Field Flow Chambers ..................................................................................................................126-4 3. Coaxial Continuous PEF Chambers ..................................................................................................126-4 4. Enhanced Electric Field Continuous Treatment Chambers ..............................................................126-4 C. Special Design Flow-Through Chambers ................................................................................................126-4 1. Continuous Chamber with Ion Conductive Membrane ....................................................................126-4 2. Chamber with the Electrode Reservoir Zones ..................................................................................126-4 Mechanisms of Microbial Inactivation ............................................................................................................126-4 A. Electrical Breakdown ..............................................................................................................................126-5 B. Electroporation ........................................................................................................................................126-5 Events of Electroporation and Microbial Lysis ..............................................................................................126-5 A. Electric Field-Induced Transmembrane Potential ..................................................................................126-5 B. Kinetics of Electroporation in Cell Membranes ......................................................................................126-6 C. Colloid Osmotic Lysis ............................................................................................................................126-6 D. Electroosmosis in Electropores ................................................................................................................126-6 Microbial Inactivation Kinetics ......................................................................................................................126-7 A. Microbial Factors in Efficacy of PEF Processing ....................................................................................126-7 1. Type of Microorganisms ....................................................................................................................126-7 2. Growth Stage of Microorganisms ....................................................................................................126-7 B. PEF Microbial Inactivation ......................................................................................................................126-7 PEF Process Calculations and Variables ........................................................................................................126-8 A. Electric Field Intensity..............................................................................................................................126-8 B. Treatment Time ........................................................................................................................................126-8 C. Pulse Waveshape ......................................................................................................................................126-8 D. Treatment Temperature ............................................................................................................................126-9 E. Electrochemistry of a Highly Polarized Electrode/Food Product Interface ............................................126-9 Mathematical Model of Continuous Operation ............................................................................................126-10 Process Calculations ......................................................................................................................................126-11 Physical Properties of Food Products for PEF Processing ..........................................................................126-11 A. Conductivity, pH, and Ionic Strength ....................................................................................................126-12 B. Particulate Foods ....................................................................................................................................126-12 Application of PEF in Food Preservation ....................................................................................................126-12 A. Processing of Apple Juice and Cider ....................................................................................................126-12 126-1

126-2


B. Processing of Orange Juice ....................................................................................................................126-12 C. Processing of Cranberry Juice ..............................................................................................................126-13 D. Processing of Milk ................................................................................................................................126-13 E. Processing of Eggs ................................................................................................................................126-13 F. Processing of Green Pea Soup ..............................................................................................................126-14 G. Processing of Yogurt Based Product ......................................................................................................126-14 H. Processing of Rice Pudding ..................................................................................................................126-14 X. PEF as a Hurdle Technology ..........................................................................................................................126-14 References ................................................................................................................................................................126-16

Pulsed electric field (PEF) processing is a non-thermal method used to maintain food safety and increase shelf life of foods by inactivating spoilage and pathogenic microorganisms. Many researchers have investigated this problem, including Sale and Hamilton (82, 83), Mizuno and Hori (64), Jayaram et al. (36), Qin et al. (75), and Pothakmury et al. (71). PEF processing is advantageous over other methods because the changes in product color, flavor, and nutritive value during the treatment are minimized (19, 40–42). A high intensity pulsed electric field processing involves the application of pulses of high voltage (typically 20–80 kV/cm) to foods placed between two electrodes. PEF treatment is conducted at ambient, sub-ambient, or slightly above ambient temperatures for less than 1 s, and energy loss due to heating of foods is minimized. For food quality attributes, PEF technology is considered superior to traditional heat treatment of foods because it avoids or greatly reduces the detrimental changes of the sensory and physical properties of foods (78). Although some studies have concluded that PEF preserves the nutritional components of foods, effects of PEF on the chemical and

nutritional aspects of foods must be better understood before PEF can be used in food processing (74). Some important aspects in pulsed electric field technology are the generation of high electric field intensities, the design of chambers that impart uniform treatment to foods with minimum increase in temperature, and the design of electrodes that minimize the effect of electrolysis. The large field intensities are achieved through storing a large amount of energy in a capacitor bank (a series of capacitors) from a DC power supply, which is then discharged in the form of high voltage pulses (108). Studies on energy requirements have concluded that PEF is an energy-efficient process compared to thermal pasteurization, particularly when a continuous system is used (73).

I. TREATMENT CHAMBERS AND EQUIPMENT Currently, there are only two commercial systems available (one by PurePulse Technologies, Inc. and one by

PEF

Continuous flow systems

Batch systems

Parallel plate chamber

Coil chamber

Co-field flow chambers

Parallel plate chambers

Co-axial flow chambers

Constant channel chambers

FIGURE 126.1 Classifications of PEF treatment chambers.

Field enhancement chambers


126-3

Thomson-CSF). Different laboratory- and pilot-scale treatment chambers have been designed and used for PEF treatment of foods. They are classified as static/batch (U-shaped polystyrene and glass coil static chambers) or continuous (chambers with ion conductive membrane, chambers with baffles, enhanced electric field treatment chambers, and coaxial chambers), see Figure 126.1. A diagram for PEF processing of foods is depicted in Figure 126.2. The test apparatus consists of seven major components (25): a high-voltage power supply, an energy storage capacitor, a treatment chamber(s), a pump to conduct food though the treatment chamber(s), a cooling device, measuring devices (voltage, current, and temperature measurements), and a computer to control operations.

A. BATCH TYPE PROCESSING (“STATIC”) CHAMBERS

(Figure 126.3a). Different spacers regulate the electrode area and amount of food to be treated. The brass blocks are provided with jackets for water recirculation and controlling temperature of the food during PEF treatment. This chamber could support a maximum electric field of 30 kV/cm. The second chamber model designed by Dunn and Pearlman (19) consists of two stainless steel electrodes and a cylindrical nylon spacer. Another model (3) consists of two round-edged, disk-shaped stainless steel electrodes, with polysulfone used as an insulation material. The effective electrode area is 27 cm2 and the gap between electrodes can be selected at either 0.95 or 0.5 cm. The chamber can support up to 70 kV/cm. Water circulating at pre-selected temperatures through jackets built into electrodes provides cooling of the chamber. 2. Glass Coil Static Chambers

1. Parallel Plate Electrode Chambers This model consists of two carbon electrodes supported on brass blocks placed in a U-shape polystyrene spacer

A model proposed by Lubicki and Jayaram (59) uses a glass coil surrounding the anode. The volume of the chamber is 20 cm3, which requires filling liquid with high conductivity and similar permittivity to the sample (media — NaCl

High voltage power supply

Initial product

Treated product

Pump

Heat exchanger

Treatment chamber

FIGURE 126.2 Flow chart of PEF food processing. +

+ a

–

(b)

–

(c)

+

–

+

–

+ +

–

+

–

–

(a)

(d)

+

(e)

FIGURE 126.3 Different PEF treatment chambers: a — parallel plate chamber, b — continuous flow parallel plate chamber, c — co-field flow chamber, d — coaxial continuous chamber, e — enhanced electric field continuous treatment chamber.

126-4


solution, σ ⫽ 0.8 to 1.3 S/m; filling liquid (water) ⬃10⫺3 S/m) to be used because there is no inactivation with a non-conductive medium (silicone oil).

B. CONTINUOUS FLOW PEF CHAMBERS Continuous flow PEF treatment chambers (77) are suitable for large-scale operations and are more efficient than the static chambers. 1.

Parallel Plate Chambers (Figure 126.3b)

The first experimental chambers were designed to treat a confined, static volume. Some of the first designs incorporated parallel plate geometry using flat electrodes separated by an insulating spacer. The major disadvantage of this type of design is the low productivity of these chambers. Due to the electric field strength limitation it is difficult to increase product load and make this chamber more efficient. 2.

Co-Field Flow Chambers

Co-field chambers described by Yin et al. (106) have two hollow cylindrical electrodes separated by an insulator to form a tube through which the product flows (Figure 126.3c). Field distribution in a co-field chamber is not expected to be uniform, though some useful advantages may be gained by special shaping of the insulator. The primary advantage of co-field chambers is that they can be designed to operate in PEF systems at lower currents than the coaxial chambers.

(a)

Coaxial chambers are generally composed of an inner cylinder surrounded by an outer annular cylindrical electrode that allows food to flow between them, see Figure 126.3d. A protruded outer electrode surface enhances the electric field within the treatment zones and reduces the field intensity in the remaining portion of the chamber. The electrode configuration was obtained by optimizing the electrode design with a numeric electric field computation. Using the optimized electrode shape, the prescribed field distribution along the fluid path without an electric field enhancement point was determined. This treatment chamber has been used successfully in the inactivation of pathogenic and non-pathogenic bacteria, molds, yeasts, and enzymes present in liquid foods such as fruit juices, milk, and liquid whole eggs (3). 4. Enhanced Electric Field Continuous Treatment Chambers Yin et al. (106) applied the concept of enhanced electric fields in the treatment zones by development of a continuous co-field flow PEF chamber with conical insulator

–

(b)

–

+

–

+

–

FIGURE 126.4 Special design chambers: a — continuous treatment chamber with ion-conductive membranes separating the electrode and food, b — continuous treatment chamber with electrode reservoir zones.

shapes to eliminate gas deposits within the treatment volume (Figure 126.3e). The conical regions were designed so that the voltage across the treatment zone could be almost equal to the supplied voltage.

C. SPECIAL DESIGN FLOW-THROUGH CHAMBERS 1.

Continuous Chamber with Ion Conductive Membrane

One design by Dunn and Pearlman (19) consists of parallel plate electrodes and a dielectric spacer insulator (Figure 126.4a). The electrodes are separated from the food by conductive membranes made of sulfonated polystyrene and acrylic acid copolymers. An electrolyte is used to facilitate electrical conduction between electrodes and ion permeable membranes. 2.

3. Coaxial Continuous PEF Chambers

+

+

Chamber with the Electrode Reservoir Zones

Another continuous chamber described by the same authors (19) is composed of electrode reservoir zones instead of electrode plates (Figure 126.4b). Dielectric spacer insulators have slot-like openings (orifices) between which the electric field enhances. The average residence time in each of these two reservoirs is less than 1 min.

II. MECHANISMS OF MICROBIAL INACTIVATION The application of electrical fields to biological cells in a medium (for example, water) causes build-up of electrical charges at the cell membrane (84). Membrane disruption in many cellular systems occurs when the induced membrane potential exceeds a critical value of 1 V, which, for example, corresponds to an external electric field of about 10 kV/cm for E. coli (15). Several theories have been proposed to explain microbial inactivation by PEF (2, 7, 44, 85, 86, 100, 105). Among them, the most studied (see Figure 126.5) are electrical breakdown and electroporation or disruption of cell membranes (113).


126-5

FIGURE 126.5 Schematic diagram of reversible and irreversible breakdown: pore development and cell membrane disruption.

A. ELECTRICAL BREAKDOWN

B. ELECTROPORATION

Zimmermann (112) explains what electrical breakdown of cell membrane entails. The membrane can be considered as a capacitor filled with a dielectric (Figure 126.5). The normal resisting potential difference across the membrane Vm is 10 mV and leads to the build-up of a membrane potential difference V due to charge separation across the membrane. V is proportional to the field strength E and radius of the cell. The increase in the membrane potential leads to reduction in the cell membrane thickness. Breakdown of the membrane occurs if the critical breakdown voltage Vc (of the order of 1 V) is reached by a further increase in the external field strength. It is assumed that breakdown causes the formation of transmembrane pores (filled with conductive solution), which leads to immediate discharge at the membrane and thus decomposition of the membrane. Breakdown is reversible if the product pores are small compared to the total membrane surface. With electric field strengths above critical and long exposure times, larger areas of the membrane are subjected to breakdown. If the size and number of pores become large in relation to the total membrane surface, reversible breakdown turns into irreversible breakdown, which is associated with mechanical destruction of the cell membrane. The corresponding electric field is Ecritical ⫽ Vcritical /fa, where a is the radius of the cell and f is a form factor that depends on the shape of the cell (84). For spherical cells f is 1.5; for cylindrical cells of length l and hemispheres of diameter d at each end, the form factor is f ⫽ l (1 ⫺ d )/3. Typical values of Vcritical required for the lysing of E. coli are of the order of 1 V. The critical field strength for the lysing of bacteria with a dimension of approximately 1 µm and critical voltage of 1 V across the cell membrane is therefore on the order of 10 kV/cm for pulses of 10 microseconds to milliseconds in duration (84).

Electroporation is the phenomenon which occurs when a cell exposed to high voltage electric field pulses temporarily destabilizes the lipid bilayer and proteins of cell membranes (15, 43, 45, 87, 114). The plasma membranes of cells become permeable to small molecules after being exposed to the electric field, and permeation then causes swelling and eventual rupture of the cell membrane (46, 88, 91, 92). The main effect of an electric field on a microorganism cell is increasing of the membrane permeability due to membrane compression and poration (31, 79, 103–105). Kinosita and Tsong (50) demonstrated that an electric field of 2.2 kV/cm induced pores in human erythrocytes of approximately 1 nm in diameter. They suggested a 2-step mechanism for pore formation in which the initial perforation is a response to an electrical suprathreshold potential followed by a time-dependent expansion of the pore size (Figure 126.5). Large pores are obtained by increasing the intensity of an electric field and pulse duration or reducing the ionic strength of the medium (26, 43).

III. EVENTS OF ELECTROPORATION AND MICROBIAL LYSIS A.

ELECTRIC FIELD-INDUCED TRANSMEMBRANE POTENTIAL

We now know that when a cell (radius ⫽ Rcell) suspended in a medium is exposed to external electric field (direct current of strength Eappl), there is a rapid redistribution of cations in the vicinity of the plasma membrane, thus generating a transmembrane potential ∆ψmembr with a rise time, τmembr: ∆ψmembr ⫽ 1.5RcellEappl cos θ [1 ⫺ exp(⫺t/τmembr)] (126.1)

126-6


τmembr ⫽ RcellCmembr (rint ⫹ rext /2)

(126.2)

Here θ is an angle between the field line and the normal from the center of the spherical cell to a point of interest on the membrane surface; Cmembr, rint, and rext are the membrane capacitance (per unit area), and the resistivities of the cytoplasmic fluid and the external medium respectively (17). For biological cells of micrometers in diameter, τmembr ⬍ 1 µs and the exponential term in equation (126.1) approaches zero within 1 µs. Cells of larger diameters have τmembr ⬎ 1 µs (49). The maximum transmembrane potential generated in a cell with the DC electric pulse a few times longer than τmembr is ∆ψmembr, max ⫽ 1.5RcellEappl

B. KINETICS OF ELECTROPORATION CELL MEMBRANES

(126.3)

IN

The plasma membrane of a cell is the first site of the electric interaction. Beside lipids, there are proteins, carbohydrates, and other types of molecules, most of which are either charged or polarizable. Channel proteins are especially sensitive to the ∆ψmembr, and each type of channel has a range of ∆ψmembr in which it becomes conductive. The range of ∆ψmembr for opening protein channels is approximately 50 mV, considerably smaller than the dielectric strength of the lipid bilayer, which is in the range of 150–400 mV. Like a lattice defect of the lipid bilayer, once a protein channel is forced to open, a strong current greatly exceeding the normal conductance of the channel will generate local heat sufficient to denature the protein. This denaturation could be reversible or irreversible, depending on the extent of temperature change and the properties of the channel. The time of opening/closing of the protein channel is in the submicrosecond time range (94). Thermal denaturation of a protein takes milliseconds to seconds. Renaturation of a protein occurs in seconds (48). Electropores in lipid domains will reseal within seconds (93). Closing of PEFperforated protein channels should transpire in milliseconds. However, repairing of a PEF-damaged cell membrane will take minutes to hours (50).

C. COLLOID OSMOTIC LYSIS A major difference between electroporation of lipid vesicles and that of cells is the colloid osmotic lysis of cells (50). A PEF-perforated cell membrane loses its permeation barrier to ions and small molecules but not necessarily to proteins. The electroperforated membrane becomes semipermeable to cytoplasmic macromolecules. The osmotic pressure of these macromolecules causes the cell to swell.

+

–

FIGURE 126.6 Electroosmosis-induced hydrodynamic flow toward the negative electrode regardless of whether electropores are in positive-facing or negative-facing hemispheres.

This process, known as colloidal swelling, eventually leads to a rupture of the plasma membrane because of the excessive osmotic pressure imposed on the cells. Colloidal osmotic pressure in the PEF-treated red blood cells was identified as the main cause of the electric field-stimulated hemolysis. Colloidal swelling depends on the osmotic imbalance of the cytoplasm and the suspending medium. When the difference is large, PEF-treated cells will swell in the minute time range. During this swelling phase, electropores in cell membranes also begin to reseal. If the resealing takes place faster than the swelling, cells will shrink again and recover their original volume, thus averting membrane rupture. If, on the other hand, the resealing is slower than the swelling, the plasma membrane of cells will be ruptured. The colloidal osmotic lysis may be prevented by balancing the osmotic pressure of the cytoplasm and the medium.

D. ELECTROOSMOSIS

IN

ELECTROPORES

An electric field parallel to the surface/liquid interface will cause a net hydrodynamic flow in the appropriate direction as long as there is an imbalance in the numbers of the two charges in the layer of liquid adjacent to the charged surface. If electropores, which are expected to be induced closer to the “poles” of the cell that face the electrodes, are viewed as cylinders with an average net negative (from ionized headgroups of phospholipids and ionized amino-acid side chains on integral proteins) charge on this surface and with their axis perpendicular to the plane of the membrane, then a hydrodynamic flow existence would be expected during the electric field pulse (Figure 126.6). It was predicted and experimentally demonstrated that the overall permeabilization difference between both hemispheres would be less than originally thought if an electroporation experiment were conducted to take electroosmosis into account (90).


IV. MICROBIAL INACTIVATION KINETICS Three types of factors that affect the microbial inactivation with PEF have been identified:

126-7

TABLE 126.1 Inactivation Models Hülsheger’s Model (32)

冢冣

t S⫽ ᎏ tc ●

●

●

the process factors (electric field intensity, pulse width, treatment time and temperature, and pulse waveshapes); microbial entity factors (type, concentration, and growth stage of microorganisms); and treatment media factors (pH, antimicrobial and ionic compounds, conductivity, and medium ionic strength).

Hülsheger and Niemann (32) were the first to propose a mathematical model for inactivation of microorganisms with PEF. Their model was based on the establishing dependence of the survival ratio S ⫽ N/No (the ratio of living cell count before and after PEF treatment) on the electric field intensity E by the following expression: ln S ⫽ ⫺bE(E ⫺ Ec)

(126.4)

where bE is the regression coefficient, E is the applied electric field, and Ec is the the critical electric field value obtained by extrapolating E for 100% survival. The regression coefficient reflects the gradient of straight survival curves and is a microorganism-media depending constant. The critical electric field was found to be the function of cell size and applied pulse duration. Hülsheger et al. (34) proposed an inactivation kinetic model that relates microbial survival fraction (S) with PEF treatment time (t) in the form of t ln S ⫽ ⫺bt ln ᎏ tc

(126.5)

where bt is the regression coefficient, t is the treatment time, and tc is the critical treatment time, or extrapolated value of t for 100% survival. The model proposed by Peleg (69) describes a sigmoid shape of the pathogen survival curves generated by the PEF inactivation. The model represents the percentage of surviving organisms as a function of an electric field and the number of pulses applied. This model is defined by the critical electric field intensity that corresponds to 50% survival (Ed), and the kinetic constant K that is a function of the number of pulses representing the steepness of the sigmoid curve. Generalized equations for both models are combined in Table 126.1. Small values of the kinetic constants for both models indicate a wide span in the inactivation rate curves and hence lower sensitivity to PEF, whereas large values imply a steep decline or higher susceptibility to PEF.

Peleg’s Model (69) 1 S⫽ E⫺Ed 1 ⫹ eᎏ K

⫺(E ⫺ Ec) ᎏᎏ K

E – electric field; t – treatment time; Ec – critical electric field; tc – critical time; K – kinetic constant.

Ed – electrical field when 50% of population is inactivated; K – kinetic constant.

Lower Ec (or Ed) values indicate lesser resistance of pathogens to the PEF treatment.

A. MICROBIAL FACTORS PEF PROCESSING 1.

IN

EFFICACY

OF

Type of Microorganisms

Among bacteria, gram-positive ones are more resistant to PEF treatment than gram-negative (33). In general, yeasts are more sensitive to electric fields than bacteria due to their larger size, although at low electric fields they seem to be more resistant than gram-negative cells (74, 82). A comparison between the inactivation of two yeast spp. of different sizes showed that the electric field intensity needed to achieve the same inactivation level was inversely proportional to cell size. These results are logical but inconsistent with the results obtained by Hülsheger et al. (33). Further studies are needed to better understand the effect of microorganism type on microbial inactivation effectiveness. 2. Growth Stage of Microorganisms Bacterial cells in logarithmic phase are more sensitive to various stresses than cells in lag and stationary phases. Microbial growth in logarithmic phase is characterized by high proportion of cells undergoing division, during which cell membrane is more susceptible to the applied electric field. Gaskova et al. (23) reported that the killing effect of PEF for S. cerevisiae in the logarithmic phase is 30% greater than for those in stationary phase of growth.

B. PEF MICROBIAL INACTIVATION Numerous publications on microbial inactivation present data on vegetative cells, the majority of them from a few genera. Extensive microbial inactivation tests have been conducted to validate the concept of PEF treatment as a non-thermal food pasteurization process (15, 73, 74, 98, 99, 108). An applied intensive pulsed electric field produces a series of degradative changes in blood, algae, bacteria, and yeast cells (15). The changes include electroporation

126-8


TABLE 126.2 Some Bacteria Effectively Inactivated with PEF Bacillus cereus (70, 81) Bacillus subtilis (18) Bacillus subtilis spores (28) Candida famata (102) Escherichia coli (95, 96) Listeria innocua (13, 14) Listeria monocytogenes (Scott A) (1, 21, 95, 96) Lactobacillus leichmannii (80, 95, 105)

Pseudomonas aeruginosa (52) Pseudomonas fluorescens (11, 97) Saccaromyces cerevisiae (111) Salmonella (37, 61) Staphylococcus aureus (ATCC 25923) (77) Yersinia enterocolitica (60)

and disruption of semipermeable membranes, which lead to cell swelling and/or shrinking, and finally to lysis of the cells. The mechanisms for the inactivation of microorganisms include electric breakdown, ionic punch-through effect, and electroporation of cell membranes (75). The inactivation of microorganisms is primarily caused by an increase in membrane permeability due to compression and poration (99). Castro et al. (15) reported a 5-log reduction in bacteria, yeast, and mold counts suspended in milk, yogurt, orange juice and liquid egg treated with PEF. Zhang et al. (109) achieved a 9-log reduction in E. coli suspended in simulated milk ultrafiltrate (SMUF) and treated with PEF by applying the converged electric field of 70 KV/cm strength, and a short treatment time of 160 µs. These processing conditions and results are adequate for commercial food pasteurization that requires 6- to 7-log reduction cycles. Table 126.2 presents the bacteria reported to be successfully inactivated by the pulsed electric field treatment.

V. PEF PROCESS CALCULATIONS AND VARIABLES To treat foods with PEF in a continuous system, the liquid food product is pumped through a series of treatment zones in the chamber with high voltage electrodes on one side of each zone and a low voltage electrode on the other side. The PEF process conditions are defined by an applied electric field strength and a treatment time.

A. ELECTRIC FIELD INTENSITY It is one of the main factors influencing microbial inactivation (20, 32). The microbial inactivation increases with an increase in the electric field intensity, above the critical transmembrane potential (72). This is consistent with the electroporation theory, in which the induced potential difference across the cell membrane is proportional to the applied electric field. The critical electric field Ec (an electric field intensity below which inactivation does not occur) increases with the transmembrane potential of the

cell. Trans-membrane potentials, and consequently Ec, are larger for larger cells (39). Pulse width (duration) also influences the critical electric field; for instance, with pulse widths greater than 50 µs, Ec is 4.9 kV/cm. With pulse widths less than 2 µs, Ec is 40 kV/cm (84).

B. TREATMENT TIME Treatment time is defined as the product of the number of pulses and the single pulse duration. An increase in any of the two variables improves microbial inactivation (82). As noted above, pulse width influences microbial reduction by affecting Ec. Longer widths decrease Ec, which results in higher inactivation; however, an increase in pulse duration may also result in an undesirable food temperature increase. Optimum processing conditions should therefore be established to obtain the highest inactivation rate with the lowest heating effect (24, 51). The inactivation of microorganisms increases with the treatment time (33). In certain cases, however, the number of pulses that increase inactivation rate reaches saturation. This is the case in Saccharomyces cerevisiae inactivation by PEF that reaches saturation with 10 pulses of an electric field at 25 kV/cm (3). Critical treatment time also depends on the electric field intensity applied (1, 80, 107). At electric field values above Ec, critical treatment time decreases with electric field increase. Barbosa-Cánovas et al. (3) reported that for the electric field strength 1.5 times higher than Ec, the critical treatment time would remain constant.

C. PULSE WAVESHAPE Electric field pulses may be applied in the form of exponentially decaying, square-wave, oscillatory, bipolar, or instant reverse charges (16, 77). Oscillatory pulses are the least efficient for microbial inactivation, and square-wave pulses are more energy and lethally efficient than exponentially decaying pulses (5, 107). Bipolar pulses are more lethal than monopolar pulses, because PEF causes movement of charged molecules in the cell membranes, and reverse in orientation or polarity of the electric field causes a corresponding change in the direction of charged molecules movement (29, 75). The difference between bipolar and monopolar pulses was reported in Bacillus spp. (30) and E. coli (75) inactivation studies. With bipolar pulses, the alternating changes in the movement of charged molecules cause a stress in the cell membrane and enhance its electric breakdown. Bipolar pulses also offer the advantages of minimum energy utilization, reduced deposition of solids on the electrode surface, and decreased food electrolysis (3). A study conducted by Zhang et al. (110) showed the effect of square-wave, exponentially decaying, and instant-charge-reversal pulses on the shelf life of orange


juice. Square wave pulses were more effective, yielding products with longer shelf lives than those treated with exponentially decaying and charge reverse pulses. In agreement with this study, Love (58) quantitatively demonstrated the stronger inactivation effect of squarewave pulses over all other wave shapes.

D. TREATMENT TEMPERATURE Experimental results have demonstrated that, in challenge tests, both treatment temperatures and process temperatures impact microbial survival and recovery (8, 107). PEF treatments at moderate temperatures (50 to 60°C) have been shown to exhibit synergistic effects on the inactivation of microorganisms (19, 36). At the constant electric field strength, pathogen inactivation increases with an increase in treatment temperature (13). Since application of an electric field causes increase in the temperature of the treated foods, proper cooling of treatment chamber is necessary to maintain food temperatures far below those existing during a thermal pasteurization process (10, 57, 68). Additional effects of high treatment temperatures include changes in cell membrane fluidity and permeability, which increase the susceptibility of the cell to mechanical disruption (34). Also, a low trans-membrane potential decreases Ec and therefore increases inactivation (39).

E. ELECTROCHEMISTRY OF A HIGHLY POLARIZED ELECTRODE/FOOD PRODUCT INTERFACE Usually, PEF processing is considered as “zero chemistry” treatment with no chemical reactions involved. However, reported changes in the sensory and physical attributes of processed foods are not solely the result of Joule heating and high electric current that passes through the food product. All treatment chambers in existing PEF systems have extremely high electrode surfaces-to-treatment volume ratio due to power supply and electric field strength limitations. Therefore, electrode materials are directly involved in the PEF treatment process. They interact with treated food products by electrochemical reactions that occur at the surface of highly polarized electrodes, and electric double layer assisted reactions of food particulates (solid phase of food product) with the electrode surface. These interactions include (6, 12, 101): ● ●

● ●

● ●

adsorption of organic and inorganic anions; changes in the chamber capacitance due to changes of electric double layer relaxation time; electrophoretic deposition of food solids; electrocoagulation of solid phase at the electrode surface; electrodissolution of electrode material; hydrogen/oxygen evolution due to electrode reactions.

126-9

Electrode surface interactions with food matrix components have been previously underestimated by the researchers and rarely investigated (5). Due to the importance of the electrode surface properties to the food product behavior in the electrode vicinity and PEF treatment process, electrochemical polarization characteristics of electrode material play the critical role. This is also important because electrical impulses can accelerate electrochemically induced changes in electrode polarization and food properties. The potential scanning measurements of various food products in the range from 0.5 to 3 V for two widely used electrode materials –– aluminum (alloy 2024) and stainless steel — have been performed. Electrochemical potential is the thermodynamic potential that characterizes reaction ability of an electrode in the solution. Increasing of thermodynamic potential by 1 V is equivalent to changing the reaction temperature by 103 K. Electrode polarization in the range between 2 and 3 V is considered as extremely high polarization. Despite the general similarity, both electrodes demonstrate different behavior in acid media (orange juice) (Figure 126.7). In low polarization region aluminum shows the more stable behavior and less corrosive activity. However, to increase electric current through the aluminum electrode one should maintain very high voltage. The stainless steel electrode can support higher current due to lower adsorbance of HO3 ions at the electrode surface. In the high polarization region both electrodes demonstrate similar volt-ampere characteristics. However, stainless steel has the potential dynamic curve shifted in the direction of higher values of electric current, and therefore is more suitable for PEF applications. For low electric field applications (ohmic heating) aluminum electrodes are more preferred. The food product composition is a significant factor influencing electrodic processes. Potential dynamic characteristics of stainless steel electrodes in orange juice, whole milk, and tomato soup are depicted in Figure 126.8. Tomato soup has classical corrosion-type characteristics that include Taffel region (adsorption) and electrodissolution (corrosion) of electrode material (12). Orange juice has similar characteristics except the two regions of current-voltage instability, which can be explained with polarized pulp aggregation at the electrode surface. The most unusual potential dynamic curve corresponds to the electrochemical treatment of milk. This type of curve usually corresponds to the passivated metal electrode. At low polarization potentials the behavior of stainless steel electrode in milk does not differ from the other food products. Potential rise leads to the deposition of milk constituents onto the electrode and blockage (passivation) of its surface. In the high polarization regime all three products behave similarly.

126-10


3

2.5

2

Potential, V

Aluminum 1.5

1

0.5 Stainless steel 0

–0.5 –7 10

10

–6

10

–5

–4

10 Current, A

10

–3

10

–2

10

–1

FIGURE 126.7 Polarization of stainless steel and aluminum electrodes in orange juice. 3.5 3

2.5

Potential, V

2 Orange juice

1.5 1 Milk

0.5 Tomato soup

0

–0.5 –7 10

–6

10

–5

10

–4

10

–3

10

–2

10

–1

10

Current, A

FIGURE 126.8 Potentiodynamic curves of polarized stainless steel electrode for various food products.

V. MATHEMATICAL MODEL OF CONTINUOUS OPERATION (22) From an engineering point of view, it should be of interest to differentiate between the single pass and recirculation modes of operation of PEF treatment chamber. In both cases, the mathematical model consists of energy and

mass balances, kinetic equations, and equilibrium conditions. It is possible to build a large and complicated mathematical model, but that would not be useful. In order to simplify the model, some assumptions may be adopted. Accordingly, plug type flow in the PEF chamber, perfect mixing in the tank, and a first order kinetics for the inactivation of microorganisms were assumed.


A simplified scheme of PEF installation operating in a single pass mode is depicted in Figure 126.2. It can be assumed that the concentration of microorganisms in the feed tank cT (microorganisms/L) is the same as that at the PEF chamber inlet, and that the rate of microorganism destruction r (microorganisms/(L/s)) follows the first order kinetics with respect to microorganism concentration c (microorganisms/L): r ⫽ ⫺kc

(126.6)

where k (s⫺1) is the kinetic constant of microorganism inactivation. Assuming stationary state and plug-type flow in the PEF chamber (56), the microorganism balance gives the following expression: q ln(c/cT) ⫽ ⫺kVr t

(126.7)

where q (L/s) is the fluid flow rate and Vr (L) is the PEF chamber volume. According to the last equation, the relation between the outlet microorganism concentration c (microorganisms/L) and time t(s) is exponential. ⫺kVr

c ⫽ cT e

ᎏ q

(126.8)

The energy balances are more complex. The energy E (J) dissipated during the discharge of the capacitor C (µF) at a voltage V(V) is given by the following equation: 1 E ⫽ ᎏ CV 2 2

(126.10)

However, only one part φ of this energy will heat the liquid food (flow q (L/s), density ρ) that passes through the PEF chamber. This ratio φ must be less than 1, and strongly depend on the electrical conductivity of the food product. Energy balance for the PEF chamber after the stationary state is reached is represented by: qρCp(T ⫺ TT) ⫽ φQ

(126.11)

where q (L/s), ρ (kg/L), and Cp (J/kg°C) are the flow rate, density, and specific heat of the liquid food product, respectively; TT and T (°C) are the temperature of food sample in the feed tank and in the chamber, respectively. Consequently, the increase in the temperature T⫺TT of the liquid food can be estimated as: 1 φfCV 2 (T ⫺ TT) ⫽ ᎏ ᎏ 2 qρCp

TABLE 126.3 PEF Process Variables Process Variable Electric field strength Total treatment time Number of electrode pairs in treatment chamber Treatment zone diameter Mean liquid velocity Product electrical conductivity Product density Product specific heat

Notation

Dimension

E t n

V/m s

D µ σ ρ Cp

m m/s Sm kg/m3 kJ/kg°C

VII. PROCESS CALCULATIONS The total possible temperature change per pair of electrodes in treatment chamber (∆T ), total energy input during treatment per electrode pair (P), and Reynolds number (NRe) can be calculated using the following equations: ∆T ⫽ (E 2tσ/ρCp)/n P ⫽ E 2tσ/n NRe ⫽ ρDu/µ

(126.13)

The process variables used in the equations are described in Table 126.3.

(126.9)

Taking into account frequency f(s⫺1) of the pulses, the energy dissipation per second during the liquid flow through the chamber Q (J/s) is: 1 Q ⫽ ᎏ fCV 2 2

126-11

(126.12)

VIII. PHYSICAL PROPERTIES OF FOOD PRODUCTS FOR PEF PROCESSING Physical properties of foods that are the most critical for PEF treatment efficacy are the electrical conductivity, density, specific heat, and viscosity of the product. Some useful data can be found in reference (5). Liquid foods contain several ion species that carry an electrical charge and conduct electricity. At a given voltage, the electrical current flow is directly proportional to the electrical conductivity of the food product (108). An increase in the electrical conductivity causes an increase in the overall energy input and change in the product temperature during processing. The density and specific heat of food product affect the temperature change during PEF treatment. As the density of product decreases, the total temperature change increases (108). Similarly, a decrease in product specific heat also increases the temperature change during PEF processing. The viscosity of the product determines flow characteristics, which are calculated based on the Reynolds number. For the Reynolds number greater than 5000, the product flow is turbulent, which provides uniform velocity profile in the treatment chamber that, in turn, is likely to provide uniformity of PEF process (8, 57).

126-12


A. CONDUCTIVITY, pH, AND IONIC STRENGTH The electrical conductivity of a medium (σ Ohm⫺1/m), which is defined as the ability to conduct electric current, is an important variable for PEF processing. Electrical conductivity is the reciprocal of resistivity (r), which is measured in Ohm/m. Foods with large electrical conductivities generate smaller peak electric fields across the treatment chamber, and therefore are not susceptible for PEF treatment (3). Studies on inactivation of Lactobacillus brevis with PEF showed that as the conductivity of the fluid increased, the resistance of the treatment chamber was reduced (36), which in turn reduced the pulse width and decreased the rate of inactivation. Since an increase in medium conductivity results from increase of its ionic strength, the latter leads to the decrease in bacteria inactivation rate. Furthermore, increased difference between the conductivities of a medium and microbial cytoplasm weakens the membrane structure due to an increased flow rate of ions across the membrane. Thus, the inactivation rate of microorganisms increases with decreasing conductivity, even at equal input energy (36). Yet another study performed by Dunne et al. (20) showed that the resistivity had no influence on E. coli and L. innocua PEF inactivation effectiveness. These controversial results may be due to the microorganisms or media used. Vega-Mercado et al. (99) studied the effect of pH and ionic strength of the medium (SMFU) during the PEF treatment. The inactivation ratio increased from not detectable (zero) to 2.5-log cycles as ionic strength of the solution was adjusted from 168 to 28 mM. At 55 kV/cm (8 pulses), as the pH was reduced from 6.8 to 5.7, the inactivation ratio increased from 1.45- to 2.22-log cycles. The PEF treatment and ionic strength of the solution were responsible for electroporation and compression of the cell membrane, whereas the pH of the medium affected the cytoplasm when the electroporation was complete. Dunne et al. (20) reported that, depending on the microorganism, acidic pH enhanced microbial inactivation, although no specific details were provided (what microorganisms were affected or what range of pH was used).

B. PARTICULATE FOODS Inactivation of microorganisms in liquid-particulate systems has been studied by Dunne et al. (20). E. coli, L. innocua, Staphyloccocus aureus, and Lactobacillus acidophilus were suspended in a 2 mm diameter alginate beads model system, and the effects of PEF process variables on microbial inactivation were tested. The researchers concluded that the process was effective in killing microorganisms embedded in particulates. However, to achieve more than a 5-log cycle reduction, high energy inputs were needed (70 –100 J/ml, depending on the bacteria treated). Qin et al. (76) reported that dielectric breakdown occurs when air or liquid vapors are

present in the food because of the difference in dielectric constants between liquid and gas.

IX. APPLICATION OF PEF IN FOOD PRESERVATION PEF has been mainly applied to preserve the quality of foods, such as to improve the shelf life of apple juice (Evrendilek et al., 2000a; Simpson et al., 1995), cranberry juice (Evrendilek et al., 2001a), skim and chocolate milk (Evrendilek et al., 2001a), orange juice (Qui et al., 1998; Yeom et al., 2000), liquid eggs (Hermawan, 1999), and pea soup (Vega-Mercado et al., 1996a).

A. PROCESSING

OF

APPLE JUICE AND CIDER

Simpson et al. (1995) reported that apple juice from concentrate treated with PEF at 50 kV/cm electric field strength, 10 pulses, 2 µs pulse duration, and maximum processing temperature of 45°C had a shelf-life of 28 days compared to a shelf life of 21 days of fresh-squeezed apple juice. There were no physical or chemical changes in ascorbic acid or sugars in the PEF-treated apple juice, and a sensory panel found no significant differences between untreated and electric field treated juices. Vega Mercado et al. (1997) reported that PEF treatment extended the shelf life of fresh apple juice, and apple juice at 22–25°C had a shelf life more than 56 days or 32 days, respectively. There was no apparent change in its physicochemical and sensory properties. Evrendilek et al. (2000a) indicated that PEF treatment of apple juice and PEF ⫹ mild heat treatment of apple cider improved the shelf life quality of the products compare to control samples at 4, 22, and 37°C without degradation of vitamin C and change in the color measured by L (white if L ⫽ 100, black if L ⫽ 0), a (⫺a ⫽ green, a ⫽ red), and b (⫺b ⫽ blue, ⫹b ⫽ yellow) values.

B. PROCESSING

OF

ORANGE JUICE

Sitzmann (1995) reported the reduction of native microbial flora of freshly squeezed orange juice by 3 log cycles with an applied electric field of 15 kV/cm without significantly affecting its quality. The shelf life of reconstituted orange juice treated with an integrated PEF pilot plant system consisted of a series of co-field chambers evaluated by Qui et al. (1998) and Zhang et al. (1997). It is reported that total aerobic counts were reduced by 3 to 4 log cycles under 32 kV/cm electric field strength. When stored at 4°C, both heat- and PEF-treated juices had a shelf life of more than 5 months. Vitamin C losses were lower and color was generally better preserved in PEF-treated juices compared to the heat-treated ones up to 90 days (storage temperature of 4 or 22°C) or 15 days (storage temperature of 37°C) after processing. In the study of Yeom et al. (2000) orange juice was treated by PEF, and with an


126-13

5

log cfu/ml

4 3 2 1 0 0

14

28

71

90

119

Storage days

FIGURE 126.9 Total plate count in chocolate milk during storage at 22°C. 䊊 ⫽ PEF ⫹ 112°C; X ⫽ PEF ⫹ 105°C; ▲ ⫽ control.

increase in electric field strength longer shelf life is obtained. Compared to heat treatment more flavor components were retained in PEF treated orange juice.

C. PROCESSING

OF

CRANBERRY JUICE

Cranberry juice was treated either by high voltage pulsed electric field at 20 kV/cm and 40 kV/cm for 150 µs, or by thermal treatment at 90°C for 90s. Higher electric field and longer treatment time reduced more viable microbial cells. The overall volatile profile was not affected by PEF treatment, but it was affected by heat treatment. Compared to control samples PEF treatment caused no color change in the samples. When treatment conditions were 40 kV/cm for 150 µs, there was no mold and yeast growth at both 22 and 4°C and no bacterial growth at 4°C (Jin and Zhang, 1999). PEF (32 kV/cm electric field strength, 500 pps frequency, 1.4 µs pulse duration, and 47 µs total treatment time) and PEF ⫹ heat (60°C for 32s) processing of cranberry juice revealed that shelf life cranberry juice stored at both 4 and 22°C increased significantly (for 197 days). The PEF and PEF ⫹ heat treatments did not cause any significant differences in the color retention of the samples (Evrendilek et al., 2001a).

D. PROCESSING

OF

MILK

Inactivation of Salmonella dublin and shelf life study with homogenized milk was performed by the electric field strength of 36.7 kV/cm and treatment time of over a 25 min (Dunn and Pearlman, 1987a). S. dublin was not detected after PEF treatment or after storage at 7–9°C for 8 days. The naturally occurring milk bacterial population increased to 107 cfu/ml in the untreated milk, whereas the treated milk showed approximately 4 ⫻ 102 cfu/ml. Further studies by Dunn (1987b) indicated less flavor degradation and no chemical or physical changes in milk quality attributes for cheesemaking. Fernandez-Molina et al. (2001) studied the shelf life of raw skim milk (0.2% milk fat), treated with PEF at 40 kV/cm, 30 pulses, and 2 µs treatment time using

exponential decaying pulses. The shelf life of the milk was 2 weeks when it is stored at 4°C; however, treatment of raw skim milk with 80°C for 6 s followed by PEF treatment at 30 kV/cm, 30 pulses, and 2 µs pulse width increased the shelf-life up to 22 days, with a total aerobic plate count of 3.6 log cfu/ml. Reina et al. (1998) studied the inactivation of Listeria monocytogenes Scott A in pasteurized whole, 2%, and skim milk by PEF. L. monocytogenes was reduced 1 to 3 log cycles at 25°C and 4 log cycles at 50°C, with no significant differences being found among the three milks. The lethal effect of PEF was a function of the field intensity and treatment time. CalderonMiranda (1999) studied the PEF inactivation of Listeria innocua suspended in skim milk and its subsequent sensitization to nisin. The microbial population of L. innocua was reduced by 2.5 log after PEF treatments at 30, 40, or 50 kV/cm. The same PEF intensities and subsequent exposure to 10 IU nisin/ml achieved 2, 2.7, or 3.4 log reduction cycles of L. innocua. Similar to cranberry juice, chocolate milk was processed by PEF (35 kV/cm electric field strength, 600 pps frequency, 1.4 µs pulse duration, and 45 µs total treatment time) and PEF ⫹ heat (105 and 112°C for 31.5 s) by pilot plant PEF processing system (Figure 126.4). Compare to control samples, the shelf life of chocolate milk treated by PEF ⫹ 105°C and PEF ⫹ 112°C increased significantly at 4, 22, and 37°C (Figure 126.9). The PEF ⫹ heat treatments did not cause any significant differences in the color retention (Evrendilek et al. 2001a).

E. PROCESSING

OF

EGGS

PEF studies in liquid eggs, on heat-pasteurized liquid egg products, and on egg products with potassium sorbate and citric acid (added as preservatives) were conducted by Dunn and Pearlman (1987b). Comparisons were made with regular heat-pasteurized egg products with and without the addition of food preservatives when the eggs were stored at low (4°C) and high (10°C) refrigeration temperatures. The study focused on the importance of the hurdle approach in shelf-life extension. Its effectiveness was even more evident during storage at low temperatures,

126-14


where egg products had a final count around 2.7 log cfu/ml stored at both 10°C and 4°C. The samples maintained a low count for 4 and 10 days, respectively, versus a few hours for the heat pasteurized samples. In addition to color analysis of eggs products, Ma et al. (1997) evaluated the density (indicator of egg protein-foaming ability) of fresh and PEF-treated LWE (liquid whole egg) as well as the strength of sponge cake baked with PEF-treated eggs. The stepwise process used did not cause any difference in density or whiteness between the PEF-treated and fresh LWE. The strength of the sponge cakes prepared with PEF-treated eggs was greater than the cake made with non-processed eggs. This difference in strength was attributed to the lower volume obtained after baking with PEF-treated eggs. The statistical analysis of the sensory evaluation revealed no differences between cakes prepared from PEF processed and fresh LWE. A study reported by Hermawan (1999) indicated that there is a 90% of reduction of Salmonella enteritidis inoculated into LWE with circulation mode fluid handling system using 200 pps pulse repetition rate, 2.12 µs pulse duration, and 25 kV/cm electric field strength.

F. PROCESSING

OF

GREEN PEA SOUP

Vega-Mercado et al. (1996) exposed pea soup to two steps of 16 pulses at 35 kV/cm to prevent an increase in temperature beyond 55°C during PEF treatment. The shelf life of the PEF-treated pea soup stored at refrigeration temperature (4°C) exceeded 4 weeks, and 22 or 32°C was found inappropriate to store the product. There were no apparent changes in the physical and chemical properties or sensory attributes of the pea soup directly after PEF processing or during the 4 weeks of storage at refrigeration temperatures.

G. PROCESSING

OF

OF

X. PEF AS A HURDLE TECHNOLOGY In general, controlling the combination of factors (hurdles), such as pH, ionic strength, and antimicrobial compounds of the solution, during PEF treatment can effectively aid in microorganism inactivation. The term hurdle technology covers an intelligent use of multiple preservation procedures in combinations specifically relevant to particular types of foods. The concept is pertinent to the control of pathogenic and food spoilage microorganisms, and to almost all food commodities and products. Furthermore, hurdle technologies have been traditionally employed in all countries of the world, although with greatly differing emphasis depending on the history and social characteristics of different cultures (55). Preservation technologies are based mainly on the inactivation of microorganisms or on the delay or prevention of microbial growth. Consequently, they must operate through those factors that most effectively influence the survival and growth of microorganisms (35). Such factors are not numerous. They include a number of physical factors, some chemical ones, and some that are essentially microbial in that they depend on the nature of microorganisms present in particular products. The most widely quoted classification of those factors derives from the original proposals of Mossel and Ingram (67), updated by Mossel (66). They include: -

YOGURT BASED PRODUCT

PEF and mild heat (60°C for 30 s) processing of yogurt based products similar to a dairy pudding dessert and yogurt based drink revealed that the combination of PEF plus mild heat significantly increased the microbial stability of the product at 4 and 22°C without any difference in the sensory attributes. Sensory evaluation of the products indicated that there was no significant difference between control and processed products. Color, pH, and °Brix were not significantly affected by the processing conditions (Evrendilek et al. 2001b; Yeom et al. 2001).

H. PROCESSING

showed that total plate count and a value for color measurement of the PEF treated and control samples were significantly different. PEF treated rice pudding has a shelf life of 94 days, whereas, control samples were spoiled in 10 days (Ratanatriwong et al. 2001).

RICE PUDDING

Due to its higher viscosity, rice pudding was preheated to 55°C for 30 s before PEF treatment. Processing conditions were 33 kV/cm electric field strength, 100 L/h flow rate, 1.47 µs pulse duration, and 500 pps frequency. Monopolar negative pulse was applied. Shelf life studies of the product

-

-

-

-

Intrinsic factors: Physical and chemical factors that exist within a food product, and with which contaminating microorganisms are inextricably in contact. Processing factors: Procedures that are deliberately applied to foods in order to achieve improved preservation. Extrinsic factors: Factors that influence microorganisms in foods, but which are applied from or exist outside the food; they also act during storage. Implicit factors: Factors related to the nature of microorganisms present in food product, and to their interactions among themselves and with the environment during growth. Net effects: These take into account that many of the factors strongly influence each other, so that the overall effect of combinations of different factors may not be obviously predictable, but may be usefully greater than the perceived effects of the single factors.


Combination (hurdle) effects are the focus of many of the recent developments in the predictive modeling of microbial growth and survival in foods. The limits presented at which these different preservative factors inactivate or inhibit relevant microorganisms must be used to evaluate the effects of these factors on spoilage and food poisoning microorganisms. However, it has to be remembered that the limits listed only apply if all other factors are optimal for the microorganisms in question. But this is hardly the case in any foodstuff. If more than one of the preservative factors (hurdles) is present then an additive or even synergistic effect results, and this is the basis of the hurdle effect and the intentional hurdle technology. The effective hurdle technologies typically employ multiple hurdles to preserve foods. In the use of such multiple hurdles, a consideration of the stress reactions and adaptations that microorganisms undergo underpins the logic of employing hurdles that affect different targets in the microbial cell. Ideally, the targets should be complementary to gain synergism rather than simply additive effects (55). If mild heating can be applied to the food, then the injury that would impair the membranes functionality may represent a further sensible target, which should amplify the effects of the previously applied hurdles relying on properly functioning membranes. The potential value of the multitarget approach can therefore be appreciated easily, and perhaps built on more logically in the future. An example of a multitarget novel process is PEF food treatment, which damages the cell membrane, in combination with the application of nisin, so the membrane cannot be repaired due to the membrane-active action of nisin (13, 65). Overall, therefore, homeostasis is interfered with by attacking two distinct targets. It has been suspected that different hurdles in a food might not just have an additive effect on microbial stability, but could act synergistically (53). A synergistic effect could be achieved if the hurdles in a food hit, at the same time, different targets within the microbial cells and thus disturb the overall homeostasis of microorganisms present in several respects. If so, the repair of homeostasis as well as the activation of “stress shock proteins” in microorganisms becomes more difficult. Therefore, employing simultaneously different hurdles in the preservation of a particular food should lead to optimal stability (54). In practical terms, this could mean that it is more effective to employ different preservative factors (hurdles) of small intensity than one preservative factor of larger intensity, because different preservative factors might have a synergistic effect. The multitarget preservation of foods is a promising research area, because if small hurdles with different targets are selected, a minimal but most effective preservation of foods could be accomplished. It is anticipated that the targets in microorganisms of different preservative

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factors will be more fully elucidated, and that hurdles could then be grouped in classes according to their targets. A mild and effective preservation of foods, i.e., a synergistic effect of hurdles, is likely to be achieved if the preservative measures are based on intelligent selection and combination of hurdles taken from different target classes (54). This approach is probably valid not only for the traditional food-preservation procedures, but also for modern processes of food irradiation, ultra-high pressure, or pulsed electric field, or light beam treatments, in combination with conventional hurdles.

a. PEF ⫹ hydraulic pressing Mechanical expression is widely used in the food industry for extraction of fruit juices and vegetable oils, dewatering of fibrous materials, etc. (89). Efficacy of this process can be increased by raw material plasmolysis, cellular damage, or permeabilization prior to its expression. Various methods are traditionally used to increase the degree of raw material plasmolysis: heating, osmotic drying or freezing dehydration, alkaline breakage, enzymatic treatment, etc. (4). Earlier on, the method of electric field treatment (both DC and AC) was also proposed for cellular material plasmolysis. The methods of electro-plasmolysis were shown to be good for juice yield intensification and for improving the product quality in juice production (62), processing of vegetable and plant raw materials (27), foodstuff processing (63), winemaking (47), and sugar production (38). But all these electric field applications are usually restricted by the high and uncontrolled increase in food temperature and product quality deterioration because of electrode material electrolytic reactions, etc. Bazhal et al. (9) investigated the influence of PEF applied simultaneously with pressure treatment on juice expression rate from fine-cut apple raw material. Three main compression phases were observed in the case of mechanical expression. A unified approach was proposed for juice yield data analysis allowing a reduction in data scattering caused by the differences in the quality of samples. PEF application at the moment when the presscake’s specific electrical conductivity reaches the minimum and the pressure achieves its constant value is reported to be the most optimal. The combination of pressing and PEF treatments significantly enhances the juice yield and improves its quality in comparison with samples untreated by PEF. The PEF treatment intensifies pressing whenever it is applied. The best juice excess yield results at the lowest value of the applied field may be obtained when PEF is applied after some pre-compression period. Such pressure pretreatment before PEF application is necessary for structuring uniformity of the press-cake, removing excess moisture, and decreasing the electrical conductivity of the material. In Bazhal et al. (9) the precompression period of 300–400 s and PEF treatment after that period were found

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to be optimal for the quality of juice, which was confirmed by its coloration and transmittance. The simultaneous pressure application and PEF treatment reveal the passive form of the PEF-induced cell plasmolysis, which develops very slowly under a low electric field without pressure application. The pressure promotes damage of defective cells, enhances diffusion migration of moisture, and depresses the cell resealing processes.

b. PEF ⫹ bacteriocins Microorganisms in the presence of PEF suffer cell membrane damage, and nisin is a natural antimicrobial known to disrupt cell membrane integrity. Thus the combination of PEF and nisin represents a hurdle for the survival of Listeria innocua in the liquid whole egg, which has been investigated by Calderon et al. (13). L. innocua suspended in liquid egg was subjected to two different treatments: PEF and PEF followed by exposure to nisin. The selected frequency and pulse duration for PEF was 3.5 Hz and 2 ms respectively. Electric field intensities of 30, 40, and 50 kV/cm were used. The number of pulses applied to the liquid whole egg was 10.6, 21.3, and 32. The highest extent of microbial inactivation achieved with PEF was 3.5 log cycles (U) for an electric field intensity of 50 kV/cm and 32 pulses. Treatment of liquid egg by PEF was conducted at relatively low temperatures, 36°C being the highest. Exposure of L. innocua to nisin after the PEF treatment exhibited an additive effect on the inactivation of the microorganism. Moreover, a synergistic effect was observed as the electric field intensity, number of pulses and nisin concentration increased. L. innocua exposed to 10 IU nisin/ml after PEF exhibited a decrease in population of 4.1 U for an electric field intensity of 50 kV/cm and 32 pulses. Exposure of L. innocua to 100 IU nisin/ml following PEF treatment resulted in 5.5 U for an electric field intensity of 50 kV/cm and 32 pulses. The model developed for the inactivation of L. innocua by PEF followed by the exposure to nisin (13) was established to be successful in predicting the extent of microbial inactivation resulting from the combination of PEF and nisin. The combination of these two preservation factors proved to be a hurdle against the survival of L. innocua in the liquid whole egg. When energy conservation is a goal, inactivation of L. innocua in liquid egg products can be accomplished by selecting the proper combination of PEF intensity and nisin concentration. Carvacrol was used in another study as a third preservative factor to further enhance the synergy between nisin and pulsed electric field treatment against vegetative cells of Bacillus cereus (70). Applied simultaneously with nisin, carvacrol (0.5 mM) enhanced the synergy found between nisin and PEF treatment (16.7 kV/cm, 30 pulses) in potassium-N-2-hydroxy-ethylpiperazine-N-ethanesulfonic acid (HEPES) buffer. The influence of food ingredients on bactericidal activity was tested using skimmed

milk that was diluted to 20% with sterile demineralized water. The efficacy of PEF treatment was not affected by the presence of proteins, and the results found in HEPES buffer correlated well with the results obtained in milk. Nisin showed less activity against B. cereus in milk, and carvacrol was not able to enhance the synergy between nisin and PEF treatment in milk, unless used in high concentrations (1.2 mM). This concentration in itself did not influence the viable count, but carvacrol did act synergistically with PEF treatment in milk, and not in HEPES buffer. This synergy was not influenced by milk proteins, since 5% milk still allowed synergy between carvacrol and PEF treatment to the same extent as 20% milk.

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Nanotechnology and Its Applications for the Food Industry


CONTENTS I. Introduction ......................................................................................................................................................127-2 II. Nanotechnology in Food Science ....................................................................................................................127-2 III. New Properties of Materials at Nanoscale........................................................................................................127-3 A. Effects of Size Constraints........................................................................................................................127-4 B. Shift of Characteristic Time Scales ..........................................................................................................127-4 C. Magnetic Properties ..................................................................................................................................127-4 D. Thermal Properties....................................................................................................................................127-4 E. Energy Conversion and Transport ............................................................................................................127-4 F. Friction Control at Nanoscale ..................................................................................................................127-5 IV. Controlled Synthesis and Processing at Nanoscale ..........................................................................................127-5 A. Nanotechnology: Drawing Inspiration from Nature ................................................................................127-5 B. New Manufacturing Paradigm ..................................................................................................................127-6 C. Synthesis of Individual Building Blocks ..................................................................................................127-7 1. Nanocrystals ......................................................................................................................................127-7 2. Nanotubes and Rods ..........................................................................................................................127-7 3. Polymeric Electronic Materials: Dendrimers and Block Copolymers ..............................................127-7 D. Templated Growth and Assembly ............................................................................................................127-7 E. Driven Systems..........................................................................................................................................127-7 F. Phase Transformations ..............................................................................................................................127-7 G. Directed Synthesis of Nanoparticles, Nanotubes, and Nanostructured Materials ....................................127-8 H. Nanomechanics and Nano-to-Micro Assembly ........................................................................................127-8 V. Nano-Bioengineering ........................................................................................................................................127-8 VI. Control and Measurement Paradigm at Nanoscale ..........................................................................................127-9 A. Nanoscale Instrumentation........................................................................................................................127-9 B. Nanosensors ............................................................................................................................................127-10 C. Smart Systems Integration: Sensing, Localization, Reporting, and Control ..........................................127-11 VII. Applications of Nanotechnology in Food Industry ........................................................................................127-11 A. Nanodevices for Identity Preservation and Tracking..............................................................................127-11 B. Nanodevices for Smart Treatment/Delivery Systems ............................................................................127-11 C. Nanoparticles Technology ......................................................................................................................127-12 D. Nanoemulsion Technology......................................................................................................................127-12 E. Packaging ................................................................................................................................................127-12 VIII. Conclusion ......................................................................................................................................................127-13 References ..................................................................................................................................................................127-13

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I. INTRODUCTION Nanotechnology — a term introduced in 1974 to describe ultrafine machining of matter — now can be applied to a wide scope of small-scale engineering (133). Nanotechnology research has emerged as one of the most revolutionary scientific topics in decades. Nanotechnology focuses on the physical/biological structures smaller than 100 nm, which result in unique material properties because of their nanosize. Some of these structures can be manipulated and converted into nanomachines able to perform functions previously not possible. Nanotechnology arises from the exploitation of new properties, phenomena, processes, and functionalities that matter exhibits at intermediate sizes between isolated atoms or molecules (⬃1 nm) and bulk materials (over 100 nm). The reason that nanoscale materials and structures are so interesting is that size constraints often produce qualitatively new behavior. When the sample size, grain size, or domain size becomes comparable with a specific physical length scale such as the mean free path of the molecules, the domain size strongly affects the corresponding physical phenomena. Figure 127.1 represents the fundamental science and engineering disciplines endowing in the nanoscience and nanotechnology development in their current state. The objective of this chapter is to show the potential of the nanotechnology field to the food science and technology community. Food manufacturing will benefit greatly from future developments in nanoscale science, engineering, and technology. For instance, nanoscale synthesis and assembly methods are expected to result in significant improvements in energy-efficient food processing; stronger, lighter materials that increase transportation efficacy; greatly improved chemical and biological sensing; use of low-energy chemical pathways to break down toxic substances for

environmental remediation and restoration; and better controls that enhance efficacy of manufacturing processes. The study of nanostructures in biological materials of plant and animal origin enables scientists to establish relationships between macroscopic and molecular properties of materials, such as molecular structure, degree of order, and intermolecular forces. Minuscule nanomachines able to circulate through the blood stream and clean out fat deposits from arteries, kill microbes, undo tissue damage, and reverse cancer, could be delivered to the human body through foods. This will put in a new perspective the health promotion role of foods (111). Generation of foods by nonbiological means using advanced nanotechnology could be another future development, meant to ensure enough nutrition for the entire human population with limited resources. In food systems it is possible to envision self-assembling molecules capable of building well-defined food structures; the manipulations of molecular conformation to deliver active compounds precisely to designated sites.

II. NANOTECHNOLOGY IN FOOD SCIENCE Nanotechnology has the potential to revolutionize world’s food system. Agricultural and food systems security, disease treatment delivery methods, new tools for molecular and cellular biology, new materials for pathogen detection, protection of the environment, and education of the public and future workforce are examples of the important links of nanotechnology to the science and engineering of agriculture and food systems. Some overarching examples of nanotechnology as an enabling tool for food industry are: ●

●

Physics

cien h. s Mat

ry

mist

c tri ec ng. l E e

Che

l ia er e at c M cien s

ce

●

al

Nano science

r te pu ce om n C scie Me dic i

ne

l ca ni a h . ec ng M e

ce

ien

c es

Lif

FIGURE 127.1 Nanoscience is the integrative combination of applied and basic sciences.

Manufacturing, processing, and shipment of food products can be made more secure through the development and implementation of nanosensors for pathogen and contaminants detection. The development of nanodevices that will enable maintaining the historical environmental records of a particular product and tracking of individual shipments. Systems that provide integration of sensing, localization, reporting, and remote control of food products (“Smart/Intelligent Systems”) that can increase efficacy and security of food processing and transportation.

Strategies to apply the achievements of nanoscience to the needs of food industry are quite different from the traditional nanotech applications. Food processing is multitechnology manufacturing with a broad range of raw materials used, high biosafety requirements, and wellregulated technological processes. Four major trust areas in food production can be significantly enhanced by nanotechnology, bringing it to the next technological level: development of new functional materials; micro- and

Nanotechnology and its Its Applications for the Food Industry

nanoscale processing; product development; and methods and instrumentation design for improved food safety and biosecurity. Possible nanotechnology applications in food industry, grouped by the target area are depicted in Figure 127.2. Employing nanoscience in food technology is the complex process not limited to improvement of individual processes and products, but considering the whole food supply chain as a continuous process sequence. Nanotechnology works at the same scale as a virus or disease-infecting particle, and thus has the potential for very early detection and eradication of pathogens. Nanotechnology holds out the possibility that “smart” treatment delivery systems could be activated long before macro symptoms appear. For example, a smart system could be a miniature device implanted in an animal that samples saliva on a regular basis. Long before an illness develops, the integrated sensing and monitoring system would detect the presence of a disease, and activate a targeted treatment delivery system. The fundamental processes in agriculture are explored through research in molecular and cellular biology. New tools for molecular and cellular biology that are specifically designed for separation, identification, and quantification of individual molecules are needed. This is possible with nanotechnology and will permit broad advances in agricultural research areas such as reproductive science and technology, conversion of agricultural and food wastes to energy and other useful by-products through enzymatic nanobioprocessing, and disease prevention and treatment in plants and animals. New materials that have special characteristics at the nanoscale will be a tremendous breakthrough in agriculture and food systems for pathogen and contaminant detection. Materials that have self-assembly and self-healing

Heat /mass transfer

Nano-scale reaction eng.

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properties will find a multitude of applications in agriculture. Packaging of food in “antimicrobial” containers would prevent food microbial contamination and facilitate food preservation, storage, and distribution. Protection of the environment through the reduction and conversion of agricultural materials into valuable products is an exciting potential area of nanotechnology advancement. The design and development of nano-catalysts for the conversion of vegetable oils into biobased fuels and biodegradable industrial solvents is one approach already under scientific examination, and would be greatly enhanced with the addition of nanotechnological abilities. Management of local and environmental emissions is another area of agriculture that could benefit from nanotechnology. Before reaching the dinner table, the lettuce, baked potato, broccoli, and warm wheat bread have survived a formidable number of challenges from the environment. Agricultural crops must be protected against the invasions of wild animals, weeds, insect pests, fungal pathogens, and the whimsy of the weather. Daily “scouting” of crops for potential problems is one of the most important tasks in the agriculture sector. Preventive monitoring and treatment of crops with nanoscale devices (sensors and delivery systems) can improve the quality of food products and durability of post-harvesting processes.

III. NEW PROPERTIES OF MATERIALS AT NANOSCALE The area of nanomaterials technology presents an unprecedented opportunity to investigate the new properties of materials at the nanoscale (158), and to exploit this knowledge to our benefit. The tools responsible for this

Nanobiotechnology

Molecular synthesis

Processing Nano-particles

Food science & technology

Product

Nano-composites

Materials

Delivery Nano-emulsions

Formulation Packaging

Nano-structured materials

Food safety & biosecurity

Nano-sensors

Nano-tracers

FIGURE 127.2 Application matrix of nanotechnology in food science.

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opportunity include synthesis methods that permit atomic and electronic structure of the material to be controlled to the atomic scale, in all three dimensions; instrumental probes that are capable of characterizing nanoscale materials, structures, and their properties on a wide range of length and time scales; and growing computational power which permits theoretical exploration of structures and properties from the atomic to the macroscopic scale. Nanomaterials can be either created through nanotechnology, or found in nature, such as nanoparticles existing in soil (clays, zeolites, imogolite, iron, and manganese oxides). They provide the potential to manipulate structures at the nanoscale, and to control and catalyze chemical reactions. The shape, structure, and aggregation abilities of individual particles at the nanoscale influence the properties of the material at the macro-level. “Smart fabrics” that can monitor the vital signs of the wearer are an example of the potential new uses envisioned for agricultural fiber products. Nanoparticles are also produced as agricultural by-products: airborne dust and aqueous runoff that cause air and water pollution. Controlling these nanoparticles is in the best interests of cost-effective and environmentally responsible agriculture. Soils are aggregates of nanoparticles, layer particles, organisms, and water. Viewing soil as a nanocomposite, and applying the paradigms and technologies of nanoscale science to it will lead to more efficient and environmentally friendly agriculture.

surface tension; and, the enhanced role of diffusion and corrosion at the large surface-to-volume ratios that exist in materials at nanoscale (106).

B. SHIFT

OF

CHARACTERISTIC TIME SCALES

Lengthscale change is accompanied by concomitant changes in the characteristic time scale of physical phenomena. In part, this is no more than the increase in characteristic frequencies that follows from the decreased time required to travel shorter distances at a fixed propagation velocity (for phonons, photons, electrons, etc.). Another time-dependent phenomenon at nanoscale is an increased rate of kinetic processes due to the increased fluctuation rate, as the reduced dimensionality of important structural features (e.g., surface-to-volume ratio) becomes important or dominant. This effect also leads to the increased effectiveness of sensor elements in biological systems.

C. MAGNETIC PROPERTIES Dramatic quantization and other effects occur in magnetic materials at the nanoscale. The ability to control thin-film growth at the near-atomic level to form epitaxial and heteroepitaxial structures has recently been extended to magnetic nanostructures, including metallic, oxide, and semiconducting phases (51, 116).

D. THERMAL PROPERTIES A. EFFECTS

OF

SIZE CONSTRAINTS

Size constraints alone are often responsible for qualitatively new behavior of materials. For example, if the nanoscale structure is smaller than the characteristic lengthscale for scattering of electrons or phonons (the mean free path), qualitatively new modes of electrical current and/or heat transport can arise (49, 148). Thermodynamic properties, including interface phenomena and phase transitions demonstrate substantial changes when the system size is comparable to the particle size or the coherence length for collective behavior (70). Systems with components sizes ranging from a few tenths to about ten nanometers lie at the fuzzy boundary between the quantum and classical domains. Such systems are also in the size range where thermal energy fluctuations and Brownian motion can have significant effects. The mechanical properties also change dramatically as the grain size in polycrystalline materials approaches the nanometer scale (3). Changes in the strength of nanoscale structural elements, changes in the nature of friction, and effective modes of fluid flow (hence, of lubrication) all require new design strategies for micromachines (87). Modes of failure also will change, as the size of devices and machines decreases toward the nanoscale. The causes include different mechanical properties that will modify fracture characteristics; the increased importance of

Thermal transport properties of nanostructured materials have received relatively little attention in the past decade. It is well known that polycrystalline materials exhibit lower thermal conductivity than low-defect single crystals of the same material. Investigators have recently realized that this could result in significantly reduced thermal conductivities of nanostructured materials (86, 129), which are expected because of a reduction in the phonon mean free path due to grain boundary scattering (43). In contrast to the reduced thermal conductivity of nanostructured thin films or coatings, opportunities exist for increasing thermal transport rates in fluids by suspending nanocrystalline particles in them. These “nanofluids” have recently been shown to exhibit substantially increased thermal conductivities and heat transfer rates compared to fluids that do not contain suspended particles (96). Food industry widely employs heat exchangers that require fluids with efficient heat transfer properties. With new “nanofluids” with increased heat transfer rates manufacturers can make heat exchange systems smaller and lighter, and reduce the amount of energy and heat transfer fluid required for the system operation.

E. ENERGY CONVERSION AND TRANSPORT Energy conversion and transport in nanostructures impacts a variety of fields and applications. Although energy


conversion and transport at macroscales is relatively well understood, it is not at all clear at the nanoscale. For example, it is well known that thermoelectric refrigerators and engines are not as efficient as other energy conversion devices because heat conduction by phonons is too high in thermoelectric materials. There is evidence that nanostructuring can improve electron transport (67–69).

F.

FRICTION CONTROL AT NANOSCALE

Nano-devices are expected to significantly improve the performance of robots, computers, communication, and other electrical/optical/mechanical devices. However, friction imposes significant limitations on the usage of these tiny devices. As a manifestation of the nano in the macro, hundreds of millions of dollars can be lost as a result of wear, friction, breakdowns, and wasted energy at nanoscale. Achievements in the research allowing friction control at the nanoscale will result in highly improved performance in the macro world and can produce significant economic savings (21). Traditional lubrication methods employ organic substances whose functional groups can adsorb onto polar surfaces to form closed-packed arrangements of almost perpendicularly oriented lubricant chains. Nano-machines lubricant selection is complicated by new considerations. Due to the built-in-place nature of nano-mechanics, lubrication by the conventional means of hidden and contacting surfaces is prohibited. Fluid lubricants may also introduce capillary and viscous shear mechanisms, which would result in energy dissipation. Despite great progress made during the past half century, many basic issues in fundamental tribology such as the origin of friction and the failure of lubrication remained unsolved. Moreover, current reliable knowledge related to friction and lubrication is mainly applicable to macroscopic systems and machinery, and will be of only limited use (if at all) in nano-systems. When the lubrication film thickness is of the same order as the molecular or atomic size, the behavior of the lubricant becomes significantly different (75). Understanding the mechanisms of friction, lubrication, and other interfacial phenomena at atomic and molecular scales can provide designers and engineers with the required tools and capabilities to control friction, reduce unnecessary wear, and predict mechanical faults and failures of lubrication in nano-devices (20, 146).

IV. CONTROLLED SYNTHESIS AND PROCESSING AT NANOSCALE Nanotechnology is comprised of the large family of phenomena and processes. Only two of them –– nanomeasurement and nanomanipulation — are currenty developed and technically proven. Molecular manufacturing is the combination of these two activities. Application of

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nanotechnology to molecular manufacturing allows that the environmentally clean, inexpensive, and efficient manufacturing of structures, devices, and “smart” products based on the flexible control of architecture and processes at the atomic or molecular scale can be feasible in the near future. The ultimate goal of the molecular manufacturing is to produce complex products on demand from simple raw materials, e.g., inserting basic chemical elements in a molecular assembly factory to yield a common household appliance, perhaps with built-in sensors and actuators to respond to commands or changes in environmental conditions.

A. NANOTECHNOLOGY: DRAWING INSPIRATION FROM NATURE Living organisms are not just the collections of nanoscale objects — atoms and molecules; these objects are organized in hierarchical structures and dynamic systems, that are the results of the million years-long Mother Nature’s experiments. Tenth-nanometer ions such as potassium and sodium generate our nerve impulses. The size of vital biomolecules, such as sugars, amino acids, hormones, and DNA, is in nanometer’s range. Membranes that separate one cell from another, or one subcellular organelle from another, are about five-fold bigger. Proteins can be tens of nanometers across. Every living organism on Earth exists because of the presence, absence, concentration, location, and interaction of these “nano-structures.” The uses of biological molecules are split between two categories: ●

●

Biological molecules can be used in conjunction with other structures to perform just as they do in organisms. Biological molecules can be used in conjunction with other structures to perform in a novel manner, quite distinct from their natural function.

Functional nanostructures can incorporate individual biological molecules. For example, biosensors can use natural sugars or proteins as target recognition groups (30). Modified biological structures can be used to act in MEMS devices — for example, modified photosynthetic membranes can split water to hydrogen and oxygen (54). Functional multi-component structures can use molecules in unusual ways. The filament protein actin, found in muscle, can be attached to the enzyme ATP synthase, which is involved in the production of most of the cell’s ATP, its “energy currency” (115), and act as a molecular motor. Many specific functions performed by living systems employ nanometer-size structures in particularly intriguing ways that we would very much like to emulate: ●

Living systems utilize self-assembling multiple individual parts in a precisely defined functional structure.

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Living systems are hierarchically organized into complex structures. For example, collagen, a fiber that rivals steel in strength, is built through successive aggregation of single amino acid strands into triple helices, triple helices into microfibrils, microfibrils into fibrils, and fibrils into fibers. Living systems use template-based elements, such as DNA, for reproduction and recovery (125). Organisms can sense their molecular surroundings, having developed exquisitely sensitive nanometer-size sensors on their outer surfaces. Using the principle of molecular recognition, only the specific desired target can bind to the surface-mounted “receptor” molecules. Upon binding, these receptors change shape in a manner that alerts other components of the system to the presence of the target (78). Living systems use nanometer-size structures to act as highly selective pumps. The electron transport chain, which is central to the trapping of the energy content of nutrients, pumps protons from one side of the mitochondrial membrane to the other, against the chemical gradient (114). Neurons pump sodium ions out and potassium ions into the cell to prepare it for the next impulse (139). Living systems use nanometer-size structures as switches. Within 200 msec of the binding of a repellant molecule to a receptor, a phosphoryl group is transferred to a protein and the rotation of the bacterial “tail” is switched from counterclockwise to clockwise. This turns the organism around and allows it to swim away from the repellant (92). Living systems use nanometer-size structures to perform catalysis with specificity, selectivity, and rate enhancements that are hardly achieved artificially. Enzymes can be selective enough to catalyze a reaction with only one particular molecule from a mix of many, ignoring even its mirror image. Enzymes can selectively catalyze

Provide a power system

Process materials/components at the nanoscale

FIGURE 127.3 Manufacturing at nanoscale.

only one of many chemically allowed reactions with that molecule, with rate enhancements up to 1016 fold (164).

B. NEW MANUFACTURING PARADIGM The concept of manufacturing at the “nano” or atomic scale dates to more than three decades ago. Many developments in biotechnology, chemistry, computational tool building, electrical engineering, and physics have moved the scientific and engineering community closer to operating smoothly on the nanoscale. Manufacturing of new nano-materials with pre-determined functionality is a sequence of macroscale processes combined with the microscale control and energy delivery (see Figure 127.3). All individual steps in the suggested manufacturing sequence already exist at least in the lab-scale experiments. The aim of the next decade is the integration of individual steps and components into the working system. Extensive molecular manufacturing applications, if they become cost-effective, will probably not occur until well into the future. However, some products benefiting from molecular manufacturing technology may be developed in the near term. As initial nanomachining, novel chemistry, and protein engineering (or other biotechnologies) are refined, initial products will likely focus on those that substitute for existing high-cost, lower-efficiency products. Likely candidates for these technologies include a wide variety of sensor applications; biomedical products including diagnostics and therapeutics; extremely capable computing and storage products; and unique, tailored, smart materials, including those for food processing and biosecurity applications. Areas that are important to the future of molecular nanotechnologybased advanced manufacturing, and in which successful discoveries could serve other applications, include the following: ●

● ●

Macromolecular design and folding (32, 59, 94, 172); Self-assembly methods (79, 124, 174, 181); Catalysis (inorganic, enzyme, and other) (135, 160, 161);

Control of process variables and environment at nano-scale

Order nano-components into structure

Interface the system components with environment

Assembling nano-scale parts into the system


●

●

●

Dendrimers, fullerenes, and other novel chemical structures development (48); Bioenergetics, nanobatteries, and ultrasounddriven chemistry (17, 31, 33, 38, 134); Semiconductor-organic/biological interfaces studying (36, 53, 62).

C. SYNTHESIS

OF INDIVIDUAL

BUILDING BLOCKS

The first step of nanoscale manufacturing process is the fabrication of individual components (building blocks for the entire system) at nanoscale. These components can greatly differ in nature and required processing conditions, but all are suitable for the high-volume production. 1. Nanocrystals In the last decade there have been significant advances in the preparation of nanocrystals (26, 104). Many common materials, such as metals, semiconductors, and magnets, can be prepared as nanocrystals using colloidal chemistry techniques, which lead to a wide range of applications in unexpected areas, such as in biological tagging (25, 29). 2. Nanotubes and Rods The exciting discovery of the fullerenes was followed closely by the discovery of nanotubes of carbon (156). Carbon-based nanotubes have the potential to act as a hydrogen storage medium that could exhibit very high storage density per unit weight, which is critical for hydrogenbased transportation systems. A crucial issue is whether or not the hydrogen could be extracted efficiently from such a storage medium at relatively low temperatures. Nanotubes also show tremendous promise as building blocks for new materials. 3. Polymeric Electronic Materials: Dendrimers and Block Copolymers Tremendous advances in the preparation of organic building blocks of considerable complexity have been made through the last decade (105, 152, 159).

D. TEMPLATED GROWTH AND ASSEMBLY High surface area materials with nanoscale dimensions, i.e., small particles and clusters with very high surface-tovolume ratio, can be attained by creating materials where the void surface area (pores) is high compared to the amount of bulk support material (119). Nano-porous inorganic oxides can be an example of such materials. Properties of these materials, e.g., chemical reactivity, magnetic moment, polarizability, and geometric structure exhibit the strong dependence on surface dimensions.

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Nanoscale surfaces as well as nano-particles have considerable utility as controlled drug delivery systems and biosensitive molecules carriers (109). Increased surface area of a nano-structured material leads to the increasing of the surface chemical reaction rate and intensification of the electron transfer (i.e., biosensor output signal) through the solid-liquid interface. Furthermore, ordered nanoscale structure of a surface substrate stimulates ordering and self-assembly of deposited specific biological components at the molecular level. The use of nanoscale fabrication techniques for low dimensional devices is being investigated to complement the more traditional fabrication methods (40). These nano-synthesizing techniques take advantage of the self-patterning of natural systems, where the biointerface material is synthesized in the size and shape of the desired nano-structure. These methods include fabrication of nano-structure arrays using selfassembled epitaxial growth (14), chemical synthesis of colloidal nano-structures (2), synthesis of nano-structures in glass and polymer materials (155), and template based chemical synthesis of nano-structures (136). Preparation of mesoporous inorganic solids has been greatly advanced by Antonelli (4). The initial work showed that it is possible to use organic surfactant molecules to prepare complex patterns. These patterns can serve as the templates for the formation of an inorganic phase.

E. DRIVEN SYSTEMS A very promising area for processing of three-dimensional bulk nanoscale structures originates with the recent discovery that mesoscopic structures can be obtained by nonequilibrium processing, such as ion irradiation, implantation, and mechanical working (ball milling, etc.) (11, 88, 103). It is worth mentioning that similar processing schemes perform very important functions in biological systems. Self-assembly is one of the tools required to build ordered nanostructures. The dynamics underlying the selfassembly process is now well understood (24, 117). A material system embedding enormous complexity dynamically transits through a variety of states, squeezing out free energy along the way, to arrive at a functional (and desirable) configuration. The spontaneous organization of a vesicle and the folding of a protein are notable examples of this phenomenon. Self-assembly, however, is not a completely enabling and sufficient tool. Biological systems make extensive use of both self-assembly and dissipative processes to make important structures or effect adaptive changes (166, 167).

F.

PHASE TRANSFORMATIONS

Many processing schemes involve transformations between different phases of the material. Although thermodynamics ultimately determines the equilibrium phase for a given set of conditions, the answers as to whether and how this phase

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is achieved from the metastable precursor phase depend on system kinetics. Since most phase transitions involving nanostructured materials occur under conditions far from equilibrium, the kinetic pathways available to these systems are numerous and not well understood. This problem is further complicated by the large contributions to the free energy of nanoscale materials from interfaces, which substantially shift phase boundaries. It will be important for making progress in the areas of synthesis and processing of nanomaterials to develop better understanding of nanochemistry and the broader general issues of nucleation and growth. One example of how phase transformations are used in the processing of nanostructured materials has already been noted, namely the formation of metallic nanoparticles in silicate glass for non-linear optical devices (98). The processing of bulk nanostructured materials from bulk metallic glass provides another example of using phase transformations in nanotechnology (65, 179). Both materials are now commercially available.

H. NANOMECHANICS AND NANO-TO-MICRO ASSEMBLY The future of nanoscale devices depends upon the abilities of scientific community to physically manipulate the nano-sized parts and to build and integrate larger micro-to-mini scaled devices (42). However, relative significance of fundamental physical forces changes as parts are reduced in size. Figure 127.4 shows the variation of different forces with respect to parts scale. For parts with features of the order of a few millimeters and above, classical mechanical phenomena such as mass and friction dominate the manipulation processes. However, when part sizes are reduced below one millimeter threshold, surface effects such as adhesion due to surface condensation and the electrostatic potential between parts start to dominate in manipulation over the classical inertia effects present in large-scale moving structures.

V. NANO-BIOENGINEERING

G. DIRECTED SYNTHESIS OF NANOPARTICLES, NANOTUBES, AND NANOSTRUCTURED MATERIALS

The use of natural biological processes to create a desired compound or material from a defined feedstock, e.g., compost material from plant and animal wastes, is called bioprocessing. Nanobioprocessing focuses on and utilizes nanoscale technology to achieve the goal of bioprocessing with greater efficacy. The use of molecular probes or development of assays that allow rapid identification of microbes present in a feedstock are examples of the research at nanoscale that can increase the efficacy of bioprocessing. The product itself may be the bulk material or nanomaterial. Nano-engineering of a biological system is focused on the assembly of nanomaterials to create or enable a specific biological function and/or the subsequent characterization of that function. This can be viewed as a separate

Nanostructured materials also promise greatly improved structural properties in comparison with conventional metal alloys. For example, small-diameter bundles of single-walled carbon nanotubes have been predicted and observed (126, 141). They have the largest strength-toweight ratio of any known material with an elastic modulus ⬃1 TPa, which is approximately 102 times that of steel but with only 1/6 its weight. Such materials offer almost unimaginable economic benefits and product opportunities, if only they can be cross-linked to overcome the low shear modulus (141). 10−1

Electrostatic forces between charged bodies

10−4 Surface tension force

Force, m

10−7 vander Waals forces

10−10

10−13

Gravitational force

10−16 10−7

10−6

10−5

10−4

Part scale, m

FIGURE 127.4 Forces variation with scale.

10−3

10−2


project from the study of nano-biomaterials themselves, which is defined by their isolation and characterization or their synthesis from basic building blocks (57, 151, 152). The extension of nano-engineering to biology includes patterning of 2D templates to direct cell or tissue response for biocompatability or biosensor applications. Applications in medical science include implants, prosthesis, drug delivery, and diagnostics. Other examples include creating a biological input/output device using nano-fabrication techniques to enable communication with individual cells for information technology applications (53, 62, 110, 170). The field of computer science is struggling with the problem of how to integrate information science with biology (73). One approach has been through bioinformatics, where traditional computer science methodology and hardware is applied to manage the enormous amount of information now available from biotechnology (173). Another approach envisions the integration of the biology with computer science in creating new hardware and technology to enable direct communication with the biology. If successful, this will provide the platform for the treatment of biology as just another peripheral for sensing, data storage, and information security functions. Other applications of this hybrid system could include communication with individual cells to switch on and off the biomanufacturing of drugs in vivo, or the construction of biological–nonbiological hybrids for robotics studies and applications (63, 149, 153). Nanopatterned surfaces are the environment and location on which most chemical and biological interactions occur (12, 143). A bioselective surface has either an enhanced or reduced ability to bind or hold specific organisms or molecules (128). Bioselective surfaces are important to the development of biosensors, detectors, and catalysts, in the separation or purification of mixtures of biomolecules, and in the processing and packaging of foods (37, 182). The primary objective of nanoengineering is the engineering of the biological interface (66). The success of two-way communication between electrically active cells and microelectronic devices depends on the proper registration of cells with the microelectrode and their close association or “seal” to this microelectrode (80). The “seal resistance,” Rseal, is measured between the electrode and the grounded recording media. Low values of Rseal are associated with poor electrode-to-cell adhesion resulting in an attenuated and distorted bioelectrical signal, a problem inherent in most extra-cellular recording systems. In contrast, Rseal values in the GOhm range, which are typically achieved using glass micropippettes during electrophysiology measurements, permit clearer resolution of bioelectric signals. In tissue this seal is accomplished by the interactions of proteins and other biological macromolecules with the glycocalyx, or outside surface of the cells. The first step in recreating this interface is nanoengineering of the non-biological surface with biologically compatible materials such as proteins, peptides, and

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biologically active functional groups (47, 83, 89). Surface modification allows a large variety of biomolecules to be attached to a microelectrode surface, which facilitates cell– microelectrode adhesion, increases Rseal, and allows for geometric placement of cells (131). For example, it is possible to control nutrients supplied to cells, so that they produce desirable surface groups that allow binding of those cells to specific nonbiological surfaces (175). The immediate benefit is that if a cell is associated with a single microelectrode, it becomes an individual sensor permitting multiple independent assays that then enable statistical analysis. Extracellular recordings of these individual sensor elements would allow long-term, multi-site measurements of electrically active cell bodies and processes for building information technology devices (144). Modern chemistry and material sciences allow the systematic and parallel patterning of matter on the nanoscale (2). The controlled positioning of atoms within small molecules is of course routinely achieved by chemical synthesis of moles of identical molecules. Nanometer-size objects are much larger entities, comprised of thousands or even millions of atoms. There are many powerful new approaches to patterning on the nanoscale, including atom manipulations by scanning probe tips, and electron beam lithography (132, 165).

VI. CONTROL AND MEASUREMENT PARADIGM AT NANOSCALE A. NANOSCALE INSTRUMENTATION Progress in nanotechnology requires the appropriate tools to observe, characterize, and control phenomena at the nanoscale. A whole new generation of analytical instrumentation and nanoscale devices, capable of providing information about physical, chemical, and mechanical phenomena, and material properties at nanoscale must be developed. Nanotechnology is already benefiting from novel instrumentation developed during the past two decades, for example scanning probe microscopes (SPM, see Figure 127.5), and the new generation of synchrotron x-ray sources capable of studying materials at the nanoscale. On the other hand, recent discoveries in nanoscale science and engineering provide the basis for the development of unprecedented new tools. Thus, both opportunities and challenges in developing instruments for synthesizing nanostructures, as well as for characterizing of existing nanostructures and measuring properties of nanomaterials, will exist during the next decade. Scanning probe microscopy is a family of techniques, which provide images of the surface topography and, in some cases, surface properties on the atomic scale. The inventions of the scanning tunneling microscope (STM) (16) and the atomic force microscope (AFM) (15) have spawned the development of a variety of new scanning probe

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2

Y X

FIGURE 127.5 Schematic representation of Scanning Probe Microscope 1 — Laser source, 2 — tip, 3 — canteliever, 4 — photodetector array.

microscopes (SPMs)(168). Whereas the STM and AFM have found applications mainly in topographical imaging, the other SPMs added more functionality. Examples include the near-field scanning optical microscope (NSOM) (13), scanning thermal microscope (SThM) (99, 100), scanning capacitance microscope (SCM) (171), magnetic force and resonance microscopes (MFM) (72, 138), and the scanning electrochemical microscope (SECM) (7). Some current applications of SPM for nano-materials analysis include a sensor that can measure atomic-level forces that develop between two surfaces as they approach each other and come into contact also has been developed (74). The Interfacial Force Microscope (IFM) utilizes a feedback force sensor that eliminates the snap-to-contact event that is inherent in other scanning force microscope designs. The IFM allows measurement of the full range of adhesive interactions and can be used to image surfaces by controllably hovering out of range of contact. Understanding of how an atom travels over a surface at different temperatures, and ultimately incorporates into the surface, is crucial to making smaller, faster, smarter nanodevices. A new instrument called “Atom Tracker” (154) can observe an individual atom in motion, and track atomic motions up to 1000 times faster than a conventional STM. Continuous monitoring of the motion of an individual atom as it binds to various sites on a surface allows this diffusing atom itself to become a probe of the surface structure and properties. In order to understand processes used to produce nanosized particles deposited on surfaces, the new aerosol instrumentation is needed. Significant progress has been made recently, with instruments now available for detecting particles as small as several nanometers in diameter (145, 150). Nano-sensors have a high potential for deployment in areas and media that are not readily amenable to probing by traditional devices (177). An example is medical operations requiring minimally invasive surgery. A collection of

specialized devices may be needed to provide all necessary functionalities. One can imagine an ensemble of micro- or nanorobots that cooperatively explore, assess, and operate on various locations in an organ. This poses formidable challenges in signal processing, control, and interconnectivity (178). Consequently, as non-destructive, real-time measurements of the physical properties of nanoparticles and nanostructured materials evolve, and as their use to monitor nanomaterials processing is developed, they will provide exceptional opportunities for fundamental and applied investigations and problem-solving at nanoscale (27, 62).

B. NANOSENSORS Conventional sensors now can provide an abundance of information about the environmental conditions such as temperature and weather, data on air, land, and sea transportation, chemical contaminants, deceleration for release of airbags in automobiles, and countless other parameters. Biological organisms also have the ability to sense the environment. For example, humans sense the environment through sight, touch, taste, smell, and sound. In living organisms, various sensors operate over a range of scales from macro- (ear drum vibrations), to micro- (nerve cells impulses), to nanoscale (molecules binding to sensors in our noses). The exciting possibility of combining biology and nanoscale technology into sensors holds the potential of increased sensitivity, and therefore a significantly reduced response time to potential problems (120, 121, 162, 176). It is possible to design a bio-analytical nanosensor that could detect a single virus long before it multiplies and the symptoms become evident in a plant or an animal (133, 163). The potential applications for bioanalytical nanosensors include detection of pathogens (85), contaminants (55), heavy metals, particulates or allergens, and environmental conditions ( 35). Thundat et al. (157) has demonstrated that the interactions of antigen molecules with their corresponding antibodies, attached to surface of an AFM cantilever, can provide sufficient surface stress to bend the cantilever beam. Such a device is an example of hybridization and integration of nanostructures at several levels, because along with nanocomponents (antibodies bound to a cantilever surface) it has microcomponents (cantilever beams), which can be delivered in a chip (millimeter scale components), that integrates biology and biochemistry with engineering. Boxer (19) has recently developed the method of the deposition of lipid membranes into lithographically defined corrals. Early results suggest that this approach may allow electrochemical addressing of photo-defined membrane cells. Consequently, this is the first step towards integrating the functional nanotechnology demonstrated by living cells into robust machine architecture. The design


and assembling of specific structures at near-atomic scales requires precise controlling of the materials according to macro- and mesoscopic specifications, which are actually decided at the quantum level (180). The development of chips/sensors for rapid detection of biological pathogens is a critical area with applications in the food handling/processing industry, in biological/ chemical warfare, and in emerging biosecurity systems with early warning for exposure to air- and water-borne bacteria, viruses, and other antigens (9, 107). Microfabricated chips for DNA analysis (93, 97) and for the detection of polymerase chain reaction (91) have already been demonstrated. The µChemLab (50) has performed the research to incorporate similar structures into a fully autonomous analytical system that can be integrated into on-a-chip architectures. For example, organically functionalized mesoporous structures have been successfully integrated on a micromachined heating and flow stage to provide 1000-fold chemical preconcentration for on-a-chip analysis of chemical warfare agents.

C. SMART SYSTEMS INTEGRATION: SENSING, LOCALIZATION, REPORTING, AND CONTROL The nanotechnologies can only reach their full potential through integration. The “Smart Systems Integration” is similar to designing and building the logic of a “nervous system” that allows the individual parts to work together (56). Integration of the nanotechnologies into a working food safety management system (whether remotely or automatically controlled) requires successful electronic communication between several components, including sensing systems, reporting systems, localization systems, and control systems (101, 122). The logic to control the subsystems (control algorithms) must be developed and eventually translated into a computer language (35).

VII. APPLICATIONS OF NANOTECHNOLOGY IN FOOD INDUSTRY A. NANODEVICES FOR IDENTITY PRESERVATION AND TRACKING Application of nanotechnology in agriculture brings the opportunities that once were only possible in science fiction. Identity preservation (IP) is a system that provides customers with information about the practices and activities used to produce a particular crop, food, or other agricultural product. Regulatory agencies can take advantage of IP as a more efficient way of recording, verifying, and certifying agricultural practices. Through IP it is possible to provide stakeholders and consumers with access to information, records, and supplier protocols regarding the food product’s farm of origin, environmental practices used in its production, food safety and quality, and animal welfare issues.

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Each day multiple shipments of different agricultural products are moved all over the world. Currently, there are financial limitations of the number of inspectors that can be employed at critical control points for the safe production, shipment, and storage of foods and agricultural products (64). Quality assurance of agricultural products’ safety and security can be significantly improved through the IP. Nanoscale IP holds the possibility of the continuous tracking and recording the history of manipulations, processing steps, and transformations which a particular agricultural product experiences. In the future, these nanoscale intelligent monitors can be linked to recording and tracking devices to improve identity preservation of foods and agricultural products (35). Originally developed by the 3M Corporation, the MICROTAGGANT® brand identification particle is a microscopic, traceable, anti-counterfeit device that is highly versatile in its applications. The MICROTAGGANT is a distinct numeric code sequence represented in multiple colored layers format. The code becomes a unique “fingerprint” to which the meaning is assigned. Optional fluorescent, magnetic, and other qualities may be added that are detectable by scanners and sensors providing enhanced coding and identification capabilities. These particles can be easily detected in the field using UV/VIS light, 100X magnification glass, magnetic, or laser scanners. The color code sequence in every particle is identical for each specific color code lot (108).

B. NANODEVICES FOR SMART TREATMENT/ DELIVERY SYSTEMS MEMS technology is based on techniques used in the semiconductor fabrication industry and has generated significant enthusiasm among physicians and surgeons in recent years. At their most basic levels, MEMS are devices with dimensions of micrometers to a few millimeters that combine electrical and mechanical components to acquire data or do work. Implantable and transdermal drug delivery microsystems allow patients both accurate and continuous dosing of medication and allow delivery of drugs directly to their intended sites of action (18, 23). Today, the application of agricultural fertilizers, pesticides, antibiotics, probiotics, and nutrients is typically performed by spray or drench methods to soil or plants, or through feed or injection systems to animals. Delivery of pesticides or medicines is either provided as “preventive” treatment, or once the disease has multiplied and symptoms are evident in a plant or animal. Nanoscale devices are envisioned to have the capability to detect and treat an infection, nutrient deficiency, or other health problem long before the symptoms become evident at the macroscale. This type of treatment can be targeted to the specific area affected by the disease.

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“Smart delivery systems” for agriculture can be defined as a combination of the following: timecontrolled, spatially-targeted, self-regulated, remotely regulated, preprogrammed, or multifunctional release of treatment to avoid biological barriers for successful pathogens inactivation (90). Smart delivery systems also can have the capacity to monitor the effects of the delivery of pharmaceuticals, nutraceuticals, nutrients, food supplements, bioactive compounds, probiotics, chemicals, insecticides, fungicides, or vaccinations to people, animals, plants, insects, soils, and the environment (35). Smart treatment delivery systems are envisioned as bioactive systems: drugs, pesticides, nutrients, probiotics, nutraceuticals, and implantable cell bioreactors (34).

C. NANOPARTICLES TECHNOLOGY There is currently considerable research interest in the use of microparticles as carriers for poorly bioavailable drugs and vaccines via mucosal (particularly oral) routes (39, 41, 76, 140). A variety of therapeutic moieties, including peptides and proteins, have shown enhanced oral uptake when entrapped within various types of microparticulate system constructs, and this approach has also been used successfully for the oral, nasal, and rectal delivery of a variety of vaccines (8, 48, 71, 123, 137). Numerous investigations have shown that both tissue and cell distribution profiles of anticancer drugs can be controlled by their entrapment in submicronic colloidal systems (nanoparticles) (22, 123, 137). The rationale behind this approach is to increase antitumor efficacy, while reducing systemic side effects. Naturally occurring antioxidants in raw fruits and vegetables are thought to provide significant health benefits, such as reduced risk of heart attack, stroke, neurodegenerative diseases, and cancer. Processed foods, which are statistically more likely to be consumed by the high-risk individuals, as opposed to raw fruits and vegetables, typically lose some or all of the potency of their natural antioxidant content. Heat-sensitive nutrients like beta-carotene, Omega-3 fatty acids, and other anti-oxidants are significantly or totally degraded upon pasteurization and canning. In foods that do not naturally contain anti-oxidants, introduction of anti-oxidant compounds is difficult due to these compound’s high susceptibility to heat, pH variations, and other conditions existing during food processing. Now nanoscale engineered materials can protect antioxidants and other health-promoting food components from degradation during manufacturing and storage. Nanoparticles (46) and various types of nano-containers –– silica-shell (61) and “nanocochleate delivery vehicles” –– offer protection for a wide variety of nutrients that currently cannot be delivered in high-temperature, adverse pH, or other conditions. BDSI’s Nanocochleates offer the following benefits for processed food nutrients: pressure

and shear resistance; protection from oxidants (air, free radicals); protection from temperature extremes; protection from photodegradation (10).

D. NANOEMULSION TECHNOLOGY The method of infection control with conventional disinfectants requires a tradeoff: to ensure microorganisms are killed, the toxic chemical must be present at levels that create health and contamination risks. Nanoemulsion formulation works very differently (112, 118, 174). Nanospheres of oil droplets ⬍1 µm are suspended in water to create a nanoemulsion requiring only miniscule amounts of active antimicrobial ingredient (60, 113). The nanospheres carry surface charges that efficiently penetrate the microorganisms’ membranes (44, 84, 127, 169). Namoemulsions are effective against a variety of food pathogens including Gram-negative bacteria (58). The nanoemulsions can be rapidly produced in large quantities and remain stable for many months at room temperatures (6). Nano-emulsions are composed of ingredients, which are either food, e.g., vegetable oils, or are on the FDA GRAS list of food ingredients. They are proved to be effective for decontamination of food processing plants, and for reduction of surface contamination of meat and poultry products (28, 118).

E. PACKAGING No longer is packaging expected just to safely contain a product — it may now capture the history of a package, interact with the consumer, have functions other than dispensing (e.g., heating/cooling), communicate with appliances, or allow itself to be tracked through the supply chain (1). Nanotechnology has rised a new packaging paradigm — Smart Packaging (5) — which includes active packaging (147), smart tagging/labeling (52), self-venting films, anticounterfeiting and tamper-proof materials/technologies, RFID devices (81, 82), self-opening packages,

FIGURE 127.6 Nano-patterned aluminum barrier film — multifunctional substrate for sensor/packaging applications.

its Applications for the Food Industry Nanotechnology and Its

diagnostic and freshness indicators, responsive labels, timetemperature indicators, self heating/cooling packages, etc. The addition of reinforcing agents is widely used in the production of packaging films (77, 102, 142). It is expected that the reducing of the added particles size to nanoscale could enhance the performance of these materials (95, 130). The new polymer nanocomposite materials, which can be produced by adding the nanoscale ceramic powders to commercial products, are aimed to substitute more expensive barrier plastic films in food industry (45, 84).

VIII. CONCLUSION Many other examples can be given to illustrate the close link between fundamental studies of nanoscale phenomena and their technological applications. Although observed at nanoscale changes in material properties can dominantly affect the nanoscale structures, we still have remarkably little experience or intuition for the anticipated phenomena and their practical implications, except for the case of electronic systems. The physics, chemistry, and biology of phenomena occurring at nanoscale is effectively the new subject with its own set of physical principles, theoretical descriptions, and experimental techniques, which we are only in the process of discovering. Thus, there is an urgent need for broad investigations of the phenomena associated with nano-systems and structures, especially in materials and structural contexts where the implications are not at all well understood. Implementation of nanoscience methods and nanoscale materials in the food industry can bring it to the next technological level. Fundamental changes in food manufacturing and technology due to scientific advances add new qualities into the established industrial practice: ● ● ●

●

●

●

control of mass and heat transfer at nanoscale; improved nanomaterials; vastly increased manufacturing capacity due to the miniaturization and combination of processes; improved logistics due to the high performance computing and integration of “smart” nanodevices into products packaging and transportation; better access to resources and control of energy consumption by introducing micro- and nanoscale power sources and more efficient processes; total food quality control by managing food additives and health promoting food components interacting with the product matrix at nanoscale.

In short, nanotechnology has the potential to make as much difference as the discovery of agriculture, steel, germ theory, the assembly line, the colonization of America, electricity, the airplane, computers, and genetic engineering all

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put together — without further environmental damage! And this is expected to happen within the next few decades.

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128

Biosensor Technology for Food Processing, Safety, and Packaging


CONTENTS I.

Needs of Food Quality/Safety Control ............................................................................................................128-2 A. Food Processing ........................................................................................................................................128-2 B. Food Contamination ..................................................................................................................................128-2 C. Sources/Raw Materials ..............................................................................................................................128-3 D. Food Substitutes and Genetically Modified Foods (GMF) ......................................................................128-3 E. Food Industry Hygiene ..............................................................................................................................128-3 II. Sources of Information for Biohazards Detection ............................................................................................128-3 III. Biosensors: General Facts ................................................................................................................................128-4 IV. Types of Biosensors ..........................................................................................................................................128-5 A. Mechanical (Resonant) Biosensors ..........................................................................................................128-5 1. Sensors Based on Electromagnetic Waves ........................................................................................128-5 B. Optical Detection Biosensors ....................................................................................................................128-6 1. Surface Plasmon Resonance ..............................................................................................................128-6 C. Electrochemical Biosensors ......................................................................................................................128-6 D. Impedimetric/Conductometric Biosensors ................................................................................................128-6 E. Amperometric Biosensors ........................................................................................................................128-7 F. Potentiometric Biosensors ........................................................................................................................128-7 1. Field Effect Transistors (FET) and Ion-Selective Field Effect Transistors (ISFET) ..........................128-7 G. Cell-Based Biosensors ..............................................................................................................................128-8 H. Lab-on-a-Chip Systems and DNA Detection Devices ..............................................................................128-8 I. DNA-Based Sensors/Assays ....................................................................................................................128-8 V. Applications of Biosensors in Food Science and Manufacturing ....................................................................128-9 A. Sensors for Pathogens Detection ..............................................................................................................128-9 B. Sensors to Monitor Food Packaging and Shelf-Life ..............................................................................128-10 1. Foreign Body Detection ....................................................................................................................128-10 C. Biosensors for Food Quality/Additives Control ......................................................................................128-11 D. Biosensors for Sensory Evaluation of Food Products ............................................................................128-12 VI. Role of Biosensors in the Food Safety Management System ........................................................................128-13 A. Biosensors and Biosecurity ....................................................................................................................128-13 B. Biosensors and HACCP ..........................................................................................................................128-14 VII. Future of Biosensing: Detection of Cellular Response with Nanosensors ....................................................128-15 References ................................................................................................................................................................128-16

Biosensor technology is a powerful alternative to conventional analytical techniques, harnessing the specificity and sensitivity of biological systems in small, low cost devices. Despite the promising biosensors developed in research

laboratories, there are not many reports of real applications in food safety and quality monitoring. A sensor is the device that can detect a property or group of properties in a food product and respond to it by a signal, often an electric 128-1


TABLE 128.1 Sensor Operation Modes

log

ns si o cl u In

hno

rce

Tec y

Food safety & quality or ns Se

C

y

n

tio

H

a ul

i yg

signal. This signal may provide direct information about the quality factor(s) measured, or may have known relation to the quality factor. Usually, sensors are classified according to their mode of use (see Table 128.1).

ns io at l) in a m ic ta em on (ch

Off-line

Sensors/detectors operate directly in the process stream, giving a real-time signal related to the quality factor of concern. ⬍30 min Sensors are used, in split-flow measurements, requiring reagent additions or sufficient time for the equilibration or chemical reaction to occur 1 hr–24 hr Sensors/assays are used in the laboratory with extensive periods of time required to performing the measurements

C

At-line

Environment

⬍5 min

Sou

On-line

on (b tam io i lo na gi ti ca on l) s

128-2

e en

rm

Fo

I. NEEDS OF FOOD QUALITY/SAFETY CONTROL Food quality control is essential in the food industry; nowadays, an efficient quality assurance is becoming increasingly important. Consumers expect adequate quality of food product at a fair price, long shelf-life, and high product safety, while food inspectors require safe manufacturing practices, adequate product labeling, and compliance with the FDA regulations. Further, food producers are increasingly demanding the efficient control methods, particularly through on-line or at-line quality sensors to satisfy consumers’ and regulatory requirements, and also to improve the feasibility of automated food processing, quality of sorting, and to reduce the production time (increase throughput) and the final product cost. Extensive development of biosensors for food safety and quality control were stimulated by acquiring several new food safety and key quality concepts during the last decade: Hazard Analysis Critical Control Points (HACCP), Total Quality Management (TQM), ISO 9000 Certifications. The wave of terrorist acts and foodborne diseases outbreaks has raised the importance of the food traceability and authentication (77, 99). There are specific safety problems (pathogenic microorganisms, BSE, GMF, pollutants, etc.), which require intensive control, data logging, and data treatments and can be effectively controlled only with the new generations of biodetection systems (46). All these tasks require in-time and on-line sensors for new data analysis systems, warning systems, tight feedback loops for automated processing, etc. Figure 128.1 explains needs for biosensors in food safety and quality management, showing the sources of biohazard contaminations in foods and their influence on technological, “shelf-life,” and perception properties of food products. The major sources of undesirable contaminants and changes in foods can be combined in five groups by their localization and occurrence. Three of them are food

FIGURE 128.1 Needs for food safety and quality control: sources of pollution and contamination.

manufacturing-related: technology (processing and sequence of process operations), industrial hygiene (food safety management at the plant level, HACCP), and formulation (product development, interactions of food additives/ingredients with food matrix, bioavailability). The sources of food raw materials and their quality are the issue of biosafety/biosecurity in the agricultural processing including post-harvesting technologies and logistics. The fifth source of biohazards is the environment in the broadest sense, including pollution, climate changes, and anthropogenic environmental factors. Below is the brief description of some possible sources of undesirable contaminations and/or changes in food products.

A. FOOD PROCESSING It is well known that many important nutrients are denaturalized, altered, or even destroyed by the faulty processing of foods. Food can also become contaminated during processing, handling, distribution, and consumption. Many undesirable or even harmful substances can enter the food as additives and toxic metabolites during its processing and preservation.

B. FOOD CONTAMINATION Food can become contaminated during every step of food processing sequence, from cultivation to consumption. The contaminants may be: Microbiological: viral, bacterial, parasitological, and fungal;


Chemical: pesticide residues, nitrates, nitrites, highsalinity, fluorides, arsenic compounds, lead, and other heavy metals. These pose serious and longterm health threats; Harmful metabolites and biological toxins (e.g., methyl alcohol, estrogen-like substances, hormones, biotoxins including mycotoxins especially aflatoxins, allergens, and carcinogens).

C. SOURCES/RAW MATERIALS The major aspect in the area is the utilization of food sources, which were previously wasted or not used. This is mainly to enrich fodder, thus ensuring better recovery for human consumption indirectly.

D. FOOD SUBSTITUTES AND GENETICALLY MODIFIED FOODS (GMF) There has been incredible progress in new biotechnology with commercialized products of insulin, human growth hormones, interferon, and recombinant vaccines using human cell culture or “novel” bacteria (90). Unfortunately, some people are allergic to some food ingredients. A reliable system of diagnosis and treatment of infant’s milk intolerance exists in all countries. Allergies to natural foods are less common and less serious compared to those to food additives and untraditional or inedible food varieties.

E. FOOD INDUSTRY HYGIENE In addition to the need for development of appropriate policy related to health (135), agriculture, trade, manufacture, and licensing, the rational consumer protection regulatory systems are to be developed and enforced (30). The next level of the diagram in Figure 128.1 represents the major types of changes in foods caused by the sources of undesirable contaminants. They can be instrumentally controlled, hence represent the primary targets for biosensors development and design. Indeed, the great challenge is to develop the real-time and on-line sensors and data systems suitable for surveying processes and products, controlling automated processes and the raw material stream, sensing the final products quality, typing the product labels with nutritional and health information, and much more.

II. SOURCES OF INFORMATION FOR BIOHAZARDS DETECTION The detection of biohazards can be performed directly (see Figure 128.2) by measurements of the pollutants/ pathogens concentration in a food product with specifically aimed biosensors. Another (indirect) approach to determining the presence/level of biohazard is through the measurements of changes in processing parameters (temperature, pressure, water activity, etc.) that lead to variations in

128-3

Indirect detection

Direct detection

Bio-hazard detection

External information

FIGURE 128.2 detection.

Internal knowledge base

Sources of information for biohazards

microbial contamination levels. External information can alert the food safety management system on possible increase of bacterial contamination or risk of bioterrorists’ attack and/or environmental pollution splash. This information, i.e., expectations of high contamination level, can be used to perform changes in screening and sampling procedures, and extend the range of pathogens to be detected. Internal knowledge base (“sensor-free detection”) is the set of accumulated data/records giving the correlation between the properties of raw materials, process parameters, and biohazard level in manufactured products. Today, the most important quality parameters and concepts in food production control are: ●

●

●

●

●

●

●

● ● ●

●

Sensory: appearance, flavor, taste, texture, stability, etc.; Nutritional, including health implications, such as “high in fiber,” “low cholesterol,” “GMF free,” etc.; Composition and labeling: additives lists, quality and ethical claims (e.g., ecological information), etc.; Pollutants record: environmental pollutants, veterinary drugs, agricultural chemicals, BSEprions and mycotoxins; Detection of foreign bodies, such as stones, glass, or metal fragments; Microbial safety, in particular Listeria, Salmonella, Campylobacter, E. coli, and Yersinia; Shelf-life: microbial, sensory, chemical, sterility testing, F0-values; Production hygiene: cleaning, decontamination; HACCP: traceability and authentication; Process parameters control: machine settings, temperature, pressure, flow, aseptic conditions, and many others; Packaging: integrity, pinholes, gas permeability, migration control.

128-4


III. BIOSENSORS: GENERAL FACTS Biosensors usually are small analytical bio-electronic devices that combine a transducer with a sensing biological component (biologically active substance). The transducer, which is in intimate contact with the biologically sensitive material, can measure weight, electrical charge, potential, current, temperature, or optical activity of the substance. The biologically active species include enzymes, multi-enzyme systems, antibodies or antigens, receptors, populations of bacterial or eukaryotic cells, or whole slices of mammalian or plant tissue, to name a few. Substances such as sugars, amino acids, alcohols, lipids, nucleotides, etc. can be specifically identified and their concentration measured by these sensors. A schematic functional representation of a biosensor and the detection principle is depicted in Figure 128.3. The biosensor consists of a biological sensing element integrated with a signal transducer; together they produce a reagent-free sensing system specific for the target analyte.

Biodetection

Biosensor

Analyte

Bio-selective element

Transducer

Signal processing

Data analysis

FIGURE 128.3 Schematics of biodetection and biosensor design.

The biological component of a biosensor used for the molecular detection is made of highly specialized macromolecules or complex systems with the appropriate selectivity and sensitivity. Biosensors can be classified according to the biocomponents used for the detection. The biodetection principle can be schematically described as follows. A chemical, biological, or physical sensor produces a signal (e.g., voltage, absorbance rate, heat, or current) in response to a detectable event, such as binding between two molecules. In case of a biological or chemical sensor this event typically involves a receptor (e.g., macrocyclic ligand, enzyme, or antibody) binding to a specific target molecule in a sample. Physical sensors, on the contrary, measure inherent physical parameters of a sample, such as current or temperature, which can change due to reactions occurring in it. In any case, the signal is then transduced by passing it to a circuit where it is digitized. The obtained digital information can be stored in a memory, displayed on a monitor, or made accessible via digital communications port. Since it is essential that the sensor’s response be detected, it is necessary that an appropriate transduction mode for electrochemical signals, optical signals utilizing changes in the fluorescence or absorbance rate of a sample, or plasmon resonance be available. With most sensors, transduction is accomplished electrochemically or optically. The transducer transforms the physicochemical variations occurring in the biosensing element as the result of a positive detection event into an electric signal, which is then amplified by an ad hoc designed electronic circuit, and used for the control of external devices. The transducers can be electrochemical (amperometric, potentiometric, conductometric/impedimetric), optical, piezoelectric, or calorimetric. Very often this classification is used to identify the type of biosensor (see Figure 128.4).

Biosensors

Bio-element

Transducer

Antibody Enzyme

Principle of operation Fluorescence

Molecular

Optical

Surface plasma resonance Adsorbance/reflectance

Nucleic acid

Piezoelectric

Cell-based Mechanical

Surface acoustic wave Cantilever resonance frequency

Tissue-based

Amperometric

Electrochemical

Potentiometric Impedimetric

FIGURE 128.4 Biosensors classification.


The bio-specific elements of the biosensor and transducer can be coupled together in one of the four possible ways (80), schematically shown in Figure 128.5: membrane entrapment, physical adsorption, matrix entrapment/porous encapsulation, covalent bonding. In the membrane entrapment scheme, a semi-permeable membrane separates the analyte and the bioelement, and the sensor is attached to the bioelement (collagen membranes, synthetic preactivated membranes (102), cellulose-acetate membranes). The physical adsorption scheme is depending on a combination of van der Waals forces, hydrophobic forces, hydrogen bonds, and ionic forces to attach the biomaterial to the sensor surface (52). The porous entrapment scheme is based on forming a porous encapsulation matrix around the biological material that helps in binding it to the sensor (nylon net (60), carbon paste (36) or graphite composites (3)). In the case of covalent bonding the sensor surface is treated as a reactive group to which the biological material can bind (108). One of the bioselective elements most frequently used in biosensors is an enzyme. These are large protein molecules that act as catalysts in chemical reactions, but remain themselves unchanged at the end of reaction.

B B

B

B

B B

B

Semipermeable membrane

Sensor (a) Membrane entrapment B

B B B B B B B Membrane Sensor

128-5

IV. TYPES OF BIOSENSORS A. MECHANICAL (RESONANT) BIOSENSORS In this type of biosensor an acoustic wave transducer is coupled with an antibody (biosensitive element). When the analyte molecules (antigens) attach to the membrane (cantilever, Figure 128.6), the membrane mass changes, resulting in a subsequent change in the resonant frequency of the transducer (57). This frequency change is detected and measured (80). 1. Sensors Based on Electromagnetic Waves Electromagnetic sensors may be classified by the wavelength of the electromagnetic waves they use: visible (400–700 nm), ultraviolet (10–400 nm), infrared (700–30,000 nm: NIR (95), FTIR (78), MRI (68)) waves, microwaves (37) (1–10 cm), radiofrequency (59) (1–10 m), X-rays (2) (100 pm–1 nm). Each sensor class may be further sub-divided according to the molecular information that can be obtained through the interaction. For instance, infrared sensors may be subdivided into near-infrared (700– 2500 nm), mid-infrared (2500–30000 nm), far-infrared (up to 1,000,000 nm), and thermography (1–15 µm) sensors, which all extract different information from the molecules (sample) interacting with the waves. We may also classify these sensors according to their precise type of interaction: absorbance, transmittance, or reflectance of light. Sensors based on interactions with electromagnetic radiation waves have been on the market for many years, in particular for laboratory purposes. On-line examples of such sensors are also numerous: x-rays used for foreign body detection (94), visible light sensors for color recognition or machine vision inspections (22), near-infrared sensors for quality inspection and temperature measurements (81), or microwave sensors for the detection of water content (15).

(b) Physical adsorption B B B B B B B B

Porous encapsulation

Sensor (c) Matrix entrapment B B B B B B B B Covalent bond Sensor

(d) Covalent bonding

FIGURE 128.5 on a substrate.

Coupling of biomaterial with the transducer

FIGURE 128.6 biomolecules.

Cantilever-based

systems

can

detect

128-6


B. OPTICAL DETECTION BIOSENSORS

D L

Strictly speaking, optical biosensors belong to the larger class of electromagnetic detectors, but due to their importance and broad use they are usually considered as a separate group of biosensitive devices. The output signal measured in this type of biosensors is a light signal (119). These biosensors can be made based on optical diffraction or electrochemiluminescence (41).

P

S

1. Surface Plasmon Resonance Surface plasmon resonance (SPR) is another optical phenomenon used in new sensors, often in those involving antibodies or enzymes. The optical range used is most often in the visible part of the spectrum, but may also be in the NIR range. Traditionally, SPR devices (see Fig. 128.7) detect minute changes in the refractive index of the sensing surface and its immediate vicinity. They may detect these changes by a diffraction grating, or with a prism on a glass slide, or through an optical waveguide carrying a thin metal layer (gold). The metal layer carries a sensitizing layer, e.g., immobilized antibodies or other molecules binding the analyte specifically; this layer is in contact with the sample. Inside the device, a collective excitement of electrons in the metal film occurs, and leads at a specific wavelength to a total absorption of light at a particular angle of incidence. This angle depends on the refractive indices on either side of the metal film. Specific molecules binding to the sensitizing layer change the refractive index, thus changing an angle of total absorption; this angle is measured and correlated to the concentration of the analyte. The SPR detection technique has been used by Hellnaes (69) for on-line and at-line detection of veterinary drug residues (hormones and antibiotics) in dairies and slaughterhouses. Clenbuterol and ethinyl-estradiol in bovine urine, sulfamethazine (SMT) and sulfadiazine (SDZ) in porcine bile, and SMT, SDZ, and enrofloxacin in milk have been successfully detected by the technique. The developed biosensor operates in real time and can simultaneously detect up to 8 different veterinary drugs with a throughput of up to 600 samples per day. The project participants have established a new company to produce and further develop the sensor systems, and several new and elegant designs of SPR sensors are now under development by other groups. The SPR sensor principle has also been used by Patel (113). The sensor developed as a result of this research has been applied to the quantification of mycotoxins, Listeria, and markers for growth hormones (recombinant bovine somatotrophin, rBST). Most current research is focused on the NIR/VIS sensors, SPR sensors (surface plasmon resonance), and NMR sensors (pulsed and low resolution); some work has also been done on fluorescence sensors, MIR and Raman sensors, Fourier transform NIR sensors, thermography

+

−

− +

+

−

+

+

F +

FIGURE 128.7 Surface plasmon resonance detection unit. L: light source, D: photodiode/photodiode array, P: prism, S: sensor surface, F: flow cell.

based sensors, and sensors combining two or more sensor principles.

C. ELECTROCHEMICAL BIOSENSORS Electrochemical biosensors are mainly used for the detection of hybridized DNA, DNA-binding drugs, glucose concentration, etc. The underlying principle of these biosensors is that many chemical reactions produce or consume ions or electrons which in turn cause some changes in the electrical properties of the solution; these changes can be sensed out and measured (75). The electrochemical biosensor can be classified based on the measured electrical parameter as conductimetric, amperometric, or potentiometric (126).

D. IMPEDIMETRIC/CONDUCTOMETRIC BIOSENSORS Many biological processes involve changes in the concentrations of ionic species. Such changes can be utilized by biosensors, which detect changes in electrical conductivity. The measured parameter is the electrical conductance/ resistance of the solution. When electrochemical reactions produce ions or electrons the overall conductivity/resistivity of the solution changes (47). This change is measured and calibrated to a proper scale. Conductance measurements have relatively low sensitivity. The electric field is generated using sinusoidal voltage, which helps in minimizing undesirable effects such as Faradaic process, double layer charging, and concentration polarization (17). Impedimetric biosensors utilize changes in the electrical conductivity in the frequency domain (impedance) of a biological system for sensing and detection (4, 52, 71). Impedance spectroscopy provides a powerful tool for investigating a variety of bioelectric processes for both electrical and non-electrical applications. In impedance spectroscopy


128-7

1

1

(a)

2

2

3

3 (b)

2

(c)

FIGURE 128.9 Amperometric enzyme electrodes. (a) “Clark’s” electrode — dialysis membrane electrode with soluble enzyme, (b) entrapped enzyme, and (c) enzyme, membrane electrode. (1) Transducer, (2) enzyme, (3) dialysis membrane.

reaction (13). The simplest potentiometric technique is based on the concentration dependence of the potential, E, at reversible redox electrodes according to the Nernst equation (17): FIGURE 128.8 Conductometric biosensor — interdigitated electrode arrangement.

the current flowing through a sample cell containing a nanoscale patterned bio-interface and the voltage across this cell are measured as a function of frequency (5, 8, 20). Design of impedimetric sensors is very similar to conductivitybased sensors (see Fig. 128.8) (61, 75, 86, 107, 117, 122). Enzyme/antibody immobilization on electrode surface makes these sensors highly selective and sensitive (128).

E. AMPEROMETRIC BIOSENSORS This highly sensitive biosensor can detect electroactive species present in biological test samples. Enzymecatalysed redox reactions can form the basis of a major class of biosensors if the flux of redox electrons can be determined (104). Normally, a constant voltage is applied between two electrodes and the current, due to the electrode reaction, determined. The first and simplest biosensor was based on this principle. It was for the determination of glucose and made use of the Clark oxygen electrode (Figure 128.1). In case of amperometric biosensors, the measured parameter is an electric current. Some of the most recent applications of amperometric biosensors include: glucose sensor for meat freshness (106); glucose sensor for use in fermentation systems (124); rapid cell number monitor (19); monitor for herbicides in surface waters (24, 85); amperometric ELISA method based on the self enzyme amplification system (84); amperometric and novel fluorescent DNA probes (150).

F.

POTENTIOMETRIC BIOSENSORS

In this type of sensor the measured parameter is an oxidation/reduction potential of an electrochemical

RT E ⫽ E0 + ᎏ ln as nF where E0 — standard redox potential, R — gas constant, T — absolute temperature, F — Faraday constant, n — number of exchanged electrons of the substance S, and as — activity of the substance S. Changes in ionic concentrations are easily determined by use of ion-selective electrodes (13). This forms the basis of potentiometric biosensors (4). Many biocatalysed reactions involve charged species, each of which will absorb or release hydrogen ions according to their pKa and the pH of the environment (88). This allows a relatively simple electronic transduction using the commonest ion-selective electrode (see Fig. 128.9), the pH electrode (121). 1. Field Effect Transistors (FET) and IonSelective Field Effect Transistors (ISFET) Potentiometric biosensors can be miniaturized by the use of field effect transistors (FET). Ion-selective field effect transistors (ISFET) are low cost devices that are in mass production (148). A recent development from ion-selective electrodes is the production of ion-selective field effect transistors (ISFETs) and their biosensor use as enzymelinked field effect transistors (ENFETs). Enzyme membranes are coated on the ion-selective gates of these electronic devices, the biosensor responding to the electrical potential change via the current output. Thus, these are potentiometric devices although they directly produce changes in the electric current. Figure 128.10 shows a diagrammatic cross-section through an npn hydrogen ion responsive ISFET with a biocatalytic membrane. The build-up of positive charge on this surface (the gate) repels the positive holes in the p-type silicon causing a depletion layer and allowing the current to flow. In (1) Langmuir-Blodgett films containing butyrylcholinestrase

128-8


Reference electrode Encapsulant

+ −

FIGURE 128.10 biosensor.

Biocatalytic membrane

n

H+ sensitive Ion selective membrane membrane

Gate

Source

n Drain

p Silicon

An ion sensitive FET-based potentiometric

(BuChE) are fabricated to realize an ion-sensitive fieldeffect transistor (ISFET) for the detection of organophosphorus pesticides in water.

G. CELL-BASED BIOSENSORS Cell-based biosensors have been implemented using microorganisms, particularly for environmental monitoring of pollutants (109). Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner that can offer insight into the physiological effect of an analyte (33, 66). Several approaches for transduction of cellular signals (67) are described in the literature: measures of cell metabolism, impedance (129), intracellular potentials, and extracellular potentials (72). Among these approaches, networks of excitable cells cultured on microelectrode arrays (9, 33, 43, 103, 109) are uniquely poised to provide rapid, functional classification of an analyte and ultimately constitute a potentially effective cell-based biosensor technology. Keese and Giaever (82) have designed a biosensor that can be used to monitor cell morphology in tissue culture environment. The sensing principle used is known as electric cell-substrate impedance sensing (ECIS). In this process, a small gold electrode is immersed into tissue culture medium. After cells attach and spread over the electrodes, the electric impedance measured across the electrode chamber changes. These changes in impedance can be used for understanding cell behavior in the culture medium. The attachment and spreading of the cells are important factors for successful use of this biosensor. Unfortunately, some types of cells, e.g., cancerous cells, can grow and reproduce freely in a medium without being attached to any substrate/surface; that makes them impossible to detect with these sensors. Proposed in (34) biosensor mimics biological sensory functions and can be used with most types of receptors, including antibodies and nucleotides. The technique is very flexible and even in its simplest form it is sensitive to pico-molar concentrations of proteins.

H. LAB-ON-A-CHIP SYSTEMS AND DNA DETECTION DEVICES Significant advances have been made in the development of micro-scale technologies for biomedical and drug discovery applications. The first generation of microfluidicsbased analytical devices (Lab-on-a-Chip (141)) have been designed and are already functional. Microfluidic devices offer unique advantages in sample handling (45, 65, 142), reagent mixing (23, 79, 140), separation (54, 89, 98), and detection (27). They include, but are not limited to: devices for cell sampling (7), cell trapping and cell sorting devices (9, 12, 16, 44, 152), flow cytometers (49, 105, 142), devices for cell treatment: cell lysis, poration/gene transfection and cell fusion devices (145). Biosensors used for DNA detection are used to identify small concentrations of DNA (of microorganisms such as viruses or bacteria) in a large sample. The detection relies on comparing sample DNA to a DNA of known microorganism (probe DNA) (28). Since the sample solution may contain only a small number of microorganism molecules, multiple copies of the sample DNA need to be created for proper analysis (31). This is achieved with an aid of the polymerase chain reaction (PCR). PCR starts by splitting the sample’s double-helix DNA into two parts by heating it. If the reagents contain proper growth enzymes, each of these strands would grow the complementary missing part and form the double-helix structure again. This happens after the temperature is lowered. Thus, in one heating/cooling cycle the amount of sample DNA is doubled (10). In general, PCR is very power-consuming, so it was previously not possible to fabricate portable biodetectors able to perform PCR. But, using newly developed MEMS devices, such biodectors (also known as lab-on-achip systems) have been created. In these MEMS-based devices the amount of reagent used is scaled down (115).

I. DNA-BASED SENSORS/ASSAYS The general principle of DNA probe assay is similar to the immunoassay described in Figure 128.11. Indeed, even the applications of DNA probes and monoclonal antibody immunoassay frequently overlap, thus establishing a “competition” between the two possible approaches. One of the most important applications for DNA probes is the testing for virus infections (96). For probes of infectious disease, it is assumed that all strains can still contain a common DNA sequence region, and thus be identified by a single probe. Recognized by the cell as a foreign body, viruses will induce an antigenic reaction causing antibody generation so they can also be detected in an immunoassay (73). Another type of biosensor developed by the Naval Research Laboratory (10) uses magnetic field instead of optics or fluorescence. This sensor equipped with magnetic


128-9

important food processing parameters (62), monitoring animal fertility, and screening therapeutic drugs in veterinary testing are well-described in another work (138). (a)

(b)

(d)

(c)

(e)

FIGURE 128.11 DNA probe assay. (a) Deposit sample organism on immobilization matrix; (b) release DNA; (c) immobilize DNA to matrix and separate strands; (d) add labeled DNA probes and hybridize; (e) read label.

sensors and microbeads (131) is able to detect the presence and concentration of bioagents. The magnetic sensor (group of sensors) is coated with single-stranded DNA probes specific for a given bioagent or sample DNA. Once a single strand of DNA probe and a single strand of sample DNA find each other, they form a double stranded (double-helix) structure, which in turn binds a single magnetic microbead. When a magnetic bead is present on a sensor surface, its resistance decreases which can be detected and measured.

V. APPLICATIONS OF BIOSENSORS IN FOOD SCIENCE AND MANUFACTURING Detailed description of all existing biosensors for food applications requires a separate book and is definitely out of the borderlines of this chapter. Instead, this chapter aims to compare needs for biosensors in food safety/ biosecurity management systems and existing biosensor technologies. The general classification of sensors, principles of their operation, and some practical examples given in this chapter accomplish this goal. To find more information about specific biosensors and applications readers should refer to other papers. Biosensors for food safety applications are reviewed in references (64, 75, 111, 114). Additionally, the author can recommend a good description of existing market for food safety applications (6). The general review of electrochemical biosensors for food pathogens detection can be found in references (91, 126, 134). Specific details are presented in the following articles about biodetection in poultry industry (93), and pathogen detection in muscle foods (42). The needs for fast, on-line, and accurate sensing, e.g., in situ analysis of pollutants in crops and soils, detection, and identification of infectious diseases in crops and livestock, on-line measurements of

A. SENSORS

FOR

PATHOGENS DETECTION

The broad spectrum of foodborne infections keeps changing dramatically over time, as well-known pathogens have been controlled or eliminated, and new ones have emerged. The burden of foodborne diseases remains substantial: one in four Americans is estimated to have a significant foodborne illness each year. The majority of these illnesses is not caused by known pathogens, so more of them remain to be discovered. Among the known foodborne pathogens, the recently identified predominate, suggesting that as more and more is learned about pathogens, they would come under control. In addition to the emergence or recognition of new pathogens, other trends include global pandemics of some foodborne pathogens, the emergence of antimicrobial resistance, the identification of pathogens that are highly opportunistic, affecting only the most high-risk subpopulations, and the increasing identification of large and dispersed outbreaks. New pathogens can emerge because of changing ecology or technology that connects a potential pathogen with the food chain. They also can emerge by transferring the mobile virulence factors, often through bacteriophage (133). Over the past decade many improvements have been seen in both conventional and modern methods of pathogenic bacteria detection in foods (75). Modification and automation of conventional methods in food microbiology involve sample preparation, plating techniques, counting, and identification test kits. ATP bioluminescence techniques are increasingly used for measuring the efficacy of surfaces and utensils cleaning. Cell counting methods, including flow cytometry and the direct epifluorescent filter technique, are suitable for rapid detection of contaminating microorganisms, especially in fluids. Automated systems based on impedance spectroscopy are able to screen high numbers of samples and make total bacterial counts within 1 day. Immunoassays in various formats make a rapid detection of many pathogens possible. Recently, there have been important developments in the nucleic acid–based assays and their application for the detection and subtyping of foodborne pathogens. The sensitivity of these methods has been significantly increased by employing the polymerase chain reaction and other amplification techniques. Alternative and rapid methods must meet several requirements concerning accuracy, validation, speed, automation, sample matrix, etc. Both conventional and rapid methods are used in the frame of biohazard analysis critical control point programs. Further improvements especially in immunoassays and genetic methods can be expected, including applications of biosensors and DNA chip technology (38).

128-10


In recent work of Bokken (18) a surface plasmon resonance biosensor was used to detect a Salmonella pathogen through antibodies reacting with Salmonella group A, B, D, and E (Kauffmann-White typing). In the assay designed, anti-Salmonella antibodies immobilized onto the biosensor surface were allowed to bind injected bacteria, followed by a pulse with soluble anti-Salmonella immunoglobulins to intensify the signal. No significant interference was found for mixtures of 30 non-Salmonella serovars at 109 CFU ml⫺1. A total of 53 Salmonella serovars were successfully detected at 107 CFU ml⫺1, except those from groups C, G, L, and P, as expected. Another sensor technology recently developed uses a micro-electrophoretic system (mFFE) that separates and concentrates the analyte in question by a number of electrophoretic methods: preparative zone, interval zone, isotachophoresis, or isoelectric focusing. The mFFE system can be designed as a plain glass substrate 1.5 mm thick, and a cross-linked polydimethyl-siloxane (PDMS) top layer with micromachined sample channels. The central separation chamber (12 ⫻ 4 ⫻ 0.15 mm) is connected to 34 inlet channels for sample injection, and 36 outlet channels for sample collection. The detector unit can be based on several principles. In the case of Listeria, the detector unit may be a well-known ATP luminescence detector. For other analytes, the SPR detection system may be used with an immobilized bio-specific layer, e.g., antibodies (111). A new ion-channel biosensor based on supported bilayer lipid membrane for direct and fast detection of Campylobacter species has been reported (76). The sensing element was composed of a stainless-steel working electrode, covered with an artificial bilayer lipid membrane (BLM). Antibodies to bacteria embedded into the BLM are used as channel-forming proteins. The biosensor has a strong signal amplification effect, which is defined as the total number of ions transported across the BLM. The biosensor has demonstrated a very good sensitivity and selectivity to Campylobacter species. A novel assay system for the detection of Escherichia coli O157:H7 has been recently developed. The detection is based on the immunomagnetic separation of the target pathogen from a sample and absorbance measurements of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the “sandwich” structure complexes (antibodies coated onto micromagnetic beads — E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface (92). The selectivity of the system has been examined, and no interference from other pathogens including Salmonella typhimurium, Campylobacter jejuni, and Listeria monocytogenes was observed. The sensor’s working range is from 3.2 ⫻ 102 to 3.2 ⫻ 104 CFU/ml, with the relative standard deviation of 2.5–9.9%. The total detection time is less than 2 hours.

An improved antibody-coated sensor system based on quartz crystal microbalance analysis of Salmonella spp. has been developed, using thiolated antibody immobilization onto the gold electrode of the piezoelectric quartz crystal surface (110). The best results in sensitivity and stability were obtained with the thin layer of a thiol-cleavable, heterobifunctional cross-linker. The long bridge of this reagent can function as a spacer, facilitating antibody–Salmonella interaction on the gold electrode. The sensor’s response was detected for the microbial suspension concentrations ranging from 106 to 1.8 ⫻ 108 cfu/ml. A label-free immunosensor for the detection of pathogenic bacteria using screen-printed gold electrodes (SPGEs) and a potassium hexacyanoferrate (II) redox probe has been reported by Susmel (130). Gold electrodes were produced using screen-printing, and the gold surfaces were modified by a thiol-based self-assembled monolayer (SAM) to facilitate antibody immobilization. In the presence of analyte a change in the apparent diffusion coefficient of the redox probe was observed; it can be attributed to impedance of the diffusion of redox electrons to the electrode surface due to the formation of the antibody-bacteria immunocomplexes. No change in the diffusion co-efficient was observed when a non-specific antibody (mouse IgG) was immobilized and antigen added. The system has been demonstrated to work with Listeria monocytogenes and Bacillus cereus.

B. SENSORS TO MONITOR FOOD PACKAGING AND SHELF-LIFE In recent work (147) a cell-based biosensor has been used to control meat freshness. Samples of fresh meat stored at 5°C were periodically removed from storage and washed with water for periods of up to 2 weeks. The water was then charged into a flow injection analysis (FIA) system combined with the microbial sensor using yeast (Trichosporon cutaneum) as a sensitive element. This sensor has been specifically developed in this work for monitoring the freshness of meat. Relationships between the sensor signals obtained by the FIA system, the amounts of polyamines and amino acids produced from the meat, and the number of bacteria that had been multiplying in the meat during the aging process were investigated. The sensor response has been found to correspond to the increase in amino acid levels and viable counts in the meat during the first stage of aging. This is due to the fact that amino acids produced initially by enzymes in the meat serve as a nutrition source for septic bacteria, and as a result, the amount of bacterial cells increases with an increasing level of amino acids. 1. Foreign Body Detection The presence of foreign bodies in processed food is of major concern to the producers. Mechanical separation


techniques based on size and weight of different components have been used for many years to help find foreign bodies in powdered and flowing products. Optical inspection techniques were able to extend the range of detectable foreign objects in free-flowing materials with regard to their shape and color. Metal detectors enabled metallic particles inside the product to be found. With recent achievements in sensor technologies advanced foreign body detection systems are becoming available (55). The working principle and design of an ultrasonic transducer system with auto-alignment mechanism was first described by Zhao (151). The proposed system has been used for detecting foreign bodies in beverage containers. Variations in reflection amplitude were analyzed as a function of the ultrasound beam incident angle to the beverage container surface. It has been concluded that a quadratic relationship exists between the strength of the reflected signal and the incident angle. Furthermore, a calculation for effective angular increment for searching the normal to a curved surface was introduced. Experiments conducted using the sensor prototype have demonstrated that foreign bodies are detectable in containers of various juices. This sensor design is also applicable to nondestructive inspection of canned food products for the presence of foreign bodies.

C. BIOSENSORS CONTROL

FOR

FOOD QUALITY/ADDITIVES

Existing food processing equipment frequently includes microprocessors that are activated by electronic or biological sensors. Recent advances in electronic vision and computer technology have opened the research horizons for greater accuracy in process control, product sorting, and operation. The development of new sensors and instruments in this area is focused on measuring/evaluating the product’s internal and external quality and flavor (138). The aim of food additives control and measurement is to develop, extend, and enhance the instrumental methods in order to improve consumer-perceived macroscopic quality factors. For quality assessment, grading, and sorting of food products, several types of electronic sensors that can provide rapid and non-destructive determination of product internal qualities have been investigated and described in the literature. A near infrared sensing technique can rapidly determine the sugar content of intact peaches. This technology has been extended to a number of other commodities, including testing avocados for oil content, and kiwifruits for starch and sugar content. NMR method, for example, can be used for nondestructive detection and evaluation of internal product quality factors, such as existence of bruises, dry regions or worm damage, stage of maturity, oil content, sugar content, tissue breakdown, and the presence of voids, seeds, and pits (see Figure 128.12).

128-11

FIGURE 128.12 MRI image of blueberry.

Machine vision for postharvest product sorting and grading is being investigated for a number of commodities. Recent research has included development of a high-speed prune defect sorter, color and defect detector for freshmarket stone fruits, raisin grading, and flower grading machines. In this technology, electronic cameras are used for monitoring the product in various packing-line handling situations. Quality features are computed from digitized images, and the control system allows for product grading and sorting. The NIR/VIS region has been used in several different sensors. Thus, Crochon in his work (35) has presented the design of a glove-shaped apparatus equipped with various miniaturized sensors providing information on fruit quality parameters, i.e., sugar content, maturity, mechanical properties (firmness, stiffness), and internal color. The sugar content and internal color were measured by a miniaturized spectrometer (NIR/VIS) coupled with optical fibers. A sound sensor evaluated the mechanical properties, and the size was measured by a potentiometer placed at the hand aperture. These sensors were coupled to a microcomputer that delivered processed information about the fruit overall quality grade, based on previously established variety and quality classes. The weight of the glove prototype was 400 g, and the electronic devices were held in a rucksack weighing 1000 g. The glove may be used before harvest to control the growth and to estimate the harvest date, at harvest to select fruits with specific qualities, or after harvest to control and measure the quality of the crop. In (11) chlorophyll fluorescence and reflectance in the NIR/VIS spectrum has been used for the mechanical quality factors assessment of green beans, broccoli, and carrots. Biosensors have been used for evaluating the effects of pasteurization on vegetable quality by measuring the remaining enzymatic activity. Use of mid-infrared (MIR) spectroscopy, as well as Raman scattering, for on-line quality assessment in bakeries, breweries, dairies, and fruit farms has been reported (56).

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Another method working in the NIR/VIS range, called time-resolved diffuse reflectance spectroscopy (TDRS), has been used to measure the internal quality of fruits and vegetables (149). The group has developed statistical models for the analysis of relationships between the TDRS signals and the firmness, sugar, and acid content of kiwifruit, tomato, apple, peach, nectarine, and melon. They have also developed the classification models to sort apples, peaches, kiwifruits, and tomatoes into quality classes. Using a pulsed laser diode (70–200 pico-seconds/pulse), the single measurement time was about 100 milliseconds. The absorption coefficient was related to the tissue constituents, while the scattering coefficient was related to the firmness and fiber content. A real-time sensor in the NIR/VIS range can be also used to measure product quality traits, such as maturity, flavor, or internal diseases and defects in potatoes, apples, and peaches (26). Among the optical sensor systems developed and demonstrated in industrial environments are machine or artificial vision sensors. The system for olives sorting, using a traditional vision camera and three CCD color sensors for the shape, size, and color evaluation, has been described (118). The new algorithm allowed the olives to be sorted into four classes with the speed of 132 olives/sec, and 6 images/sec. The molecular imprinted polymers (MIP) technology is the new technology used for the development of biosensor substrates (97). The polymers are produced by imprinting the recognition sites of predetermined specificity into cross-linked synthetic polymers. The polymer is consequently able to selectively re-bind the imprinted molecule (100). These sensor materials are called “artificial antibodies” (123). The MIP technology has particular strengths for small molecular analytes up to about 400 Dalton; it may be used to bind and detect many chemicals polluting food products, e.g., pesticides and veterinary drugs in meat and dairy products. This technology has been successfully employed to develop and optimize plug-in detection cartridge supporting the molecularly imprinted polymer assay (83) for detection of different β-lactam antibiotics in milk. The sensor consists of a micro-fabricated column accommodating an optical detection window. Molecular imprinted polymers in the form of beads were used as packing materials and recognition elements; analyte binding was detected by the fluorescence. The same MIP technology has been used in several other studies, the overall objective of which was to develop novel and robust MIP-based technology that can be used in sensors for real-time measurements of food product contaminants (112, 114, 136). The results of the study indicate that MIP can be used to prepare both selective and general recognition matrices for either individual analytes or groups of compounds, with very good detection reproducibility and stability (136). SPR based sensor shows similar results for dairy product quality applications (48). The

MIP developed for clenbuterol has been successfully applied in preparing a novel sensor comprising MIP as the selective element and amperometric detector as the transducer (97). The responses from several sensors were determined to have a variability of 10%. The feasibility for an oxacillin MIP-based sensor was also demonstrated. At-line immunological sensors using amperometric detection of the resulting antibody-antigen complexes were described (125). The target quality factor assessed in this project was the presence of toxic chlorophenolic fungicides and their chloroanisole breakdown products in potable water, wine, and fruit juices. The electrochemical immunosensor uses monoclonal antibody preparations. The investigations of the effects of liquid food matrices on electrochemical transduction processes indicated that horseradish peroxidase is a suitable label for interrogation of the analyte-antibody immune complex, using amperometry and in-house fabricated screen-printed electrodes. The detection of hormonal substances for growth promotion, also based on immunosensors has been recently reported by Guilbault (58). The sensor has to be used prior to slaughtering, and can detect and measure testosterone, methyltestosterone, 19-nortestosterone, stanozolol, and trenbolone levels in biological fluids (blood). Analysis time achieved was about 30 minutes, compared to 24–36 hours for tests used in laboratories today.

D. BIOSENSORS FOR SENSORY EVALUATION FOOD PRODUCTS

OF

“Electronic noses” (139, 146) and “electronic tongues” (32) are the common names of devices responding to the flavor/ odor (volatiles) or taste (solubles) of a product using an array of simple and non-specific sensors, and the pattern recognition software system (50). Historically, the sensors used were advanced mass spectrometers or gas/liquid chromatographs, producing a unique fingerprint of the analyte. Nowadays, these sensors have been substituted by arrays of simple electric and/or frequency sensors, or sensors measuring changes in voltage or frequency as a response to the food contact. Electronic noses and tongues are used in food production and quality control of different products, typically for laboratory tests or at-line control, but may be further developed for in-line operation in the future. Testing times are often in the range of a few minutes, and the largest drawback of these devices is the lack of sensor stability. Examples of claimed successful applications include (14): ●

●

●

Discrimination between single volatile compounds; Tracking of aroma evolution of ice-stored fish or meat; Tracking of the evolution of cheese aroma during aging;


● ●

● ● ● ● ●

Classification of wines; Determination of boar odor (androsterone) in pork fat; Classification of peaches and other fruits; Differentiation of spices by the area; General raw materials control; Testing of coffee, soft drinks, and whisky; Control of beer quality and faults.

Essentially, each odor or taste leaves a characteristic pattern or fingerprint on the sensor array, and an artificial neural network is trained to distinguish and recognize these patterns (see Figure 128.13). Pattern recognition is gained by building a library of flavors from known flavor mixtures given to the network. Thus, e-noses and tongues are the devices intended to simulate human sensory response to a specific flavor, sourness, sweetness, saltiness, bitterness, etc. (14, 132). The potentiometric chemical sensors such as ion selective sensors are most often used in the electronic noses. Considerable interest exists in the development of cheap, portable electronic noses to detect, on-line or at-line, odor quality of many foods. For instance, olive oil producers would tremendously benefit from the possibility of detecting oil quality and shelf-life, and classifying the oils by their quality (e.g., Extra Virgin olive oil). This was the objective of a course project in which scientists from olive producing countries developed electronic noses especially for the olive production plants, and tested them with great success (25). In (40) different tea samples were used to evaluate the applicability of electronic noses for sensory studies. A metal oxide sensor-based electronic nose has been used to analyze tea samples with different qualities, namely, drier month, drier month again over-fired, well fermented normal fired in oven, well fermented overfired in oven, and under fermented normal fired in oven. Electronic tongues are also widely used to assess taste quality of various products. An electronic tongue based on voltammetry measurements, and a multichannel lipid

128-13

membrane taste sensor based on potentiometry were compared using two aqueous solutions: detergent and tea (74). The electronic tongue consists of four electrodes made of different metals, a reference electrode and a counter electrode. The measurement principle is based on pulse voltammetry technique in which an electric current is measured during the amplitude change of the applied potential. The taste sensor consists of eight different lipid/polymer membranes. The voltage difference between the electrodes and an Ag/AgCl reference electrode is measured when the current is close to zero. The multichannel electrochemical (potntiometric) sensors have demonstrated better sensitivity, faster dynamic response, but lower reproducibility of the results. In study performed by Legin (87) the electronic tongue based on a sensor array comprising 23 potentiometric cross-sensitive chemical detectors, and pattern recognition and multivariate calibration data processing tools, has been applied to the analysis of Italian red wines.

VI. ROLE OF BIOSENSORS IN THE FOOD SAFETY MANAGEMENT SYSTEM A. BIOSENSORS AND BIOSECURITY Food industry is one of the major potential targets for bioterrorism. The most damage can be attained through: (1) final product contamination using either chemical or biological agents with an intent to kill or cause illness among consumers; (2) disruption of food distribution systems; (3) damaging the food producing cycle by introducing devastating crop pathogens or exotic animal diseases such as foot-and-mouth disease, which could severely impact the food system. Efforts to develop recognizing preparedness and response strategies for protecting the nation’s food supply pose substantial challenges for a number of reasons, including the following (70, 127, 144): ●

●

Electronic nose

Odor molecules

Sensor array

Pattern recognition system

Odor ID

Food sample

FIGURE 128.13 Principle of the electronic nose operation (42).

●

The food system encompasses many different industries; A great variety of biological and chemical agents could potentially contaminate the food supply, and the possible scenarios for deliberate contamination are essentially limitless; The public health system is complex, and responsibilities for foodborne diseases prevention and control may overlap, or much worse, fall in the “gray area” between authorities of different agencies.

To achieve an adequate food supply chain and agricultural security, improvement is needed in the activities on bioterrorism prevention, detection, and response. In

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addition, appropriate areas for applied research must be identified: ●

●

●

Recognition of a foodborne bioterrorism attack. This may be delayed because of background levels of foodborne diseases and the potential wide distribution of the contaminated product or ingredient. Rapid diagnostic methods for identifying foodcontaminating agents. They are not yet consistently available, and coordinated laboratory systems for pathogens detection are not fully operational. Rapid trace-back procedures for potentially contaminated products.

B. BIOSENSORS AND HACCP Timely detection of unsafe foods is the main issue that the food safety system should address, providing guidance for the design and integration of such system into the existing food safety management structures, i.e., HACCP. The preventive detection of the biohazard can be accomplished by direct measurements with the biosensors, or indirect detection by the process/environment monitoring and control. Such detection is based on the data from physical and chemical sensors, which are very reliable and allow scale-down, which means the possibility of easy integration into the existing information carriers. The HACCP system for food safety management is designed to identify health hazards, and to establish strategies to prevent, eliminate, or reduce their occurrence. An important purpose of corrective actions is to prevent potentially hazardous foods from reaching consumers. Where there is a deviation from the established critical limits, corrective actions are necessary. Therefore, corrective actions should include the following elements: (a) determine the disposition of non-compliant product; (b) determine and correct the cause of non-compliance; (c) record the corrective actions that have been taken. Currently, the use of HACCP is voluntary, but it is widely used in the food processing industry as a successful component of comprehensive food safety program. HACCP is a food safety management system in which food safety is addressed through the analysis and control of biological, chemical, and physical hazards from raw material production, procurement and handling, to manufacturing, distribution, and consumption of the final product. The terms “HACCP” and “food safety” are used interchangeably in the food industry, implying that HACCP may be the only approach to achieving food safety. HACCP is designed for use in all segments of the food industry from growing, harvesting, processing, distributing, and merchandising, to preparing food for consumption (135). However, there is a need for enhancement and integration of the existing HACCP system into the total

quality management system, and food safety/biosecurity management on higher levels. The system currently includes the mechanisms to decrease the potential for contamination of or damage to the food supply from farm to table (i.e., prevention activities); systems to ensure early detection of deliberate food contamination at any point along the production pathway, including surveillance, rapid laboratory diagnostic, and communication systems; systems to ensure a rapid and thorough response if a bacterial contaminant is detected, including protection of workers and consumers (i.e., emergency response, control, trace-back, and mitigation activities). The ultimate goal is the integration of sensors and sensor networks into the food safety management structure (see Figure 128.14). Such integration will allow one to perform on-line and “on-shelf” control of the internal and/or external food product quality and package environment. The integrated sensor information system combines data from multiple sensors (from different packages and/or products) and the information about environmental and process conditions to achieve highly specific information that cannot be obtained by using a single, independent microbiological assay. The emergence of new information carriers and advanced processing methods will make the food safety management system increasingly dependable. A successful biohazard detection system should be able to: (a) identify potential hazards; (b) identify hazards that must be currently controlled; (c) conduct hazard analysis; (d) recommend control factors, critical limits, and procedures for hazard monitoring and verification; (e) recommend appropriate corrective actions if a deviation occurs. Based on a comprehensive model for multisensor data processing, developed by the US Joint Directors of Laboratories (JDL) Data Fusion Group on DoD request (63), the integrated concept of multiple sensors data processing has been developed for the existing HACCP system of food safety monitoring and biohazard prevention (see Figure 128.14). This model is specifically adapted to the HACCP workflow and utilizes the principle of information Bio-security information

Decision making

HACCP management

Verification

Decide Critical limits confirmation

Corrective actions

Orient

Act

Signal processing

Records/ documentation

Observe Monitoring

Product/sensor Sensing/detection data

Product/process management

FIGURE 128.14 Schematic process model for integrated HACCP and biohazard detection system.


system cyclic interaction with the environment. The four major steps, including observation/ detection, hazard recognition, decision making, and corrective actions, strictly correspond to the seven HACCP principles. Integration of such a system does not require the redesigning of existing manufacturing and control processes. The new integrated sensors are able to monitor HACCP control points with corresponding material packaging flow on a continuous basis, or with pre-determined monitoring frequency. Statistically designed data collection or sampling systems lend themselves to this purpose. Issues that need to be addressed when considering implementation of an integrated food safety monitoring system include: where the system would be established; how it would be funded; how the data would be generated, analyzed, summarized, and disseminated; and how “snap surveys” could be utilized as a part of the system. Microbiological tests are rarely effective for food safety monitoring due to their time-consuming properties and problems with ensuring detection of contaminants. Physical and chemical measurements are preferred because they are rapid and usually more effective for the control of microbiological hazards. For example, the safety of pasteurized milk is based upon the measurements of heating time and temperature rather than on testing the processed milk for the absence of surviving pathogens. In order to address the issues of connectivity between biosensor devices, the Connectivity Industry Consortium (CIC) has been formed to set up the standardized communication platform for all devices (29). The CIC has identified five requirements: bidirectionality, connection commonality, commercial software interoperability, security, and QC and regulatory compliance (120). Under these standards, new devices should seamlessly link into the existing data management system without additional expenses. Traditionally, food quality monitoring units consist of a sensor for the particular analyte, an electronic unit to convert the response into a digital signal, and a cable to communicate with the base station. Advances in technology now enable sensors to be integrated with the base station through wireless communication that frees sensors from being physically attached to it. An interest in such freestanding monitoring units is growing rapidly, since they offer the potential for developing integrated networks of sensing devices that can detect, diagnose, and monitor various food safety problems. The merging of computing with wireless communication systems and sensors has led to an increased accessibility to the real-time information in digital form. Due to achievements in communications and connectivity, data from these sensors can even now be easily accessed via personal digital assistants, PCs, mobile phones, and networks. On the other hand, the communications network that has assembled over the past decade and continues to attract huge investments will fuel demands for more sources of health-related information and data.

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New technologies do not come into existence easily. It is not just the matter of making conventional laboratory instruments smaller or putting a sensor into the human body. The new sensor devices and networks must satisfy food industry needs by delivering new benefits to users, offering new ways of monitoring food product properties/ contaminations, developing tests that are cheaper, or creating devices that have significant advantages over those already available. It has been predicted that the trend in biodetection systems development lies in the autonomous sensing technology with the next-generation handheld, portable sensing devices, “smart” sensing, and in-line biodetection. The only limitation to the fast progress in this area is the fact that the sensors — especially chemical and biological ones — lag behind the electronics. In the future, the evolution of integrated food safety management system may lead to the emerging of a food processing control “nervous system,” which will comprise multitudes of sensors and sensing technologies. Such systems could provide the information “nodes” for food safety management and control applications.

VII. FUTURE OF BIOSENSING: DETECTION OF CELLULAR RESPONSE WITH NANOSENSORS Cells are the smallest functional and integrating communicable units of living systems. Cultured cells transduce and transmit a variety of chemical and physical signals by producing specific substances and proteins throughout their life cycle within specific tissues and organs. Hence, cells and their responses might be usefully employed in screening tools to obtain important information for both pharmaceutical and chemical safety, and drug efficacy profiles in vitro. However, cellular signals are very weak and cannot be easily detected with conventional analytical methods. By using novel micro- and nanobiotechnology methods and integrated on-a-chip devices, higher sensitivity to cellular responses and better signal amplification have been achieved (12, 53, 101). Micro- and nanotechnology are now rapidly evolving to suggest new combinations of methods with improved technical performance (53), helping to resolve challenging bioanalytical problems including detection sensitivity, signal resolution, and specificity by interfacing these technologies in micro-scale format in order to confirm specific cellular signals (21, 33, 51, 116, 137, 143). Receiving cell signals in rapid time and small space, and importantly, integration of signals from different cell populations (communication and system modeling), will permit more valuable measuring of the dynamic aspects of cell responses to various chosen stimuli (39). This represents the near future for cell-based biosensing (67).

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Concerns over biosafety and security have accelerated the implementation of biohazard control processes, including hazard identification, assessment of its impact on human health, and determination of when, where, and how it would have an impact. Continuous biosafety control is used for assessing the exposure to a hazard, and predicting the necessary dose response.

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129

Genetically Modified Organisms in Food Industry

N. Gryson

Department of Biotechnology, Center for Applied Research and Services

K. Messens

Department of Biotechnology, Center for Applied Research and Services

K. Dewettinck

Department of Food Technology and Nutrition, Ghent University

CONTENTS Abbreviations................................................................................................................................................................129-2 Foreword ......................................................................................................................................................................129-3 I. Historical Background ......................................................................................................................................129-3 II. Transformation Methods....................................................................................................................................129-4 A. Agrobacterium tumefaciens ......................................................................................................................129-4 B. Biolistics ....................................................................................................................................................129-5 C. Electroporation ..........................................................................................................................................129-5 D. Microinjection ............................................................................................................................................129-5 E. Polyethylene Glycol ..................................................................................................................................129-5 F. Silicon Carbide Fibres................................................................................................................................129-5 III. Transgenes ........................................................................................................................................................129-5 A. The Promoter..............................................................................................................................................129-5 B. The Terminator ..........................................................................................................................................129-6 C. Structural Genes ........................................................................................................................................129-6 D. Marker Genes ............................................................................................................................................129-6 IV. Strategies for Genetic Manipulation ..................................................................................................................129-7 A. Sense Strategy ............................................................................................................................................129-7 B. Transgene Silencing ..................................................................................................................................129-7 V. Application of Genetic Manipulation in the Food Industry ..............................................................................129-7 A. Benefits of GM Foods................................................................................................................................129-7 B. Potential Risks of Genetically Modified Foods ........................................................................................129-8 1. Food Safety..........................................................................................................................................129-8 2. Food Related Concerns........................................................................................................................129-9 3. Environmental Concerns ....................................................................................................................129-9 4. Other Concerns..................................................................................................................................129-10 C. Genetically Modified Microorganisms ....................................................................................................129-10 1. Lactic Acid Bacteria ..........................................................................................................................129-10 2. Enzymes ............................................................................................................................................129-10 3. Yeasts ................................................................................................................................................129-11 4. Fungal Factories for Enzyme Production..........................................................................................129-11 VI. Detection of GMOs..........................................................................................................................................129-11 129-1

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A. Labelling of GMOs ..................................................................................................................................129-11 B. Detection Based on DNA ........................................................................................................................129-11 1. DNA Isolation....................................................................................................................................129-11 2. PCR — Principle ..............................................................................................................................129-12 3. PCR Strategies for GMO Screening and Identification ....................................................................129-12 4. Quantitative Detection Methods Based on DNA ..............................................................................129-13 C. Detection Based on RNA ........................................................................................................................129-13 D. Detection Based on Protein......................................................................................................................129-14 1. Western Blot ....................................................................................................................................129-14 2. ELISA................................................................................................................................................129-14 3. Lateral Flow Strip..............................................................................................................................129-15 4. Other Immunoassays ........................................................................................................................129-15 E. Other Detection Methods ........................................................................................................................129-15 1. Chromatography ..............................................................................................................................129-15 2. NIR Spectroscopy ............................................................................................................................129-15 3. Microarray ........................................................................................................................................129-15 4. Nucleic Acid Lateral Flow Immunoassay (Nalfia) ..........................................................................129-15 VII. The Effect of Food Processing on the Detection of GMOs by PCR ..............................................................129-16 References ..................................................................................................................................................................129-17

ABBREVIATIONS

HPLC

ACC

kb LAB mRNA MS Nalfia

A. rhizogenes ARM APCI A. tumefaciens B.C. Bp Bt CaMV cDNA CE Ct CTAB CTP DNA ds EDTA ELISA EPSPS EU FDA FRET GM GMO(s) GUS

1-amino-cyclopropane-1-carboxylic acid Agrobacterium rhizogenes antibiotic resistance marker atmospheric pressure chemical ionisation Agrobacterium tumefaciens Before Christ basepair Bacillus thuringiensis Cauliflower Mosaic Virus complementary DNA capillary electrophoresis cycle threshold cetyltrimethylammonium bromide chloroplast transit peptide deoxyribonucleic acid double stranded ethylen diamine tetra acetic acid enzyme linked immunosorbent assay 5-enol-pyruvylshikimate-3-phosphate synthase European Union Food and Drug Administration fluorescence resonance energy transfer genetically modified genetically modified organism(s) β-glucuronidase

NASBA NIR nm nos nptII PCR PEG PG QC-PCR rDNA Ri RNA RT sam-k SDS spp. ss Taq T-DNA Ti UV Vir

high performance liquid chromatography kilobase lactic acid bacteria messenger RNA mass spectrometry nucleic acid lateral flow immunoassay nucleic acid sequence-based amplification near infrared nanometer nopalin synthase neomycin phosphotransferase II polymerase chain reaction polyethylene glycol polygalacturonase quantitative competitive PCR recombinant DNA root inducing ribonucleic acid reverse transcriptase S-adenosylmethionine sodium dodecyl sulphate species single stranded Thermus aquaticus transfer DNA tumor inducing ultra violet virulent


FOREWORD The application of biotechnology in food industry is not entirely new. Traditional biotechnology has played a key role in the production of food for thousands of years. For many centuries, the process of fermentation has used microorganisms (yeasts and bacteria) to produce beer, yoghurt and cheese. Naturally occurring microorganisms occur in bread making, beer brewing and vegetable pickling and nowadays, traditional biotechnology techniques are still widely used in the production and preservation of foods. The basis for modern biotechnology, also referred to as gene technology, is DNA. All organisms are composed of cells containing DNA. This DNA contains the genetic information of an organism. Each organism has its own genetic fingerprint made up of DNA, which determines the regulatory functions of its cells, and thus the characteristics that make it unique. Prior to genetic engineering, the exchange of DNA was possible only between individual organisms of the same species or closely related parent plants to produce offspring, having desirable traits such as disease resistance. The limitations of traditional or conventional biotechnology are time and precision; considerable time may be necessary to achieve the desired traits and the offspring may or may not exhibit the trait of interest, hence the lack of precision. However, due to improvements in scientific techniques and the advent of genetic engineering in the 70s, scientists have been able to identify specific genes associated with desirable traits in one organism, and transfer those genes beyond the species boundary into another organism. For example, genes from bacteria, viruses or animals may be transferred into plants to produce genetically modified plants with desired characteristics. Through the use of modern biotechnology precision increased and the time to reach the desired trait or characteristic in a cell, animal or microorganism was reduced. The impact of genetic engineering on the contemporary life has reached unseen heights. Biotechnology continues to be a growing choice among farmers worldwide as the global acreage of crops enhanced through biotechnology increased by 15 percent, or 22 million acres in 2003, according to a report released from the International Service for the Acquisition of Agri-biotech Applications (1). For the seventh consecutive year, farmers worldwide adopted biotech crops at a double-digit pace, with 2002 global biotech acreage reaching 167 million acres. More than one-fifth of the global crop area of soybeans, corn, cotton and canola acres are now biotech. Nearly 6 million farmers in 18 countries chose to plant biotech crops in 2003, up from 5 million farmers in 13 countries in 2001. The aim of this chapter is to provide enough information and some examples to give the reader a sound knowledge

129-3

of the use of plant biotechnology for food purposes. It is not intended to provide an encyclopaedic coverage of the subject though. The text describes the most common technologies that enable the genetic manipulation of crop plants and some applications in food industry. Furthermore, attention is focussed on the detection of genetically modified organisms in food products.

I. HISTORICAL BACKGROUND Since life began, genes have crossed the boundaries of related and unrelated species in nature. Biotechnology applications by humans date back to 1800 B.C., when people began using yeast to leaven bread and ferment wine. By the 1860s, people started breeding plants through deliberate cross pollination. They moved and selected genes to enhance the beneficial qualities of plants through crossbreeding without knowing the traits for which the genes coded. Most foods, including rice, oats, potatoes, corn, wheat and tomatoes, are the products of traditional crossbreeding. This time-tested practice continues to produce crops with desirable traits. However, traditional cross-breeding has its limitations. It can only occur in the same or related plant species, so genetic resources available are limited. Moreover, when plants are cross-bred, all plant’s genes are mixed, producing random combinations. Since traditional plant breeders ultimately want only a few genes or traits transferred, they typically spend 10 to 12 years backcrossing hybrids with the original plants to obtain the desired traits and to breed out the tens of thousands of unwanted genes. Clearly, this process is not speedy nor precise. With the advent of recombinant DNA technology in the 1970s, the genetic manipulation of plants entered a new age. Traits previously unavailable through traditional breeding could be acquired through the advance of recombinant DNA technology, developed in 1973. The technique allowed for effective and efficient transfer of genetic material from one organism to another. Genetic engineering of plants began in 1983 when researchers reported that the Ti plasmid of Agrobacterium tumefaciens, a common soil bacterium, could be modified to allow transfer of foreign DNA into the plant genome (2, 3). The researchers introduced new genes into plants with the aid of the Agrobacterium and also introduced a marker gene for kanamycin resistance to select the transformed cells (2, 4, 5). The production of genetically modified plants rapidly became an important tool for scientific investigation, and transformation methods for a wide variety of crops were subsequently developed (6–11). Many technological breakthroughs in the laboratory soon followed, including engineered resistance to plant viruses, insect resistance based on expression of Bacillus thuringiensis (Bt)

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proteins, tolerance to various herbicides, control of fruit ripening and softening in tomatoes, engineered male sterility and restoration, modified carbohydrate composition and altered oil composition (12). Elite plant varieties from all of these discoveries at the laboratory bench have now been developed. They have been approved by regulatory agencies and are being prepared or grown for commercial applications. In the 1990s, the first genetically engineered foods were made available to the public. In 1990, Pfizer Corporation’s genetically engineered form of rennet used in making cheese was approved, but it received little public attention. Only four years later, in 1994, the Food and Drug Administration (FDA) gave approval for Calgene Corporation’s Flavr Savr Tomato, the first genetically engineered whole food approved for the market (13).

II. TRANSFORMATION METHODS A transgenic plant is a plant that has received a DNA segment or gene(s) from another organism. The foreign segment of DNA is incorporated into the plant’s genome through natural systems present in plant cells. Numerous methods to introduce foreign DNA into plant cells have been developed. A transformation system should allow for (14): – – – –

ability of Agrobacterium spp. to transfer bacterial genes into the plant genome (15). Virulent strains of A. tumefaciens and A. rhizogenes contain a large plasmid (more than 200 kb), respectively known as the Ti-plasmid (tumor inducing) (Figure 129.1) and the Ri plasmid (root inducing). These bacteria possess the exceptional ability to transfer T-DNA, a particular mobile DNA segment of the Ti or Ri plasmid, into the nucleus of infected cells where it is then stably integrated into the host genome and transcribed, causing the crown gall disease (Ti) and hairy roots (Ri) respectively (16, 17). The process of T-DNA transfer is mediated by the cooperative action of proteins encoded by genes determined in the Ti plasmid virulence region (vir genes) and in the bacterial chromosome. The initial results of the T-DNA transfer process to plant cells demonstrate three important features for the practical use of this process in plant transformation. Firstly, the tumor formation is a result of the integration of T-DNA into the plant cells and the subsequent expression of the T-DNA genes. Secondly, the T-DNA genes are transcribed only in plant cells and do not play any role during the transfer process. Thirthly, every DNA sequence can be transferred to plant cells, no matter where it comes from. These well-established facts allowed the construction of

T-DNA

stable integration into the host genome without structural alteration of the foreign DNA, integration of a distinct number of copies of the transforming DNA, stability of the new phenotype over several generations, eventual tissue and development specific regulation of the introduced gene.

o R

onc R

Among the array of genetically engineered plants which currently have been approved, the transformation of choice has been the use of modified plasmids of Agrobacterium. Other transformation methods are based on physical and chemical principles. vir

A.

AGROBACTERIUM TUMEFACIENS

Plant transformation mediated by Agrobacterium tumefaciens, a soil plant pathogenic bacterium, has become the most used method for the introduction of foreign genes into plant cells and the subsequent regeneration of transgenic plants. Jozef Schell and Marc Van Montagu were the first to discover that the bacterium A. tumefaciens transfers a copy of parts of its genetic material into cells of wounded plants, causing the formation of crown gall tumors (3). The ability to cause crown galls thus depends on the

FIGURE 129.1 The Ti plasmid of A. tumefaciens. [R: repeat (border) sequence, O: coding for an opine-synthesizing enzyme, onc: coding for enzymes that are involved in the biosynthesis of plant hormones, vir: controls the transfer of the T-RNA to the host (plant) chromosome.]


the first vector and bacterial strain systems for plant transformation (18).

B. BIOLISTICS Particle (gun) bombardment, or biolistics, is the most important and most effective direct gene transfer method in regular use. In this method, rapidly propelled tungsten or gold microprojectiles coated with DNA are blasted into target plant material, where the DNA is released and can integrate into the genome (6, 19). The integration of the transgenic DNA though is infrequently. In order to generate transgenic plants, the plant material, the tissue culture regime and the transformation conditions have to be optimised quite carefully and in many cases tissue regeneration is necessary. Shortly after its discovery, researchers demonstrated the effectiveness of the microprojectile mediated system by successfully transforming monocots, the first of which was Black Mexican Sweet corn (10, 20). This new ability to transform and regenerate monocot plants marked a significant advance in plant transformation.

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of divalent cations (usually calcium). The PEG and the divalent cations destabilise the plasma membrane of the plant protoplast and render it permeable to naked DNA. Once inside the protoplast the DNA enters the nucleus and integrates into the genome (24). PEG treated protoplasts for the most part have been abandoned as a genetic delivery system, because of the low effectiveness. An advancement to PEG mediated transformation was the liposome mediated transformation technique. Foreign DNA is encapsulated in a spherical lipid bilayer termed a liposome (25). In the presence of PEG, the host protoplast will bind and envelop the liposome through endocytosis (26). After endocytosis, the DNA is free to recombine and integrate with the host genome. The liposomes are formed from neutral lipids similar to those which compose the plasma membrane and can be produced in a variety of sizes ranging from 30–50 nm with a volume of approximately 2 ml. The DNA is packaged in vitro and then combined with the target protoplasts. As with other transformation systems, a variety of vectors including viral vectors can be incorporated into this system.

F. SILICON CARBIDE FIBRES C. ELECTROPORATION The electroporation of cells can be used to deliver DNA into plant cells and protoplasts. The vectors used can be simple plasmids. The genes of interest require plant regulatory sequences, but no specific sequences are required for integration. Material is incubated in a buffer solution containing DNA and subjected to high voltage electrical pulses. The DNA then migrates through high voltage induced pores in the plasma membrane and integrates into the genome (21). Electroporation has been successfully used to transform all the major cereals, particularly rice, wheat and maize.

With this technique, plant material is introduced into a buffer containing DNA and silicon carbide fibres, which is then vortexed. The fibres penetrate the cell wall and plasma membrane, allowing the DNA to gain access to the inside of the cell (27). Although the procedure has been utilised with friable callus from maize (28), this type of friable callus is limited only to a few genotypes of maize and oats. Many cereals produce an embryogenic callus that is hard and compact and therefore not easily transformed with this technique. Recently though, some progress has been made in transforming such material, and procedures are being developed to allow transformation of cereals such as rice, wheat, barley and maize without the need to initiate cell suspensions (29).

D. MICROINJECTION Simmonds and coworkers reported the use of microcapillaries for the introduction of plasmid DNA into the germ line precursor cells of apical meristems by microinjection (22). Despite a positive indication by PCR amplification of DNA isolated from injected apices, this approach has failed to yield any transgenic plants (23).

III. TRANSGENES

E. POLYETHYLENE GLYCOL

A. THE PROMOTER

This procedure revolves around the use of protoplasts and their totipotent ability to regenerate into mature plants. Protoplasts are plant cells whose cell walls have been removed leaving only a plasma membrane around the cells. Plant protoplasts can be transformed with naked DNA by treatment with polyethylene glycol (PEG) in the presence

Promoters mediate the initiation of transcription in a manner that is dependent on tissue type and sometimes other signals. Promoters of variable strength and tissue specificity are available. They can be constitutive (typical) or inducible by environmental or chemical stimuli (rare). The most widely used promoter is the promoter driving

Transgenes are not necessarily different from endogenous genes. However, transgenes are often chimeric, that is cobbled together from elements found naturally in different genes. The most important element is usually the protein coding region, which consists of one open reading frame.

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expression of the 35S Cauliflower Mosaic Virus. CaMV 35S is a strong promoter that is active in essentially all dicot plant tissues. Promoters have also been constructed that are inducible by heat shock, copper ions, glucocorticoid hormones, alcohol, antibiotics and other stimuli. Native plant promoters are usually less than 1 kb in length. A plant promoter will often work in many different plant species, but yeast, human or bacterial promoters do not function in plants (15).

B. THE TERMINATOR The terminator serves as a transcriptional stop signal to the polymerase. Various terminators are in widespread use, e.g., one derived from CaMV and one derived from the nopaline synthase (nos-3) gene of Agrobacterium. The various terminators are basically equivalent (30).

C. STRUCTURAL GENES The structural genes are the DNA sequences which contain the information encoding the protein of interest. Distinction should be made between target genes and reporter genes (see marker genes). Many genes have been used for the generation of the currently approved transgenic crops. Some of these genes, such as accd, accS and sam-k (tomatoes) and some genes coding viral coat proteins have only been used in one particular genetically engineered product. As a consequence, the identification of sequences of one of these genes in food would represent a product-specific detection method, provided the actual sample did not contain the natural sources of these sequences (e.g., from bacteriophages or plant viruses) (30). More common is the endotoxin gene from Bacillus thuringiensis (insect resistance — corn, potato, tomato) or the bar/pat gene, originally isolated from Streptomyces hygroscopius, coding for the enzyme phosphinothricin acetyltransferase (herbicide tolerance — chicory, corn, soybean, oilseed rape, sugar beet, rice). Variants of the CP4 EPSPS gene from Agrobacterium (herbicide resistance — corn, soybean, cotton, oilseed rape, sugar beet, potato), the β-lactamase gene (tomato) and the polygalacturonase gene (tomato) have also been introduced. Furthermore, the gene encoding for barnase from the bacterium Bacillus amyloliquefaciens encodes a ribonuclease which catalyzes the hydrolysis of single stranded RNA molecules. The gene is expressed in the anther only and causes male sterility (chicory, corn, oilseed rape) (30, 31). Other genetically modified (GM) foods currently available are: –

melon: reduced accumulation of S-adenosylmethionine (SAM), and consequently reduced ethylene synthesis, by introduction of the gene encoding S-adenosylmethionine hydrolase,

–

–

–

papaya: papaya ringspot virus (PRSV) resistant papaya produced by inserting the coat protein (CP) encoding sequences from this plant potyvirus, wheat: selection for a mutagenised version of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase, squash: cucumber mosiac virus (CMV) and/or zucchini yellows mosaic (ZYMV) and watermelon mosaic virus (WMV) resistant squash produced by inserting the coat protein (CP) encoding sequences from each of these plant viruses into the host genome.

D. MARKER GENES One of the technical problems encountered in attempts at gene transfer is knowing whether a particular gene has actually been introduced into a new host cell and, if transferred, whether it is directing the synthesis of protein. To overcome this problem, reporter or marker genes have been developed, which can be transferred to the plant cell using Agrobacterium. The choice of a selectable marker gene depends on the plant species and the specific genotype of the plant. In general, antibiotic resistance genes make good selectable markers for many dicotyledonous species, such as tobacco or Arabidopsis thaliana. In contrast, many monocot species are quite resistant to common antibiotics, and herbicide resistance genes are preferred in this case. Herbicides are also cheaper than antibiotics and they can be applied to soil grown plants. Marker genes tend to be developed from bacterial genes coding for easily assayed enzymes (32–34). A typical marker gene is the neomycin phosphotransferase II gene (nptII), with kanamycin resistance, as used in the Flavr Savr™ tomato (30, 35, 36). An alternative system uses the gene for a naturally derived enzyme, phosphomannose isomerase (37, 38). This particular enzyme enables plant cells to use mannose as a source of energy. The cells that manage to grow in the presence of mannose have acquired the marker gene and have therefore also taken up the other genes of interest. This system, and similar ones based on other sugars, should allay the fear that GM poses a danger to human health. These should allow a refocusing of effort to tackle the overuse of antibiotics in intensive farming and their overprescription in medicine which pose a far greater threat to our health. GUS, the Escherichia coli β-glucuronidase gene (39, 40), and the luciferase gene, which is obtained either from fireflies (Photinus pyralis) or the marine bacterium Vibrio harveyi have been very successful as reporter genes too (41). In the near future, it can be expected that the selection of markers for antibiotic resistance will be avoided. There is no current list though of antibiotic resistance markers that


cannot be used in the genetic modification of plant crops. In Europe, Article 4(2) of Council Directive 2001/18/EC refers to the phasing out of genes expressing resistance to antibiotics which may have adverse effects on human health and the environment or are of use in medical or veterinary treatment. This phasing out must take place by 31 December 2004 in the case of GM crop plants for marketing and by 31 December 2008 in the case of the release of GM crop plants for research and development purposes. In accordance with this requirement the commission has established an expert working group to address the use of antibiotic resistance marker (ARM) genes that genetically modified organisms (GMOs) may contain and will aim to produce a list of ARMs which must be phased out.

IV. STRATEGIES FOR GENETIC MANIPULATION Enzymes are the products of the majority of transgenes introduced into the currently approved genetically engineered agricultural crops. The expression of these enzymes has conferred novel traits to the respective plants. Proteins without an enzymatic activity, such as toxins, or antisense constructs have also been expressed.

A. SENSE STRATEGY In order to add a new trait to a crop, one or more genes or their complementary DNA (cDNA) should be added to the genome of the host plant. For this purpose, the gene or cDNA is cloned in a sense orientation in between promotor and terminator. This results in the expression of messenger RNA to a protein.

B. TRANSGENE SILENCING Instability of transgene expression is still a problem encountered in many experiments involving transgenic plants and is often referred to as gene silencing. Gene silencing can involve a variety of methods and is still relatively poorly understood. When the chromosomal locus harboring a transgene is analyzed carefully, one can find either a single T-DNA or multiple copies of the T-DNA. If multiple copies are present, they can be arranged as direct repeats (sense) or inverted repeats (antisense), or partial copies may be present next to complete copies. Transgene loci with multiple copies, especially inverted repeats, are often associated with gene silencing, meaning that the transgene(s) are poorly expressed. Gene silencing is thought to represent a highly sequence specific plant genome surveillance mechanism. The plant is able to recognize certain nucleic acids as foreign and it has means to suppress the expression of such genes. Two mechanisms can be distinguished. Either the

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Sense

5′

3′

GAATG

CTAAG

CTTAC

GATTC

3′

5′

GAAUG

5′

3′

3′

CTTAG

CATTC

GAATC

GTAAG

CUUAG

GAUUG

3′

CUAAG

Sense-mRNA

Antisense

5′

5′

5′

Antisense-mRNA

3′

3′

5′ GAAUG CUUAC

CUAAG GAUUC

5′

3′ Polygalacturonase

FIGURE 129.2 Construction of the antisense mRNA technology for the polygalacturonase vector.

rate of transcription is reduced (transcriptional gene silencing) or the mRNA is destabilized (post-transcriptional gene silencing). Transcriptional gene silencing occurs when genes share homology in their promoter regions. It usually results in altered methylation patterns and altered chromatin conformation, which results in gene silencing by repressing transcription (42). Sometimes, even endogenous genes that are similar in sequence to the transgene are silenced along with the transgene (co-suppression). This is usually undesirable, although the effect has been exploited to some advantage as well. For example, the delayed fruit softening in the Flavr Savr™ tomato is controlled by co-suppressing the endogenous gene for ethylene production with a transgene of related sequence (43). This tomato contains a gene that is transcribed into a messenger RNA anti-sense to the mRNA from the polygalacturonase (PG) gene (Figure 129.2). The complementary in vivo base pairing of these two molecular species results in inhibition of the expression of the gene, with a dramatically decreased PG activity in the transgenic tomatoes. The enzyme PG degrades pectin, a major constituent of the cell wall of the fruit. Its inhibition increases the shelf-life of the tomatoes and prevents them from becoming soft (44). Unfortunately, it is almost impossible to control whether single or multiple T-DNAs are integrated. One has to generate several independent transgenic lines and screen them for stable gene expression, or stable silencing, as the case may be. It is also possible to find T-DNAs at several unlinked sites in the genome (normally 1–4). Multiple sites are also associated with gene silencing. In this case, genetic backcrossing to wild-type plants should reduce the number of transgene loci and may overcome silencing.

V. APPLICATION OF GENETIC MANIPULATION IN THE FOOD INDUSTRY A. BENEFITS

OF

GM FOODS

Most of the research in the application of gene technology on food crops has sought to improve product quality and

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5′

CaMV P35S promotor CTP

CP4-EPSPS

3′

nos terminator

Plant DNA

Plant DNA

FIGURE 129.3 Constructs introduced in the glyphosate resistant Roundup Ready soybean.

agronomic traits and develop a better resistance to the environment. Genetic engineering can be used to increase crop yield and reduce crop loss by making plants tolerant to pests, weeds, herbicides, viruses, insects, salinity, pH, temperature, frost and drought. Insect resistant corn (see frame 1), soybean, cotton, potato and apples, virus resistant cantaloupes, squash, papaya, cucumbers and herbicide tolerant corn, tomatoes, potatoes, canola and soybean (see frame 2) have all been produced (45–47). These crops with improved agricultural qualities are considered as the first generation of GMOs, introduced on the market from 1995. Nowadays, more attention has been paid to the development of GMOs with a clear, direct and significant advantage for the consumer. This second generation of GMOs should gain consumer’s interest, trust and acceptance. These foods may have one of the following benefits: – –

–

–

–

improved shelf-life, e.g., the Flavr Savr tomato, and organoleptic quality of foods (48), improved nutritional quality and health benefits, e.g. oils with an improved fatty acid profile (49), higher lycopene levels in tomato and peppers (47, 50), golden rice with provitamine A (50–54), allergen free rice and peanuts, …, improved protein quality and/or quantity (cassava) or increased content in essential amino acids (55–59), increase in food carbohydrate content, e.g., potato with a high solids content, which makes it useful for making French fries (60, 61), edible vaccines and drugs, e.g., banana with proteins that may be used as vaccines against hepatitis, rabies, dysentery, cholera, diarrhoea or other gut infections prevalent in developing countries (62, 63).

Examples Bt corn Bt is a naturally occurring soilborne bacterium that is found worldwide. A unique feature of this bacterium is its production of crystal-like proteins that selectively kill specific groups of insects (Cry proteins). Plant molecular biologists created Bt corn by inserting selected exotic DNA into the corn plant’s own DNA. Proteins have been found with insecticidal activity against the Colorado potato beetle (for example, Cry3A, Cry3C), corn earworm (Cry1Ac, Cry1Ab), tobacco budworm (Cry1Ab) and the European corn borer (Cry1Ab, Cry1Ac, Cry9C).

Glyphosate-tolerant soybean Roundup Ready® soy (Monsanto), the first biotechnologically improved soybean to be marketed, became commercially available in 1996. Glyphosate, the active ingredient in Roundup herbicide, controls weeds by inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is an enzyme in the shikimate pathway for aromatic amono acid biosynthesis in plants. Roundup Ready soybean event 40–3–2 was produced by particle acceleration transformation of active EPSPS isolated from Agrobacterium sp. strain CP4 (CP4 EPSPS) into the genome of the soybean cultivar A5403. The insertion of DNA (Figure 129.3) includes the Cauliflower Mosaic Virus (CaMV)-derived 35S promotor with duplicated enhancer, the petunia-derived chloroplast transit peptide (CTP) region, which is responsible for the correct processing of the protein in the cell, the EPSPS gene and the nopaline synthase (nos) sequence to terminate the transcription of the genetic construct.

B. POTENTIAL RISKS OF GENETICALLY MODIFIED FOODS The critics of genetic engineering of foods have concerns, not only for safety, allergenicity, toxicity, carcinogenicity and altered nutritional quality of foods, but also for the environment. The use of marker genes has been restricted to prevent the development of antibiotic resistance. Furthermore, genetic pollution, the creation of superweeds and superpests have to be considered. 1. Food Safety The introduction of modified foods has led to a shift in the food safety assessment towards a greater need for whole food safety assessment. An important feature in determining the potential risks is whether or not the GMO is able to cause disease to humans, animals or plants. In the United States, it is the responsibility of the Food and Drug Administration (FDA) to provide oversight for all foods, including those derived from GMOs. More than 15 years of laboratory research and field trials with rDNA-engineered plants indicate that the risks posed by these plants are not any greater than or different from the risks posed by plants produced by traditional breeding methods used for more than 100 years (64). Various organisations and the biotechnology industry have been working together since 1990 to design a safety assessment strategy for genetically modified crops and


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Genetically modified plant and products

Molecular characterisation of inserted gene construct

Molecular characterisation data

Direct toxicity as predicted from the genetic modification

Data from classical toxicity studies

Unwanted indirect effects from the genetic modification

Data from comparative analytical studies

Morphology and phenotypical behaviour

Comparative field test data

Rejection

Food safety dossier

Regulatory review

Approval

FIGURE 129.4 The current approach for the safety assessment of genetically engineered food (Pedersen et al. 2001).

their derived products. The EU legislation has elaborated different rules covering the safety of genetically modified foods: the Directive on the deliberate release into the environment (2001/18/EC) and the Regulation concerning novel foods (258/97/EC). The food safety assessment of genetically engineered foods should determine whether the modified food is as safe as its traditional counterpart. As a starting point for the safety assessment the concept of ‘substantial equivalence’ was introduced as a means of establishing a benchmark of safe food. The potential risks associated with the use of a GMO are determined by the characteristics of the organism which receives the modification, the characteristics of the used genetic material and the circumstances under which the GMO is applied. The food safety assessment of genetically engineered foods is considered to consist of the following parts: (1) a molecular characterization of the insert, (2) determination of any unwanted direct toxicological effects as can be predicted from the nature of the inserted sequences, (3) determination of any unwanted indirect toxicological consequences resulting from the modification and (4) a morphological and behavioral analysis of the plant under relevant field conditions. The concept of substantial equivalence was applied for the first time in the safety assessment of the Flavr Savr™ tomato before it was placed on the USA market in 1994. In the following years, a lot of experience with the safety assessment of a large variety of genetically modified plants has been gathered. In the EU food ingredients derived from herbicide tolerant soybeans and from several insect and/or herbicide tolerant maize lines, and refined oils derived from several herbicide tolerant rape seed lines were registered and approved according to the legal requirements that have been put in place since 1990 and 1997 respectively.

2. Food Related Concerns One of the major concerns regarding food safety is the potential allergenicity of genetically modified foods. Well known is the methionine rich protein (MRP) soy from Pioneer Hi-bred International. To increase the protein content of its animal feed, the company incorporated Brazil nut genes into soybeans. This gene modification caused allergic reactions to consumers who were allergic to Brazil nut, so this product was voluntarily recalled in 1996 (65). It is also believed that foreign genes might alter nutritional value of foods in unpredictable ways by decreasing levels of some nutrients while increasing levels of others. Moreover, genetic modification could inadvertently enhance natural plant toxins by switching on a gene that has both the desired effect and capacity to pump out a poison (66). 3. Environmental Concerns Environmentalists are concerned that transgenic crops will present environmental risks when they are widely cultivated (62). Genetically modified crops with herbicide and insect resistance could cross-pollinate with wild species, creating superweeds (63, 67). These superweeds can become invasive plants with the potential to lower crop yields and disrupt natural ecosystems. A critical and very controversial aspect of the antibiotic resistance issue is the utilization of antibiotic resistance genes as the selection marker in genetically modified organisms (GMOs). The main safety concern relates to the escape or transfer of the antibiotic resistance genes to sensitive bacterial strains when these GMOs are introduced into the environment. Moreover, the extended exposure to plant produced pesticides could result in the development of a resistance mechanism in the target organism (63). Plants engineered to contain virus particles as part of a strategy to enhance resistance could facilitate the creation

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of new viruses in the environment (53). Plants engineered to express potentially toxic substances such as drugs and pesticides will present risks to other organisms that are not intended as targets. One example includes pollen from transgenic corn, which has been suggested to kill the Monarch butterfly larvae. It has been shown that hybrid corn expresses a bacterial toxin in its pollen, which is then dispersed over 60 meters by wind. In this range, the corn pollen is deposited on other plants near cornfields where it can be ingested by non-target organisms including the Monarch butterfly. These butterflies have been found to eat less, have a slower growth rate and higher death rate (68). On the other hand, research has proven that the Monarch butterfly population has not been affected (69). Since Bt-176 corn is no longer available in US maize varieties, the risk to the Monarch butterfly populations from current Bt maize varieties is low (15). 4. Other Concerns The debate involves not only scientific but also political, socio-economic, ethical, religious and philosophical issues (63, 70–73). Some critics of genetic modification argue that patenting which allows corporations to have monopoly control of genetically altered plants or animals violates the justice of life (74).

C. GENETICALLY MODIFIED MICROORGANISMS Council Regulation 258/97 defines a novel food as food which has not been used for human consumption to a significant degree within the European Community. Within this scope fall foods and food ingredients consisting of or isolated from microorganisms, fungi or algae. Developments in modern biotechnology facilitate the production of bacterial strains with particular properties. The introduction of such microorganisms into food raises several issues that need to be addressed as part of the safety evaluation. These include: (1) the risk of infectivity and pathogenicity, (2) the potential for colonization and gene transfer within the gastrointestinal tract, and the consequences thereof, (3) their effects on microflora composition and function, including the production of deleterious metabolites and (4) their effects on gastrointestinal mucosa and function (75, 76). There is huge potential for using biotechnology to develop foods with improved processing qualities. Biotechnology is also likely to be used to produce improved microorganisms, both to improve conventional fermentation processes and to develop new ones. These could include microorganisms for the production of foods (e.g., bread, wine, yoghurt and cheese), or for a wide range of fermentation products for use in food processing (e.g., enzymes, vitamins, amino acids and high-grade chemical additives such as citric acid, a flavouring and acidifying agent).

The major areas currently attracting attention are described in this section. 1. Lactic Acid Bacteria The vast majority of bacteria used in the food and dairy industries belong to the group known as the lactic acid bacteria (LAB). In general, genetic manipulation of LAB is achieved either by the inactivation of a gene or by the expression/overexpression of a gene (77). Such manipulation may affect a biochemical pathway resulting in different end products or altered yields of end products. This in turn affects the taste, texture, yield or quality of the fermented food. A commercially important area is the production of new strains of lactic acid bacteria which carry out a faster, more efficient fermentation. For example, using plasmid technology, new strains of Lactobacillus spp. have been produced to provide improved starter cultures for cheese production. The potential for the more rapid production and maturation of cheeses, and a whole range of other fermented products, is being exploited. Another important achievement is the introduction into lactic acid bacteria of genes resistant to destructive bacteriophages. Additionally, lactic acid bacteria are excellent producers of peptidases, which are already widely used in food technology. This characteristic is thought to have enormous potential, not least because these bacteria, being associated with the production of traditional foods, are largely recognised as being ‘safe.’ GM lactic acid bateria have also been used in meat and sausage fermentation (78, 79). 2. Enzymes Enzymes are very important in food processing. Apart from enhancing nutritional value, they can be used to influence flavour, aroma, texture, appearance and speed of production. Microorganisms are the most important sources of these enzymes. There is no reason why the ability to synthesise enzymes normally associated only with plants and animals should not be engineered into microorganisms for culture in fermenters. This was achieved some time ago for the enzyme chymosin (rennin), used in cheese manufacture. Chymosin from transgenic yeast was the first enzyme from a genetically modified organism to gain regulatory approval for food use in 1988. Three such enzymes are now approved in most European countries and the USA derived from Escherichia coli, Kluyveromyces lactis and Aspergillus niger. These proteins behave in exactly the same way as calf chymosin, but their activity is more predictable and they have fewer impurities. Such enzymes have gained the support of vegetarian organisations and of some religious authorities. Chymosin obtained from recombinant organisms has been subjected to rigorous tests to ensure its purity (80).


3. Yeasts Much attention is currently being paid to the possible use of yeasts as a vehicle in which to express transferred genes. Yeast is an important microorganism in the food industry and has applications in brewing, baking and in the production of fermented foods such as soya sauce. In recent years, much research has been conducted into the genetic manipulation of Saccharomyces cerevisiae in order to enhance endogenous characteristics, such as ethanol tolerance and to obtain expression of foreign genes and the secretion of foreign proteins, some of which are useful to the food industry (81–83). The yeast Saccharomyces cerevisiae var. diastaticus, whilst not itself suitable for use in brewing, produces an amylase capable of hydrolysing starch residues which normally remain in the brew. The high calorie starch residues are thus converted into fermentable sugar. A gene coding for the enzyme is transferred, via a plasmid, to normal brewing strains of yeast. The transgenic yeast can be used to produce a high alcohol premium product or, alternatively, a greater volume of low calorie ‘lite’ beer. New yeast strains that are tailored to the barley and hops that are grown in different regions of the world are being developed for use in the brewing industry. 4. Fungal Factories for Enzyme Production Fungi are of major economic importance as opportunistic pathogens and spoilage organisms but they also have a number of positive uses in food and non-food industries, e.g., the production of catalase, glucose oxidase, lipase and pectinesterase. These useful fungi may be modified genetically to improve their efficiency and enzymesecreting capacity (84).

VI. DETECTION OF GMOs A. LABELLING

OF

GMOS

There is a need for processors and traders to meet emerging mandatory GMO-labelling requirements in certain countries, in particular the EU, but also in Switzerland, Australia, New Zealand, Japan, etc. The tolerance levels for labelling may differ among countries or still have to be decided. EU legislation on labelling is summarised in the following section. Since 18 April 2004 two new regulations (1829/2003 and 1830/2003) (86, 87) concerning traceability and labelling of GMOs entered into force in the EU. A harmonised community system is set up to trace GMOs, the labelling of GM feed is introduced, the current labelling rules on GM food are reinforced and a streamlined authorisation procedure for GMOs in food and feed and their deliberate release into the environment is established. Today, labelling of GM food or feed is mandatory, even when the

129-11

specific DNA or protein of the GMO can no longer be identified in the final product. Adventitious and technically unavoidable presence of GMO are not subject to these labelling provisions as long as this presence is below a 0.9% threshold of the food ingredient individually considered. For the presence of GM material that is not approved in the EU but benefits from a favourable scientific risk assessment by the Scientific Committees or the European Food Safety Authority, a 0.5% threshold is established. These laws also extend the labelling requirements in order to cover food and food ingredients produced from GMOs under the level of detection, e.g., soya or maize oil produced from GM-soya or GM-maize and biscuits with maize oil produced from GM-maize. It is obvious that the development and application of reliable and quantitative analytical detection methods are of utmost importance for the implementation of these labelling rules. In general, detection methods for GMOs are based on DNA or protein level. The first uses the polymerase chain reaction (PCR), the latter is based on immunoassays. Other detection strategies will be discussed briefly.

B. DETECTION BASED

ON

DNA

1. DNA Isolation Provided that the laboratory sample is representative for the field sample, batch or lot of the product and that is has been adequately homogenised, even small aliquots of vegetal material are sufficient for DNA extraction, usually between 100 mg (87) and 350 mg (88). For the extraction of DNA from plant tissues and food products, a vast range of methods is available. An overview has been given by Anklam et al. (31). Currently, three different approaches to DNA isolation from plant material and plant-derived products are favoured for GMO detection: the CTAB method, DNA binding silica columns (various commercially available kits) (89) and a combination of these two. In general, DNA extraction from plant material has to accomplish the following steps (31): 1. the breakage of cell walls is usually achieved by grinding the tissue in dry ice or liquid nitrogen, 2. the disruption of cell membranes is achieved by using a detergent (e.g., CTAB or SDS), 3. inactivation of endogenous nucleases is achieved by the addition of detergents and/or EDTA, which binds Mg2, an obligatory co-factor of many enzymes. Proteinase K may be added for inactivation and degradation of the proteins, particularly in protocols using DNA binding silica columns, 4. separation of inhibitory polysaccharides is possible due to the differential solubility of polysaccharides and DNA in the presence of cetyltrimethylammonium bromide (CTAB),

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5. separation of hydrophobic cell constituents, e.g. lipids and polyphenols is attained by extraction with an organic solvent like chloroform, 6. finally, the separation from the detergent and concentration of DNA is carried out by alcohol/ salt precipitation. 2. PCR — Principle The polymerase chain reaction allows the millionfold amplification of a target DNA fragment in a highly sensitive and specific manner. DNA fragments with a length of 100 to 1000 base pairs are amplified with the aid of the polymerase enzyme and two primers. Primers are oligonucleotide sequences complementary to either one of the two strands from the double stranded DNA target. The kinetics of the PCR are determined by the temperature profile used. Because of the high temperatures required for the denaturation of DNA, the use of a thermostable DNA polymerase is necessary. For this purpose, a polymerase isolated from the thermophylic bacterium Thermus aquaticus (Taq-polymerase) is used. The PCR protocol exists of a 20 to 50 cycle program, each consisting of the following steps (Figure 129.5): –

–

–

denaturation: the single stranded DNA molecules are obtained by heating the DNA solution to a temperature of 94–95°C, enough to break the hydrogen bonds between the strands, annealing: by decreasing the temperature to around 55°C, the primers bind to their complementary DNA sequence. Template DNA, primers and DNA polymerase are included in the reaction from the beginning of the PCR, extension: a polymerisation step is carried out at around 72°C under the action of the DNA polymerase.

The primer extension products synthesised in one cycle will serve as a template in the next. The repetitive series

of cycles results in the exponential accumulation of a specific fragment whose termini are defined by the primers. After the PCR, the length of the amplification products has to be checked using the electrophoresis technique, although other separation methods such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) have been used (90, 91). For the separation of large DNA fragments agarose gels are used, while polyacrylamid gels are more suitable for the separation of small fragments. For GMO detection, gel electrophoresis is preferred. Under an electric field, the negatively charged DNA molecules will move through the gel at different rates depending on their size, resulting in a segregation of the different fragments of DNA. The DNA is stained with a fluorescent molecule that binds to DNA and is visualized under UV light. 3. PCR Strategies for GMO Screening and Identification Any PCR based detection strategy depends on a detailed knowledge of the transgenic DNA sequences and the molecular structure of the GMOs in order to select for the appropriate oligonucleotide primers. Before conducting a GMO specific or screening PCR assay, the presence of amplifiable DNA in food samples must be determined by using species specific primers. Several (GM) food ingredients have been analyzed using PCR: soy (92), wheat (93), canola, potatoes (94), rice, papaya (40), alfalfa (39), maize, sugarbeet and tomatoes (95). Nowadays, routine GMO is focused on the detection of the Cauliflower Mosaic Virus (CaMV) 35S promotor (P-35S) and the Agrobacterium tumefaciens nos terminator (nos3), which are present in many GMOs currently on the market. However, additional target sequences are needed in order to guarantee a complete identification procedure. Moreover, these target sequences may occur as natural contaminants in the sample (from plant viruses and bacteria). Therefore, specific sequences that are characteristic for the individual transgenic organism should be targeted,

Double stranded DNA

Denaturation

5′

3′

3′

5′

5′

3′

3′

5′

Extension

Hybridisation of the primers 3′

5′

5′

3′

5′

3′ 5′ 3′

FIGURE 129.5 The polymerase chain reaction.

3′ 5′

5′ 3′

Y

3′ 3′

X

5′

5′


such as the cross border regions between the integration site and transformed genetic element of a specific GMO, or specific sequence alterations due to truncated gene insertions (i.e., cDNA, or altered codon usage) (96). If there is any doubt about the fragment identity, the amplified fragment can be checked more precisely using specific endonucleases (restriction fragment analysis). These enzymes cut only the expected DNA sequence into two fragments of known size (97). With Southern blotting, the sample DNA is isolated and fixed onto nitrocellulose or nylon membranes and probed with double stranded labelled nucleic acid probes specific for the GMO. Hybridisation can be detected radiographically, fluorometrically or chemiluminescently. Recently, an alternative Southern blot technology has been attempted with near infrared (NIR) fluorescent dyes (emitting at ⬃700 and 800 nm) coupled to a carbodiimidereactive group and attached directly to DNA in a 5 min reaction. The signals for both dyes are detected simultaneously by two detectors of an infrared imager (98, 99). Another strategy for GMO identification recently discussed makes use of amplified fragment length polymorphism (AFLP), a DNA fingerprinting method, which has already been used successfully to discriminate between and identify plant varieties, including processed agricultural materials (100, 101). Other methods to confirm PCR results are: hybridisation (96), direct sequencing of the PCR product (98, 102, 103), nested PCR (97, 104, 105), anchored PCR (106, 107) and mass spectrometric detection of PCR products (108). 4. Quantitative Detection Methods Based on DNA Two kinds of PCR strategies are currently being used for the quantification of GMOs in food: end-point PCR (quantitative competitive PCR) and real-time PCR.

a. Quantitative competitive PCR (QC-PCR) The principle of quantitative competitive PCR is the (co-) amplification of internal DNA standards together with target DNA. A small difference between target and control sequence (40 bp) makes it possible to distinguish between the two reaction products. Each sample is amplified with increasing amounts of competitor, while keeping the sample volume/concentration constant. PCR products are separated by an appropriate method, such as agarose gel electrophoresis and subsequently quantified by photometric methods. At the equivalence point, the starting concentration of internal standard and target are equal (i.e., the regression coefficient is 0.99 and the slope of the regression line ⬇1) (109). Although the presence of PCR inhibitors will be noticed immediately because the amplification of both internal standard and target DNA will be simultaneously affected, competition between the

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amplification of internal standard DNA and target DNA generally leads to loss of detection sensitivity. QC-PCR has been developed for Roundup Ready soybean, Bt and Maximizer maize, the P-35S promotor and nos3 terminator (110–114).

b. Quantitative real-time PCR (RT-PCR) The real-time PCR technique was originally developed in 1992 by Higuchi and co-workers, allowing to follow the amplification of the target DNA sequence during the whole reaction by indirect monitoring of the product formation (115). Real-time detection strategies rely on the continuous measurements of the increments in the fluorescence generated during the PCR. Therefore, several formats can be used: (1) the ds-DNA-binding dye SYBR Green I, (2) hybridisation probes or fluorescence resonance energy transfer (FRET) probes, (3) hydrolysis probes (TaqMan® technology) and (4) molecular beacons (116). The number of PCR cycles necessary to generate a signal statistically significant above the noise is taken as a quantitative measure and is called the cycle threshold (Ct). As long as the Ct value is measured at the stage of the PCR where the efficiency is still constant, the Ct value is inversely proportional to the log of the initial amount of target molecules. More than 150 food products containing GM soy (e.g., baby food, diet products, soy drinks, desserts, tofu and tofu products, cereals, noodles, fats, oils and condiments) have been analysed by TaqMan®, proving this method to be sensitive (112, 117–120). Other research has been done on GM maize (121). c. PCR-ELISA PCR-ELISA uses the strategy of real-time PCR and can be quantitative when the PCR is stopped before a significant decrease in amplification efficiency occurs (i.e., before the plateau phase is reached). Then ELISA can be used to quantify the relatively low amounts of PCR products (122, 123).

C. DETECTION BASED

ON

RNA

The RNA based methods rely on the specific binding between the RNA molecule and a synthetic RNA or DNA molecule (primer). The primer must be complementary to the nucleotide sequence at the start of the RNA molecule. Usually binding between the RNA molecule and the primer is followed by conversion of the RNA to a DNA molecule through reverse transcription. Finally the DNA can be multiplied with PCR or translated into as many as 100 copies of the original RNA molecule and the procedure can be repeated by using each copy as a template using nucleic acid sequence-based amplification (NASBA). The specific primers needed for the procedure cannot be developed without prior knowledge of the composition of the RNA molecule to be detected (124).

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+ Antibody

+ Antigen

+

Chromogenic Antibody/ enzyme complex substrate

Colour reaction

FIGURE 129.6 ELISA principle.

The first consideration when using reverse transcriptase PCR for mRNA analysis is RNA isolation. The RNA should be high quality and free from genomic DNA contamination. However, since most reverse transcriptase PCR methods amplify only a few hundred bases rather than the complete mRNA sequence, the sample RNA can be slightly degraded. The major problem for the use of reverse transcriptase PCR for GMO analysis though is the chemical instability of the RNA molecule and the ubiquitous presence of RNAses.

D. DETECTION BASED

ON

PROTEIN

GMOs are characterised by an altered genome which may lead to the expression of new proteins. Therefore GM foods might be identified by testing for the presence of the introduced DNA or by detecting expressed novel proteins encoded by the genetic material. Immunoassay technologies with antibodies are ideal for qualitative and quantitative detection of many types of proteins in complex matrices when the target analyte is known. Both monoclonal (highly specific) and polyclonal (often more sensitive) antibodies can be used depending on the amounts needed and the specificity of the detection system (e.g., antibodies to whole protein or specific peptide sequences), depending on the particular application, time allotted for testing and cost (125). On the basis of typical concentrations of transgenic material in plant tissue (10 µg per tissue), the detection limits of protein immunoassays can predict the presence of modified proteins in the range of 1% GMOs (126). Both Western blot and enzyme-linked immunosorbant assay (ELISA) techniques have been used for the analysis of protein products of transgenic crops. 1. Western Blot The Western blot is a highly specific method that provides qualitative results for determining whether a sample contains the target protein below or above a predetermined threshold level (127), and is particularly useful for the analysis of insoluble protein (128). Although developed for the detection of modified soy (129, 130), this method is preferred for research purposes rather than for routine analysis. 2. ELISA ELISA is the most common type of immunoassay. Antibodies, raised against proteins derived from GMOs, are

TABLE 129.1 Main Characteristics of DNA and Protein Based GMO Tests (170) Characteristics Test sensitivity Contamination sensitivity Test complexity Test speed Universal markers Test design flexibility Markers availability Automation chance Quantification chance Complex matrices detection

DNA (PCR) High High High Medium Yes Yes Yes Yes Yes/no High

Protein (ELISA/Lateral Flow) Medium Low Medium/low Medium/high No No Low (antibodies) Yes/no Yes/no Low

coated on a microwell plate. For the detection of GMOs, several approaches can be used. In a sandwich ELISA setup (Figure 129.6), the protein extract is spread on the microwell and a specific antigen–antibody binding takes place. After removal of the excess of protein extract, a second enzyme labelled antibody is added, which binds the target antigen. Unbound enzyme labelled antibodies are removed and an enzyme specific substrate is added, which results in a colour reaction if all of the previous reactions have taken place. The intensity of the signal is a measure for the amount of GMO present in the tested sample. One of the major drawbacks of immunochemical assays is that their accuracy and precision can be adversely affected in a complex matrix, such as those found in many processed agricultural and food products. The possible causes for interference from the matrix have been attributed to nonspecific interaction with the antibody by proteins, surfactants (saponins) or phenolic compounds, antibody denaturing by fatty acids and the presence of endogenous phosphatases or enzyme inhibitors. Moreover, detection and measurement may be rendered difficult by low levels of expression of transgenic proteins, the degradation associated with thermal treatments or pH changes, a poor antibody affinity or the commercially available source of antibodies and standards (31, 128, 131). In summary, the main characteristics and peculiar differences between the DNA based and protein based tehniques for GMO detection are listed in Table 129.1. Nonetheless, several immunoassay-based methods have so far been developed, such as for the neomycin phosphotransferase II (nptII), the EPSPS enzyme (Roundup Ready soy), Bt insecticide Cry1Ab and PAT proteins


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tube. The particles with bound reactants are separated from unbound reactants in solution by a magnet. Advantages of this format are superior kinetics because the particles are free to move in reaction solution and increased precision owing to uniformity of the particles (128).

Filter cover

Au

E. OTHER DETECTION METHODS

Result protein

Membrane

1. Chromatography

Immobile antibody

Reservoir Au Test

Antibody coated on gold

FIGURE 129.7 Schematic representation of the strip test.

(132, 133, 130, 134). Several commercial immunoassay methods are currently available for detection and quantitation of biotech crops expressing Cry1Ab, Cry1Ac, Cry3A, Cry2A, Cry9C, CP4 EPSPS and PAT proteins. 3. Lateral Flow Strip A variation on ELISA uses strips instead of microtiter wells. A typical strip consists of a reservoir, a result window and a filter cover (Figure 129.7). The reservoir contains (gold) coated antibodies against the target protein. Once the strip has been put into the test solution and this solution reaches the reservoir, the labeled antibodies bind the target proteins. When this complex reaches the area of the second (immobilised) antibodies, a sandwich complex is formed and a colour reaction is observed on the strip, while antibodies are immobilised on the strip. As a positive control, a second band (control line) must be visualised. The lateral flow strip gives results in 5 to 10 min, is economical, consumer friendly and suitable as an initial screening method early in the food chain (125). 4. Other Immunoassays In addition to microplate ELISA and lateral flow devices, other immunoassay formats are being developed, i.e., in combination with instrumental techniques. For example, in addition to the hyphenated methods, such as immunoassay–mass spectrometry, considerable advances in relative observation of antibody binding to target molecules using biosensors have been reported. Furthermore, immunoassays can be performed with magnetic particles as the solid support surface. The magnetic particles can be coated with the capture antibody and the reaction carried out in a test

Where the compositon of GMO ingredients, e.g. fatty acids or triglycerides is (significantly) altered, conventional chemical methods based on chromatography can be applied for detection of differences in the chemical profile. This has been demonstrated with oils derived from GM canola for which high performance liquid chromatography (HPLC) coupled with atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) has been applied to investigate the triglyceride patterns (135). 2. NIR Spectroscopy Recently, NIR has been used in attempts to distinguish Roundup Ready soy from conventional soybean (136). In this study, spectral scans were taken from three spectrometers of whole grains. Results varied slightly, but were promising in alle cases. However, the capability of NIR to resolve small quantities of GMO varieties in non-GMO products is assumed to be low, as is true for the chromatographic methods. 3. Microarray With the microarray or DNA chip technology microscopic arrays of single stranded DNA of the specific transgene of interest are spotted on a solid support (probe DNA). DNA isolated from the sample of interest (target DNA) is amplified using multiplex PCR. Addition of an exonuclease transforms the double stranded PCR products into single stranded DNA, which is able to hybridise with the spotted ss-DNA. After incubation, fluorescent signals are observed where a positive reaction occured. Analysis of the resulting pattern of spots with a significant degree of hybridisation, and therefore with a significant fluorescent signal, reveals the presence and, depending on the spotted sequence, the identity of GM varieties present in the sample (137). The method allows fast and simultaneous analysis of several thousand nucleic acids within the very small area of the chip. Therefore, it is very cost saving while maintaining high precision and reproducibility. 4. Nucleic Acid Lateral Flow Immunoassay (Nalfia) With the Nalfia technique, DNA is amplified through PCR, using primer pairs with different labels. After PCR,

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several microliters of the PCR product is pipetted onto a filter (strip), containing an enzyme which interacts with one label of the amplified DNA (originating from one of the introduced labelled primers). The sample then migrates through a nitrocellulose membrane containing another protein, which is able to bind the second labelled primer. If the sample under investigation contains the target gene, the PCR products will contain both labelled primers, which will be sandwiched by the two enzymes from the strip and a colour signal is observed. For the screening of GMOs a prototype of this test has been developed for Roundup Ready soy (138).

VII. THE EFFECT OF FOOD PROCESSING ON THE DETECTION OF GMOs BY PCR DNA is the preferred analyte for almost any kind of sample (raw materials, ingredients, processed foods) due to the fact that DNA is a rather stable molecule and the most common DNA based detection method, namely the polymerase chain reaction (PCR), is highly sensitive. The efficiency of the PCR reactions depends on the quality, quantity and purity of the extracted DNA. These factors vary according to the material, the degree of processing of the sample and the DNA extraction method. The DNA quality is determined by its fragment length and its degree of damage due to processing. The quantity of the DNA is determined by the food products itself, the degree of processing and the extraction method applied. The purity of the DNA usually depends on the method of extraction. The detectability of the DNA fragment is also dependent on the PCR approach (nested PCR, real-time PCR, multiplex PCR) and the choice of the primers. For routine PCR diagnostics in processed foods, amplicon length should be situated below 300 basepairs (30). Damage within the DNA fragments is believed to be caused by the exposure to heat, enzymatic degradation by nucleases, temperature, ionic strength, chemical agents and pH values (139–141). The mechanism of DNA destruction by heat is based on depurination or deamination. At temperatures above 100°C a significant strand scission and irreversible loss of secondary structure occurs (142, 143). The influence of pH may be limited due to cell wall structures protecting the DNA from cleavage. Detectability of DNA template after prolonged incubation at low pH suggests that after initial cell lysis and preliminary DNA destruction, the enzymes responsible for DNA degradation (endogenous nucleases) are destroyed quicker than DNA itself and its further breakdown is avoided (143). In food the rates of these DNA degradation reactions are strongly affected by matrix properties as well as the processing and storage conditions.

Basically no difference between the stability of DNA of a wild type and recombinant organism can be identified. Modifications resulting from methylation or association with DNA binding compounds (e.g., histones, polyamins) might cause minor effects on DNA stability, but these factors apply to any DNA. These conclusions are supported by studies of DNA stability in food such as dairy products, maize polenta, fermented sausages, tofu from soybeans or breads (144). It is clear that the purity of DNA can be affected by various contaminants in food matrices. These contaminants may originate from the material under examination, e.g., polysaccharides, lipids polyphenols (97, 145, 146) or chemicals used during the DNA extraction procedure, such as CTAB (88, 147). Furthermore, nitrite salts used in sausages (78) and dairy products (148) have been shown to be potent inhibitors of the PCR. A long list of salts, carbohydrates and other compounds frequently used in buffer solutions also decrease the performance of PCR (149, 150). The choice and optimisation of DNA extraction procedures, which eliminate potential inhibitory components may thus be of crucial importance for the success of a given PCR method (151). Different succesfull DNA extractions have already been published for several GM foodstuffs (30, 85, 88, 92, 97, 141, 146, 152–161). Although the basic reactions contributing to DNA degradation are already known and many methods have been developed for the detection of DNA sequences of GMOs in foods according to the current legislations, only limited data are available about the release of DNA from cells, as well as its presence and stability during processing and storage of foods. Bauer et al. (144) investigated the kinetics of degradation of plasmid DNA by the process parameters acidic pH and/or temperature using a tomato serum (pH 4.3 and temperature of 65°C). The highest degradation was found for the combined effect of acidic conditions and heating. Thermal treatment of corn meal at 100°C and potatoes at 80°C contributed to degradation of DNA to fragments smaller than 585 bp and 792 bp respectively (162). A similarly effective thermal DNA degradation was also described for dry corn grains by Chiter et al. (163) and for corn gluten and flaked corn by Forbes et al. (164). Specific attention has been paid to the degradation of DNA during alkaline boiling (pH 11.0) of corn meal. Alkaline-cooked corn, called nixtamal of corn masa, is an instant product for the production of Mexican corn-based foods such as tortillas, corn chips, taco shells and tamalas. Kharazmi et al. (162) reported the failure of amplification of fragments greater than 585 bp while Hupfer et al. (140) still detected 1914 bp DNA fragments of corn after boiling at pH 9.0 for 60 min. In a real-time quantitative PCR approach, Quirasco et al. (165) were able to detect and


quantify up to 0.1% StarLink corn, in spite of progressive degradation of genomic DNA during processing. In soybean flour, a rapid decrease in the maximal detectable length of plant DNA under acidic conditions (pH 4.75) was found, even at ambient temperature (144). This is in agreement with the results found by Hupfer et al. (140) which illustrated that DNA fragments of 1914 bp were no longer detectable after boiling of Bt-176 maize flour for 5 min at pH 2.0 to 3.0, whereas after boiling for 60 min at pH 8.5 to 9.5 the fragments remained detectable. It was shown for the production of feed that DNA was degraded during ensilaging of Bt-176 maize (pH 3.9–4.1) and fragments of 1,914 bp were no longer detectable after 106 days. For production of soy milk and tofu, DNA degradation during heat treatment does not significantly contribute to the DNA degradation, the mechanical step of grinding of soaked soybeans is a more crucial DNA degrading step (162). A similar extent of degradation was also observed by Hupfer et al. (140) during chopping of whole corn plants. It can be assumed that during grinding or chopping DNA is released and becomes sensitive to the attack of nucleases. Such nucleolytic activities were shown to occur in food matrices, e.g., in bread dough (166) or sugar beet raw juice (167) and it was observed that their action depends on the processing temperature. Klein et al. (167) showed that DNA is completely removed during the production of sugar and the overall efficacy of DNA elimination was calculated to 1014. In addition, the degradation of the plant DNA during production of bread was monitored indicating that temperature and pH are the major effective factors (141, 166). Still, for some refined and highly processed food products, the detection of DNA remains difficult, resulting in the impossibility to perform a GMO analysis. This category of products contains among others: starch, sugar cane, caramel, dextrose, sorbitol (155, 158), bread with soy sauce (158), refined soybean oil (159, 168), refined corn oil (158), tomato concentrate, tomato puree and tomato ketchup (158), cocoa drinks containing lecithin (97). For these food products, GMO analysis is impossible, or a higher sample volume should be required (160, 161, 169).

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Part O Packaging

130

Food Packaging: New Technology

Vanee Komolprasert

Division of Food Processing and Packaging, U.S. Food and Drug Administration

CONTENTS I. II. III. IV. V. VI. VII.

Introduction ....................................................................................................................................................130-1 Evolution of Packaging ..................................................................................................................................130-1 Food Packaging ..............................................................................................................................................130-2 Recent Developments of Barrier Packaging Systems ....................................................................................130-2 Barrier Materials ............................................................................................................................................130-3 Barrier PET Containers ..................................................................................................................................130-5 Multi-Layer Structures ....................................................................................................................................130-6 A. Sealica ....................................................................................................................................................130-6 B. Nylon-Based Nanocomposites ................................................................................................................130-6 C. Passive-Active Barrier Systems ..............................................................................................................130-7 1. SurShield Barrier ..............................................................................................................................130-7 2. Oxbar Barrier ..................................................................................................................................130-7 VIII. Surface Coating Technologies ........................................................................................................................130-7 A. Interior Coating of PET Bottles ..............................................................................................................130-8 1. Actis (Amorphous Carbon Treatment on Internal Surface) ............................................................130-8 2. Plasma Nano Shield (PNS) ..............................................................................................................130-8 3. Glaskin ............................................................................................................................................130-8 4. Plasmax ............................................................................................................................................130-8 B. External Coating of PET Bottles ............................................................................................................130-8 1. Bairocade ..........................................................................................................................................130-8 2. BestPET ............................................................................................................................................130-8 3. Combustion Chemical Vapor Deposition (CCVD) ..........................................................................130-9 IX. Outlook for Food Packaging ..........................................................................................................................130-9 References ................................................................................................................................................................130-10

I. INTRODUCTION Food packaging is constantly changing to meet new challenges in the market and new needs of the consumer. New technologies continue to emerge with innovations in new packaging materials and packaging techniques that offer new possibilities for manufacturing, packaging, and marketing a wide variety of foods. Today’s food package provides not only basic functions (contains and protects) but also offers convenience, facilitates product use, and communicates with intended buyers of the product. The package label informs the consumer of nutrition facts, sells the product through colorful graphics, and addresses environmental concerns such as source-reduction and recycling.

Among the innovations and recent developments, major efforts have focused on new barrier materials and technologies for flexible and rigid food containers. Better barrier technology is desirable to enhance quality and safety of food as well as to extend its shelf life. Barrier packaging technology is the main focus in this chapter.

II. EVOLUTION OF PACKAGING Packaging roles have continuously evolved with social and community development (1). Initially bulk packaging was used to deliver goods to retailers where it was received and bagged for merchants. As food processing 130-1

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technology advanced individual container for consumer products such as canned foods were widely used. By the middle of the 20th century, most consumer goods were individually packed and branded. The small community store that originally sold products manufactured locally evolved into a modern supermarket equipped with refrigeration and accommodated more products. As the number of products increased to hundreds or thousands, the storekeeper could not aid and influence the consumer’s purchase. The consumer was left face-to-face with the package. The package had to sell the product as well as to inform the purchaser, calling for the package’s motivational and informational roles. To attract the purchaser, package design and graphics needed to be attractive, thus creating a new profession, package design, as well as prompting an evolution in printing and decorating arts. The post WW II baby boom affected population structure and trends, and subsequently food product and package design. The 1950s marked the emergence of fast food outlets that created a demand for new kinds of packaging, including disposable single-serve packaging and bulk packaging for ready-to-cook food portions. Fast food outlets boomed along with a growing trend toward eating out. This led to formation of the hotel, restaurant, and institutional sector (HRI). The 1960s marked the growth of convenience and prepared food packages, as thermoplastics became available as packaging materials. As use of these plastics increased, the consumer’s concern toward environmental problems increased. The 1970s and early 1980s brought changes such as labeling laws, ozone-depleting chlorofluorocarbons (CFCs), standards for the acceptance of new packaging materials, and microwave ovens and microwaveable packaging. Changing demographics of the 1990s were reflected in changes to product packaging. The nutrition labeling law required the package label to include nutrition facts. The aging population became heath conscious and desired food and food products that promote health and living wellness. Families became smaller and the single-person household became common. Married couples have professional careers and higher income levels, calling for “convenient” food products. The food package is required to be environmentally friendly, tamper-proof, and convey detailed information on the nutritional values and ingredients of the food product. Currently, there is a proposed country-of-origin labeling law. Once the law is passed, it will be effective in September 2004 (2) and will apply to packaging of meat, seafood and produce, both fresh and frozen. Food packages will continue to evolve in the 21st century.

III. FOOD PACKAGING Food packaging is the process of wrapping food with a suitable package. The package may be made of one or more materials that provide proper functionalities and

properties for holding and protecting the food from the point of production to the consumer, while the quality and safety of the food are maintained. Holding and protecting the food are two major functions for packaging of food. Food is generally sensitive and susceptible to environmental abuse, and deteriorates by chemical, biochemical, and/or microbiological changes that are usually accelerated by environmental factors such as oxygen, water, light, and temperature. With a suitable package, these changes can be prevented or delayed. A suitable package can also prevent contamination by foodborne pathogens, which render the food hazardous and unwholesome for consumption. Food packaging provides wholesome, high-quality, and nutritious food products. Packaging technology is dynamic as a result of the new challenges and new technologies developed to accommodate new needs in the changing society. Discovery of new packaging materials, new processes, and new techniques have shaped the way we package, deliver, and consume products. The thermoplastics developed in the 1950s have revolutionized the packaging industry. Although many foods are still packaged in traditional glass bottles and tin cans, newer plastic, multilayer, and composite packaging materials are creating opportunities for improved product convenience, presentation, quality, and safety, as well as innovative food product development. An ideal food package is designed to meet many requirements of the food itself, the processing or preservation methods, distributor and retailer needs, and consumer expectations and acceptance. Modern packaging is driven toward more intensive marketing and globalization. Packaging plays a significant role in motivating purchase. Among competing products that are similar in performance and quality, their packages are different. Package design is then critical in competition, and a new package often helps create uniqueness for its brand. An increase in market globalization requires a product with an extended shelf life. Better barrier packaging materials and techniques are needed to achieve a desirable shelf life for export. There are two factors influencing the development of food packaging with increased barrier properties. First are the regulatory initiatives that are intended to limit waste generation. Source reduction via downgauging of flexible packaging is one way of reducing plastics’ content in solid waste. Second is the improved food preservation that barrier flexible packaging can offer in preventing food losses and spoilage during storage.

IV. RECENT DEVELOPMENTS OF BARRIER PACKAGING SYSTEMS A barrier can be defined in many ways depending on the desired level of protection from physical damage and chemical and biological changes that affect food quality and safety. Since most food packages are plastics, a barrier


is conceived to be for control of permeation of gases and vapor through the package. Barrier technology has been designed and developed for both flexible and rigid food containers. A desired barrier level can be achieved by using one or more barrier materials for food packages, or by incorporating this barrier material using multi-layer structure, lamination, or coating techniques. Years of research and development have resulted in new barrier technologies for various foods and food products.

V. BARRIER MATERIALS Although polyethylene terephthalate (PET), high-density polyethylene (HDPE), low-density polyethylene (LDPE), polypropylene, (PP), polyvinyl chloride (PVC), and polystyrene (PS) are widely used plastics for food packaging, they provide inadequate barrier properties. Newer barrier materials were desirable. Several barrier materials of current industry interest include polyvinylidene chloride (PVDC), ethylene vinyl alcohol copolymer (EVOH), nylon (PA), modified nylon (MXD6, Selar PA), liquid crystal polymer (LCP), polyethylene naphthalate (PEN), adhesive barrier materials, nanocomposites, and oxygen scavengers. These materials are high priced, so they are used in as small amounts as possible to give the desired barrier properties. Barrier material can be incorporated into a lower cost material by using lamination for a multilayer structure or coating onto a monolayer material. PVDC is a favored choice for an improved barrier of a food package. It was usually used in multi-layer films and containers before the arrival of EVOH in the 1970s. PVDC and EVOH could be used in co-extrusion of 5-, 7-, and 9- layer cast barrier sheet structures for shelf-stable and retortable food packaging. The basic 7-layer barrier structure is typically a symmetrical arrangement of polyolefin/ regrind/tie layer/EVOH, PVDC, or PA/regrind/tie layer/ polyolefin. EVOH was used for a ketchup bottle that was developed in 1984, and the first multi-layer packaging for retortable packages and microwaveable soup bowls was commercialized in 1987. Food packaged in shelf-stable, retortable containers is at least one-third more expensive than similar products in metal cans, mostly due to the price of packaging. Despite high packaging prices, the market for the multi-layer cast barrier sheet for shelf-stable and retortable packaging has continued to grow. The growth is especially high for specialty foods, where the package cost is outweighed by the features and benefits that the products provide and the need they meet (3). For example, single-service containers of applesauce offer healthier alternatives to most snack foods consumed at school or work, and microwaveable baby foods offer nutrition and convenience in pre-measured, hot meals. Emerging end uses for the multi-layer barrier containers include aseptic packaging for low acid food with particulates, and modified atmosphere packaging

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(MAP). MAP of meats is going into supermarket chains. Typical MAP is a foamed PS/ tie layer/EVOH/PE or EVA structure with one regrind layer. Shipping meat from fabricating plants in MAP containers could eliminate the need for butcher shops in supermarkets. The expense of packaging, labor, and freight of MAP could be a slightly lower-cost alternative to the in-store butchers. In the early 1990s, flexible barrier packaging shifted from metalized films, laminates, and rigid containers to films incorporating barrier resins, especially EVOH, along with films produced by new coating techniques that deposit oxides of silicon or metal to obtain clarity (4). EVOH use has grown rapidly as a substitute for PVDC coated films and metalized films that are considered less recyclable. Since its introduction, EVOH grades have been developed to overcome delamination problems when used in multilayer structures. Eval Co. of America (Evalca) developed a third-generation delamination-resistant EVOH barrier resin, Eval grade XEP-567, which has better adhesion to PET without a tie layer than the previous grades, MDX6, and other nylons. The XEP-567 grade offers about 50% lower O2 permeability and about 40% lower CO2 permeation than Eval XEP-562, a second generation delamination-resistant resin introduced in 1999 (5), and offers carbonated soft drinks a shelf life of 16 weeks. MXD6 is a modified nylon developed by Mitsubishi Gas Chemical in 1986. Selar PA is modified nylon developed by DuPont. Both are often used in multi-layer structure food packages. MXD6 nylon can provide ca. 19–20 times greater barrier capacity than PET. Since it has similar processing temperature to PET, it can be blended with PET but has a drawback of high haze (6). MXD6 and liquid crystal polymer (LCP) blend is then preferred for use in multi-layer PET bottles. Liquid crystal polymer (LCP) is a barrier material superior to PVDC and EVOH. It functions at one-fifth the thickness of EVOH, resulting in overall material saving (7). Superex Polymers Inc. developed counter-rotating die technology for biaxially oriented and extruded LCP (8). Packaging applications of LCP include beer bottles and blown films. It has more than 200 times greater O2 barrier than PET and is not moisture sensitive. A monolayer PET bottle is unsuitable for beer because it is neither an efficient oxygen nor flavor barrier, so product shelf life and taste stability are problems. A thin layer of LCP on the PET eliminates both problems. Since LCP has a high price, its use in packaging is minimal and depends on desired product shelf life. A three-layer structure (PET/tie layer/LCP) less than 0.5 mm thick and containing LCP less than 5% of the bottle weight could provide beer with a one-year shelf life. Less LCP could be used if a 6-month shelf life is acceptable, and thus reduce bottle cost. Biaxially oriented LCP film could be laminated or coextruded to produce a lower cost material for barrier packaging.

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PEN, a new polyester that has been blended with PET, improves the CO2 and O2 barrier properties of the monolayer PET bottle used for beer. As PEN has a glass transition temperature (Tg) of 122°C, far higher than for PET, PEN blends allow the monolayer to sustain pasteurization temperatures for beer. However, the high price of PEN and PET/PEN blends is a major obstacle preventing its widespread use for single-use containers such as beer bottles. Nevertheless, PEN is cost-competitive with refillable glass bottles and less costly than one-way PET bottles when PEN bottles are refilled at least three to five times (9). Monolayer PEN bottles are used for marketing of multi-trip refillable beer and water bottles in countries where refillable bottles are accepted, i.e., European countries, Latin America, South Africa, and China. Blox is the trade name of a new series of clear, tough, highly adhesive barrier polymers from Dow Plastics. They are poly-amino ethers made by polymerizing liquid epoxy resins with an amine to create solid, low melting thermoplastics (10). Blox resins can be extruded into films, injection molded, or blow molded. One series of Blox resins can be co-extruded as a barrier layer of multi-layer bottles for juices, beer, or carbonated soft drinks. Their O2 and CO2 barriers are about ten-fold higher than that of PEN and in the range of EVOH or MXD6 nylon, depending on humidity level. The clarity and toughness of Blox materials are higher than those of EVOH and MXD6. Blox resins have better adhesion to PET, permitting design of more complex bottle shapes without the risk of delamination, and no tie layer is required. Nanocomposites are materials where nanometer particles are dispersed in a polymeric matrix, which can be single or multiple phase. A nanoscale particle is a particle with at least one dimension in the nanometer range. Natural and synthetic clays are mainly used as nanoparticles in plastic composites (11). However, mixing plastics and clays is not a simple process because the materials are immiscible and tend to form a very light packing of individual clay layers. As a result, clays must be treated with an organic intercalant to improve interactions between clay platelets as well as dispersion of organoclays. An intercalant is an oligomer or polymer that is sorbed between platelets of the layered material and complexes with the platelet surface to form an intercalate. The original concept for these plastic composites began with the invention of polyamide-clay composites by the Toyota Research Corporation in 1985; these were used to make under the hood heat resistant automotive parts that were lighter than metal. The technology has advanced and can be applied to various plastics including thermoplastic olefin (TPO), thermoplastic elastomer (TPE), PP, PET, and nylon. The nanocomposites offer improved mechanical, electrical, gas, and liquid barrier properties. Nanocomposite technology has been migrating from automotive parts to other applications including rigid and flexible packaging. Nanocomposite plastics are usually enhanced by fillers

derived from the industrial clay called bentonite (12). The fillers form flat platelets that disperse into a matrix of layers, which force gases to follow a tortuous path through the polymer. By increasing the path of diffusion of gases and other molecules, the clay platelets slow gas transmission and increase the barrier properties of the plastics. Nylon is a preferred nanocomposite additive for making a barrier layer in multilayer PET containers, which are increasingly used to package oxygen sensitive foods and beverages. Nylon has better inherent adhesion than EVOH (an alternative barrier resin), thus sealants are not needed between the nylon and PET layers. With nanoclay additives, the barrier properties of nylon can be doubled or tripled, making it an alternative barrier material to the superior barrier EVOH resins. However, getting nanoparticles to disperse properly to make the tortuous path principle work is not easy. The most promising way is to introduce the clay additive during polymerization, such as with the Aegis’s nanocomposite nylon. Eastman Chemical Co., in cooperation with nanoclay producer Nanocor, has developed a nylon composite barrier material, Imperm (5). Imperm was designed for use in multi-layer bottles, providing 50–100 times greater O2 barrier than PET, compared to 10–20 times barrier improvement of MXD6 nylon over PET. A 20-g three-layer PET/Imperm/PET bottle with 4% Imperm in the bottle wall has a 3–5 times greater O2 barrier than PET and less than 8% haze. A bottle with 10% Imperm has a 6–11 times greater O2 barrier and less than 10% haze. Several oxygen scavenger systems have been developed using either oxidizable metal such as iron, various oxidation promoters, and fillers or metal free absorbent systems such as mixtures of organic compounds including quinines, glycol, and phenolics (13). In the early 1990s, Toyo Seikan introduced a non-conventional technology, Oxyguard (iron salt-based), for incorporating a high oxygen barrier into blow molded food containers and other rigid packaging (14). Oxyguard is an alternative to the EVOH or PVDC coextrusion of a multi-layer structure, where barrier properties decrease under elevated relative humidity and temperature such as during retorting, and they cannot remove oxygen in the headspace of the bottle between the contents and closure. In contrast, Oxyguard’s barrier is claimed to be capable of trapping and holding oxygen coming from the headspace instead of simply blocking it. Amosorb oxygen scavengers developed by Amoco are available in pellet concentrates of PP, PE, PET, and elastomer resins. The oxygen scavenger resins are designed for either retort and hot-fill food applications or non-retort and refrigerated food and beverage packaging, and can be employed in a wide range of packaging structures including rigid containers, films, and closure liners. They are claimed to be heat stable to 320°C and activated by moisture. Amosorb 3000 copolyester was developed for beer, tomato products, fruit juices, and teas. It is an iron-based system that bonds permanently with oxygen that permeates


the package wall, is present in the internal headspace, or is dissolved in the packaged contents. It is transparent and compatible with other polyesters. A multi-layer package containing one or more core layers of Amosorb 3000 is claimed to offer better protection than glass or metal for even the most oxygen-sensitive products. Hybrid BA-030 copolyester from Mitsui Chemicals was developed for use in beer bottles as part of a 5-layer structure (PET outer layer/BA-030/O2 scavenger/UV barrier/PET inner layer). The grade is claimed to reduce acetaldehyde levels to as low as 3 ppm, and match or exceed PEN properties when combined with PET (15). AmberGuard polymer from Eastman Chemical was developed for UV light protection. It can be used in a multilayer container with Eastman’s Imperm nanocomposite polymer to provide UV protection and O2 and CO2 barriers. Oxygen absorbing organic ingredients can be copolymerized with monomers of existing packaging polymers to create inherently absorbent structures. Carnaud Metal Box and Crown Cork & Seal developed Oxbar, which is a non-iron-based system that uses nylon and a cobalt salt formulation incorporated into a polyester base, as a chemical trap (14). It is used in three-layer PET bottles for short term applications e.g., single-serve fruit juice and beer as well as wide mouth containers for tomato-based sauces and condiments (16). Southcorp technology developed ZERO2, which is a non-metallic oxygen scavenger system that is activated by UV light after it is incorporated into packaging structures of materials such as PE, PP, PET, and ethylene vinyl acetate (EVA) (13). It is used in conjunction with vacuum packing and barrier films.

VI. BARRIER PET CONTAINERS Polyethylene terephthalate (PET) is one of the most widely used polymers. It was the first polymer to successfully recycle, generating reclaimed materials for a wide range of non-food and food applications. The recyclability of PET material is a factor promoting its use beyond the carbonated soft drink market to include other foods and beverages. It is replacing glass bottles and some bottles made from other plastics such as HDPE, PP, and PS. PET’s success is a result of its better barrier and clarity than the plastics being replaced, as well as technological development of the processes used to convert PET into flexible and rigid packages at high outputs, which is crucial for the minimization of packaging costs. Use of PET resins continues to rise because of new applications, as well as the innovation of barrier technologies that help enhance the barrier properties of PET, and thus making it suitable for other demanding applications including use with oxygen sensitive foods. PET is now a commodity polymer competing directly with polyolefins and styrenics in the markets for food and beverage packaging, as well as for other products.

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Monolayer PET packages are generally suitable for many food applications but are not suitable for beverages and food products that require better protection or gas barriers. The ideal approach to improve the gas barrier of PET is to design a monolayer PET structure that will provide package design freedom. This approach requires blending a barrier resin, oxygen scavenger, or both with the PET. The monolayer solutions are less practical mainly because suitable materials are high-priced. However, this approach has been the subject of a recent development by Interbrew in cooperation with M&G Group. Interbrew has launched a single-layer PET barrier bottle, Pivopack, for a Russian beer, Klinskoye brand. Pivopack is claimed to be the first monolayer, barrier-enhanced PET bottle, which uses M&G’s new Acti TUF, a PET resin made by a proprietary oxygen-scavenging technology that is triggered to react with oxygen only when a container is filled with beverage (17). The monolayer PET provides the advantages of allowing the preforms to be manufactured on standard machines. The pricing of these resins is in a range of Eur330–650/ton above that of standard PET (18). Two other approaches for improving gas barriers are multi-layer structures and surface coatings. Advancements in multi-layer and surface coating technologies are making PET bottles cost competitive with glass bottles and metal cans for beer, carbonated soft drinks (CSDs), oxygen sensitive juices and hot-filled foods. PET bottles for beer were developed in 1999 with at least nine plastic beer bottle programs underway (19). Bottling beer in plastic is difficult due to beer’s extreme sensitivity to light and oxygen. Converting beer bottling from glass to PET requires a barrier against carbon dioxide egress and oxygen ingress, while retaining clarity and strength. Beer in bottles requires 120 days of shelf life with less than 15% loss of CO2 and no more than 1 ppm gain of O2 (20). A major obstacle is a unit cost that is much higher for barrier PET bottles compared to glass. Shifting to barrier PET bottles requires breweries and blow molders to invest heavily in new development. It is not feasible for brewers to drop PET beer bottles into glass bottle lines capable of a high-speed production at a rate of tens of thousand bottles per hour. Another obstacle is that the PET beer bottles fail when they are exposed to thermal stress and pressure at temperatures beyond 62°C, typically used for tunnel pasteurization by 80% of the world’s beer filling operations. One solution is heat setting the PET to increase the crystallinity of the material during blow molding. The process produces heavier preforms and slows the process, but the resultant bottle has a thicker wall that can withstand pasteurization at 65°C. Besides a higher cost than glass and cans, one large hurdle to the growth of PET for beer is consumers’ perception that beer tastes better in glass (21). As PET beer bottles with multi-layers may disrupt the existing monolayer PET bottle recycling stream because of the layers of nylon and PET, the amber color, aluminum cap, and metalized label, some plastic

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beer bottle programs have been discontinued. Although recyclability is technically not a problem because studies demonstrated the multi-layer PET beer bottles could be recycled and reused, most recyclers still cannot economically separate the materials (22). Due to these complexities, use of both multi-layer and coated PET beer bottles is currently restricted to low-volume breweries, and to concerts and sporting events where there are public safety issues. The barrier PET beer bottles are not as successful as anticipated, but blow molders remain confident that the PET beer bottle will re-emerge in the future. Blow molders are shifting their efforts toward designing barrier PET bottles for less demanding applications in juices, carbonated soft drinks, and hot-filled products.

VII. MULTI-LAYER STRUCTURES Multi-layer food packaging structures have been used for many decades. Ethylene vinyl alcohol (EVOH) co-extruded with polypropylene (PP) was commonly used in the 1970s. As demand for bottle transparency increased, co-injection blow molding of polyester (PET) with EVOH was developed during the 1990s. However, clarity improvement of multi-layer PP containers is still emerging. Pechiney Plastic Packaging, Inc. (PPPI) has developed a family of PP barrier containers that are claimed to be as clear as multi-layer PET bottles, and to be a cost-effective alternative to multi-layer PET bottles. The three-layer (PP/EVOH/PP) barrier containers are made using modified reheat stretch-blow mold machines (23). Today, PET and PP are competing to determine which of these two base polymers, with other barriers, will dominate the barrier plastic bottle market (24). Multi-layer structures are far more prevalent than coatings and account for about 70% of barrier PET bottles (25). The technology has succeeded for a decade in the PET ketchup bottle. It is claimed that the multi-layer PET food bottle is the optimal solution in barrier performance, functionality, and cost. Higher productivity currently favors multi-layer preform co-injection systems over coatings. Multi-layer containers can be engineered to survive pasteurization successfully by new bottle design features, processing techniques, and materials modifications. In a multi-layer structure, a core layer or layers containing higher priced barrier materials are sandwiched by PET structural layers. There are several five-layer PET bottles designed for beverages. A five-layer PET bottle is used for Reallife® line of new-age beverages, non-carbonated flavors. The bottle incorporates virgin PET, EVOH for barrier properties, and post-consumer recycled (PCR) resin within the five-layer structure (virgin PET/EVOH/PCR-PET/ EVOH/virgin PET); no adhesives or tie layers are used. Two thin layers of EVOH provide the necessary barrier (26). Instead of EVOH, MXD6 is used in a five-layer structure (virgin PET/MXD6/virgin or PCR-PET/MXD6/virgin

PET) for Coca-Cola bottles (27). Continental PET Technologies supplies a five-layer structure (PET/O2 scanvenger/PET/O2 scavenger/PET) single-serve PET bottle for Miller beer (28). Krones has developed a five-layer structure (PET/nylon 6/PET/nylon 6/PET) PET beer bottle for a Swiss brewery, while Bass developed a multi-layer (PET/ EVOH/PET) PET bottle for Carlsberg Black Label beer.

A. SEALICA Tetra Pak’s patented two-stage process, Sealica, was developed for molding a multi-layer PET preform using a new thermoplastic epoxy barrier resin called Blox from Dow Plastics (20). Blox is a resin made by the reaction of resorcinol diglycidyl ether (RDGE), a resorcinol derivative used extensively in high performance composites, with monoethanolamine to yield extreme barrier performance (29). The preforms are injection molded using less PET, only 60–70% as thick as normal, and subsequently they are overmolded using a thermoplastic barrier material that fills the remaining 30–40% of the preform cavity. The other half of the mold and its cavities are then injected with PET to complete the molding process. The thickness of the barrier can be adjusted to suit the application. The multi-layer approach is using promising new barrier materials such as nylon-based nanocomposites and passive-active barrier systems. The latter are dual use of a passive barrier material and an active oxygen scavenger that blocks oxygen entry and absorbs this gas from headspace and content.

B. NYLON-BASED NANOCOMPOSITES Nanocor in alliance with Mitsubishi Gas Chemical has melt-compounded its own nanoclay additives with MXD6 nylon for making nanocomposite (M9) for use in barrier PET bottles and films. M9 nanocomposite is claimed to improve the CO2 and O2 barrier of standard MXD6 by 50% and 75%, respectively, while retaining high clarity and delamination resistance equal to standard MXD6 (25). A three-layer (PET/M9/PET) structure extends the shelf life of beer to 110 days in the US and 180 days in Europe. The structure uses a thinner M9 layer than if plain MDX6 were used; this provides a cost saving for a processor even though M9 costs more than MXD6. Honeywell developed several nylon 6-based nanocomposites, Aegis products, to cover the full spectrum of high barrier food bottles and film applications. Aegis products are reactor-made or melt-compounded blends of nylon 6 with low levels (2%) of nanoclay platelets (25). The platelets act as a tortuous path barrier to CO2 and O2 gases. To improve the O2 barrier to the level of glass, Honeywell uses a proprietary oxygen scavenger that involves a polydiene entity dispersed in the nylon 6 without impairing the matrix properties. Aegis OX is a nanocomposite grade that


contains the O2 scavenger, offering sufficient passive and active CO2 and O2 barriers to protect beer. Aegis CSD and HFX are grades optimized for carbonated soft drinks and hot-filled foods, respectively. Aegis CSD extends the shelf life of carbonated soft drinks in 0.5 L containers from 9 to 14–16 weeks. Aegis HFX is a passive-active system that provides a greater O2 resistance for foods. Both grades also have improved delamination resistance.

C. PASSIVE-ACTIVE BARRIER SYSTEMS Use of an oxygen scavenger in multilayer packaging is emerging. 1. SurShield Barrier Owen Illinois has developed a patented process, SurShot, for co-injection molding a patented five-layer plastic bottle for beer (30). The outer, middle, and inner layers of the bottles are made of virgin PET. Sandwiched between them are two layers of proprietary passive-active barrier material, SurShield. This barrier system includes MXD6 nylon and an oxygen scavenger in two super-thin layers sandwiched between outer PET layers and a core layer incorporating up to 35% recyclate (25). This structure is claimed to improve the CO2 barrier up by 40%. The bottles are designed for food applications from ketchup to beer. 2. Oxbar Barrier Developed by Constar International, this uses a threelayer structure with a barrier layer of MDX6 nylon and O2 scavenger (25). This passive-active barrier system is aimed at juices, flavored alcoholic beverages, hot-filled foods, and beer. The Constar bottles are claimed to survive tunnel pasteurization by using extended necks that expand to relieve pressure and base designs that retain shape and strength.

VIII. SURFACE COATING TECHNOLOGIES Coating basic plastic structures is not new, but the materials and application methods used to achieve desired properties are. Plastic bottle coating technology is evolving. PVDC coating of polyester bottles exists commercially but its use has been slow because there are too many economic issues and environmental concerns, especially in Europe. A surface coating can be applied to a PET bottle to improve gas barriers. The coating technologies apply a super thin barrier to one surface of a monolayer PET bottle. Coating systems use monolayer PET bottles for their economies, but require costly equipment for integration into complex, high speed filling-capping-labeling lines. Coatings are less prevalent than multi-layer structures because they offer an intermediate barrier and cannot give the barrier of a multi-layer (31). However, coated bottles have advantages.

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They are less expensive on a per bottle basis than the multilayer ones. Coatings can be applied by end users. Coated bottles that use only one resin are considered far easier to recycle than multi-layers ones. They are less likely to delaminate during handling, avoiding problems in material handling in retail venues like vending machines. Several barrier-coating technologies are available and differ by the type of coating materials, coating placement (interior or exterior), and application method. Methods for increasing barrier properties in packaging while reducing material use by applying a microscopic layer of silicon on film emerged in the 1990s (32). These methods were achieved by coating or by vacuum or plasma deposition on a substrate, and then sandwiching the silicon for protection in a laminated structure. The silicon layer acts almost as a glass-like barrier. Lawson Mardon Packaging Inc. claimed that silicon monoxide (SiOx)coated ceramic films yield substantial reductions in oxygen and water vapor permeation. Because the silicon coating is so thin (400–1000 angstroms), recyclability of the films is not an issue. When used in appropriate laminates, the SiOx barrier maintains a high degree of flex-crack resistance. Silicon coatings can be applied on PET films for retortable and nonretortable laminates, and on other plastics including PP, PS, and polyamide (PA). Applications for films include pouches for dry foods, and liquid and high-moisture content foods. Another process pioneered by AIRCO and PC Materials, Inc. uses a low temperature (40–50°C) plasmadeposition process that applies silica under a low or soft vacuum (750 milli-torr range). The system is a batch, air-vacuum-air process that produces a coating less than 40 nanometers thick. The low-vacuum nature of the process puts less thermal stress on substrates and reduces wear on the coating chamber. Another silicon-oxide QLF (quartzlike-film) barrier coating for films of PET, oriented PP, LDPE, or biaxially oriented nylon was developed by BOC Coating Technology (33). QLF coatings are applied in a low temperature, plasma-enhanced chemical vapor deposition (PECVD) process. The clear, colorless silica coatings are only 20–40 nanometers; it is claimed that they improve the O2 barrier by 120-fold and the moisture barrier by 45 fold of 12.5 micron PET films. These developments have led to the current technology called plasma-enhanced chemical vapor deposition (PECVD). PECVD is most commonly employed to apply silicon oxide barrier coatings on films, sheets, and bottles. The process applies a microscopically thin (40–60 nanometers thick) layer of silicon oxide on plastic surfaces. Under a low vacuum, a silicon containing chemical such as silane, i.e., hexamethyl disiloxane (HMDSO), is exposed to microwave or radiofrequency energy to convert it to a plasma (34). The silicon oxide bonds to the plastic and creates a coating that blocks the permeation of gases, water vapor and flavor. Plasma coating may be applied using carbon or silicon oxide on interior or exterior surfaces of packaging.

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A. INTERIOR COATING


OF

PET BOTTLES

1. Actis (Amorphous Carbon Treatment on Internal Surface) Sidel developed the Actis cold plasma technology that was originally aimed for beer containers. The process uses acetylene gas as a source of carbon coating. The gas is excited by a microwave-assisted process into plasma, which deposits a layer of hydrogenated amorphous carbon about 100 nanometers thick on the bottle’s interior. Actis is claimed to improve the CO2 barrier seven-fold and the O2 barrier thirty-fold. It reduces acetaldehyde migration to one-sixth of normal (19). The Actis system is a stand-alone rotary-style coating machine and offers an advantage over multi-layer structure machine. Actis Lite is a new Actis system designed for less demanding juice, CSD, and hotfilled applications. Actis-treated bottles have a light amber color, providing some UV protection for beer packaging (35). Actis coated bottles cost ca. 20% less than multilayer PET bottles with comparable barriers. Actis received a letter of non-objection from the FDA for beverage contact and commercial application for Mountain Dew’s Code Red product to prolong CO2 retention.

the interior of PET bottles. The process is based on the plasma impulse chemical vapor deposition (PICVD) technology of Scott HiCotec (36). The technology uses a pulsed, cold plasma process that deposits a thin-layer (0.01–1.0 microns thick) of silicon oxide on the interior of PET bottles. The process occurs in a vacuum chamber, where the bottles are held neck down. The bottles are filled with gaseous hexamethyl disiloxane, and are microwaved to decompose this gaseous precursor to form a deposit of SiOx on the bottle. Byproducts, CO2 and water, are removed by the vacuum system. The application process uses a rotary coater that enables the system to be integrated into end users’ filling line. A transparent adhesive layer is applied before the coating cycle, allowing for good bonding of the barrier layer to the interior wall of the bottle even in case of asymmetrical or other complex bottles shapes. The process creates a barrier to O2, CO2, moisture, and chemicals. The SiOx layer is claimed to improve the oxygen barrier more than ten-fold, and the CO2 barrier more than seven-fold.

B. EXTERNAL COATING

OF

PET BOTTLES

Instead of internal coating, an exterior coating can be applied to the PET bottles.

2. Plasma Nano Shield (PNS) PNS was formally called Diamond-Like-Coating (DLC), was developed by Kirin/Mitsubishi of Japan. The process uses a radiofrequency source plus internal and external electrodes to ionize the gas to produce a coating 20–40 nanometers thick on the internal surface of PET bottles (34). The coating offers an excellent gas and water vapor barrier. Coated bottles outperform PEN bottles in reduced color and flavor sorption and may be refillable (15).

1. Bairocade

Tetra Pak developed Glaskin, a silicon-dioxide plasmacoating system for the interior of PET bottles. The coating is created by reacting hexamethyl disiloxane with oxygen (34). Microwave energy is used to excite a gas, depositing a thin (10–20 nanometers), clear layer of silicon oxide, essentially glass, on the bottle’s interior wall. It is claimed to deliver CO2 and O2 barriers equal to glass. The coating is elastic and ensures barrier integrity with crack resistance despite expansion and contraction during the bottle filling operation. It is designed for beer, juice, carbonated soft drink, and hot-fill applications. It delivers 4–12 months of shelf life for beer and juice (29) with excellent flavor retention (28).

The Bairocade system was developed and is supplied by PPG Industries. It is an epoxy-amine coating applied by electrostatic spray, and cured in an infrared oven to thermoset the material on the exterior of the PET bottles. The cross-linked, 1–6 micron thick coating is glossy, offers excellent O2 and CO2 barriers, and survives pasteurization (25). It is claimed that the shelf life of certain products can be tripled. The coating’s lubricity facilitates the treatment of tens of thousands of bottles per hour in a continuous operation. The coating can be removed by aqueous cleaners commonly used in washing reclaimed PET bottle flake. The system can color the clear coating such as amber for beer. Bairocade bottles are currently used most often in hot-filled applications. The technology allows end users to create the barrier for desired shelf life via coating thickness in a range of 6–8 microns, which will increase by 3–5 times the barrier compared to the untreated PET bottles (37). It comes in three formulations: 1) one for carbonated soft drinks that keeps CO2 inside the bottle, 2) one for juice that keeps O2 outside the bottle, and 3) one for beer that prevents egress of CO2 and ingress of O2. Graham Packaging uses the coating on 12- to 20-oz juice bottles. Pepsi uses the coating on single serve CSD bottles sold in Saudi Arabia.

4. Plasmax

2. BestPET

SIG Corpoplast and Schott HiCotec developed Plasmax, which is another silicone oxide (SiOx) coating system for

The process was co-developed by Krones in cooperation with user Coca-Cola. The system uses an energy-intensive

3. Glaskin


evaporative process to generate ions of silicone-oxide (glass) that coat the exterior of PET bottles for CSD (34). This glass coating is claimed to retain good clarity while improving CO2 and O2 barriers. A new BestPET Plus version has a topcoat to protect the outside glass coating, and is used for single serve CSD, juice, beer and hot-fill containers. 3. Combustion Chemical Vapor Deposition (CCVD) The process was developed by Micro-Coating Technologies. CCVD is a new coating system. It is an openatmosphere, flame-based system that deposits a thin coating made of several organic or inorganic materials onto the exterior of cans, plastic bottles, or films (34). Plastic surfaces do not require pre-treatment to achieve an adhesion that is better than conventional plasma deposition. The process atomizes a low vapor pressure coating solution that contains the precursors into a mist in a flame, where they are combusted to generate the coating material. Heat from the flame provides the energy to evaporate the mists and for the precursors to react and to deposit the vapor on the package material substrate, to dry and simultaneously to cure. Exposure time to the flame is so short that no significant increase of substrate temperature is observed. It is claimed to be simpler to operate, have lower cost per bottle, and be more environmentally friendly than other coating systems. CCVD coatings can be thinner to achieve the same barrier as alternative technologies, and improve CO2 and O2 barrier properties without impairing clarity. The company’s organic coating is adequately flexible to resist much more physical abuse in packaging and distribution than the conventional silica coating. Markets for barrier coatings include various types of beverages. Carbonated soft drinks currently using monolayer PET bottles represents one of biggest potential markets for coated PET. Soft drink bottlers have two potential advantages when using coated bottles: increased shelf life and decreased material cost. With coatings, the potential exists for downgauging of monolayer PET. If the total saving from downgauging are more than the cost of coatings, the bottlers will use coatings. Another major market for coated PET bottles is beer. Unlike multi-layer structures, interior coatings provide the protective barrier next to the product. Several European brewers have used barrier coating systems including Actis and Glaskin. The coated PET bottles can also be used with other demanding beverages such as nutraceutical or healthy drinks, which are emerging.

IX. OUTLOOK FOR FOOD PACKAGING Food packaging technology and food package design have rapidly evolved since the 1950s and 1960s when new

130-9

container alternatives, including flexible packages and new decorating methods, became part of the packaging choices (38). Food package design is becoming a focus for branding, the consumer, and public health. Use of multiple-color printing on all package surfaces has increased to improve a marketer’s shelf visibility. The development of healthier foods and nutraceuticals is emerging in response to the consumer demand. There are several reasons for designing a new package, but the main one is for a new food product. A company uses packaging to introduce new products, as well as to give a new life to the company’s existing brand products. Packages are redesigned to meet the changing needs of the target consumer group. Today the number one need driving new designs for food products is convenience, for preparation and consumption either at home and/or at work. Consumers want products that are easy to handle and quick to prepare. Many food packages designed for convenience are using barrier packaging systems (39). Technical aspects of barrier packaging systems are related to the type of processing, including hot filling, retorting, aseptic, and controlled atmosphere package and modified atmosphere package (CAP-MAP). A hot-fillable, heat set PET container is an innovative wide-mouth jar. The jar incorporates an easy-grip pinched-in middle to aid pouring, thus allows the container to withstand the vacuum generated as the hot product cools after filling (40). MAP is used with ready-to-eat salad mixtures for the consumer who has neither time nor patience to wash and chop salad ingredients. Borden Foods has launched the first shelf-stable cooked pasta that eliminates the need for refrigeration or freezer storage, and requires minimal preparation and cooking time. The pasta is contained in a rotary thermoformed, polypropylene tray sealed with clear, peelable lidstock that holds pouches of cooked pasta and tomato sauce (39). Both pouches use the same clear barrier material. This barrier structure and Borden’s proprietary processing techniques yield a nine-month shelf life. For the vending channel, Welch Foods has launched 16-ounce bottles for white grape, grape, and 100% apple juices as the first commercial applications of an epoxy/amide barrier coating (Bairocade), which doubles product shelf life compared to monolayer PET. Del Monte Foods developed shelf-stable, ready-to-eat fruits called Fruit To-Go. The fruit is packaged in a clear, 4-oz. plastic cup that is flushed with a mixture of gases and provides up to an 18-month shelf life when stored at room temperature. The fruit is coldfilled and then retorted in a package made of polypropylene to provide heat resistance during retorting, while ethylene vinyl alcohol in the package acts as an oxygenbarrier layer to help maintain product quality and safety. Innovations in all food package designs must fulfill the basic requirements: to hold the food being contained and provide suitable protection to the food, to offer suitable functional barriers to the permeation of gases and vapors

130-10


that cause deterioration of the food, and to be user friendly (ease in opening the package while being tampering-proof). Companies have a significant interest in improving their food products. Like food products themselves, packaging materials are constantly evolving to meet the latest demands of the marketplace. New packaging materials as well as new packaging techniques that offer optimal barrier properties will definitely help the companies meet the challenge of keeping products fresh and extending their shelf life. Trends predict expansion and a promising market for evolving technologies including new barrier resins and oxygen scavengers, low-cost surface coatings, and high-output multilayer PET preforms molding systems (25).

REFERENCES 1. W Soroka. Fundamentals of Packaging Technology. Institute of Packaging Professionals, Herndon, VA, pp. 7–9, 1995. 2. K Bertrand. Food packagers ponder proposed countryof-origin labeling regulations: the new rules will affect packaging of meat, seafood and produce — fresh and frozen — when they become mandatory. Food and Drug Packaging, pp. 34–41, April 2003. 3. PA Toensmeier. Modern Plastics, pp. 44–47, December 1995. 4. Mapleston P. Modern Plastics, pp. 62–63, June 1992. 5. M Knights. PET Processing Enhancements Highlight Packaging Conference. Plastics Technology, pp. 44–47, April 2000. 6. M Knights. Beer in plastic — so many ways to get there. Plastics Technology, pp. 58–59, April 1999. 7. Anon. Food Processing, p. 16, April 1996. 8. B Miller. LCP developments progressing. Packaging World, pp. 16–17, August 1996. 9. MT Defosse. Modern Plastics, pp. 51, September 2002. 10. Anon. Newest barrier resin is thermoplastic epoxy. Plastics Technology, p. 25, January 2000. 11. P Mukhopadhyay. Emerging trends in plastics technology. Plastics Engineering, pp. 28–37, September 2002. 12. P Demetrakakes. Nanocomposites raise barriers, but also face them: clay-based additives increase the barrier qualities of plastics, but obstacles to commercialization must be overcome. Food and Drug Packaging, pp. 54–55, December 2002. 13. G Graff. Modern Plastics, pp. 69–72, February 1998. 14. R Leaversuch. Modern Plastics, pp. 39–42, April 1992.

15. MT Defosse. Modern Plastics, pp. 24–25, December 1998. 16. MC Gabriele. Modern Plastics, p. 73, October 1999. 17. Anon. News perspective — single-layer PET packs Russian beer. Packaging Digest, p. 4, April 2003. 18. Anon. Plastics Engineering Europe, p. 29, May 2003. 19. M Knights. Plastic beer bottles are no longer just a dream. Plastics Technology, pp. 39–41, April 1999. 20. M Knights. Prospects brighten for PET beer bottles. Plastics Technology, pp. 35–36, January 2000. 21. Anon. Food and drug packaging, pp. 50–53, December 2002. 22. MT Defosse. Modern Plastics, p. 42, February 2003. 23. R Leaversuch. Super-clear PP barrier bottles are now stretch-blow molded. Plastics Technology, pp. 47, February 2003. 24. A Brody. Brand Packaging, p.11, March 2003. 25. R Leaversuch. Barrier PET bottles — no breakthrough in beer, but juice & soda surge ahead. Plastics Technology, pp. 48–60, March 2003. 26. Anon. Food and Drug Packaging, p.11, February 1997. 27. Anon. Plastics Technology, p. 9, December 1997. 28. Anon. Food and Drug Packaging, p. 9, July 1999. 29. MT Defosse. Modern Plastics, pp. 26–27, March 2000. 30. Anon. News perspective — experience the plastic brick. Packaging Digest, p. 32, December 2002. 31. P Demetrakakes. Barrier coatings may overcome PET’s barrier. Food and Drug Packaging, pp. 56–59, March 2003. 32. PA Toensmeier. Modern Plastics, pp. 17–18, February 1995. 33. LM Sherman. Plastics Technology, pp. 17–21, December 1995. 34. A Brody. Thou shalt not pass — barrier plastic packaging. Food Technology, 57(1): 75–77, 2003. 35. MT Defosse. Modern Plastics, pp. 23–24, August 2000. 36. M Knights. Barrier coating, hot filling and more PET bottle machinery news. Plastics Technology, pp. 32–34, December 2002. 37. R Lingle. PET barrier coating technology “juiced up” bottles make U.S., world debuts. Packaging Digest, pp. 74–80, November 1999. 38. J R Parcels. Food packaging: from the cracker barrel to the Internet. Packaging Digest, pp. 52–57, December 1999. 39. http://www.preparedfoods.com/archives/2000/2000_04/ 0004package.htm 40. Anon. Jarring move to PET bodes well for Knouse. Packaging Digest, pp. 26–30, June 1999.

131

Food Packaging: Plastics

Susan E.M. Selke

School of Packaging, Michigan State University

CONTENTS I. Introduction ....................................................................................................................................................131-1 II. Basic Plastic Structure and Properties ............................................................................................................131-2 III. Plastics Commonly Used in Food Packaging ................................................................................................131-4 A. High Density Polyethylene ....................................................................................................................131-5 B. Low Density Polyethylene ....................................................................................................................131-5 C. Linear Low Density Polyethylene ..........................................................................................................131-6 D. Polypropylene ........................................................................................................................................131-7 E. Polyethylene Terephthalate ....................................................................................................................131-8 F. Polystyrene ............................................................................................................................................131-8 G. Polyvinyl Chloride ................................................................................................................................131-9 H. Nylons ....................................................................................................................................................131-9 I. Polycarbonate ......................................................................................................................................131-10 J. Ethylene Vinyl Alcohol ........................................................................................................................131-10 K. Polyvinylidene Chloride ......................................................................................................................131-10 L. Ionomers ..............................................................................................................................................131-11 M. Other Polymers ....................................................................................................................................131-11 IV. Additives ......................................................................................................................................................131-11 V. Basic Plastic Forming Processes ..................................................................................................................131-12 VI. Multilayer Packages ....................................................................................................................................131-13 VII. Permeability and Shelf Life ..........................................................................................................................131-13 VIII. Migration and Scalping ................................................................................................................................131-15 IX. Information Sources ....................................................................................................................................131-15 References ................................................................................................................................................................131-15

I. INTRODUCTION Use of plastics as packaging materials has grown rapidly during the last several decades (Figure 131.1) (1). The development of new plastic resins and the combination of resins in multilayer structures has allowed plastics to substitute for glass and metal, in particular, in a variety of applications. Such changes generally result in smaller and lighter packages that take less space and consume less energy in manufacture, storage, and distribution. Plastics have also substituted for paper in a significant number of applications. In other cases, a combination of paper and plastics, sometimes with aluminum foil as well, has replaced glass or metal. The area of flexible packaging has been a major source of growth for the use of plastics. However, plastics are certainly not confined to such uses.

Plastic is also used in crates, boxes, and trays where it usually substitutes for corrugated board, and in pallets where it substitutes for wood. Plastic bottles, drums, and other containers are also widely used. The focus of this chapter is on the use of plastics for food packaging, and our concentration therefore will be on the primary package — the package that directly contacts the food. First, we will discuss the properties and food-related uses of some of the major packaging plastics. Common plastics additives will be covered briefly. The major processing methods for forming plastic resins into food packages will also be described. For many food products, the barrier ability of the package, especially to water and oxygen, is critical, so we will also discuss package permeability and its relationship to product shelf life. 131-1

131-2


45 40

Million tons

35 30 25 20 15 10 5 0 1960

1970

1980

Glass packaging

Steel packaging

Paper packaging

Plastics packaging

1990

2000

Aluminum packaging

FIGURE 131.1 Packaging material use in the United States (1).

II. BASIC PLASTIC STRUCTURE AND PROPERTIES Plastics are characterized by being formed by the joining together of small building-block molecules (monomers) into a large chain-like or network structure, in the process known as polymerization. At some point in their manufacture, plastics are capable of being deformed by a combination of heat and pressure. Most of the plastics we use in packaging are thermoplastics, which can be repeatedly softened by the application of heat, and hardened by cooling (think of melting and cooling butter). Most thermoplastics used in packaging are linear, although some branched polymers (low density polyethylene, for example) are very important packaging materials. A few packaging plastics are thermosets. These plastics undergo a chemical reaction when they are heated, in a process known as curing, which forms them into a network structure, usually three-dimensional, resulting in extremely large molecules, which are no longer capable of flow. These materials cannot be softened and melted once cured (think of cooking an egg). Thermosets are found primarily in can coatings (although some coatings are thermoplastics), and occasionally in rigid closures (although the vast majority of closures are thermoplastics). Because of the preponderant position of thermoplastics in food packaging, we will concentrate on this category of plastics. Plastics that are formed from only one type of monomer, and hence have a regular repeating unit in their structure, are called homopolymers. Polymers that are formed from more than one type of monomer, so that the units that make up the molecule differ from place to place in the structure, are called copolymers. Copolymerization is used fairly widely as a way to alter the properties of the basic polymer, to fit its performance to application requirements.

Another categorization of plastics is as addition or condensation polymers. In addition (or chain-growth) polymers, generally the whole monomer is added into the polymer structure. The monomers usually have double bonds, and the “opening” of the double bonds allows the monomers to join together in a chain. Condensation (or stepwisegrowth) polymers are formed by more “ordinary” chemical reactions such as those between acids and amines, or acids and alcohols, in which a small by-product molecule is eliminated in each step. Despite the definitions of homopolymers and copolymers above, the majority of condensation polymers are made from two different monomers, each containing two identical functional (reactive) groups. Because this results in a polymer with a single repeating unit (formed by the reaction of one type of group with the other and the elimination of the byproduct molecule), these are generally classified as homopolymers. A condensation polymer that is considered a copolymer will be formed from three or more different monomers, so that there are differences between the repeating units. The processes we use for forming plastics depend, for the most part, on our ability to melt the materials and shape them as desired. An important characteristic of plastics in this regard is their viscoelasticity. Viscoelasticity can be thought of as a plastic’s tendency to exhibit viscous flow behavior, characteristic of a liquid, and elasticity, characteristic of a solid, at the same time. In many plastics forming processes, we depend on the ability of a plastic to “hold together” when it flows (melt strength), so that we can shape it in the desired manner while it is in or near a liquid state. At the same time, this flowing plastic has “elastic memory” so that when we remove the deforming force, there is often a response by the plastic to return partially to the shape and dimensions it had before we exerted that force. In the solid state, viscoelasticity is responsible

131-3

Weight fraction


Size of molecule

Mn Mw

FIGURE 131.2 Example of a molecular weight distribution. — — M n is the number average molecular weight; M w is the weight average molecular weight.

for our ability to deform the plastic to such an extent that we get limited “flow” of the molecules, resulting in permanent deformation of the plastic, allowing us to modify its shape. Here, too, when the force producing the deformation is removed, we will get an elastic response from the plastic that removes some of the deformation we have imposed. Plastics generally are also characterized by having a viscosity that depends on flow rate, not just on temperature. In other words, they are non-Newtonian fluids. The viscosity of most plastics decreases as the rate of flow (or more accurately of shear) increases. Another important point about plastics is that many of their properties are influenced by their molecular weight. Unlike materials made up of ordinary small molecules, such as sugar, a plastic resin will contain molecules that differ in size. Whenever we talk about the molecular weight of a polymer, we really mean the average molecular weight. To further complicate matters, these averages can be calculated in different ways, with some being more closely related to certain performance variables than others. Often, — — we use a viscosity-average molecular weight, M v or M η, because it is the easiest to determine, and is reasonably closely related to the performance properties of interest for packaging applications. This average is closely related — to the weight average molecular weight, M w, and is often used to approximate it. In general, when the average molecular weight of a plastic increases, its strength, stiffness, and other mechanical properties improve, while its resistance to flow (its viscosity) increases, as does its cost. Processing is easier for materials with lower viscosity (as long as viscosity is not too low). Therefore, users often must compromise between performance of the finished material and cost and ease of processing. The behavior of a plastic resin is influenced by its molecular weight distribution, as well as its molecular weight average. Molecular weight distribution (Figure 131.2) refers to the range of sizes of molecules found in the plastic resin. Small molecules contribute to decreased

viscosity by, in essence, “lubricating” flow. Large molecules contribute to strength. The proportion of smaller and larger molecules and how much they differ from the average affects performance in much the same ways as does the molecular weight average. Distributions can be characterized as narrow or wide. Polymers with narrow molecular weight distributions tend to have higher strength and other mechanical properties, along with higher viscosity (and cost) compared to polymers with wide distributions. While normally molecular weight distributions approximate the classical normal distribution bell-shaped curve, some polymer resins, typically formed by combining two or more batches of the basic polymer, have bimodal distributions. This is one way to better optimize the mix of mechanical and flow properties of the resin for a particular application. Plastics can be characterized as crystalline or amorphous. Crystallinity implies the arrangement of molecules (or parts of molecules) in a regular repeating pattern. While materials composed of small molecules such as salt and sugar can be totally crystalline, because of the large size of the molecules in plastics, it is not possible to totally crystallize the material. Thus a crystalline plastic is characterized by crystalline regions linked together by non-crystalline (amorphous) regions. When we refer to a plastic as crystalline, therefore, we mean that it has some significant degree of crystallinity. We classify as amorphous plastics that have no significant crystallinity. Another difference between plastics and materials composed of small molecules is their melting behavior. While materials such as water melt at a precise temperature, polymers do not. In fact, if a polymer is amorphous, the softening as temperature increases is so gradual that we are unable to clearly distinguish between solid and liquid. Therefore, for amorphous plastics, the melting temperature is not defined. For crystalline plastics, we define the melting temperature as the temperature at which the crystallites (small crystalline regions) break up. Since the size of the crystallites influences how much energy (and therefore what temperature) is required to disrupt the structure, and since the size of the crystallites inside a given plastic resin varies, crystalline plastics melt over a narrow range of temperature, rather than at a precise point. The ability of a polymer to crystallize is determined by its chemical structure. To arrange in a regular repeating order, there has to be a degree of orderliness of the underlying structure. Therefore, some polymers cannot crystallize. If the structure permits crystallization, the way a plastic is processed can have a profound impact on the amount of crystallinity that actually develops. Plastics can crystallize only over a certain temperature range, and the rearrangement of molecules into a crystalline array takes time. If a polymer is cooled rapidly to a temperature below the lower limit for crystallization, it may not develop any significant crystallinity, while the same plastic resin, cooled more slowly, may be highly crystalline. Another factor is that growth of crystals

131-4


depends on initiation (nucleation). Think of a supersaturated salt solution that has no crystals. If a seed crystal is dropped into the solution, all at once there can be massive crystallization. Similarly, providing sites that facilitate crystal formation (adding nucleating agents) results in more rapid crystallization. Another important temperature for understanding polymer behavior is the glass transition temperature, Tg. If a plastic is below its glass transition temperature, it tends to behave like a stiff, brittle material, while above its Tg, it tends to behave like a soft, flexible material. The Tg of a plastic, therefore, serves as a guide to its behavior at any given temperature. All thermoplastics are stiff and brittle if they are cold enough, and soft and flexible if they are warm enough — but the temperatures at which this behavior occurs vary widely. One important consequence of a change in molecular weight or molecular weight distribution is its effect on melting temperature, and in particular on sealing temperature and sealing temperature range. Polymers with a narrower molecular weight distribution tend to soften and flow (melt) over a narrower range of temperatures. Polymers with a higher average molecular weight tend to soften and flow (melt) at a higher temperature. Since heat sealing fundamentally depends on the flow of molecules, or parts of molecules, from one layer into the adjoining layer, the differences in melting temperature influence the temperature required to produce a seal. At the same time, if the plastic gets too hot, there will be too much flow, resulting in weakening of the material, or even the development of holes. Therefore, if the heat seal range is narrow, greater control over the sealing temperature is required to ensure that it does not deviate too far from the optimum value. Rather than specifying molecular weight average, molecular weight distribution, or even viscosity, plastic resins are often characterized by their melt index (or melt flow index). The melt index refers to the amount of plastic that will flow through a small orifice of a prescribed size under specified temperature and pressure conditions. Therefore, it is an indirect measure of viscosity; a high viscosity (resistance to flow) means a low melt index, and vice versa. The units normally used for melt index are g/10 min. Plastic resins are often available with a wide variety of melt index. For example, high density polyethylene (HDPE) resins used for milk bottles typically have a melt index of less than one, often in the 0.5 to 0.7 range. HDPE resins are even available that have a melt index of 0 at the usual conditions, so must be subjected to higher pressure to get a measurable value (the result is referred to as a high load melt index). An HDPE resin used for injection-molded margarine tubs, on the other hand, would likely have a melt index of at least 4, and maybe even 90 or so. As would be expected, a high melt index is associated with a wide molecular weight distribution and with a low average molecular weight (low viscosity), and vice versa.

Other 8% PET 17%

LDPE/LLDPE 26%

PS 2%

PVC 3% PP 12%

HDPE 32%

FIGURE 131.3 Plastic packaging used in the United States, 2000 (1).

TABLE 131.1 The SPI Coding System for Plastic Containers Resin Type Polyethylene terephthalate (PET) High density polyethylene (HDPE) Polyvinyl chloride (PVC) Low density polyethylene (LDPE and LLDPE) Polypropylene (PP) Polystyrene Other plastics, including multilayer

Number

Symbol

1 2 3 4 5 6 7

PETE HDPE V LDPE PP PS OTHER

III. PLASTICS COMMONLY USED IN FOOD PACKAGING The most-used packaging plastics, by far, are high density polyethylene (HDPE) and low density polyethylene (LDPE) (Figure 131.3). Polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC) are considered the other major packaging plastics. A number of other plastics are used in smaller quantities, for specialty applications or as one component in a multi-resin structure. Important examples include ethylene vinyl alcohol (EVOH), polyvinylidene chloride copolymers (PVDC), nylons, polycarbonate, ionomers, and ethylene vinyl acetate (EVA). In most states, plastic bottles 16 oz. and larger, and other plastic containers 8 oz. and larger, are required to be marked with their resin type, using the Society of the Plastics Industry (SPI) coding symbol. This symbol consists of a triangle formed by three chasing arrows, with a number inside and a letter code below the triangle. Table 131.1 shows the letters and codes used for the various types of resins used for plastic containers.


131-5

A. HIGH DENSITY POLYETHYLENE High density polyethylene (HDPE) is an addition polymer of ethylene, with a predominantly linear structure that can be represented as –(CH2 –CH2)n–. The few branches that it contains are short, and do not much influence its properties. Polyethylene has a Tg of about ⫺120°C (estimates vary, for reasons too complex to go into here). Therefore, at the vast majority of use temperatures, HDPE is in the soft, flexible range of behavior. The density of HDPE is actually quite low, about 0.94–0.97 g/cm3, less than water. It is characterized as high density polyethylene only because its density is higher than that of low density polyethylene, which will be discussed next. The melting temperature, Tm, of HDPE is also relatively low, at about 128–138°C. Therefore, a characteristic of HDPE is that it maintains its flexibility well at cold temperatures, such as those used for frozen food, but it is too soft to be used for hot-filled products. As the existence of a Tm implies, HDPE is a crystalline plastic. It is able to crystallize over a wide range of temperatures, and generally is 65–90% crystalline. The crystallinity and density of polymers are correlated, as crystalline regions pack a larger number of atoms (and therefore higher mass) into a unit volume of space than do amorphous regions, with their greater degree of disorder. HDPE has excellent chemical and oil resistance. It is a good water vapor barrier, but a poor barrier to gases such as oxygen and carbon dioxide. Its transparency is poor. For the most part, crystalline polymers tend to have inferior transparency to amorphous polymers, as the crystallites tend to scatter light, interfering with its transmission. The largest use of HDPE is in containers, especially bottles, although it is also used in film. Its single most common use is for plastic milk bottles. The hazy appearance of these bottles is an example of HDPE’s natural appearance. For many applications, HDPE is pigmented, making the containers opaque. This can be done to provide protection for the product against light-induced degradation, or for marketing reasons, to make the container, and hence the product, more attractive. HDPE is produced by polymerization at moderate temperatures and pressures, using a catalyst to facilitate the reaction. The traditional catalyst systems are in the ZieglerNatta family. In recent years, there has been increasing use of a new catalyst family, metallocenes. These are capable of providing plastic resins with a narrower and more controllable molecular weight distribution, as well as having other desirable attributes.

B. LOW DENSITY POLYETHYLENE Low density polyethylene (LDPE) is also an addition polymer of ethylene (C2H4), but it is polymerized at high temperature and pressure, resulting in a polymer with a highly

HDPE

LDPE

LLDPE

FIGURE 131.4 Illustrations of the structure of HDPE, LDPE, and LLDPE.

branched structure, containing both long and short branches (Figure 131.4). While its Tg is the same as HDPE, it is softer and more flexible because of its lower crystallinity, 40–60%. This also gives it better transparency, although it still has a significant degree of haze. Like HPDE, it has excellent oil and chemical resistance, and it is a reasonably good water vapor barrier. Its barrier properties are inferior to HDPE, however. Permeation occurs almost exclusively through the amorphous areas in a polymer, as the crystallites do not have wide enough spacing between polymer molecules to allow the passage of the permeant molecules. Therefore, if other factors are the same, plastics with higher crystallinity will be better barriers. LDPE tends to have lower tensile strength than HDPE, but higher impact strength. Tensile strength is increased by HDPE’s increased crystallinity, since the crystallites resist both deformation and fracture. On the other hand, impact strength is strongly affected by the ability of the polymer molecules to rearrange without fracture, and in doing so absorb the impact energy. This is facilitated by a greater preponderance of amorphous regions, since the crystallites have very little ability to rearrange without producing fracture. Applications for LDPE range from stretch wrap for pallet loads of products to bread bags to squeezable drink bottles, but are predominantly in the area of flexible packaging. A common household food bag illustrates the somewhat hazy appearance of LDPE. LDPE is also found in many multimaterial food packages, where it often serves as a heat seal layer. It may also serve as a moisture barrier, or as protection against interaction or chemical attack. For example, in drink box structures, a layer of LDPE on the outside protects the printed paper from exposure to moisture and abrasion, and a layer of LDPE on the inside prevents direct contact of the aluminum foil and the product, as well as providing the ability to form the package by heat sealing. An additional layer of LDPE within the structure serves as an adhesive to bond the foil and paper into a single structure (Figure 131.5). Discussion of LDPE is complicated somewhat by frequent grouping of the next polymer in our list, linear low density polyethylene (LLDPE), with the type of LDPE we have been discussing, using LDPE to refer to both polymers. The highly branched LDPE described here is produced by

131-6


LDPE

Aluminum foil

LDPE

Paper

LDPE

FIGURE 131.5 Structure of a package for aseptic packaging of juice drinks (juice box) (not to scale).

polymerization at high temperature and pressure, and results in the highly branched structure described, having both short and long chain branches.

C. LINEAR LOW DENSITY POLYETHYLENE Linear low density polyethylene (LLDPE) is a polymer that is formed by a process similar to that used for HDPE, polymerization at moderate temperatures and pressures, with a catalyst, resulting in an essentially linear structure. However, LLDPE has levels of crystallinity, and consequently density, in the same range as LDPE. This is possible because LLDPE is a copolymer. When LLDPE is polymerized, in addition to ethylene, either butene, hexene, or octene are introduced. Where these comonomers are present in the structure, they leave a “tail” hanging off the chain, that looks (and acts) like a short chain branch (see Figure 131.4). The branches in LDPE cause it to have lower crystallinity than HDPE, because they interfere with the orderly arrangement of the chains — the branches don’t fit into the crystal lattice. In just the same way, these side groups in LLDPE interfere with crystallization because they do not fit into the crystal lattice. Therefore, LLDPE is produced with the same range of densities, and the same proportions of crystallinity, as LDPE. The reduction in density depends on both the size and the number of the incorporated comonomer groups. However, because only one type of comonomer is generally used, all the “branches” are identical in size, and their average proportion is readily controlled by varying the amount of comonomer introduced. The use of moderate temperatures and pressures with a catalyst also permits a greater degree of control over the breadth of the molecular weight distribution. HDPE and LLDPE, in general, have narrower molecular weight

distributions than LDPE. This greater degree of uniformity, along with the lack of long chain branches, imparts some significantly different properties to LLDPE than are found in LDPE. Melting temperatures are higher by about 10–15°C, compared to LDPE of the same density. Both tensile strength and impact strength are higher, as well. Because the comonomers used to produce LLDPE are higher priced than ethylene, LLDPE costs more per pound than LDPE. However, the improvement in performance often means that thinner LLDPE films can be used than would be required to get acceptable performance with LDPE. This downgauging means that fewer pounds of LLDPE are required to package the same amount of goods. The price per unit, as a result, is often less using LLDPE, even though the price per pound is greater. The cost savings that can be gained by using LLDPE have led to it replacing LDPE in a variety of applications. The primary area where LLDPE is inferior to LDPE is in heat-sealing. The lower melting temperatures of LDPE and broader molecular weight distribution mean heatsealing temperatures can be lower, and the heat-seal range is wider. Further, entanglement of long-chain branches across the interface between materials occurs faster than entanglement of linear molecules, facilitating the rapid development of sufficient strength in the seal to permit release from the sealing mechanism, and without requiring cooling of the material (hot tack). This combination of improved mechanical properties from LLDPE and better heat seal performance from LDPE has led to these two materials being combined in many applications, either in discrete layers or as blends. Therefore, the term LDPE, as mentioned, sometimes means only the highly branched “true” LDPE, and sometimes means both LDPE and LLDPE, along with materials that are a combination of both resins. The use of metallocene catalysts, along with other catalysts that are often characterized as “single-site” catalysts, has resulted in the production of a whole new generation of LLDPEs. When Ziegler-Natta catalysts are used, the active sites on the catalyst vary in their reactivity. Some sites produce “average” molecules. Others are not as fast at adding in new monomers to a growing chain, but tend to be relatively better at adding in the larger comonomers, so they produce, on average, smaller molecules with a higher proportion of comonomer groups. Other sites are more efficient at adding in ethylene, but less efficient at adding in comonomers, so they produce, on average, larger molecules with a lower than average proportion of comonomer groups. The “single site” catalysts have active sites that all have identical chemistry and geometry, so they are all equally reactive. Therefore, the polymers produced with these catalysts, although they still vary in molecular weight, etc., are more uniform, both in terms of size and composition, than those produced through typical Ziegler-Natta polymerization.


One other attribute of metallocene catalysts is that they are able to add in monomers that are bigger than octene, which Ziegler-Natta catalysts cannot do. By incorporating larger monomers with a double bond on one end (higher alpha-olefins), it is possible to produce linear polymers with the functional equivalent of long-chain branches. Therefore, better heat-sealing varieties of LLDPE can be produced. Of course, these higher alpha-olefins are also more costly than the smaller ones. There are also polyethylenes, produced either with Ziegler-Natta or metallocene catalysts, that have a density lower than the range defined as low density polyethylene. These very low density polyethylenes (VLDPEs) incorporate a greater proportion of comonomer, resulting in a lower degree of crystallinity and consequently a lower density. They are used to produce very soft, flexible films, and have so far had only limited applications in food packaging.

D. POLYPROPYLENE Polypropylene (PP) is a close relative of polyethylene, and is a linear addition polymer of propylene. Consequently it has a methyl group attached to every other carbon. While its physical structure is regular, when the molecules are looked at three-dimensionally, three different physical configurations are possible. When the carbons are stretched out in a linear fashion (the fully extended chain conformation), the methyl groups may all be on one side of the chain (isotactic), they may alternate from one side of the chain to the other (syndiotactic), or the side of the chain on which they appear may be random (atactic) (see Figure 131.6). Although it is not obvious from the two-dimensional view, it is impossible to convert one of these configurations to another without breaking and reforming chemical bonds. If there is no pattern to the placement of the methyl groups, their presence interferes with crystallization in much the same way as does the branching in LDPE. Because the groups appear on every other carbon, this is sufficient to prevent crystallization all together. The result is that atactic PP has very poor performance properties, and is not a desirable packaging material. Fortunately, the Ziegler-Natta catalysts, as well as the metallocenes, tend to add the monomers in a way that results in a preponderantly isotactic configuration (the methyl groups all on the same side of the fully extended chain). Therefore, when we talk about PP for packaging applications (as well as for use in other products), we are almost always talking about isotactic PP, sometimes denoted iso-PP. It used to be very difficult to make syndiotactic PP, and there was little motivation to do so, since for most applications its performance is inferior to the more readily available, and less expensive, iso-PP. With the advent of metallocene catalysts, some interest has emerged in making syndiotactic PP for certain applications. Syndiotactic PP crystallizes less than iso-PP,

131-7

H

C

CH3

H

C

CH3

H

C

CH3

H

C

H

H

C

H

H

C

H

H

C

CH3

CH3

C

H

H

C

CH3

H

C

H

H

C

H

H

C

H

H

C

CH3

H

C

CH3

CH3

C

H

H

C

H

H

C

H

H

C

H

H

C

CH3

CH3

C

H

H

C

CH3

H

C

H

H

C

H

H

C

H

H

C

CH3

H

C

CH3

CH3

C

H

H

C

H

H

C

H

H

C

H

H

C

CH3

CH3

C

H

CH3

C

H

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C

H

H

C

H

H

C

H

Isotactic PP

Syndiotactic PP

Atactic PP

FIGURE 131.6 Illustrations of the structure of isotactic, syndiotactic, and atactic polypropylene.

thereby producing a polymer with increased flexibility and transparency. Like its cousins in the PE family, PP tends to be chemically inert, have good grease resistance, be a good water vapor barrier, and a poor barrier to gases such as oxygen and carbon dioxide. Its melting point is significantly higher than that of HDPE, about 160–175°C. Its glass transition temperature is much higher than PE, about ⫺10°C. Therefore, PP is significantly stiffer than HDPE. At typical frozen food temperatures, PP is very near its Tg and therefore subject to brittleness which can lead to package failure under impact, such as dropping a frozen microwaveable dinner on the floor. On the other hand, PP can be used at higher temperatures than PE without undergoing excessive deformation. In particular, hot-filling products in PP is possible. The enhanced stiffness of PP also makes it suitable for threaded closures (caps). HDPE cannot be used in such applications because, under load, it will creep too much, causing loosening of the cap and loss of sealing efficacy. PP is much less subject to such deformation, and is by far the most commonly used plastic in closures of all types. Uses of PP in packaging are divided approximately equally between film, containers, and closures. The stiffness of PP is also an advantage in some film applications. It got its major start replacing cellophane in high-speed packaging lines such as those used for cigarettes. LDPE was too soft for such applications. PP also has better transparency than LDPE; thin PP films generally have excellent transparency. In thicker gauges, PP often has a somewhat cloudy appearance. This can be modified by blending nucleating

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agents into the film. By providing multiple sites for the initiation of crystallite growth, PP with nucleating agents tends to have a larger number of crystallites, but they are smaller, as adjacent crystallites interfere with each other’s growth. These small crystallites interfere less with light transmission than larger ones, so the net result is improvement in transparency. The tendency of PP to be brittle at cold temperatures can be alleviated in two basic ways. One common approach for film is biaxial orientation. Orientation is a process of stretching a plastic film (or container) to partially align the polymer molecules in the direction of the stretch. This tends to enhance mechanical properties and barrier by producing a greater degree of regularity in the structure. Biaxial orientation stretches the plastic in two perpendicular directions, producing alignment in the plane of the stretch. Biaxially oriented PP is called BOPP. Another way to reduce brittleness is to copolymerize propylene with a small amount of ethylene. This introduces ethylene units into the molecule, which bring with them increased flexibility, lowering the Tg of the plastic and reducing its brittleness.

E. POLYETHYLENE TEREPHTHALATE Polyethylene terephthalate (PET) is the plastic that has been growing in use most rapidly. Its largest use remains plastic bottles for carbonated soft drinks, but it is increasingly being used in bottles for a variety of applications, including drinking water, salad dressing, peanut butter, etc. Within the last several years, PET surpassed HDPE as the plastic most often used for bottles. PET also is used in trays, for products varying from croissants to fresh vegetables to meat. PET films and coatings are also used for food packaging applications. One of the earliest uses of PET was as a coating for ovenable paperboard, paperboard trays designed to be useable in both microwave and conventional ovens. PET is a polyester, and a member of the condensation polymer family, and has the chemical structure shown in Figure 131.7. While there are a large number of different polyesters, products made from PET are often referred to simply as polyester. For example, polyester carpet, polyester clothing, and polyester fiberfill are all made from PET — and often incorporate PET recycled from beverage bottles. Because condensation polymers have non-carbon atoms (oxygen, in the case of PET) in their main chain, they tend to be more susceptible to chemical reactions leading to rapid decrease in molecular weight than most addition polymers, which generally have only carbon in the main chain. In particular, PET is subject to hydrolysis when exposed to water at high temperature and shear. Therefore, it is important to keep PET dry during processing. PET is a significantly better oxygen and carbon dioxide barrier than HDPE and PP, but not as effective as a water vapor barrier. The polar bonds in PET result in stronger intermolecular forces, which reduce permeation of non-polar

H

[O

CH2 CH2

O

O

O

C

C] n

O CH2 CH2 OH

FIGURE 131.7 Polyethylene terephthalate (PET).

substances. However, they also permit greater interaction with water and other polar molecules. Most PET is biaxially oriented for improved performance. As discussed, this orientation improves both strength and barrier properties. Biaxially oriented PET bottles have sufficient CO2 barrier to provide an adequate shelf life for carbonated soft drinks. While the O2 barrier of PET is much better than that of HDPE and PP, it is still not sufficient to provide adequate protection for many oxygen-sensitive products, such as ketchup. The most common structure for plastic ketchup bottles, and for plastic containers of other oxygen-sensitive products, combines PET with a barrier resin, usually ethylene vinyl alcohol. PET has a Tg of 73–80°C. Therefore, at normal use conditions it is on the stiff and brittle side of its behavior. PET is capable of crystallizing, but has a narrow temperature window for crystallization. This permits the degree of crystallinity that develops in PET to be greatly modified by changing the processing conditions. PET films and most PET containers develop only a low degree of crystallinity, with small crystallites that do not interfere substantially with light transmission, resulting in packages with excellent transparency. If increased crystallinity is desired, nucleating agents can be added to facilitate crystallization, resulting in opaque CPET (crystalline PET). Long residence time at temperatures within the crystallization range also produces an opaque white material. Some bottles designed for hot-filling have PET bodies that are transparent, but finishes (the threaded neck area that accepts the closure) that have been crystallized and are opaque white. This results in a bottle neck that is less subject to deformation during the hot-filling process, providing improved sealing. Because typical hot-fill temperatures are near or even above its Tg, biaxially oriented PET bottles, if unmodified, can undergo a large amount of distortion during hot-filling, as some of the stresses imposed by the orientation are now able to relax. (To see an example, send an empty PET peanut butter jar through a standard dishwasher cycle.) Therefore, containers intended for hot-fill applications have to be stabilized, usually in a process known as heatsetting, where they are subjected to elevated temperature while being held in the desired shape, to allow stress relaxation without permitting deformation.

F.

POLYSTYRENE

Polystyrene (PS) is another member of the addition polymer family, with a benzene ring attached to every other carbon (Figure 131.8). Like PP, PS can be either atactic, isotactic,


131-9

H

H

C

Cn

H

FIGURE 131.8 Polystyrene (PS).

or syndiotactic. The PS used in packaging is atactic. This lack of order in the spatial positioning of the benzene ring means that the PS molecules cannot be packed into an orderly repeating arrangement, so PS is an amorphous polymer. It has a Tg of 74–105°C, so like PET it is stiff and brittle at most use conditions. Since it is amorphous, PS has excellent transparency, but it has poor barrier properties. Transparent grades of PS are often called crystal PS. It should be noted that crystal PS is highly transparent precisely because it is not crystalline! Despite its high Tg, PS is not suitable for high temperature applications, as it undergoes liquid flow at about 100°C. The brittleness and low impact strength of PS is a drawback in many food packaging applications, while its relatively low cost and ease of thermoforming are assets. Two distinct approaches are commonly used to modify its brittleness. One of these is foaming. Expanded polystyrene (EPS) uses small bubbles produced by a foaming agent to reduce the density of PS, and also to provide it the ability to better absorb stress without fracture. The low thermal conductivity produced by the bubbles also makes foam PS an excellent insulating material. While the biggest application of foamed PS is as molded or loosefill cushioning materials, it is also found in such food packaging applications as disposable cups for hot beverages, meat and produce trays, and egg cartons. Since the presence of the bubbles results in light scattering, foamed PS is opaque. Another way to improve the impact resistance of PS is to modify it with a rubber material that has high impact strength. High impact polystyrene (HIPS) is partially a copolymer and partially a blend of PS with polybutadiene, a synthetic rubber. While HIPS, like PS, is amorphous, the presence of two phases (PS regions and butadiene regions) interferes with light transmission, making HIPS, like foam PS, opaque. Two common applications of HIPS are yogurt containers and disposable cutlery.

G. POLYVINYL CHLORIDE Unlike most plastic packaging materials, the use of polyvinyl chloride (PVC) in packaging has not been growing. In fact, PVC has lost a number of its packaging markets, mostly to PET. Most of these, however, were in non-food packaging. PVC is FDA-approved only for limited food packaging applications.

PVC is an addition polymer, with a basic structure similar to PP and PS, having, in this case, a chlorine atom attached to every other carbon in the main chain. PVC has a slight tendency to be syndiotactic, but its irregularity is substantial, resulting in a degree of crystallinity so slight that it is often referred to as an amorphous plastic. Because of this low crystallinity, PVC has excellent transparency, with a slight bluish cast. As PVC ages, it tends to yellow, so it is common for PVC containers to have additional blue coloration added, to mask the yellow that will eventually develop. The Tg of unmodified PVC is 75–105°C, so it is stiff and brittle, and is a reasonably good barrier to oxygen and carbon dioxide. However, PVC used in packaging is generally modified by incorporation of plasticizer. A plasticizer is a component that acts as an internal lubricant in the plastic, getting between the polymer molecules and, by disrupting the attractions between the polymer molecules, increasing their ability to change position, thus lowering the Tg and making the plastic more flexible. PVC, because of its highly polar C-Cl bonds, has a large affinity for plasticizers, so they can be incorporated in substantial amounts. Incorporation of various amounts and types of plasticizer permits the production of a wide variety of PVC resins with significantly differing properties. One of the major uses for PVC in food packaging is in soft, highly flexible stretch films used for meat wrap, etc. In addition to increasing flexibility, incorporation of plasticizer significantly reduces barrier, so these soft PVC films are poor barriers to oxygen, carbon dioxide, etc. Over the years, PVC has been subject to a number of attacks on environmental and health grounds. The earliest major concern was potential migration of vinyl chloride monomer (the building block for PVC and a carcinogen) into foods or beverages. Changes in production technology greatly reduced the concentration of residual monomer in containers, and hence the potential for migration. The next major concern was the potential for PVC to contribute to formation of dioxins during incineration. This held up the more widespread approval for use in food packaging that was expected following the resolution of the residual monomer problem. Currently, concerns are related primarily to the potentially harmful effects of plasticizers migrating into the package contents. Along the way, the adverse effects on quality of recycled materials from even very slight PVC contamination of recycled PET have also been a concern. Regardless of whether the concerns about PVC are reasonable or not, PVC has suffered from a relatively negative environmental and health image, and PET, with its relatively positive image, has benefited from the comparison.

H. NYLONS Nylons, or polyamides, are a family of condensation polymers made by polymerization of amines and car-

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O

O

(a) p H2N (CH2)n NH2 + p HOC

(CH2)m O

→H

(NH

(CH2)n

NH

C

O (CH2)m

O (b) p H2N (CH2)n

COH → H

COH

C)p

OH + (p-1) H2O

O (NH

(CH2)n

C)p

OH

+ (p-1) H2O

FIGURE 131.9 General scheme for polymerization of nylon: (a) from diamine and dicarboxylic acid, (b) from amino acid. If monomers are linear, structures formed by (a) are named Nylon n, (m ⫹ 2), and those formed by (b) are named Nylon (n ⫹ 1).

boxylic acids, or by polymerization of amino acids (Figure 131.9). Therefore, they contain nitrogen atoms in the main chain, and attached to the N is a hydrogen atom. This means nylons exhibit hydrogen bonding, resulting in very strong intermolecular attractions, and therefore relatively high Tg and Tm. Nylon 6, for example, has a Tg of 60°C, and a Tm of 210–220°C. The properties of nylons differ, depending on their precise chemical structure. Two of the most common nylons for packaging applications are nylon 6 and nylon 11. Both of these materials are crystalline polymers; as is the case with PET, the amount of crystallinity exhibited is strongly dependent on processing conditions. Some nylon copolymers are amorphous. Nylons tend to have excellent strength and thermal stability, while maintaining flexibility and strength at low temperatures. They are good gas and oil barriers, and tend to be excellent barriers for odors and flavors. They do tend to be moisture sensitive, however. The combination of low temperature strength and high temperature stability of nylons allows them to be used for applications such as boil-in-bag frozen foods. Nylons are higher in cost than the more common packaging plastics, and therefore are often used in combination with other polymers, in multilayer structures, to reduce overall package cost while still benefiting from nylon’s properties.

I. POLYCARBONATE Polycarbonate (PC), more properly known as poly(bisphenol-A carbonate) has limited applications in food packaging, primarily due to its high cost. It is a very tough, rigid plastic, which is widely used for 5-gallon refillable water bottles. Recently, however, PET has begun to make inroads into this market. PC has also been used for refillable milk bottles, but this was never a large market and has tended to decrease over time. PC is an

amorphous polymer with good impact strength, thermal stability, heat resistance, and good low temperature performance. It is a poor barrier to gases and water vapor, and has relatively poor chemical resistance to alkalis. Like PVC, polycarbonate packaging often incorporates plasticizers, some of which are under attack as hormone mimics or disruptors.

J. ETHYLENE VINYL ALCOHOL Ethylene vinyl alcohol (EVOH) is another plastic which has grown rapidly in use. It is, in essence, a random copolymer of ethylene and vinyl alcohol, although it is actually made by hydrolysis of ethylene vinyl acetate (EVA), as the vinyl alcohol monomer is unstable. EVOH typically contains 27 to 48 mole % ethylene units. The O–H groups in the alcohol units of EVOH provide very strong hydrogen bonding between adjacent molecules, and in addition EVOH can crystallize. The –OH and –H groups can both fit the crystal lattice, so the irregularity of the structure does not prevent crystallization in this case. The result is a polymer that is an excellent barrier to gases such as oxygen and carbon dioxide. The most common reason for using EVOH is to take advantage of its excellent oxygen barrier properties. However, the same hydrogen bonding that makes EVOH a good O2 barrier also makes it highly sensitive to water, and as the polymer absorbs water, its barrier properties decrease. Therefore, EVOH is almost always found in a buried inner layer in a package structure, surrounded by other plastics that can offer protection from exposure to high humidity. Use of multilayer structures containing EVOH has permitted plastic containers to replace glass and metal in a variety of food packaging applications. The first plastic bottle for ketchup, for example, was a 6-layer structure, PP/regrind/tie/EVOH/tie/PP. The regrind layer is composed of manufacturing scrap from bottle production that is flaked and fed back into the process. The tie layers are a plastic that serves as an adhesive, bonding the EVOH and PP layers together. The current ketchup bottle structure is a 5layer structure, PET/EVOH/PET/EVOH/PET. The moisture sensitivity of EVOH is a particular concern for containers that are retorted, as during retorting the package is exposed to the combination of high temperature and high humidity. For such applications, structures have been developed that contain a desiccant in the tie layer, to absorb moisture that gets through the outside package layers and thereby reduce its effect on the EVOH.

K. POLYVINYLIDENE CHLORIDE The other major oxygen barrier plastic is polyvinylidene chloride (PVDC). The basic structure of PVDC is similar to


PVC, except that instead of a single chlorine on every other carbon, PVDC has two chlorines on every other carbon. One consequence is that PVDC can be highly crystalline. The combination of high crystallinity and strong intermolecular attractions due to the polar C-Cl bonds makes PVDC, like EVOH, an excellent barrier. Since PVDC does not contain hydrogen bonds, however, it is an excellent barrier to water vapor as well as to oxygen, carbon dioxide, odors and flavors, etc. Further, its barrier properties are not much affected by exposure to moisture. A major drawback of PVDC is that its intermolecular forces are so strong that it is very difficult to process. In practice, PVDC must be modified by copolymerization to make it processable; PVDC homopolymers are not used. The degree and type of comonomerization affects PVDC properties, decreasing its barrier. The PVDC polymers with the highest barrier tend to be used as coatings, applied as solutions or emulsions, as they cannot be melt-processed. The melt-processable grades have somewhat poorer barrier properties. Some grades, such as PVDC used for household wrap, have been plasticized, in addition to copolymerization, and consequently their barrier properties are further reduced. The oxygen barrier of the best PVDC resins is generally inferior to that of the best EVOH resins as long as the EVOH is relatively dry. At very high humidity, PVDC is generally superior in barrier to EVOH. More widespread use of PVDC is limited partially by its cost, and even more by processing difficulties. PVDC copolymers are quite heat sensitive (as is PVC), tending to degrade producing HCl. On the other hand, PVDC coatings on films (or containers) can significantly increase barrier as well as providing heat-sealability.

L. IONOMERS Ionomers are plastics that contain some interchain ionic bonding, most often produced by partially neutralizing ethylene/methacrylic acid or ethylene/acrylic acid copolymers with sodium or zinc bases. The percentage of acid groups is usually between 7 and 30 weight percent, and the amount of neutralization usually is between 15 and 80 percent. The unneutralized acid groups provide hydrogen bonding sites, while the salt ions provide ionic attractions, functioning much like reversible cross-links. Ionomers, as a result, have increased strength, toughness, tensile modulus, oil resistance, and clarity. Their impact strength and puncture resistance are outstanding. Ionomers excel in difficult heat-seal applications, and are widely used in vacuum packaging of processed meats. Here, their ability to seal through contamination means excellent seals can be obtained even if there is contamination of the seal area with grease. They are also used for packaging of cheese and snack foods. The excellent hot tack of ionomers allows them to be used in form-fill-seal packaging in cases where product is delivered into a pouch before the bottom seal has completely cooled.

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M. OTHER POLYMERS Ethylene vinyl acetate is used as an adhesive, as a heat seal layer on other polymers, and as a very flexible but tough cling film. In most food applications, the vinyl acetate content is between 5 and 20 percent. Increasing the vinyl acetate content results in improvement in clarity, increased flexibility, increased impact strength, and increased adhesion. One of the newest entries in food packaging is polymers based on lactic acid (PLA). PLA plastics tend to have properties somewhat inferior to those of PET, but have the advantage of being biodegradable and compostable. They do not yet have widespread use in food packaging, but this may change in the future. Polyethylene naphthalate (PEN) is another member of the polyester family. Its properties in general are superior to those of PET, but its cost is much higher, which has greatly limited its use. There is, however, some use of blends and copolymers of PET and PEN, to get some of the advantages of PEN without as much of an increase in cost. A number of other plastics are used in food packaging applications to a limited extent, because of the properties they can provide.

IV. ADDITIVES When a plastic resin is formulated, along with the base polymer a number of other components are often added. These additives generally have some potential to migrate to the product, and consequently are regulated by FDA as indirect food additives. It is, therefore, necessary to be sure that foodgrade resins are selected for food packaging applications. A very common class of additives is antioxidants. Some of these are compounds such as BHA and BHT that are also used in food to suppress oxidative deterioration of products. They serve much the same purpose in plastics, usually being targeted primarily at preventing oxidation during processing. These additives can, in some cases, migrate to the product during storage, providing additional product protection. In fact, they have sometimes been added to packaging plastics precisely for this purpose. Colorants, most often pigments, are used to provide desired colors to plastic packages. For example, certain types of soft drinks are packaged in green PET bottles. Pigments can also be used to prevent transmission of light, or of certain wavelengths of light, in order to minimize light-induced degradation of the product. Of increasing interest recently are various types of antimicrobial additives, most based on compounds classified as “generally recognized as safe” (GRAS), and intended to inhibit the growth of microorganisms that might otherwise lead to post-processing contamination. Some of these are effective through surface contact, and others act by migrating from the package to the food product. Examples include nisin, potassium sorbate, sorbic acid, natamycin, zeolite-based silver ions, and others.

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Other categories of additives include plasticizers, heat stabilizers, UV stabilizers, antiblock agents, slip and antislip additives, lubricants, mold release agents, nucleating agents, antifogging agents, antistatic agents, oxygen scavengers, desiccants, etc. Fillers and/or reinforcing materials are also used in some food packaging plastics. Additives may be mixed uniformly into the resin pellets. Often, however, they are supplied in the form of a “master batch” that has a high concentration of the additive. The amount of master batch added depends on the level of additive desired in the final product. Additives can also be added to the extruder when the plastic is being formed. This is common for fibers and fillers, for example.

V. BASIC PLASTIC FORMING PROCESSES Most packaging plastics are formed into the desired package shape by melting the plastic and using heat and pressure to shape it as desired. The plastic is generally fed into an extruder in pellet form. The extruder melts and mixes the plastic, so that a uniform stream of melt is available at the desired temperature and pressure for downstream processing. The plastic usually exits through a die, which shapes it to the desired profile. Plastic film is made in two major ways. For cast film, the melted plastic exits the extruder through a slit die and is brought into contact with a water-cooled chrome roller (or set of rollers). The film may, if desired, then be oriented uniaxially or biaxially. The edges are trimmed off, as relaxation in the plastic makes the edges thicker than the rest of the film. It is then rolled up, and is ready for converting operations, where it may be printed, folded, cut, sealed, etc. Blown film is made using an annular die, which produces a hollow tube of melted plastic. Increased air pressure inside the tube expands it in diameter, while it is stretched in the lengthwise direction as it is drawn through the machinery. Therefore, blown film is biaxially oriented without requiring an additional orientation step. The tube of plastic may be rolled up as-is, for use in processes such as bag or pouch-making. In this way, it is possible to produce a pouch, for example, that has only top and bottom seams. Often, the tube is instead slit on both edges, producing two rolls of flat film. Plastic sheet is too thick to be produced by the blown film process, so the cast process is used. For packaging applications, the sheet is usually further modified by thermoforming. In thermoforming, the sheet is reheated and then stretched into or over a mold, using some combination of vacuum and pressure. Foamed plastic sheets, such as polystyrene foam, can be thermoformed, as well as solid plastic sheets. Thermoforming is routinely used for trays, and can also be used for cups and similar shapes. The plastic thins as it is stretched, until it contacts the cool mold and stops stretching. Therefore, the parts of a

thermoformed package that have been stretched the most during forming will be the thinnest in the final package. There are a variety of different thermoforming methods; which method is chosen depends on the material used and the package shape being produced. Plug-assist pressure forming, in which a mechanical device (the plug) helps push the plastic into the mold, as well as additional air pressure for forming are used. This facilitates deep draws, can improve the uniformity of wall thickness, and shortens cycle times. Another way to make plastic containers, closures, etc., is using injection molding. In this process, the plastic is melted in an injection molding machine, which is essentially an extruder, but does not have a die. Instead, the melted plastic is injected into a mold. The mold usually contains multiple cavities, so that several plastic packages or package components (usually identical to each other) are produced in a single step. In injection molding, the plastic fills the entire mold cavity, so there is excellent control over the dimensions of the finished article. Injection molding is limited to shapes that can be removed from the solid core. Therefore shapes such as bottles cannot be made, since there is no practical way to get the solid center out of the molded bottle. For some shapes, innovations such as collapsing cores can be used, where the core is made of multiple pieces that can retract when a center piece is pulled back, allowing their removal. However, this is not practical for bottles. When the plastic flows into the mold block, it passes through a system of runners in order to reach the mold cavities. In packaging applications, these runners are generally heated to keep the plastic inside from solidifying, so that it can be used in the next cycle, rather than needing to be removed and discarded or used as regrind. This process is called hot runner molding. One of the most common applications of injection molding in packaging is in manufacture of threaded plastic closures. To make containers such as bottles, blow molding is used. There are two major categories of blow molding. In extrusion blow molding, the plastic is extruded as a hollow tube, much like is done for blown film. The tube, or parison, when it is the appropriate length, is cut off and captured in a mold. Air is then blown in to expand the parison into the mold shape. Excess material at the top and bottom of the container is cut off, and generally immediately recycled into the process (this material is known as regrind). Bottles with handles can easily be produced by using a parison that is wide enough to cover both the body and handle area when it is captured in the mold. These handles will be hollow, and have an open connection to the body of the container. The gap between the handle and the body is created by pinching the two plastic layers together during molding, and then cutting out this solid piece of plastic. There is often excess plastic at the sides of the neck of the bottle that must be removed, as well, especially for bottles with handles.


To achieve more uniformity in wall thickness of extrusion blow molded containers, it is common to modify the shape of the die (die shaping) to correct for ovality in the container, and to modify the size of the opening as the parison is produced (parison programming) to control for vertical asymmetry. These techniques can also be used, of course, to produce walls that are thicker at certain points, if this is desired. For example, the thickness in the finish area of the bottle will probably be more than in the body, since greater strength and stiffness are needed there. While these techniques add somewhat to cost, they generally more than make that up by permitting reduced material use. In injection blow molding, the bottle is produced in two steps. First a parison is produced by injection molding, as was described above. The bottle finish is completely formed in this step. This allows very precise control over finish dimensions. The body of the parison can also be designed to provide varying wall thickness, thus achieving greater wall thickness uniformity in the finished container. In the second step, the parison is placed into the container mold and blown into its final shape, using air pressure. These two steps may be done sequentially in the same machine. In this case, the injection-molded parison is cooled only enough to maintain its shape, with the support of the core rod that shapes the interior of the parison, during the transfer to the blow mold. It is also possible to completely cool the parison and ship it to another location for blow molding at a later time. In that case, of course, the parison must be reheated before it is blown. It was mentioned earlier that PET soft drink bottles are biaxially oriented. Blow molding naturally produces uniaxial orientation, since the plastic is stretched radially as it is blown. To achieve biaxial orientation, stretching in the vertical direction must be added. This is done by stretch blow molding. While extrusion stretch blow molding does exist, it is rarely used, so we will discuss only injection stretch blow molding. In this process, the parison is produced by injection molding, as already described. However, it is much shorter in length than the height of the final container. The parison is placed in the blow mold with a stretch rod inside. After the parison is carefully reheated to give the desired temperature profile, it is simultaneously blown with air emitted through the stretch rod, and stretched vertically as the stretch rod descends into the cavity, thus producing the desired biaxial orientation.

VI. MULTILAYER PACKAGES It has become very common to use packages that are made of more than one material. Structures that combine plastic, paper, and/or aluminum and structures that contain more than one type of plastic can efficiently perform functions that cannot be obtained from a single layer structure. There are four basic ways to obtain such multilayer structures.

131-13

The first method is coating. We can add a polymer coating to paperboard, for example, to improve its water resistance. We can add a barrier coating to a container to increase the shelf life of an oxygen-sensitive product. When plastic resins are used for coating paper or foil, the most common process used is very similar to that for producing cast film. The plastic is melted in an extruder and then emitted through a slit-shaped die. However, instead of contacting a chrome roller, the plastic goes onto the paper or foil, and adheres to it as the plastic cools. Solvent-based coatings or water emulsions of polymers are sometimes used, instead. The choice depends primarily on the polymer requirements. The second method is lamination. In this process, two rolls of material (substrates) are joined into a single material by causing them to adhere to each other. Most often the adhesion is achieved by using a third component, an adhesive. The substrates may be plastic, paper, or foil. The choice of the adhesive and how it is applied depends on the requirements of the substrates and the characteristics of the adhesive. If the adhesive is low density polyethylene, for example, it is likely extruded into the gap as the substrates are brought together. The third method, which has become very common, is coextrusion. In this process, individual plastics are melted, each in their own extruder, and the melt streams are then brought together, either in the die or in a feed block just before the die. The process of bringing the melted plastics together is done very carefully, so that the streams do not mix with each other, but instead flow uniformly in separate layers, each of the desired thickness. Next the multilayer plastic melt is shaped as described already. This can be used to produce multilayer sheet for thermoforming into multilayer structures, multilayer film through either the cast film or blown film process, or multilayer containers through extrusion blow molding. The fourth method is the newest. Producing multilayer injection-molded objects is more technically challenging than producing multilayer extruded or extrusion-blown objects. Coinjection processes fall into two basic categories. One approach is to use multiple mold cavities and multiple injections, essentially building up the object layer by layer. The more common method, in packaging applications, is to fill the injection mold with a multilayer flow of melted plastic in a single step, again starting with each plastic melted in a separate extruder. Coinjection stretch blow molding, such as is used for PET/EVOH/PET/EVOH/PET ketchup bottles, starts with a coinjection-molded parison, and then uses stretch blow molding to form the final container.

VII. PERMEABILITY AND SHELF LIFE The length of time a food product remains acceptable once it is packaged is often dependent at least in part on the ability of the package to protect it from external influences

89.9–158 10–1200

Tensile modulus (103 psi)

Elongation at break (%)

O2 permeability, 100–185 25°C (cm3 mil/100 in2 24 h atm)

WVTR (g mil/100 in d at 100°F, 90% RH) 0.32

2500–6500

Tensile strength (psi)

2

0.94–0.965

Density (g/cm )

128–138

Tm (°C)

3

⫺120

HDPE

Tg (°C)

Property

400–540

0.95–1.3

100–965

24.9–75

1200–4550

0.912–0.925

105–115

⫺120

LDPE

130–240

0.25–0.76

100–600

165–225

4500–6000

0.89–0.91

160–175

⫺10

PP

TABLE 131.2 Typical Properties of Plastics Used in Food Packaging

3.0–6.1

1.0–1.3

30–3000

400–600

7000–10500

1.29–1.40

245–265

73–80

PET

250–380

4.4–10

1.2–2.5

330–475

5200–7500

1.04–1.05

74–105

PS

9.4–600

1.9–40

14–450

600

1490–8020

1.35–1.41

212

75–105

PVC

1.2–2.6

9.9–11

300

100–247

6000–24000

1.13–1.16

210–220

60

Nylon 6

32

2.5–5.1

300–400

185

8000–9500

1.03–1.05

180–190

Nylon 11

300

4.9–5.9

110–150

345

9100–10500

1.2

265

150

PC

0.0067⫹

1.40–38.1

180–330

5400–13600

1.13–1.21

156–189

48–69

EVOH

0.02–6.9

0.02–0.61

160–400

50–80

2800–5000

1.60–1.75

160–172

⫺15 to ⫹2

PVDC



such as exposure to oxygen or gain of moisture, or from internal changes such as loss of volatile flavor compounds, loss of carbonation, or drying out. In all of these cases, the barrier capability of the plastic, its ability to retard transfer of one or more components through the package wall, is a major factor. The barrier ability of a plastic is determined by two factors: how soluble the compound in question is in the plastic, and its ability, once in the plastic, to move through it (its diffusivity). Typically, we put these two factors together and evaluate the permeability of the package or package material. A package that is a good barrier has low permeability. As we have seen, plastics can be good barriers for some components of interest, and poor barriers for others. For example, HDPE is a good water vapor barrier but a poor oxygen barrier, while EVOH is a good oxygen barrier but a poor water vapor barrier. Barrier is enhanced by increasing crystallinity, since crystallites are essentially impervious to permeating molecules. Strong intermolecular forces also increase barrier. Permeation is significantly faster in a polymer above its glass transition temperature than in the same polymer below its glass transition temperature. Above Tg, we have activated diffusion, in which segmental movements of the polymer chain tend to open up pathways for the permeating molecules. While a thorough discussion of permeability and shelf life is beyond the scope of this chapter, oxygen permeability coefficients for various plastics are presented in Table 131.2. For water vapor, it is traditional in packaging to present water vapor transmission rate (WVTR) coefficients, rather than permeability coefficients. A WVTR is a function of the conditions at which it is measured, so that condition must be taken into account and used to transform the value to a permeability coefficient before it can be used in a shelf life calculation at a differing relative humidity. WVTR values are also listed in Table 131.2. The shelf life of a product depends on how much of the compound of interest can be gained or lost without making it unacceptable, the area available for mass transfer (generally the surface area of the package), its thickness, and the driving force for transfer, in addition to the permeability coefficient. The driving force for transfer is the difference in chemical activity on the two sides of the package (inside and outside), typically expressed as a concentration (partial pressure) difference.

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VIII. MIGRATION AND SCALPING Migration and scalping also involve mass transfer, but instead of transfer between a product and the outside environment, as is the case in permeation, the transfer is between the product and the package itself. By migration, we mean transfer of a component from the package to the product. As mentioned, components that transfer in this way are classified by FDA as indirect food additives. They must either be approved as food additives, have GRAS status, or migrate in such small amounts that they have been determined to pose no real risk. Potential migration of unknown contaminants from a recycled plastic stream is one factor limiting the use of recycled plastics in food packaging. Some recycled resins have been approved for direct food contact, either alone or blended with virgin plastic. Others have been approved provided a “functional barrier” of some minimum thickness of virgin plastic is interposed between the recycled plastic and the food, in a multilayer structure. Scalping refers to the transfer of a component from the product to the package. Usually, this involves a flavor or odor component, and the transfer is undesirable. Polyethylene, for example, can readily scalp the components that give fruit-flavored cereals their desired smell and taste.

IX. INFORMATION SOURCES Additional information on plastics used for food packaging, plastic forming processes, additives, mass transfer and shelf life can be found in Refs. 2–4.

REFERENCES 1. U.S. Environmental Protection Agency. Municipal Solid Waste in The United States: 2000 Facts and Figures. Office of Solid Waste and Emergency Response. EPA530R-02-001, 2002. 2. SEM Selke. Understanding Plastics Packaging Technology. Munich: Hanser, 1997. 3. RJ Hernandez, SEM Selke, JD Culter. Plastics Packaging: Properties, Processing, Applications and Regulations. Munich: Hanser, 2000. 4. AL Brody, KS Marsh, eds. The Wiley Encyclopedia of Packaging Technology, 2nd ed. New York: Wiley, 1997.

132

Paper and Paperboard Packaging

J.M. Park

Paper Technology Information Center, Chungbuk National University

CONTENTS I. Introduction..........................................................................................................................................................132-2 II. Manufacturing of Paper and Paperboard ............................................................................................................132-2 A. Raw Materials ............................................................................................................................................132-2 B. Processes and Equipments ..........................................................................................................................132-3 III. Converting of Paper and Paperboard ..................................................................................................................132-3 A. Processes and Equipments ..........................................................................................................................132-3 1. Coating ................................................................................................................................................132-3 2. Laminating ..........................................................................................................................................132-3 3. Sizing ..................................................................................................................................................132-4 4. Metallizing ..........................................................................................................................................132-4 IV. Classification of Paper and Paperboard ..............................................................................................................132-4 A. Properties and Test Methods ......................................................................................................................132-4 B. Functions and Usage of Paper and Paperboard for Food Packaging ........................................................132-5 1. Kraft Paper ..........................................................................................................................................132-5 2. Greaseproof Paper ................................................................................................................................132-5 3. Glassine Paper ......................................................................................................................................132-5 4. Parchment Paper ..................................................................................................................................132-5 5. Waxed Paper ........................................................................................................................................132-5 6. Tissue Paper ........................................................................................................................................132-5 7. Coated Paper ........................................................................................................................................132-5 8. Paperboard ..........................................................................................................................................132-5 9. Corrugated Board ................................................................................................................................132-6 V. Manufacturing and Usage of Paper Containers ................................................................................................132-6 A. Paper Bag and Paper Sack ..........................................................................................................................132-6 B. Paper Box ....................................................................................................................................................132-6 C. Corrugated Box ..........................................................................................................................................132-6 D. Liquid Container ........................................................................................................................................132-6 E. Component Can or Drum ............................................................................................................................132-6 F. Paper Mould Container ................................................................................................................................132-6 VI. New Trends ........................................................................................................................................................132-6 References ..................................................................................................................................................................132-6

Various materials, functions, forms, and technologies of food packaging are used to protect and maintain product quality during shipping and storage. Recent concern on food safety, health, and resources conservation are evident

ever before. Paper and paperboard are made of fibers from renewable, environmental friendly resources. This chapter describes those characteristics of paper and paperboard most relevant to food packaging applications. 132-1

132-2


I. INTRODUCTION Paper and paperboard are made of fibers from easily obtained natural, renewable resources such as wood or vegetable fibers. They are useful for packaging, writing, and a variety of other purposes. There is no distinct difference between paper and paperboard. Paperboard has characteristics of thick caliper. They are pliable, relatively low cost, easy to convert into various shapes, recyclable, biodegradable, and eco-friendly materials. Depending on purpose of packaging, paper and paperboard can withstand conditions of high and low temperatures which are experienced by sterilized food and frozen or chilled foods. Excellent printability and glueability are required for certain purposes. In spite of various advantages of paper and paperboard, they have certain disadvantages such as easiness to burn, and weakness to water, which can be controlled to a certain extent. Therefore, in order to modify properties of paper and paperboard various additives are added to paper or they are laminated with other materials. Various materials for packaging are used as the science, technology, and machineries are developed, because the packaging market is changing very rapidly. Convenient, high quality, and safe packaging is sought. Share of paper and paperboard in packaging material consumption is about 40% which is higher than plastic, metal, and glass materials. With growing concerns on environment, recyclablility of paper and paperboard packaging materials appeals to customers. Properties can be controlled by changing raw materials, adding additives, and modifying papermaking processes, or using converting machines depending on purposes.

II. MANUFACTURING OF PAPER AND PAPERBOARD Paper is manufactured by Fourdrinier, cylinder, or twinwire former. Fourdriner former has an endless turning wire to distribute fibers evenly on it. Paper produced by Fourdriner former has different sides because of uneven distribution of fillers and small fibers through thickness of paper structure by downward dewatering only. By rotating a cylinder with a wire rotated partially in a fiber suspension, paper is produced on the outside wire and water is drained inward through the wire. The thickness of paperboard by multiple cylinder formers has no limit by depositing one layer on another layer by additional cylinder formers. One directional dewatering limits the speed of Fourdriner former. By impinging fibers solution between two wires and dewatering from both sides of paper, twin-wire former provides even distribution of fillers and small fibers with high speed. Corrugated medium is produced by corrugator and pasted to linerboard to produce corrugated board.

Manufacturing process of paper is composed of two steps, including adding water to fibers then removing water from the sheet structure. Paper is fibrous material of certain thickness with wide area. In order to distribute fibers uniformly, fibers are dispersed in water at low concentration of 0.3–5.0%. Then diluted fibers solution is spread out through a slot, called “headbox,” at high speed (about 100 Km/h for modern machine) across a papermaking machine. Water is removed by gravitation, mechanical pressure, and drying energy. Paperboard is a kind of thick paper with several layers of paper in one structure. It is made by making one layer of sheet first, then adding another layer by another headbox, and so on, to make 3–7 layers of sheet.

A. RAW MATERIALS Cellulosic fibers are used as raw materials for both paper and paperboard. Fibers can be obtained usually from wood or annual plants, but sometimes from animal, mineral, or synthetic for special paper. The fiber is a tubular or cylindrical element several millimeters long and less than 100 micrometers wide. If you tear a sheet of paper and look under a bright lamp at its edge then you can see individual fiber sticking out from the torn zone. Fiber properties are quite different depending on source. There are many factors that affect final properties of paper and paperboard, but the most profound effect may come from the fiber resources. It is critical to choose proper fibers for specific characteristics development. Chemical constituents of wood fibers are cellulose, hemicellulose, lignin, and extractives. Cellulose is betaglucosidic linked glucose chains. Hemicellulose is various polysaccharides which are associated with cellulose, such as glucose, mannose, galactose, xylose, and arabinose. Lignin is phenyl propane unit with complex structure. Extractives are not a part of cell wall structure, but can be removed by neutral solvent such as ether, benzene, alcohol, and water. Pulping is a process to prepare fibers from wood or vegetables by using mechanical and/or chemical energy to separate individual fibers, because fibers are assembled with lignin as paste that bind fibers together. Pulp means fibers that are separated through pulping. Mechanical pulping uses a grinder to apply friction force to separate individual fibers. Lignin is still in fibers of mechanical pulp (MP), so fibers are brown in color before bleaching. Chemical pulping uses chemicals to dissolve out lignin which binds fibers together; it gives low yield but fibers are strong. Through bleaching chemical pulp (CP) becomes bright in color. A brown grocery bag, the most common chemical pulp, is strong and made of Kraft pulp (KP). Small pieces of wood (called “chips”) and chemicals are mixed in a digester to react at certain temperature for a certain period of time, and the reaction conditions


132-3

determine the mechanical, morphological, and chemical properties of fibers.

III. CONVERTING OF PAPER AND PAPERBOARD

B. PROCESSES AND EQUIPMENTS

Most papers and paperboards are converted by impregnating, saturating, laminating, embossing, and forming processes to specific shapes and sizes for efficient usage. In food industry, various papers and paperboards are combined with other materials such as aluminum foil, plastic or metallized flexible packaging material, and extruded films. Representative surface converting processes are coating and laminating. Paper is composed of numerous fibers with many pores in its structure, so its surface is relatively rough. Very fine and white pigment particles are coated on the surface to improve smoothness and brightness of paper. To bind the particles themselves or on the fibers surface, natural or synthetic adhesives are used. Laminating is a combining process of similar or dissimilar webs to impart barrier properties against moisture, oxygen, light, odor and flavor, or to impart special properties.

Fibers are supplied in a diluted solution of water after pulping. In order to make fibers suitable for paper properties, fibers are treated by mechanical force to develop microfibrils sticking out of the surface so flexibility of fibers is increased. Fibers are mixed well, pumped, and passed through a “headbox” at a very high speed. Papermaking is basically an efficient process of removing water from the diluted solution of fibers, which involves using gravitational force by putting the fiber solution on the wire, using mechanical squeezing compressional force by passing through two rolls, and evaporization drying by contacting paper on steam heated cylinder can. Continuous paper web is wound by winder. A block diagram of the papermaking process is shown in Figure 132.1. Various pulps from softwood, hardwood, and annual plants are refined to obtain proper fiber properties. Refining is a mechanical bruising process to develop fibrils on fiber surface and delaminate internal layers to make fibers more flexible to bond easily to improve bonding strength. During refining fibers are hit by mechanical force when they pass through stationary or rotating bars. Refined fibers are diluted in water and ejected at high speed from the headbox on a continuously revolving wire. Fibers are retained on wire and water flows through wire without vacuum or with vacuum to form wet web of fibers. By squeezing wet fibers structure water is removed through porous felt and slit roll at press. After removing a certain amount of water by squeezing, mechanical force cannot remove water any more. Then heat energy at a dryer can be used to remove water by evaporation. For printability smooth surface of paper and paperboard is essential. In order to increase smoothness paper and paperboard pass between several rolls called a calender. In continuous web winder, the web is wound in order to prepare for converting in the next process. Web width is about 2–10 m depending on papermaking machine, and total length of web is about 1–10 km. Sheet cutter makes paper sheets rectangular by using a rotary knife or guillotine. For storage or transportation paper rolls or sheets are wrapped with plastic film or vinyl laminated paper.

Pulp

Wrapper

Refiner

Cutter

Headbox

Winder

Figure 132.1 Papermaking processes.

Wire

Press

Calender

Dryer

A. PROCESSES AND EQUIPMENTS 1. Coating Coating can be divided into aqueous coating and extrusion coating. Aqueous coating, or simply coating, is a process of applying a coating solution on the surfaces of paper and paperboard for enhancing smoothness and printability. Coating is like a makeup on a face for paper. Very fine mineral pigments of 0.1–2 micrometers and adhesives such as natural starch, casein, and synthetic latex are applied on the surface of the paper. To cover coating materials uniformly on paper surface, a very thin knife (called “blade”), an air knife (blowing air), a roll or rod is used to level out across the whole width of paper roll. Extrusion coating is a process that applies high molecular weight polymer on paper by extruder die assembly. It provides heat stability and resistance to water, moisture vapor, some gases, and oil for a paper. 2. Laminating There are several types of lamination, such as (1) wax and hot melt, (2) wet, (3) dry, and (4) solventless laminations. Critical conditions of lamination are temperatures of web and adhesive, tension of web, humidity of web, and adhesive conditions. Melted wax is supplied from a tank by a geared pump to die or coating station for the wax and hot melt lamination. Wet lamination combines two webs, one of which has wet adhesive on its surface. In dry lamination two webs are combined after drying water or solvent from the adhesives. For solventless lamination, 100% solid adhesives with no volatile components are used and drying is not necessary.

132-4


3. Sizing Pigments or starch solution of low concentration can be applied on paper’s surface by passing paper through two or more of rolls (called “size press”). To produce silicone coated baking paper, silicone coating of aqueous emulsion is applied on refined paper fibers at the size press. Silicone coating prohibits sticking of food to the paper during cooking and serving. Internal sizing is the treatment of fiber slurry. Surface sizing is the addition of starch solution or other materials onto the paper surface. 4. Metallizing In order to improve appearance and be a barrier to gas and light, a very thin layer of metal (usually aluminum) is deposited on a substrate surface in a vacuum. For satisfactory vacuum-metallized treatment, paper and paperboard should have less than 4% moisture content and be very smooth, and free from pores and voids. The surface of paper is usually treated with polyurethane, acrylic, or polyamide lacquer for proper metallizing.

IV. CLASSIFICATION OF PAPER AND PAPERBOARD There is no definitive distinction between paper and paperboard, but relatively thick paper is called as a paperboard. Depending on raw materials, converting process, and surface properties, grades of paper and paperboard are classified into various kinds. Usage of paper and paperboard are classified into three categories: packaging, printing and writing, and wiping. In this chapter, packaging grades are discussed.

A. PROPERTIES AND TEST METHODS There are many grades of paper and paperboard depending on purposes. Required properties of paper and paperboard vary depending on production processes, converting processes, and final usage. The moisture content of paper is very important to decide paper quality in many aspects. Therefore properties are measured under standard conditions of temperature and relative humidity since they depend on equilibrium moisture contents. Properties of paper and paperboard related to food packaging are thickness, grammage, tensile strength, tear strength, impact strength, barrier (vapor or gas barrier), capacity, curl, flexibility, stiffness, static and dynamic friction coefficient, heat sealing, vapor transmission rate, and so on. Chemical properties of food wrapping papers are important, and they must be free of harmful chemicals. Parchment paper for butter should have less than 3 ppm of copper content and 6 ppm of iron content for preventing

off-flavor. Properties may be divided into physical, mechanical, strength, chemical, and optical properties. Physical properties are basis weight, thickness, apparent density, smoothness, porosity, formation, curl, sizing, and printability. Basis weight is a mass of paper per unit area. Thickness of paper is measured as a caliper at the specific pressure as a distance between bottom and top plates where a paper is located. Because detailed contour of paper is not uniform nominal thickness is measured. Apparent density is calculated by basis weight divided by thickness, which is equivalent to mass divided by volume. Smoothness is a measure of how smooth a paper surface is. Smooth surface gives better printability because printing plate contact uniformly on smooth surface. Usually paper has two-sideness which means two sides of paper have different characteristics, because during papermaking process drainage of water occurred in one direction through wire. Top side (felt side) is smoother than bottom side (wire side). Paper structure has a lot of pores in it, and porosity of paper is determined by the degree of fiber refining and filler contents, because filler, small mineral particles, plugs in pores of paper. Porosity may be measured by how fast a specific volume of air can pass through paper structure. Formation is an extent of distribution uniformity of fibers and fillers throughout a paper. Uniformity of paper determines the quality of paper. Paper tends to curl depending on extent of difference in properties of top and bottom side. Paper requires a resistance to ink or liquid. Sizing is an extent of resistance to liquid penetration. Printability may be measured in uniformity of ink density in overall image, sharpness of image, and so on. Papermaking process is continuous in travel direction that is called machine direction (MD), and perpendicular direction is called cross-machine direction (CD). More fibers tend to align in MD, so paper is stronger in MD and elongates more in CD. The ultimate force a specimen can endure when it is in tension is expressed in tensile strength. Tensile strength may be expressed as the breaking length, which is a length of paper when it is ripped, when a paper roll is unwound to a certain length due to its own weight of paper supported at one end. Burst strength of paper measures the amount of hydrostatic pressure to rupture a piece of paper. Stiffness measures the bending moment of specimen resisting bending. Fold endurance measures number of folding under a specific tensile force before breaking. Tear strength is determined by energy required to tear several sheet of paper at a fixed distance with initial tear. Chemical contents may be analyzed by various methods of chemical analysis. Optical properties include color, brightness, opacity, and gloss. For corrugated paperboard, burst strength, edgewise compression strength, flat crush test, pin adhesion, and puncture test are usually performed. For finished


corrugated box, puncture, compression strength, impact resistance, stiffness, and drop test are performed. Recently the ISO (International Organization for Standardization) issued some global standards for pulp and paper. Each country adopts specific standards to measure the pulp, paper, and paperboard properties. Standard methods such as TAPPI (Technical Association of Pulp and Paper Industry (USA)), SCAN (Scandinavian Pulp, Paper and Board Testing Committee), PAPTAC (Pulp & Paper Technical Association of Canada), PITA (Paper Industry Technical Association), and APPITA (Technical Association of the Australian and New Zealand Pulp & Paper Industry) usually describe the definitions, specimen preparation, conditioning and measuring methods, testing equipment, and reporting method.

B. FUNCTIONS AND USAGE OF PAPER AND PAPERBOARD FOR FOOD PACKAGING Traditional purposes of paper and paperboard were printing and writing. Various packaging and wrapping usage are developed with a variety of characteristics these days. Life cycle of new products (turn-over) becomes shorter than before. There are too much versatile grades to describe here. Only representative paper and paperboard grades are described.

132-5

barrier properties. Transmission of moisture vapor can be enhanced by waxing, lacquering, or laminating the paper. It can be used as a protective wrapper for all kinds of foodstuffs and many purposes where its transparent feature is helpful. 4. Parchment Paper It is produced by passing paper of cotton fibers or pure chemical pulp through sulfuric acid solution, washing thoroughly, and drying. It is odorless, tasteless, and has good grease resistance and wet strength. It is used for packaging butter, margarine, meat, poultry, and other food products. It can also be used for interleavers for the food (such as meat slice) because of its good releasing property. 5. Waxed Paper Unsized or sized paper is impregnated or coated with molten wax to make waxed paper. It is used for wrapping or packaging bread and sandwiches, baking cups for cup cakes, and liners for cartons and cracker boxes. To make molten wax, microcrystalline wax and polyethylene are added to paraffin wax. 6. Tissue Paper

1. Kraft Paper Bleached or unbleached Kraft pulp (KP), one of the major chemical pulps with high strength, is used in more than 80% for making Kraft paper. Kraft paper is used primarily as a wrapper or packaging material, and its other usage is grocer bags, envelopes, multiwall sacks, butchers wraps, waxed paper, and all types of specialty bags and sacks. Crepe paper is produced by reducing the speed of the press roll to increase elongation rate of 35–200% of its original length. Crepe paper is used for multiwall bags. 2. Greaseproof Paper By refining chemical pulp severely, fibers bond closely and compactly, and there are few pores in its structure, scattering of light is decreased to have almost transparent appearance. Greaseproof paper is for protective packaging material having a resistance to fat penetration in packaging butter or fatty food with few pores. Greaseproof paper is suitable for lamination and coating with wax or lacquer to improve water resistance. After wax coating, it is used as a wrapping paper of potato chips, dried food, cookies, ice cream, and coffee. 3. Glassine Paper By very severe calendaring of chemical pulp, paper becomes almost transparent. It has very smooth surface, high density, and low opacity. It also has good grease resistance and gas

Low grammage thin paper is generally called tissue paper. Sometimes it is creped to enhance elongation and softness. It is used as a wrapping tissue, waxing tissue stock, fruit and vegetable wrapping tissue stock, and various specialty purposes. It can be transparent or semi-opaque. 7. Coated Paper In order to get a high quality printing surface, coating materials such as pigments, adhesives, and additives are used. Clay, calcium carbonate, and titanium dioxide can be used for pigments. Starch, latex, or protein is used for adhesives. Waterproofing agents, plasticizers, rheology control agents, dispersants, defoamers, and dyes can be used as additives to perform specific purposes. Coated paper can be used for labels or bags, and it can be laminated with other material to give special features. 8. Paperboard Relatively thick paper is called paperboard. Generally it has several layers of paper in whole structure. Properties of each layer of paper determine final properties of paperboard. Various pulps can be used for each layer to save production cost and enhance certain properties. It is classified as containerboard (which is used for corrugated boxes), boxboard (which is used to make cartons), and various paperboards.

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9. Corrugated Board Corrugated board consists of liner board on each side and corrugated medium in between. Various shapes of corrugation determine the compression strength and amount of cushion. Various structures may be produced depending on final usages and required strength. Single wall, double wall, and even triple wall structures may be produced.

V. MANUFACTURING AND USAGE OF PAPER CONTAINERS A. PAPER BAG AND PAPER SACK Paper bag is used for packaging dried foods such as sugar or flour is packed in food company and protected during distribution channels of wholesales, and retail stores. Paper bag is also used in grocery stores or retail stores. Recently a high portion of paper bag usage is replaced by plastic bag. Paper sack is made of 2–6 plies of Kraft paper.

B. PAPER BOX Paperboard of 0.3–1.1 mm is cut and punctured to produce a paper box. Paper box in folded shape is supplied to a food factory, and final shape of the paper box is produced during the packaging processes. Paper box is used for dried foods such as cookies and snacks in unit packaging. Puncture strength and strength at humid condition are required for frozen food or marine product packaging. Barrier properties and heat sealing properties may be provided by plastic lamination. Paper box is more expensive, but higher in puncture strength, than the corrugated box.

C. CORRUGATED BOX There are many shapes of corrugated board container. The container may be stored in folded shape for later use, and it is easy to handle for closing and opening. Waterproof properties may be provided for specific end use. This container is used for many purposes including food packaging to protect contained food from impact. Compression strength is required during loading and transportation of box piles. Depending on required burst strength and compression strength, proper shape of container should be provided. Much research is concentrated on reducing raw fibers and use of thinner board for slim packaging in order to reduce the box cost.

D. LIQUID CONTAINER For liquid containers, inside or both sides are coated with wax to provide water resistance. Laminated composite paper in pyramid, rectangular hexahedron, or various

shapes is used for milk and fruit juice. It is usually used with an aspetic packaging system. PE layer of outside provide protection from water or abrasion. Paper of inside provides strength to support the content.

E. COMPONENT CAN

OR

DRUM

Paper is wound in spiral or normal direction to make a component can or drum that is cheap and easy to discard. It is used to protect salt, pepper, powdered hot pepper, powder, spice, cookies, dried snack, biscuit, doughnut, and so on. Bottom and top are made of metal, plastic, or paperboard. Main body can be also laminated in aluminum foil, plastic film, or high strength paperboard for higher barrier properties and strength. Inside the component can or drum may consist of parchment, waxed paper, aluminum foil, glassine paper, or coated paper to improve protection of contents. Paperboard of 0.25–0.5 mm is used for this purpose.

F.

PAPER MOULD CONTAINER

Paper mould is produced in a similar manner as paper. Mechanical pulp, chemical pulp, and recycled pulp are used as raw materials. Screen mould is used to drain water by pressurized extrusion or suction forming method. The container is used for egg tray, shock absorbing packaging, vegetable, and high quality liquor.

VI. NEW TRENDS Regulations for packaging material are now stricter than ever before. By improving printability for more attractive appearance, increasing strength for better protection and convenient handling, developing efficient recycling method for fewer environmental problems, and reducing raw material for less amount of solid waste, paper and paperboard packaging can be more competent.

REFERENCES 1. Eldred, N. R., Package Printing, Jelmar Pub. Co., Inc., Plainview, New York, 1993. 2. Foods & Pharmaceuticals Packaging Handbook, Research Association of Packaging in 21 Century (ed.), Sammi Pub. Co., Tokyo, Japan, 2000. 3. Robertson, G. L. Food Packaging, Marcel Dekker, New York, pp.144–172, 1993. 4. Kouris, M. (ed.), Dictionary of Paper, 5th ed., TAPPI, Georgia, USA, 1996. 5. Smook, G. A., Handbook for Pulp & Paper Technologists, 3rd ed., Angus Wilde Publ. Inc., Vancouver, Canada, 2002.

133

Frozen Food Packaging

Kit L. Yam and Hua Zhao

Department of Food Science, Rutgers University

Christopher C. Lai Pacteco Inc.

CONTENTS I. II. III.

Functions of Packaging ......................................................................................................................................133-1 Deterioration Modes of Frozen Foods................................................................................................................133-2 Packaging Materials............................................................................................................................................133-3 A. Paper and Paperboard ................................................................................................................................133-3 B. Plastics........................................................................................................................................................133-3 1. Polyethylene (PE) ..............................................................................................................................133-4 2. Polypropylene (PP) ............................................................................................................................133-4 3. Polyvinyl Chloride (PVC) ..................................................................................................................133-4 4. Polystyrene (PS) ................................................................................................................................133-4 5. Polyethylene Terephthalate (PET)......................................................................................................133-4 6. Ethylene-Vinyl Acetate (EVA) ..........................................................................................................133-4 C. Barrier Properties of Plastics .....................................................................................................................133-5 IV. Packaging Technologies .....................................................................................................................................133-5 A. Vacuum Packaging and Modified Atmosphere Packaging ........................................................................133-5 B. Time-Temperature Indicator (TTI).............................................................................................................133-6 V. Concluding Remarks .........................................................................................................................................133-6 Acknowledgement ......................................................................................................................................................133-7 References ....................................................................................................................................................................133-7

I. FUNCTIONS OF PACKAGING The basic functions of the package are to contain the food, protect the food, provide convenience, and convey product information. The package protects the food against physical, chemical, and biological damages. It also acts as a physical barrier to moisture, oxygen, volatile compounds, and microorganisms that are detrimental to the food. The package provides the consumer with convenient features such as microwavability, resealability, single serving, and ease of use. The package conveys useful information such as product contents, nutritional values, and preparation instructions. All these functions are applicable to the packaging of frozen foods [1]. The food package can function best when integrated into a food packaging system, which involves certain physical components and operations. The major physical

components are the food, the package, and the environment (Figure 133.1). It is useful to divide the environment into internal and external. The internal environment refers External environment Package

Internal environment

Food

FIGURE 133.1 system.

Physical components of food packaging

133-1

133-2


to the conditions inside the package, which contains the food product and in many cases some air space (also known as headspace). The external environment refers to the conditions outside the package, and it depends on the storage and distribution of the food package. The operations are the manufacturing, distribution, and disposal of the food package. In designing the food packaging system, these physical components and operations must be considered to prevent over-packaging or under-packaging, which results in higher costs, lower quality and in some cases, health risks. There are several requirements in the selection of packaging materials for frozen foods: temperature stability, barrier properties, thermal insulation properties, consumer appeal, and machine compatibility [2]. Temperature stability is necessary since the packaging materials must be able to withstand the abuses encountered over a broad range of temperatures, including freezer temperatures during transportation and storage as well as high temperatures during the heating of the food package in the microwave or conventional oven. Barrier properties are necessary to minimize deteriorative effects of moisture, oxygen, and light to the food product. Thermal insulation helps maintain low temperatures for frozen foods during distribution, and minimize temperature fluctuations which may cause degradation of the products. Consumer appeal is necessary for successful marketing of the products; the packaging materials should allow high quality printing and graphics. Machine compatibility is necessary to ensure that the packaging materials are compatible with low cost, high speed machineries. Frozen food packages are typically made using carton machines, form-fill-seal machines, and pouchforming machines. The constructions and operations of these machines may be obtained from the manufacturers and the literature [3].

II. DETERIORATION MODES OF FROZEN FOODS In addition to mechanical damages, frozen food products can also fail due to several deterioration modes. The most important deterioration modes of frozen foods are related to the transport of moisture. The water molecules in ice exert a vapor pressure which increases with temperature. Water molecules tend to move from high concentration to low concentration. Figure 133.2 illustrates the various transport mechanisms of water molecules (small circles in figure) in a frozen food package. Diffusion of water can occur within the food if a concentration gradient exists (in most cases, in the direction from center to surface). Sublimation, evaporation of water from ice to vapor, can occur at the food surface. Precipitation of water vapor as ice crystals can occur on the food surface or on the interior package surface. Permeation is the transport of water vapor across the package

permeation

External environment Headspace

precipitation

sublimation

Frost formation Diffusion Frozen food

Package

FIGURE 133.2 Transport mechanisms of water molecules.

walls, and the water vapor transmission rate (WVTR) is determined by the permeability of the package. The transport of water molecules can result in dehydration and frost formation, two of the major deterioration modes of frozen foods. In addition to water molecules, the transports of oxygen, flavor, and odor compounds are also important to frozen foods. Dehydration in frozen foods, also known as freezer burn or desiccation, is the moisture loss at the product surface due to sublimation of ice. The moisture loss results in a drier product surface and a concentration gradient which cause water molecules to diffuse from the food center to its surface. Dehydration is a major deterioration mode in frozen food since it reduces product weight and adversely changes product appearance, texture, and taste. For example, when proteins in meat, poultry, and fish products become irreversibly dehydrated, the tissues become dry and tough. These products frequently contain considerable amount of fats and oils, and dehydration can make these fats and oils more susceptible to oxidation by opening up the tissues and thus making more surface areas available for oxidation. If the food product is unprotected (i.e., without package), the rate of moisture loss to the external environment is rapid. To retard moisture loss, protecting the product by a good moisture barrier package is necessary. In addition, the package should also have good tensile, tear, and burst strength at low temperatures; otherwise, package damages (such as holes or cuts) can occur and cripple the protective function of the package. Frost formation is a phenomenon by which water vapor precipitates as frost on the food surface or on the interior surface of the package. Frost formation contributes to the problem of freezer burn since moisture is removed from the product, and it also makes the package less appealing to the consumer. A major factor which affects frost formation is headspace volume: in the


presence of headspace, moisture loss occurs from the food surface to the headspace through sublimation, even when the food is protected by a good moisture barrier package. It is the water vapor in the headspace which is responsible for frost formation. Therefore, an effective packaging technique is to tightly wrap the food product to eliminate the headspace and its water. Another major factor which affects frost formation is temperature fluctuations. Since vapor pressure is temperature dependent, any temperature fluctuations can result in different vapor pressures at different locations, and thus a concentration gradient is created which tends to accelerate the rates of sublimation and precipitation. Oxidation is another deterioration mode for frozen foods. Although oxidation occurs slowly at freezer temperatures, it remains a problem since frozen foods are often stored for prolonged periods of time and oxygen is more soluble in food at lower temperatures. Oxidative reactions can result in rancidity, off-flavor, and pigment discoloration in frozen meat and seafood products. In general, oxidation reactions accelerate with increasing amounts of oxygen present, but there are exceptions. Different foods have different susceptibility to oxidation; for example, pork and poultry are more susceptible than beef and veal to oxygen. To protect oxygen sensitive frozen foods, the package should have low oxygen permeability. Flavor loss is also a deterioration mode for frozen foods. Some flavor compounds are volatile and exert considerable vapor pressures even at freezer temperatures. The alternation of flavor profile due to flavor loss may cause the consumer to reject the product. Odor pickup is also a deterioration mode. Trimethylamine, a compound responsible for the objectionable “fishy” flavor, is volatile at temperatures as low as ⫺23°C. Therefore, the package should also have low permeability to flavor and odor compounds. It is clear from the above discussion that packaging is vital for protecting frozen foods. Understanding the deterioration modes can help to develop packaging strategies to extend shelf life of the products.

III. PACKAGING MATERIALS Packaging materials include paper, plastics, glass, and metal. For packaging frozen foods, paper and plastics are most commonly used, metal is occasionally used (for example, as metal ends in composite cans for frozen concentrated juice), and glass is seldom used. In some package designs, combinations of paper and plastics are used: for example, a frozen meal may be placed inside a plastic tray with a lid, and the tray is placed inside a paperboard carton. The major roles of the package are to protect the products against mechanical damages and deteriorative effects of gas and vapor at low temperatures.

133-3

A. PAPER AND PAPERBOARD Paper and paperboard are mainly used to provide structural support and protect the frozen food products from mechanical damages. These materials are sometimes used as a light barrier, but their moisture and oxygen barrier properties are poor. These materials are made of wood fibers containing cellulose, hemicellulose, and polymeric residues. They have the advantages of good structural strength, low cost, recyclability, and good printability. There are several types of paper used for frozen food packaging. Kraft paper is a coarse paper, which may be used in unbleached or bleached form. Greaseproof paper and glassine paper provide good protection against oil and grease. Waxed paper is a good moisture barrier and can serve as a heat-sealable layer. Paper is sometimes used as an insert to separate individual items (such as beef patties) within the same package so that those items do not stick together due to freezing. Paperboard is commonly used for individual packages and secondary packages (e.g., a box which contains several individual packages). The waxed cartonboard, with a moisture-proof regenerated cellulose film overwrap, was used as earlier packaging for frozen vegetables and fruits [4]. There are two basic folding paperboard designs: skillet and threeflap closed carton [3]. Bleached Kraft carton is often used in packaging for the frozen foods due to its strength and good appearance. To improve moisture and oxygen barrier, paperboard is sometimes coated or laminated with plastics or aluminum. To improve appearance and printing quality, the paperboard is sometimes coated with clay and other minerals. It is quite common that a plastic bag containing a frozen food product is placed inside a paperboard carton. In this case, the plastic bag provides the gas and vapor protection, and the carton provides the structural support and mechanical protection.

B. PLASTICS Many frozen food products are packaged in plastics for moisture, oxygen, flavor, and odor protection. Plastics consist mainly of synthetic polymers and small amounts of additives (e.g., antioxidant and pigment) which can be cast, extruded, and molded into various shapes such as films, sheets, and containers. Most polymers used in foodpackaging plastics have molecular weight between 50,000 and 150,000. Plastics provide a wide range of properties relating to mechanical strength, gas barrier, printability, heat performance, and machine performance [5]. Plastics have a wide range of gas and vapor barrier properties, which offer many choices for different package requirements. The gas barrier properties of a plastic packaging material are usually quantified in terms of permeability: the lower the permeability, the better the gas barrier. Permeability is a function of the plastic material,

133-4


H H

H CH3

H Cl

C C

C C

C C

H H

H H

H H n n Polyvinyl chloride (PVC) Polyethylene (PE) Polypropylene (PP) n

H

H H

C C H H

n Polystyrene (PS)

H H C C H H

O

O

O C C O C

C

H H n Polyethylene terephthalate (PET)

COCH3 | H O C C

H H x y Ethylene-vinyl acetate (EVA) copolymer

FIGURE 133.3 Chemical structures of some common foodpacking polymers.

temperature application such as boil-in-bag applications [2]. HDPE is used in films and containers for frozen foods. LDPE normally has a density range of 0.91 to 0.93 g/cm3. It is a branched polymer with many long side chains. LDPE is used mostly as film, an adhesive in multilayer structures, or waterproof and greaseproof coatings for paperboard packaging materials. The film made from LDPE has the advantages of low cost, softness, flexibility, stretchiness, clarity, and heat sealability. LLDPE is a copolymer with many short side chains. It has LDPE’s clarity and heat sealability, as well as HDPE’s strength and toughness. Therefore, LLDPE has substituted LDPE in many food-packaging applications. 2. Polypropylene (PP) PP has the lowest density (⬃0.9) among all major plastics. It has higher tensile strength, stiffness, and hardness than PE. PP cast film is clearer than PE film and is used in applications where transparency is required. 3. Polyvinyl Chloride (PVC)

permeant gas, temperature, and in some cases relative humidity. In selecting plastic materials for frozen foods, it is necessary to select those which remain flexible at freezer temperatures. Abuse testing (usually includes a combination of shipping, vibration, compression, and drop tests) should be conducted to ensure the package is not brittle and loses its product integrity at low temperatures. The following is a general discussion of the basic food packaging polymers (Figure 133.3). These polymers are mostly used as bags or pouches for packaging frozen foods. Relatively thick films are used to protect against frozen food products (such as crab legs) which have cutting edges or sharp points. Some packaging films are coextruded or laminated multilayer films consisting of several layers of different polymers. By wisely selecting different polymers, multilayer films can offer the advantages of lower cost and/or better performance. 1. Polyethylene (PE) PE is a commonly used polymer as plastics bags for individually quick frozen (IQF) foods (e.g., vegetables, fruits, shellfish) [2]. The advantages of PE are low cost, easy processing, and good mechanical and printing properties. PE is usually classified into high-density polyethylene (HDPE), low-density polyethylene (LDPE), and linear low density polyethylene (LLDPE). These classifications differ in density, chain branching, and crystallinity. HDPE is a linear polymer with relatively few side chains. Its density is typically between 0.94 and 0.97 g/cm3. It has a higher melting point than LDPE, (135°C versus 110°C typically), and thus it is more suitable for high

PVC is a clear, hard polymer which is often modified with plasticizers (organic liquids of low volatility). Plasticized PVC films are limp, tacky, and stretchable, and the films are commonly used for packaging meat. PVC has better clarity, oil resistance, and barrier properties than those of HDPE. 4. Polystyrene (PS) PS is a clear, hard, and low impact resistance polymer. High-impact polystyrene (HIPS) is formed by modifying PS with elastomeric molecules such as butadiene. HIPS is more suitable for freezer temperature applications because it has significantly higher impact resistance. Expanded PS (EPS) of various bulk densities are manufactured by adding foaming agents in the extrusion process. Some frozen seafood products (such as lobster tails) are vacuumskinned down on an EPS tray with a coextruded film. 5. Polyethylene Terephthalate (PET) PET is the major polyester used in food packaging which can tolerate freezer temperatures and high temperatures. It also provides good resistance to grease and moisture. Biaxial orientation of PET film can improve its clarity and mechanical properties. The crystallized polyethylene terephthalate (CPET) can withstand high temperature up to 220°C, and CPET food trays are suitable for use in microwave and conventional dual ovens. 6. Ethylene-Vinyl Acetate (EVA) EVA is a copolymer containing 2 to 18% vinyl acetate. It has long chains of ethylene hydrocarbons with acetate

133-5

groups randomly throughout the chains. EVA film is tough and tacky, and thus it is often blended with polyethylene to improve sealability, stress resistance, and flex cracking resistance. EVA can be used as bags for frozen foods, and is coextruded with Surlyn ionomer and LDPE for the application in skin packaging.

C. BARRIER PROPERTIES

OF

PLASTICS

For foods that are sensitive to moisture or oxygen, gas barrier protection is the major function of the package in providing adequate shelf life, the time period during which the food maintains acceptable quality. Controlling moisture loss is important for frozen foods because moisture loss (sublimation of ice) results in freezer burn and discoloration of the product. Oxidative reaction is also important for some foods even at freezer temperatures. Transport of gases between the external environment and the headspace through the package can occur by means of leakage and/or permeation. For a properly sealed package in which leakage is not a problem, permeation is the major mechanism of gaseous transport. Gas permeation is an important consideration in packaging foods with plastics, since food packaging plastics are permeable to moisture, oxygen, carbon dioxide, nitrogen, and other gases (including those that can cause off-odor problems). The gas permeation rate of most interest for frozen foods is water vapor transmission rate (WVTR). WVTR may be defined as the amount of water vapor transmitted through the package per day [g H2O/ (day(package))] under specific conditions (usually 38°C, 90% relative humidity). WVTR can also be expressed more generally in terms of the amount of water transmitted through 100 in2 package area per day [g H2O/(day (100 in2)] where the package surface area is not specified. If WVTR is assumed a constant, the shelf life (ts) can be estimated by H2O, max ts⫽ ᎏᎏ WVTR

(133.1)

where H2O,max is maximum allowable water (g H2O) which can be determined by sensory evaluation. In practice, WVTR is not a constant but decreases with time, because headspace relative humidity decreases and concentration gradient decreases with time. Thus the actual shelf is slightly higher than that predicted by Equation (133.1). For products that are oxygen sensitive, the oxygen transmission rate (OTR) of a plastic package from the external environment to the headspace can be expressed by – PA ᎏ OTR ⫽ L (Pe ⫺ Pi) (133.2)

O2 permeability (cc mil/100in2 ⋅ day ⋅ atm) at 25°C


1000

LDPE Ionomer HDPE PP (unoriented) PS PC OPP

100

PVC (unplasticized) 10

PET

PVDC

Nylon 6 EVOH (100% RH)

1 2 0

EVOH (0% RH)

0 0

1

10

100

WVTR (g mil/100in2 ⋅ day) at 38°C, 90% RH

FIGURE 133.4 Gas barrier properties of common food packaging polymers.

– where OTR is oxygen transmission rate, cc O2/day; P is oxygen permeability, cc O2 (mil)/(100 in2 (day (atm); A is surface area of package, in2; L is thickness of package, mil; Pe is oxygen partial pressure in external environment, atm; and Pi is oxygen partial pressure in headspace, atm. The shelf life (ts) can be calculated using O2, max ts ⫽ ᎏ OTR

(133.3)

where ts is shelf life, day, and O2,max is maximum allowable oxygen, cc O2. It has assumed in Equations (133.1) and (133.3) that permeation through the package (not the rate of deterioration) is the major factor limiting the shelf life. This is a reasonable assumption when packaging materials of low permeability are used. Combining Equation (133.2) with Equation (133.3) gives O2,max L – ts ⫽ ᎏᎏ PA (Pe ⫺ Pi)

(133.4)

Equations (133.1) through (133.4) can be used to evaluate many what-if scenarios. For example, according to Equation (133.1), if the thickness of the package is decreased by 20% and the surface area is increased by 20%, then the above equation predicts that the shelf life will be decreased by 33.3%. An obstacle is that permeability values of packaging polymers are generally not available at freezer temperatures. It is mostly due to the time and cost necessary for measuring permeability at low temperatures. However, literature permeability values are available at higher temperatures, and Figure 133.4 shows the relationship between oxygen permeability and WVTR for some commonly used food packaging plastics [5]. In practice, one can use literature data as

133-6


reference when comparing different polymers; better still, one can measure the permeability of interest.

prolonged period of time. Vacuum equipment or gas flushing equipment is also required.

IV. PACKAGING TECHNOLOGIES

B. TIME-TEMPERATURE INDICATOR (TTI)

A. VACUUM PACKAGING AND MODIFIED ATMOSPHERE PACKAGING

It is critical to maintain frozen food products at constant low temperatures. Temperature abuses due to improper handling may result in lower food quality and, even worse, microbial growth if the abuse is severe. Monitoring temperature is a critical control point for frozen foods in designing a HACCP (hazard analysis critical control points) program. While temperature recorders (such as handheld electronic temperature monitoring devices) are often placed in storage rooms and trucks to monitor temperatures, these recorders are not attached directly to the food packages. A time-temperature indicator (TTI) is a small selfadhesive label that can be attached to a food package for monitoring the package temperatures from the time of production to the time of consumption. The TTI helps to determine whether the product is still fresh at pointof-purchase and at home by providing the consumer with a visual indication. An important aspect of TTI is the visual indication system which typically involves color change or size change associated with diffusion, chemical reaction, or enzymatic reaction. The visual indication (color change or size change) is correlated to the temperatures or timetemperature history. In order to use the TTI to indicate the shelf life of the product, the kinetics of the TTI and the kinetics of the food must be known, and it is also necessary to match the activation energies of the TTI and food deterioration reaction. The technical details are beyond the scope this chapter but they can be found elsewhere [9]. There are two common types of TTIs. The first type is the temperature limit indicator (or threshold indicator) which triggers an indication when a certain temperature limit is exceeded. For example, if the upper limit is set at ⫺3°C, the TTI will trigger a color indication once the temperature limit is exceeded. The second type is the time-temperature integrator which triggers an indication when the time-temperature limit is exceeded. For example, if the indicator is set at 60 days and ⫺18°C, the TTI will trigger a color indication once an equivalent of this timetemperature history is exceeded. The equivalent time-temperature history is estimated from the kinetics of the TTI. Presently several TTIs are available in the market. The LifeLines Fresh-Check® is based on polymerization reaction which responds to cumulative exposure to temperature. The 3M Monitor Mark® is based on dye diffusion which is activated by pulling out an activation strip. Upon exposure to temperatures above the threshold, the activated indicator’s window irreversibly turns blue, warning that product quality testing should be performed. The Vitsab® TTI is based on enzymatic color change. More

As mentioned earlier, the headspace is an important factor which affects several deterioration modes of frozen foods. The water vapor in the headspace can cause frost formation, and the oxygen in the headspace can cause oxidation. A technique to control the headspace is vacuum packaging, which simply involves removing air from the headspace. This technique has been shown to help maintain the quality of various frozen products including pizza, seafood, beef, and pork [6]. There are two forms of vacuum packaging depending on rigidity of the package. The first form of vacuum packaging involves a rigid package (e.g., glass jar) or a semi-rigid package (e.g., plastic container) in which most of the air is evacuated, but a headspace still remains in the package. The removal of air typically reduces the oxygen level in the headspace to as low as 1%, which significantly helps to reduce the problem of oxidation. However, frost formation and freezer burn are still problematic since the headspace exists. The second form of vacuum packaging involves a flexible package (e.g., a plastic pouch) in which not only the oxygen is removed, but also the headspace is eliminated. Thus both oxidation and frost formation are controlled. This form is also known as vacuum skin packaging, since the food is tightly wrapped by the package. The mechanical stress created by the vacuum also helps to remove air pockets inside the product. This technique has been widely used to package frozen meat and seafood products including meat balls, clam strips, lobster tails, salmon, and farmed rainbow trout [7]. Several types of materials are used for vacuum skin packaging such as a blend of Surlyn ionomer resin with low density polyethylene (LDPE) and ethylene-vinyl acetate (EVA) [8]. Modified atmosphere packaging (MAP) is a technique which involves replacing air (especially its oxygen) in the headspace by other gases such as nitrogen and carbon dioxide. Nitrogen is used as inert gas filler, and carbon dioxide is used because of its ability to inhibit microbial growth. MAP is seldom used for frozen foods because vacuum packaging is often a better alternative in terms of cost (no gas required) and effectiveness (no frost formation). However, MAP is used in some refrigerated and shelf stable food products where the benefit of carbon dioxide is justified or the products cannot withstand the mechanical stress of vacuum packaging. Both vacuum packaging and MAP requires the use of gas barrier packaging materials; otherwise, the vacuum or the modified atmosphere cannot be maintained for a


information about these TTIs can be found on the company websites.

V. CONCLUDING REMARKS Packaging is essential for protecting frozen foods from mechanical damage, moisture loss, flavor loss, odor pickup, and oxidation. Reducing headspace and air pockets inside of the package by vacuum packaging will help minimize frost formation. One needs to analyze both the distribution environment and stability of the product in order to formulate the packaging requirements for a specific product. In analysis of food stability, the dominant deterioration modes should be identified. Their kinetics and acceptable limits should also be determined. Once the food stability and required shelf life is known, package requirements for protecting the product from the dominant deterioration modes can be decided. It is helpful to work with the packaging material supplier and packaging machine manufacturer to design and manufacture the package according to the requirements decided. Other factors including those described earlier in this chapter should also be considered in the package design process.

ACKNOWLEDGEMENT The information in this chapter has been modified from “Frozen foods packaging,” by K. L. Yam, H. Zhao, C. C. Lai. In Handbook of Frozen Foods, Editors: Y. H. Hui et al., Marcel Dekker, New York, 2004.

133-7

REFERENCES 1. KL Yam, RG Saba, YC Ho. Packaging general consideration. In: FJ Francis. ed. Encyclopedia of Food Science and Technology. New York: John Wiley & Sons, 1999, pp. 1807–1811. 2. M George. Selecting packaging for frozen food products. In: CJ Kennedy. ed. Managing Frozen Foods. England: Woodhead Publishing, 2000, pp. 195–211. 3. P Harrison, M Croucher. Packaging of frozen foods. In: CP Mallett. ed. Frozen Food Technology. London: Blackie Academic & Professional, 1993, pp. 59–91. 4. GL Robertson. Food Packaging Principles and Practice. New York: Marcel Dekker, 1993. 5. KL Yam, RG Saba, YC Ho. Packaging materials. In: FJ Francis. ed. Encyclopedia of Food Science and Technology. New York: John Wiley & Sons, 1999, pp. 1824–1829. 6. VM Balasubramaniam, MS Chinnan. Roles of packaging in quality preservation of frozen foods. In MC Erickson, YC Hung. ed. Quality in Frozen Food. New York: Chapman and Hall, 1997, pp. 296–309. 7. HJ Anderson, G Bertelsen, AG Christophersen, A Ohlen, LH Skibsted. Development of rancidity in salmonoid steaks during retail display. A comparison of practical storage life of wild salmon and farmed rainbow trout. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung 191(2): 119–122, 1990. 8. B Spottiswode. Skin packaging. In: AL Brody, KS Marsh. ed. The Wiley Encyclopedia of Packaging Technology. 2nd ed. New York: John Wiley & Sons, 1997, pp. 839–843. 9. TP Labuza, B Fu. Shelf life testing: procedures and prediction methods. In: YC Hong. ed. Frozen Food Quality. Denver: CRC Press, 1997, pp. 377–415.

134

Thermal Processing of Packaged Foods

Donghwan Chung

Kangnung National University

Spyridon E. Papadakis

Department of Food Technology, Technological Educational Institution of Athens

Kit L. Yam

Department of Food Science, Rutgers University

CONTENTS I. II.

Introduction ......................................................................................................................................................134-1 Heat Penetration in Food during Thermal Processing ....................................................................................134-2 A. Estimation of Heat Penetration Parameters ............................................................................................134-2 B. Physical Meanings of f- and j-Values ......................................................................................................134-3 III. Evaluation of Thermal Processing ..................................................................................................................134-4 A. Sterilizing Value ......................................................................................................................................134-4 B. Guide to Sterilizing Value ........................................................................................................................134-4 C. Mass Average Sterilizing Value ................................................................................................................134-4 IV. Methods for Determining Sterilizing Value ....................................................................................................134-5 A. General Method ........................................................................................................................................134-5 B. Analytical Method ....................................................................................................................................134-5 C. Formula Method ......................................................................................................................................134-5 V. Factors Affecting Thermal Processing ............................................................................................................134-6 A. Food Factors ............................................................................................................................................134-7 B. Processing Factors ....................................................................................................................................134-7 C. Package Factors ........................................................................................................................................134-7 VI. Retorts and Heat Transfer Media ....................................................................................................................134-7 A. Retorts ....................................................................................................................................................134-7 B. Heat Transfer Media ................................................................................................................................134-7 VII. Retortable Packages ........................................................................................................................................134-8 A. Metal and Glass Packages ........................................................................................................................134-8 B. Plastic Packages ......................................................................................................................................134-8 References ..................................................................................................................................................................134-8

I. INTRODUCTION The term thermal processing has been widely used in food industry to describe the process of heating, holding, and cooling that are required to produce microbiologically safe packaged food products of acceptable quality [1]. Sterilization is a type of thermal processing designed for complete elimination of both spores and vegetable cells. Commercial sterilization does not require complete microbial elimination, but the degree of elimination has to be regulated and optimized under accepted criteria to ensure

product safety. Pasteurization eliminates only vegetable cells and thus does not provide shelf-stable food products without other preserving processes such as refrigeration. A general procedure for evaluating a thermal process of a food package is as follows. First, the heat penetration in the food during the thermal process is determined by measuring the temperature of the food at a specific point (often the slowest heating point) as a function of processing time. Heat penetration parameters such as f- and j-values may then be determined from this time-temperature profile. Second, a target microorganism is selected based 134-1


on the pH, water activity, and other considerations of the food. The thermal destruction properties of the target microorganism including decimal reduction time (D-value) and thermal resistance constant (z-value) may then be obtained from experiments or the literature. Based on these thermal destruction properties and other considerations such as initial microbial load, the minimum heating time (Fr) at a given reference temperature that is required to reduce the target microorganism population to a stated safe standard is determined. Third, the sterilizing value (F-value) of the thermal process is determined. This is the heating time at the reference temperature yielding a microbial lethal effect equivalent to that of the entire actual processing. For commercial thermal processing, the F-value is often set significantly higher than the Fr value to ensure microbiologically stable food products [1].

Heating medium

Thm T1 Temperature (T )

134-2

T at the slowest heating point of food

Heating phase

Cooling phase

T0 t cu

Time (t)

FIGURE 134.1 Typical temperature profiles in a batch thermal processing. log (Thm − Ths)

Figure 134.1 shows typical temperature profiles of heating medium and the slowest heating point of a packaged food during a batch thermal processing. Thm is the holding temperature of heating medium, T0 is the initial temperature of food, T1 is the temperature of the slowest heating point at the end of heating phase, and tcu is the come-up time. From these profiles, heat penetration curves of the slowest heating point for the heating and cooling phases may be constructed.

For the heating phase, the heat penetration curve is constructed by plotting log(Thm T ) versus heating time. See Figure 134.2 for the symbols used in the following discussion. The lag factor for heating phase ( jh) is defined as: (134.1)

where Ths is pseudo-initial temperature obtained by extrapolating the linear line to time zero. The slow comeup of the retort is responsible for part of the lag time (tlag). A widely accepted method to compensate for this comeup effect is to determine a new zero time at 58% of the come-up time (t58) [1, 2]. That is, 42% of the come-up time is added to the process time at Thm. With the new zero time, the jh-value is redefined as: Thm Ths jh Thm T0

Slope = –1/fh

t58 t lag

Heating time (t )

FIGURE 134.2 Heat penetration curve for heating phase.

where Ths is pseudo-initial temperature obtained by extrapolating the linear line to time t58. With the jh-value, the equation for the linear line (for t tlag) can be written as:

A. ESTIMATION OF HEAT PENETRATION PARAMETERS

Thm Ths jh Thm T0

log (Thm −T )

log (Thm − Ths') log (Thm − T0)

II. HEAT PENETRATION IN FOOD DURING THERMAL PROCESSING

(134.2)

t log(Thm T) log[ jh(Thm T0)] fh or t Thm T jh(Thm T0)10 fh

(134.3) (134.4)

For cooling phase, plotting log(T Tcm) versus cooling time also yields a negative sloped line with initial curvilinear portion, where Tcm is holding temperature of cooling medium. The lag factor for cooling phase ( jc) is defined as: Tcs Tcm jc (134.5) T1 Tcm where Tcs is pseudo-end temperature of heating phase obtained by extrapolating the line to the end time of heating phase. The equation for straight line (for t tlag of cooling phase) can be expressed as: t (134.6) log(T Tcm) log[ jc(T1 Tcm)] fc or

t f

T Tcm jc(T1 Tcm)10

c

(134.7)


134-3

The next section will show that theoretically the j-value depends on the location in the food, at which the temperature is measured, when conduction is the main heat transfer mechanism in the food, and the f-value depends on the thermal properties and the size of the food.

B. PHYSICAL MEANINGS

Comparing this model with Equation (134.3), one can obtain theoretical expressions of jh and fh for the convection-dependent thermal processing:

OF F- AND J-VALUES

For a theoretical illustration of the physical meanings of the heat penetration Equations (134.3) and (134.6) as well as the j- and f-values, two cases of thermal processing are often considered. The first case is when the food is liquidlike and convection is mostly responsible for the heat transfer within the food during thermal processing. The second case is when the food is solid-like and conduction is the main mechanism of heat transfer within the food. The heat transfer resistance due to the packaging material is not taken into account, since the material is assumed to be very thin and have much larger thermal conductivity than that of the food.

jh 1

(134.10)

2.303ρVc fh p UA

(134.11)

Equation (134.11) shows that fh is proportional to the volume and the specific heat of the food but inversely proportional to the overall heat transfer coefficient and the internal surface area of the package. In practice, the value of fh is smaller than the theoretical value during initial heating, since the initial large temperature difference between the medium and the food yields high U value, and increases gradually to constant theoretical value. Due to the initial small fh-value, the actual jh-value is somewhat less than the theoretical value of 1. The meanings of jc- and fc-values can be illustrated in a similar manner. 2. Conduction-Dependent Thermal Processing

1. Convection-Dependent Thermal Processing If the food being heated or cooled is liquid-like and sufficient convective heat transfer occurs, the temperature gradient within the food may be ignored. Due to the uniform internal temperature, this convection-dependent thermal processing can be analyzed using the lumped capacity method or Newtonian heating or cooling method, which describes the time-temperature history of a solid object when the Biot number (Bi) of heat transfer is very small (often less than 0.1) [3]. The mathematical model developed for this method, known as the Schultz-Olson model [1], had been derived by equating the rate of change in internal energy of the food and the rate of heat transfer between the medium and the food. The heat balance equation for heating phase is:

If a solid food is processed thermally, conduction controls the heat transfer within the food. As an illustration, one may assume a cylindrical food package of radius a and length l, exposed to a constant temperature environment with infinite surface heat transfer coefficient (i.e., infinite Bi) and uniform initial temperature. Analytical solution describing the time-temperature history of the cylindrical food during heating phase is found in Cowell and Evans [4] or elsewhere: 8 Thm T Thm T0 π

∞

∞

冱冱 k1 n1

J0(Pkρ) PkJ1(Pk)

sin[(2n 1)πξ] eAB (134.12) 2n 1 and

dT ρVcp UA(Thm T) dt

(134.8)

where ρ is the density of the liquid food (kg m3), V is the volume of the food (m3), cp is the specific heat of the food at constant pressure (J kg1 K1), U is the overall heat transfer coefficient at the internal side of the package (W m2 K1), and A is the internal surface area of the package (m2). By integrating Equation (134.8) from t 0 to t and T T0 to T, the Schultz-Olson model for heating phase is obtained: 0.434UAt log(Thm T) log(Thm T0) (134.9) ρVcp

r ρ a y ξ l 2

冢冣 (2n 1) π , m l

Pk A m

2

2

a

αt B l2 where J0 and J1 are Bessel functions of the first kind of order zero and one, respectively; Pk is the root of Bessel

134-4


functions; r and y are the coordinates of radial direction and length-direction, respectively; and is the thermal diffusivity of the food. For a large value of B, the firstterm approximation of Equation (134.12) may be used: 8 J0(P1ρ) Thm T sin(πξ)eA11B π P1J1(P1) Thm T0

(134.13)

where 2

冢冣 π

P1 A11 m

2

and

P1 2.4048

Comparing Equation (134.13) to Equation (134.3), theoretical expressions of jh and fh for the conduction-dependent thermal processing can be obtained: 8 J0(P1ρ) jh sin(πξ) π P1J1(P1) fh

2.303 2

π + l

2

(134.14)

(134.15)

冤冢冣冢冣冥

α

P1 a

Equation (134.14) shows that jh depends on the position in the food, at which the temperature is measured, regardless of the size and the type of the food. At center, the theoretical value of jh is 2.0397 since ρ 0 and ξ 0.5. Equation (134.15) shows that fh depends on the thermal diffusivity and the size of the food, but it is the same at any location in the food. The meanings of jc- and fc-values can be also illustrated in a similar manner.

III. EVALUATION OF THERMAL PROCESSING A thermal process should be carefully evaluated to ensure microbiologically safe food products. In most cases, the evaluation requires either of the followings: (1) determination of process sterilizing value (F-value) for a given processing time and a heat penetration curve, or (2) determination of a proper processing time required for a target F-value [1, 5].

To determine the F-value, the lethal rate (L) needs to be defined: TT F D r L r r 10冢 z 冣 FT DT

(134.16)

where Fr and FT are minimum heating times required for reducing the population of target microorganism at the critical location to a stated safe standard value at a reference temperature Tr and at a certain temperature T, respectively. And Dr and DT are D-values for the target microorganism at Tr and T, respectively. The fundamental equation for determining F-values is given by:

冢冣

F F 冱 r F T i

i

ti

冕 L dt 冕 10冢 t

t

0

0

TTr z

冣dt

(134.17)

where ti is the actual heating time at a certain temperature T, and t is the total processing time. Various methods developed for integrating Equation (134.17) is discussed in Section IV. The reference temperature Tr is decided depending on the purpose of thermal processing. For sterilization, the value of Tr is in the range 115–130°C. For example, in canning industry, 121.1°C (250°F) is often used. For pasteurization, the range of Tr is 60–100°C. For example, 60°C and 72°C are suggested for beer and milk industries, respectively [2, 6].

B. GUIDE TO STERILIZING VALUE Viability of microorganisms depends on various factors such as the type of microorganism, initial microbial load, the pattern and the stage of cell growth, processing conditions, operating procedure, physicochemical properties of food, etc. While F-value should be determined and validated for each food process, some useful guides are available. Holdsworth [1] provides general principles for selecting F-values and summarizes the recommended F-values for meat, vegetable, fish, poultry, and other food products based on various sources including Alstrand and Ecklund [7], Townsend et al. [8], NFPA [9, 10], Hersom and Hulland [11], Codex Alimentarius Commission [12], and reports from UK.

C. MASS AVERAGE STERILIZING VALUE A. STERILIZING VALUE The F-value is defined as the heating time at a reference temperature, which can yield the lethal effect equivalent to that of the actual process at a critical location, often the slowest heating point. For example, if the reference temperature is 121.1°C and the determined F-value is 3 min for a thermal process, then this means that the process can inactivate the target microorganism as much as the 3 min-heating at 121.1°C at the location. By determining the F-value, different thermal processes can be compared for their lethality.

The concept of mass average sterilizing value (Fs-value) or integrated F-value was introduced by Stumbo [13, 14] for evaluating the lethality of entire food, not just at the slowest heating point. The Fs-value is useful when the location at which the temperature is measured, mostly the geometrical center of the food, is not the slowest heating point. According to Holdsworth [1], the phenomenon is not observed for the domestic sizes of cans, but for large cans the slowest heating location could be a toroidal ring around the center. However, using the Fs-value is not quite


134-5

appropriate in evaluating the lethal effect of a thermal processing, since the microbial safety of a food product always depends on the population of a target microorganism at the slowest heating point. A more useful application of the Fsvalue can be the quality optimization of thermally processed foods [1, 15–18]. The thermal degradation of heat-sensitive food components, which are distributed throughout the food product, can be evaluated using the Fs-value.

IV. METHODS FOR DETERMINING STERILIZING VALUE A. GENERAL METHOD Once the time-temperature profile of the slowest heating point is obtained experimentally or theoretically, the lethal rate (L) at the location can be calculated using Equation (134.16) and plotted with respect to the processing time (Figure 134.3). From the definition (Equation 134.17), the F-value can be determined by calculating the area under the lethal rate curve. Various methods are available for the area calculation. The simplest method is to count squares or use a planimeter [1]. Another well-known method involves the construction of lethal rate paper [19–22]. Numerical calculations have also been used using trapezoidal rule [23], Simpson’s rule [24, 25], or Gaussian integration formula [20].

determination methods. However, the analytical solutions of Equation (134.17) are not always available, and the assumptions needed for the derivations sometimes greatly reduce the accuracy of the method.

C. FORMULA METHOD In the formula method, empirical heat penetration equations such as Equations (134.4) and (134.7) are obtained experimentally and substituted into Equation (134.17) to yield the F-value. The method was first proposed by Ball [28] and further developed by a number of researchers, including Ball and Olson [2], Stumbo [14], Gillespy [29], Jakobsen [30], and Hayakawa [5, 31]. In the following sections, the Ball’s method and the Hayakawa’s method are discussed. 1. Ball’s Method In the Ball’s original method [28], Equations (134.4) and (134.7), which are linear on the semi-logarithmic heat penetration curve (see Figure 134.2), are substituted to Equation (134.17) and integrated to determine the F-value (Fh) for heating phase and the F-value (Fcl) for cooling phase, respectively. The F-value (Fcc) for the curvilinear portion of the heat penetration curve, observed at the beginning of cooling phase, is also considered. Therefore, the F-value for the entire thermal process is calculated as: F Fh Fcc Fcl

B. ANALYTICAL METHOD Analytical solutions of Equation (134.17) could be obtained if the time-temperature profile follows analytical heat penetration equations based on heat transfer theory, such as Equations (134.9) and (134.12). Examples of the solutions were discussed by Holdsworth [1], Hicks [26], and Hurwicz and Tischer [27]. An advantage of the analytical method is that the meanings of the F-value and various processing factors can be better understood and, therefore, it can provide theoretical basis for other F-value

Food temperature

Lethal rate (L)

Temperature (T )

By the substitution and integration, the Fh-value for heating phase is given by: ThmTr fh Thm T1 Fh 10 z E1 ln10 ln10 z

Lethal rate

Area = F-value T0

0

冤冦

冢

冦

冢

冣冧

jh(Thm T0) E1 ln10 z

where

or

T1

(134.18)

E1(x)

冕

x

冣冧冥 (134.19)

ep dp p

(134.20)

∞ (1)nxn E1(x) γ ln x 冱 n1 n n!

(134.21)

Equation (134.20) is useful when x 0.01, because its solution tables are available. Equation (134.21) is used when x 0.01 and is Euler’s constant (0.577215665). Temperatures are expressed in degree Fahrenheit. For a broken heating curve, the Fh-value is given by:

冤冤冦

冢

ThmTr 1 Thm T 1* Fh 10 z fh1 E1 ln10 ln10 z

冣冧

Time (t)

FIGURE 134.3 Typical profiles of temperature and lethal rate at slowest heating point.

冦

冢

jh(Thm T0) E1 ln10 z

冣冧冥

134-6


Thm T1 fh2 E1 ln10 z

冤冦冢冣冧 (T T ) E 冦ln10 冢冣冧冥冥 z hm

* 1

1

where za is a reference z-value and zb is any z-value of interest. With Equation (134.27), the F-value can be determined for any z-value. The U-value for heating phase (Uh) and the value for cooling phases (Uh) are given by: Uh

(134.22) where fh1 and fh2 are fh-values before and after breaking point, respectively, and T *1 is the food temperature at breaking point. For cooling phase, jc 1.41 is assumed. The Fcc-value for the initial curvilinear portion of the semi-logarithmic heat penetration curve is calculated using the following equation: Fcc fc 10

T1Tr z

冤

0.789 m z

0.1435 e

0.692 m E 0.1096 e z m

冥

(134.23)

where E (2.303n)2

冕

2.14

e2.303nx 兹苶 x2苶苶1 dx

1

m T1 Tcm 0.3m n z And the Fcl-value for the linear portion is given by:

冤冦

冢

TcmTr fc 0.657m Fcl 10 z Ei ln10 ln10 z

冦

冢

冣冧

m g 80 Ei ln10 z

冣冧冥

(134.24) where g Thm T1 and Ei(x)

冕

x

(ThmT1) Kz

Uc 10

s a

冕 10 tc

冕

th

(ThmT) Kz

10

s a

dt

(134.28)

0

(T1T) Ksza

(ThmT1) Ksza

dt 10

0

Ugc (134.29)

where th and tc are heating and cooling times, respectively. The values of Uh and Uc can be conveniently obtained from the universal tables of Uh/fh and Ugc/fc, respectively. The tables were prepared based on the following two observations: (1) the Uh/fh-values are independent of fh-values when the jh- and g ( Thm T1 )-values are fixed, (2) the Ugc/fc-values are independent of fc-values when values for jc and for T1 Tcm are fixed. In the calculations for table preparation, Equations (134.4) and (134.7) were used for the linear portions of heating and cooling curves, respectively. The curvilinear portions of heating and cooling curves were evaluated using the following empirical circular formulas.

a. Formulas for the curvilinear portion of heating curve (i) 0.4 jh 1.0: Thm T (Thm T0)cot(Btπ/4) for 0 t tlag (134.30) where

π log(Thm T0) 1 B tlag arctan log{jh(Thm T0)} tlag /fh 4

冦

冤

冥

tlag 0.9 fh (1 jh)

ep dp p

(134.25)

Tables for Equation (134.25) are also available elsewhere. Ball’s original method was further modified by Ball and Olson [2], Hicks [32], Pflug [33], and many other researchers.

(ii) 1.0 jh 3.0: Thm T (Thm T0)cot Bt for 0 t tlag

冤

2. Hayakawa’s Method Hayakawa [5] introduced the special sterilizing value (U) for determining the F-value. According to his two theorems, the F-value can be calculated using the following equation. T T 冢冣 K hm

The relative z-value, Ks, is defined as: zb Ks z a

冥

tlag 0.7 fh (jh 1)

(134.26)

b. Formulas for the curvilinear portion of cooling curve The curvilinear formulas for the cooling curve can be obtained by replacing some symbols in the above formulas in the following ways.

(134.27)

fh ⇒ fc

r

sza

(134.31)

where log{jh(Thm T0)} tlag /fh 1 B arccos tlag log(Thm T0)

F (Uh Uc) 10

冧


jh ⇒ jc Thm T ⇒ T Tcm Thm T0 ⇒ T1 Tcm tlag for heating ⇒ tlag for cooling

134-7

(134.32)

The Hayakawa method is considered as one of the most versatile and reliable techniques [1, 34, 35].

V. FACTORS AFFECTING THERMAL PROCESSING The severity of a thermal processing, that is, the required F-value, is affected by a number of factors originated from the complexity of food nature and the variety of processing methods and packages. An outline of such factors is well presented in Holdsworth [1].

A. FOOD FACTORS 1. The phase and the rheological behavior of the food, the packing of food components in the package determines if the main mechanism of internal heat transfer is conduction, convection, or both, as well as the rate of heat transfer [36]. 2. As the initial temperature of the product increases, the processing time and the fh-value decrease, and the jh-value increases. The initial temperature is more important in the conduction-dependent thermal processing. Also, the initial temperature distribution in the product affects the jh-value [37, 38]. 3. Initial load of target microorganisms and their z- and D-values are important to determine the process severity. 4. The thermal diffusivity and specific heat of the food, affected by the food composition and the consistency of food components, greatly influences the f-value (Equations (134.11) and (134.15)). Therefore, the change of thermal diffusivity or specific heat with temperature may not be negligible. 5. The pH of the product determines the process severity. The products with lower pH may require lighter thermal processing. 6. Certain additives such as nitrite, salt, sugar, and including various antimicrobial agents can reduce the process severity.

B. PROCESSING FACTORS 1. As the temperature of heating medium increases, the processing time and the fh-value decrease, and the jh-value increases.

2. The rotation of product in the retort can enhance the internal heat transfer to reduce the fh-value. 3. The longer the processing time, the greater the heat penetration. 4. As the surface heat transfer coefficient between the package and the heating medium increases, the fh-value decreases and the jh-value increases [2, 39].

C. PACKAGE FACTORS 1. The thermal conductivity of the packaging material determines the rate of heat penetration. 2. The shape and dimension of the package are important. The f- and j-values depend on the package shape [37]. Equations (134.11) and (134.15) show that the fh-value depends on the size of the packaged food. The heat penetration into the package is greater for the package of larger surface area and the smaller thickness. 3. The amount of headspace is important for agitation and rotation, which can enhance the internal heat transfer. 4. The position of packages inside the retort and the type of stacking can affect the heat transfer to individual packages.

VI. RETORTS AND HEAT TRANSFER MEDIA A. RETORTS Currently, a large number of different types of retort are available. In general, the type of retort may be classified into five groups depending on the mode of operation: (1) batch-static (e.g., conventional vertical or horizontal batch retorts without rotation), (2) batch-rotary (e.g., batch retorts with internal rotation of packages), (3) continuousstatic (e.g., hydrostatic cookers without rotation), (4) continuous-rotary (e.g., hydrostatic cookers with rotating carrier bars, reel and spiral cookers), and (5) semicontinuous (e.g., Crateless retorts) [1]. Selecting a proper retort requires careful considerations on the three types of factors mentioned in Section V. The pressure balance between the inside and outside of the package is another important factor to be considered, especially for rigid and plastic packages prone to distortion at high temperatures and at high rates of cooling. Beyond the scientific evaluation of the factors, the economic factors such as available area, factory layout, the number of operator, production yield, the cost of installation, operation, and maintenance should be also considered. More detailed considerations on retort systems are presented by Holdsworth [1], Lopez [40], Rees and Bettison [41], and Footitt and Lewis [42].

134-8


B. HEAT TRANSFER MEDIA

B. PLASTIC PACKAGES

For heating media, saturated steam, steam-air mixture, hot water, and gas flame, and for cooling media, chilled water, are available. For a proper selection of heat transfer media, the factors presented in Section V, especially surface heat transfer coefficients and packaging materials, as well as various economical, environmental factors must be carefully taken into account. The heat transfer coefficient at the surface of the package in contact with the heat transfer media is one of the critical parameters to determine the efficiency of heat penetration. Its values for saturated steam, vigorously boiling water, and gas flame are very high and thus the heat transfer resistance at the package surface is often negligible, that is, the Biot number can be assumed infinite. Due to their rapid heat transferring ability, those heat transfer media are suitable for HTST (high-temperature shorttime) processes. Other media have much lower values of surface heat transfer coefficient, and therefore, the resistance at the surface is necessary to be considered in the process evaluation. The ranges of the surface heat transfer coefficient (or overall heat transfer coefficient) of the media are well reviewed in Holdsworth [1]. The type of packaging materials is also an important factor to be considered in the media selection. For heating metallic packages, all the existing media are usable. The air heating and cooling of canned products have been also developed, however, their commercial applications have not been appeared yet [1]. For heating retortable laminated pouches or plastic packages, hot water [43] or steam-air mixture [44] can be used. For glass packages, saturated steam, steam-air mixture, and hot water are suitable heating media. The cooling stage generally uses chilled water of about 10°C, and is slower than the heating stage due to the lower surface heat transfer coefficients. Relatively few studies have been reported on the cooling media and their surface heat transfer coefficients [1].

Plastic packages are sometimes used for thermal processing of foods, especially ready-to-eat military rations. These packages are usually in the form of pouches or containers. A typical retortable pouch for military rations is made of a foil layer sandwiched between two layers of plastics such as polypropylene. A typical retortable plastic container is made of an oxygen barrier layer such as EVOH sandwiched between two polypropylene layers. There are several advantages of using plastic packages for thermal processed foods [46]: (1) high surface-tovolume ratio that allows rapid heat transfer under mild thermal conditions, (2) convenience due to light weight, easy opening, easy handling, and microwavability, (3) large surface area for printing, and (4) flexibility in package design. However, plastic packages are not as reliable as metal or glass packages due to the following limitations [46]: (1) lower tolerance to heat and pressure, (2) relatively poor oxygen barrier, (3) heat seal of plastic packages is not as reliable as double seam of metal cans, and (4) seal inspection is more difficult.

VII. RETORTABLE PACKAGES A. METAL AND GLASS PACKAGES Metal cans and glass jars have been widely used in thermal processing due to their mechanical strength and thermal stability under high temperature and pressure conditions. The excellent closure integrity of these packages is especially advantageous for protecting high water activity, low acid, meat-based foods against microbial contamination. Metal cans may be two- or three-piece cans made of tinplated steel, lacquered tin-free steel, or aluminum [1, 45]. An advantage of using glass jars is the visibility of the contents; however, careful operation and handling are required to prevent shock breakage [1].

References 1. Holdsworth, S.D. 1997. Thermal Processing of Packaged Foods. Blackie Academic & Professional, London, UK. 2. Ball, C.O., Olson, F.C.W. 1957. Sterilization in Food Technology-Theory, Practice and Calculations. McGrawHill, New York, NY. 3. Geankoplis, C.J. 1983. Transport Processes and Unit Operations, 2nd ed. Allyn and Bacon, Inc., Newton, MA. 4. Cowell, N.D., Evans, H.L. 1961. Studies in canning processes. IV. Lag factors and slopes of tangents to heat penetration curves for canned foods heating by conduction. Food Technology, 15:407–412. 5. Hayakawa, K. 1970. Experimental formulas for accurate estimation of transient temperature of food and their application to thermal process evaluation. Food Technology, 24:1407–1418. 6. Kessler, H.G. 1981. Food Engineering and Dairy Technology. Verlag A. Kessler, Freising, Germany. 7. Alstrand, D.V., Ecklund, O.F. 1952. The mechanics and interpretation of heat penetration tests in canned foods. Food Technology, 6:185–189. 8. Townsend, C.T., Somers, I.I., Lamb, F.C., Olson, N.A. 1954. A Laboratory Manual for the Canning Industry. National Food Processors’ Association, Washington, DC. 9. NFPA. 1971. Processes for low-acid canned foods in glass containers. Bulletin 30-L. National Food Processors’ Association, Washington, DC. 10. NFPA. 1982. Processes for low-acid canned foods in metal containers. Bulletin 26-L, 12th ed. National Food Processors’ Association, Washington, DC. 11. Hersom, A.C., Hulland, E.D. 1980. Canned Foods. Thermal Processing and Microbiology, 7th ed. ChurchillLivingstone, Edinburgh.


12. Codex Alimentarius Commission. 1986. ALINORM 86/16, Appendix VI. World Health Organization, Rome. 13. Stumbo, C.R. 1953. New procedures for evaluating thermal processes for foods in cylindrical containers. Food Technology, 7:309–315. 14. Stumbo, C.R. 1973. Thermobacteriology in Food Processing, 2nd ed. Academic Press, New York, NY. 15. Hayakawa, K. 1969. New parameters for calculating mass average sterilizing values to estimate nutrients in thermally conductive food. Canadian Institute of Food Science and Technology Journal, 2:167–170. 16. Teixeira, A.A., Dixon, J.R., Zahradnik, J.W., Zinsmeister, G.E. 1969. Computer optimization of nutrient retention in thermal processing of conduction heated foods. Food Technology, 23:137–142. 17. Manson, J.E., Zahradnik, J.W., Stumbo, C.R. 1970. Evaluation of lethality and nutrient retentions of conduction-heating foods in rectangular containers. Food Technology, 24:1297–1302. 18. Jen, Y., Manson, J.E., Stumbo, C.R., Zhradnik, J.W. 1971. A procedure for estimating sterilization of and quality factor degradation in thermally processed foods. Journal of Food Science, 36(4):692–698. 19. Schultz, O.T., Olson, F.C.W. 1940. Thermal processing of canned foods in tin containers. III. Recent improvements in the general method of thermal process calculation. Food Research, 5(4):399–407. 20. Hayakawa, K. 1968. A procedure for calculating the sterilizing value of a thermal process. Food Technology, 22:905–907. 21. Leonhardt, G.F. 1978. A general lethal-rate paper for the graphical calculation of processing times. Journal of Food Science, 43:660. 22. Hayakawa, K. 1973. Modified lethal rate paper technique for thermal process evaluation. Canadian Institute of Food Science and Technology Journal, 6(4):295–297. 23. Patashnik, M. 1953. A simplified procedure for thermal process evaluation. Food Technology, 7:1–5. 24. Toledo, R.T. 1991. Fundamentals of Food Process and Engineering, 2nd ed. Van Nostrand Reinhold, New York, NY. 25. Murphy, R.Y., Johnson, E.R., Marks, B.P., Johnson, M.G., Marcy, J.A. 2001. Thermal inactivation of Salmonella senftenberg and Listeria innocua in ground chicken breast patties processed in an air convection oven. Poultry Science, 80:515–521. 26. Hicks, E.W. 1951. On the evaluation of canning processes. Food Technology, 5:134–142. 27. Hurwicz, H., Tischer, R.G. 1952. Heat processing of beef. I. A theoretical consideration of the distribution of temperature with time and in space during processing. Food Research, 17:380–392. 28. Ball, C.O. 1923. Thermal process time for canned foods. Bulletin No. 37, National Research Council, Washington, DC.

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29. Gillespy, T.G. 1953. Estimation of sterilizing values of processes as applied to canned foods. II. Packs heating by conduction: complex processing conditions and value of coming-up time of retort. Journal of the Science of Food and Agriculture, 4:553–565. 30. Jacobsen, F. 1954. Note on process evaluation. Food Research, 19:66–79. 31. Hayakawa, K. 1971. Estimating food temperatures during various heating and cooling treatments. Journal of Food Science, 36:378–385. 32. Hicks, E.W. 1958. A revised table of the Ph function of Ball and Olson. Food Research, 23:396–400. 33. Pflug, I.J. 1968. Evaluating the lethality of heat processes using a method employing Hick’s table. Food Technology, 33:1153–1156. 34. Hayakawa, K. 1978. A critical review of mathematical procedures for determining proper heat sterilization processes. Food Technology, 38(3):59–65. 35. Hayakawa, K., Downes, T.W. 1981. New parametric values for thermal process estimation by using temperatures and z values expressed in degree Celsius units. Lebensmittel-Wissenschaft und-Technologie, 14:60–64. 36. Jackson, J.M., Olson, F.C.W. 1940. Thermal processing of canned foods in tin containers. IV. Studies of the mechanisms of heat transfer within the container. Food Research, 5(4):409–420. 37. Olson, F.C.W., Jackson, J.M. 1942. Heating curves. Theory and practical application. Industrial & Engineering Chemistry, 34:337–341. 38. Berry, M.R.Jr, Bush, R.C. 1989. Establishing thermal processes for products with straight-line heating curves from data taken at other retort and initial temperatures. Journal of Food Science, 54(4):1040–1042, 1046. 39. Alles, L.A.C., Cowell, N.D. 1971. Heat penetration into rectangular cans of food. Lebensmittel-Wissenschaft und-Technologie, 4(2):50–54. 40. Lopez, A. 1987. A Complete Course in Canning, Vol. 1: Basic Information on Canning. The Canning Trade, Inc., Baltimore, MD. 41. Rees, J.A.G., Bettison, J. 1991. Processing and Packaging of Heat Preserved Foods. Blackie Academic and Professional, Glasgow. 42. Footitt, R.J., Lewis, A.S. 1995. The Canning of Meat and Fish. Blackie Academic and Professional, Glasgow. 43. Peterson, W.R., Adams, J.P. 1983. Water velocity and effect on heat penetration parameters during industrial size retort pouch processing. Journal of Food Science, 48:457–459, 464. 44. Kisaalita, W.S., Lo, K.V., Staley, L.M., Tung, M.A. 1985. Condensation heat and mass transfer from steam/air mixtures to a retort pouch laminate. Canadian Agricultural Engineering, 27(2):137–145. 45. Brody, A.L. 2002. Food canning in the 21th century. Food Technology, 56(3):75–79. 46. Brody, A.L. 2003. The return of the retort pouch. Food Technology, 57(2):76–79.

135

Edible Films and Coatings

S.-Y. Lee and V.C.H. Wan

Food Science and Human Nutrition Department, University of Illinois

CONTENTS I. History of Edible Films and Coatings ..............................................................................................................135-1 II. Definition ..........................................................................................................................................................135-2 III. Functions of Edible Films and Coatings ..........................................................................................................135-2 A. Retard Moisture Migration ........................................................................................................................135-2 B. Retard Gas Transfer....................................................................................................................................135-2 C. Retard Aroma Loss/Gain............................................................................................................................135-2 D. Retard Lipid Migration ..............................................................................................................................135-3 E. Improve Mechanical Properties ................................................................................................................135-3 F. Carrier of Additives....................................................................................................................................135-3 IV. Components of Edible Films/Coatings..............................................................................................................135-3 A. Film Forming Agents ................................................................................................................................135-4 1. Proteins................................................................................................................................................135-4 2. Polysaccharides ..................................................................................................................................135-5 3. Lipid ....................................................................................................................................................135-5 4. Composite or Bilayer Film..................................................................................................................135-5 B. Plasticizer ..................................................................................................................................................135-6 1. Definition and Functions ....................................................................................................................135-6 2. Selection of Plasticizers ......................................................................................................................135-6 C. Surfactant....................................................................................................................................................135-6 V. Method of Making EFC ....................................................................................................................................135-6 A. Casting........................................................................................................................................................135-6 B. Compression Molding ................................................................................................................................135-7 C. Extrusion ....................................................................................................................................................135-7 VI. Factors Affecting Film Functionalities ..............................................................................................................135-8 A. pH ..............................................................................................................................................................135-8 B. Heat Treatment and Irradiation ..................................................................................................................135-8 C. Concentration and Molecular Weight of Macromolecule..........................................................................135-8 VII. Future Research Direction ................................................................................................................................135-8 References ....................................................................................................................................................................135-9

I. HISTORY OF EDIBLE FILMS AND COATINGS The properties and applications of edible films and coatings (EFC) have been studied extensively during the past few decades. It has been demonstrated that EFC can extend shelf life and maintain quality of various food products.

In fact, applying EFC to a food product is not a new concept. Waxing on fruits to reduce water loss and to provide attractive gloss has been used for thousands of years (1). In the sixteenth century, Englishmen wrapped foods with fat, a process called “larding,” to prevent water loss (2). Furthermore, sausage casings made with collagen, which is mainly used for containment of meat batter, is one of the earliest forms of food processing. 135-1

135-2


II. DEFINITION Edible films and coatings are classified differently by definition. Edible films are defined as a pre-formed edible layer which can be placed on or between food components. Edible coatings are defined as a thin layer of edible material formed as an integrated coating on food surfaces (3).

III. FUNCTIONS OF EDIBLE FILMS AND COATINGS A. RETARD MOISTURE MIGRATION Maintaining an appropriate range of moisture level is crucial for the quality and shelf life of food products. Physical structure of bakery products would collapse if moisture is absorbed. Sensory properties such as texture and flavor are also significantly affected by the changes in moisture content. Furthermore, water loss of food products such as fruits and vegetables, decreases shelf life. One of the functions of edible coatings is to retard moisture migration. Studies have shown that some EFC are efficient moisture barriers (4). Lipid edible films and bilayer films made with lipid and protein or polysaccharide are generally better moisture barriers than protein and polysaccharide films due to higher level of hydrophobicity. Waxes have the lowest water vapor permeability (WVP) among edible film-forming materials. They have 25 times less WVP compared to common oil films and 100–200 times less WVP than for protein films (5). Raisins pretreated with starch and coated with beeswax had less moisture loss than untreated raisins after five weeks of storage (6). Chocolate brownies coated with beeswax and methylcellulose (MC) had significantly less moisture gain than uncoated brownies. This study also showed that moisture barrier property of MC coating alone is poor, since there is no difference in moisture gain between MC coated and uncoated brownies (7). Besides coating products, edible films can be placed on or between layers of food product to retard moisture migration from a higher moisture level component to a lower moisture level component. Kamper and Fennema (8) developed an edible bilayer consisting of stearic-palmitic acid and hydroxypropyl methylcellulose (HPMC), and placed it between tomato paste and crackers. It significantly delayed water transfer from tomato paste to crackers. Conventionally, waxes are applied to fruits and vegetable surfaces to reduce shrinkage due to water loss (1).

B. RETARD GAS TRANSFER The levels of carbon dioxide and oxygen that come in contact with a product have to be taken into consideration in order to retain quality of the product and consequently lengthen shelf life (9). The primary deterioration that is involved with gases is rancidity, developed by oxidation

of lipids, which results in off-flavors. Products with high content of fat such as nuts and potato chips are susceptible to rancidity (10). Protein and polysaccharide coatings which are highly impermeable to fat, oil, and oxygen are effective in preventing lipid oxidation (11). Wu, Rhim, Weller, Hamouz, Cuppett, and Schnepf (12) reported that wheat gluten, soy protein, chitosan, and carrageenan coatings effectively controlled lipid oxidation of precooked beef patties. Lee, Trezza, Guinard, and Krochta (13) showed that rancidity of whey-protein-coated peanuts was significantly lower than uncoated peanuts. It is desirable to regulate gas transfers during storage of fruits and vegetables, since respiration of fruits and vegetables depletes oxygen and increases carbon dioxide level. If the level of oxygen is too low, anaerobic respiration will occur and lead to abnormal ripening, development of off-flavors, and spoilage. In the presence of oxygen, production of ethylene increases and promotes ripening and senescence, which results in shorter shelf life (14). Internal modified atmosphere can be achieved by applying edible coatings made with materials that have low permeability to gases. Protein and polysaccharide coatings are generally good barriers to gases. Lee, Park, Lee, and Choi (15) reported lower initial respiration rate of apples coated with carrageenan or whey protein than uncoated apples. Enzymatic oxidative browning is another deterioration that can be prevented by minimizing the uptake of oxygen. When oxygen, polyphenol oxidase, and copper ion are present, enzymatic oxidative browning occurs, which turns phenol, a colorless substance, into a brown substance, melanoidins. This could, thus, lead to the decrease in acceptance of the product. Delayed browning of apple and potato slices coated with calcium caseinate or whey protein solutions were observed by Le Tien, Vachon, Mateescu, and Lacroix (16).

C. RETARD AROMA LOSS/GAIN Aroma, along with other attributes, such as appearance, taste, and texture is important to the quality of food products. Aroma is perceived when volatile compounds are dissolved in the nasal cavity and perceived by the olfactory system. Volatile compounds that are lost to or picked up from the storage environment could be regulated with edible coatings (11). Debeaufort and Voilley (17) showed that wheat gluten film is an effective barrier for 1-octen-3-ol, which represents a smell of mushrooms. They suggested that it can be used for wrapping cheeses to prevent aroma gain of 1-octen-3-ol from the refrigerator. Oranges coated with cellulose-based coating or commercial shellac coating had retained higher concentration of volatile compounds after storage up to 55 days than uncoated oranges (18). Shellaccoated apples had higher concentrations of fruit-like and apple-like volatiles than uncoated apples, which was due to the reduction in evaporation rate of these volatile


compounds (19). Nisperos-Carriedo, Shaw, and Baldwin (20) also stated that fruits coated with beeswax emulsion and TAL Pro-Long (commercial coating composed of different fatty acids and carboxymethyl cellulose (CMC) sodium salt) effectively retained and increased volatile compounds that were considered important to fresh orange flavor.

D. RETARD LIPID MIGRATION Lipid migration is a major problem in confectionary products. Liquidly lipid such as fatty acid tends to migrate to the surface of chocolate coatings, which results in soft and sticky surface (21,22). Chocolate “bloom” is a result of migration of cocoa butter from chocolate to the surface. Nelson and Fennema (21) investigated lipid barriers of five hydrocolloid films — MC, hydroxypropylmethyl cellulose (HPMC), CMC, carrageenan, and polyethylene glycol alginate (PGA) — and reported that they all were effective barriers to lipid migration. Methods of reducing fat intake during deep-frying have been investigated extensively due to the rising health concerns of consumers. Studies have shown that edible coatings that had good lipid barrier property could significantly trim down fat intake of fried products. Soy protein isolate (SPI), whey protein isolate (WPI), and MC coatings were listed as the best film-forming materials to be used to reduce fat absorption of fried dough (23,24). Rayner, Ciolfi, Maves, Stedman, and Mittal (25) reported that doughnut mix and potato fries that were coated with soy protein film had considerable fat reduction. They also showed that consumers preferred the coated fries over the uncoated. Balasubramaniam, Chinnan, Mallikarjunan, and Phillips (26) explained that the layer of thermal gel formed by HPMC film controlled the transfer of fat between the meatball and the oil medium, and consequently reduced fat intake.

E. IMPROVE MECHANICAL PROPERTIES Mechanical strength of products can be improved by EFC. The structure of extruded or molded products can be protected by coating with EFC (9). Waxing of fruits and vegetables minimizes surface abrasion during handling of these products (27). Xie, Hettiarachchy, Ju, Meullenet, Wang, Slavik, and Janes (28) investigated puncture strength of eggshell coated with SPI, WPI, CMC or WG. They showed that eggshell coated with SPI and WPI had the greatest puncture strength, which implied that the SPI and WPI EFC could minimize breakages of eggs during processing, handling, and storage. Guilbert, Gontard, and Gorris (4) stated that food filling coated with edible coating could reinforce the structure and protect the filling. In addition to improving mechanical strength of products, EFC such as mineral oil (29) and hydrocolloid coatings

135-3

such as pectinates, alginates, and starch (10) were used to minimize stickiness of food products. Improving the mechanical and handling properties can lead to a smoother process operation and reduction in product loss.

F.

CARRIER

OF

ADDITIVES

EFC can provide a cohesive surface structure for additives such as anitmicrobial agents and antioxidants. Antimicrobials are substances that are added to EFC to improve quality and shelf life of products by retarding growth of yeast, molds, and bacteria during storage and distribution. Examples of food grade antimicrobial agents are organic acids and their salts such as benzoic acid, sodium benzoate, sorbic acid (SA), potassium sorbate, propionic acid, lactic acid, and acetic acid (30,31). Cagri, Ustunol, Osburn, and Ryser (32) investigated the effect of adding antimicrobial, p-aminobenzoic acid (PABA) and/or SA into WPI coating for bologna, summer sausage and hot dogs. Zein film coatings with nisin were shown to effectively prevent growth of L. monocytogenes on readyto-eat chicken (33). Antioxidants may be added to EFC to protect products from oxidation which results in oxidative rancidity, degradation of nutrients, and discoloration. Tocopherols, carotenoids, acids (as well as their salts and esters), and phenolic compounds such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are some examples of food grade antioxidants. Antioxidants such as ascorbic acid and citric acid were added to MC edible films, which were applied to mushrooms and cauliflower. It was found that the coatings significantly decreased oxygen permeability of the vegetables and thus slowed the browning reactions (34). Lee, Park, Lee, and Choi (15) also reported that adding these two antioxidants along with oxalic acid to whey protein or carrageenan coatings helped maintain the color of minimally processed apple slices. Calcium chloride, known to be a firming agent, was incorporated into EFC to inhibit softening of fruits and vegetables. Lee, Park, Lee, and Choi (15) reported that addition of calcium chloride to whey protein or carrageenan coatings in acidic condition could minimize softening of apple slices. Firmness of kiwifruit slices were also maintained during storage by adding calcium chloride to coating (35). EFC could also serve as adherence surfaces for seasonings and flavor enhancers (10). Salt and flavorings were added to wheat gluten and dextrin coatings and modified food starches and gum Arabic coatings (36).

IV. COMPONENTS OF EDIBLE FILMS/ COATINGS EFC are made with a high molecular weight molecule as the backbone, a plasticizer, and a surfactant if needed. In this section, macromolecules including protein, polysaccharide,

135-4


and lipid that are used to make EFC will be discussed. They are listed in Table 135.1 along with the solvent they are dissolved in to make the film-forming solution.

1. Proteins

A. FILM FORMING AGENTS Film forming capability of macromolecules largely depends on their polymer backbone (37). Chain length, number of functional groups, and structure of macromolecules influence the functionality of EFC. For example, wheat gluten film is hydrophobic in nature because it has a large number of non-polar amino acids (38). Polysaccharides and proteins are hydrophilic in nature, while lipids are hydrophobic. Therefore, EFC made with polysaccharides or proteins have limited water vapor barrier properties while EFC made with lipids have good water vapor barrier properties. Table 135.2 summarizes some of the WVP values of protein and polysaccharides that are investigated in the literature. Oxygen permeability (OP), in contrast, is high in lipid films while low in polysaccharide and protein films. Krochta (42) summarized that protein EFC such as corn zein and wheat gluten films are better oxygen barriers

TABLE 135.1 Film Preparation for Different Macromolecules Macromolecule

Solvent

Protein Soy protein Corn zein Wheat gluten Whey protein Polysaccharides Methyl cellulose Hydroxypropylmethyl cellulose Hydroxypropyl cellulose Lipids Beeswax Shellac

Water 95% ethanol Water-95% ethanol Water Water-95% ethanol Water-95% ethanol Water-95% ethanol Melt 95% ethanol

TABLE 135.2 Water Vapor Permeability Values of Different Edible Films Reference 105 106 69 78 62 107 39

Composition c

d

SPI /GLY (5:3) WGe/GLY (15:6) WPIf/GLY (1.6:1) CZg/GLY MCh/PGi HPCj HPC/SAk (1.1:1)

Thicknessa

Condition

WVPb

0.000254 0.087 0.121 — 0.025 0.05 0.019

25°C, 50%RH 25°C, 50%RH 25°C, 65%RH 25°C, 50%RH 25°C, 52%RH 21°C, 85%RH 27°C, 97%RH

2.54 1.41 1.39 0.59 1.00 0.11 0.0005

Thickness in mm, bWVP in ⫻10⫺9 gm⫺1 s⫺1 Pa⫺1, csoy protein isolate, glycerol, ewheat gluten, fwhey protein isolate, gcorn zein, hmethyl cellulose, ipropylene glycol, jhydryoxypropylcellulose, kstearic acid. a

d

than polysaccharide films. In general, as the hydrophobicity of the film increases, OP decreases.

Proteins can be classified into water-soluble and waterinsoluble proteins. Soy proteins and whey proteins are examples of water-soluble proteins, while wheat gluten and corn zein are water insoluble proteins.

a. Soy proteins Soybean, in the family of Leguminosae, is believed to be originated in Eastern Asia (43). The whole soybean, with 40% protein, 21% fat, and 34% carbohydrate, has much higher protein content than other grains which usually have 8–15% protein (44). Globulin is the major protein group of soy proteins. Soy proteins are further fractionated according to the molecular weight of the protein by ultracentrifugation. The four most widely known soy protein fractions are 2S, 7S (conglycinin), 11S (glycinin), and 15S. Conglycinin and glycinin make up approximately 60–70% of the soybean globulins (45). Many researches have been conducted to determine the mechanism of soy protein film formation. Okamoto (46) suggested that heat-denatured proteins, which are partially unfolded and more hydrophobic, move to the surface of the film solution, and as water evaporates, protein molecules interact to form film structure. Rangavajhyala, Ghorpade, and Hanna (47) found that aggregation of proteins under heat treatment is through hydrogen bonds and intermolecular disulfide bonds. Kinsella (45) stated that under alkali condition, glycinin breaks down to subunits and unfolds due to disulfide bond cleavage, which is followed by gelation of the protein solution. He also suggested that 11S fraction dissociates into subunits and aggregates under heat treatment. b. Whey proteins Whey is a by-product of cheese production. There are five main components in whey proteins: α-lactalbumin, β-lactoglobulin, bovine serum albumin, immunoglobulins, and proteosepeptones. Different components can be fractionated by differential solubility, electrophoretic, and chromatographic methods. Researchers have shown that EFC can be made with β-lactoglobulin and bovine serum albumin alone (48), as well with WPI and whey protein concentrate (WPC). Denatured whey protein films are water insoluble due to the intermolecular disulfide bonds induced by thioldisulfide interchange and thiol oxidation reactions which are promoted by heat treatment (3). c. Wheat gluten Wheat gluten is the protein of wheat kernels which accounts for 8–15% of the dry weight (49). Most of the protein


consists of gliadins and glutenins which are only soluble in alcohol. Therefore, wheat gluten films are prepared by dispersing wheat gluten in a mixture of ethanol and water. The pH of wheat gluten film solutions have to be adjusted to the range of 2 to 4 or 9 to 13 to avoid extreme acidic or alkaline conditions, and to avoid isoelectric point (pI) of wheat gluten at which proteins coagulate. Gontard, Guilbert, and Cuq (50) reported that wheat gluten films made in acidic conditions (pH ⬇ 2 to 6) have better sensory and visual properties than films made in alkaline conditions. However, Gennadios, Brandenburg, Weller, and Testin (51) reported that wheat gluten films made in alkaline conditions have higher tensile strength. Wheat gluten films are cohesive and elastic (51). Due to the high glutamine content of wheat gluten, highly cooperative protein-protein interactions occur, which contribute to the cohesiveness of the film (52). Hydrogen bonds between hydrated gluten are responsible for film’s elasticity (53). Wheat gluten films are very effective oxygen barriers (54). Like other protein-based EFC, wheat gluten films have poor water barrier properties because of the hydrophilic nature of the proteins (42).

d. Corn zein Corn zein, which is the prolamine fraction of corn proteins, account for about 45–50% of corn proteins (55). It has large amounts of non-polar amino acids, which contribute to the hydrophobic nature of zein (56). The low polar amino acid content and high nonpolar amino acid content also contribute to the insolubility of corn zein in water (57). Therefore, corn zein is generally solubilized in a mixture of water and alcohol (ethanol). Shukla and Cheryan (55) concluded that corn zein films are glossy, tough, hydrophobic, and good lipid barriers. Tomatoes coated with corn zein had delayed ripening and color development, due to low permeability of oxygen of corn zein EFC, when compared to tomatoes coated with typical shrink wrap films. However, tomatoes coated with corn zein EFC exhibited higher weight loss than the ones coated with typical shrink wrap films, due to high WVP of the zein films (58). They also compared corn zein–coated tomatoes with uncoated tomatoes. Again, they showed that corn zein EFC significantly delayed color development of tomatoes. In this case, corn zein–coated tomatoes reduced softening and weight loss over a period of 8 days comparing to the uncoated tomatoes (59). 2. Polysaccharides Polysaccharides such as cellulose and its derivatives, starches and its derivatives, pectins, seaweed extracts, and gums are used to form EFC. Cellulose is a rigid material composed of plant cell walls. It is insoluble in water due to high amount of intramolecular hydrogen bonding in the cellulose polymer

135-5

(60). Therefore, cellulose is generally dispersed in water and ethanol. MC, hydryoxypropylcellulose (HPC), and HPMC are examples of water-soluble cellulose ethers that are modified by etherification. MC is the least hydrophilic among the soluble cellulose ethers (9,60). It is shown to have better moisture barrier property than other cellulose ether (24,61). Mechanical and barrier properties of MC films with different plasticizers were investigated (62). Cellulose edible films are tough and flexible (60). They are resistant to oxygen and lipid migration. Cellulose-based films exhibited lower OP than synthetic films, such as low density polyethylene film (LDPE) (40). MC, HPMC, and HPC coatings applied to fried pastry mix and fried potato ball reduced fat uptake (23,24,61). Williams and Mittal (61) showed that HPC and MC films reduced water loss of fried pastry mix. Cellulose-based EFC, like protein-based EFC, are hydrophilic. Therefore, they generally have poor water barrier properties. Park and Chinnan (40) reported that WVP of cellulose films were 100 times greater than LDPE. Cellulose-based films were supplemented with a layer of shellac to form an edible film layer to separate one food phase from another phase (63). 3. Lipid Lipids that are generally used for making EFC are waxes (i.e., paraffin and carnauba wax), mineral oil, fatty acids, monoglycerides, resins (i.e., shellac), and rosins. EFC made with lipid are cohesive and flexible (64). These characteristics are dependent on molecular weight of both hydrophilic and hydrophobic phases, branching, and polarity of lipid. As hydrophobicity of the film increases, OP decreases. Films that are made with resins such as shellac have lower permeability to oxygen and carbon dioxide than films that are made with waxes. On the other hand, waxes are more resistant to water vapor than other lipid film, due to the tight orthorhombic arrangement of the crystals. Lipid films, in general, are good water vapor barriers due to their hydrophobic nature. Some example applications of wax and oil coatings are on fruits and vegetables to prevent moisture loss (14), on raisins to prevent moisture migration from the raisin to dry cereal (10), and on confectioneries to provide gloss (64). 4. Composite or Bilayer Film Fats, fatty acids, and waxes can be added to polysaccharide or protein films to improve water barrier properties (38). There are three main types of composite films: lipid bilayer films, emulsion films, and composite films made of polysaccharide and protein. Bilayer films composed of HPMC and solid lipids such as beeswax (2), and corn zein with a layer of oil or waxes (65) had significantly lower WVP than protein or polysaccharide films alone. Emulsion films developed with whey proteins, beeswax, and glycerin showed

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reduction in WVP by 50% compared to films made of whey proteins alone (41). Krochta (42) stated that emulsion films had even lower WVP than bilayer films due to the optimum orientation of fatty acids in emulsion films. SPI and wheat gluten composite film was made by mixing the macromolecules with the plasticizer and solvent to form the film-forming solution. The film was shown to have lower WVP and improved TS than film made with SPI alone (38).

B. PLASTICIZER 1. Definition and Functions Plasticizer is defined as “a substantially nonvolatile, high boiling, nonseparating substance, which when added to another material changes the physical and/or mechanical properties of that material” (37). Plasticizers induce flexibility of films by reducing the degree of hydrogen bonding and increasing intermolecular spacing of the polymers (66). 2. Selection of Plasticizers Selection of plasticizers is based on compatibility of the plasticizer and the substance which they plasticize. For example, water-soluble substance should be plasticized with compound(s) containing hydroxyls (37). The number and position of hydroxyl groups and the number of hydrogen bonds capable of forming with the macromolecule affect the efficiency of plasticization (67). Cho and Rhee (68) stated that hydrophilicity and concentration of plasticizers affect moisture sorption of SPI films. SPI films plasticized with glycerol, which is more hydrophilic than sorbitol, absorbed more moisture than sorbitol-plasticized films. Plasticizer concentration also affected plasticizing effect of whey protein films (69). Addition of plasticizer increases permeability of the film due to the increase in free volume (66). Studies showed that plasticizers increased oxygen and/or water vapor permeability of MC films (62), gellan films (70), whey protein films (71), and gelatin films (72). Increased concentration of plasticizer further increased permeability of edible films (73,74). Water and polyols such as glycerol, propylene glycol (PG), polyethylene glycol (PEG), and sorbitol are commonly used plasticizers. Glycerol is the most widely used plasticizer, probably because it has small molecule weight, which enables it to incorporate into polymer matrix very easily. Studies have been conducted to investigate the effect of incorporating different plasticizers into polymers on mechanical and permeability properties of edible films. McHugh and Krochta (71) reported that sorbitol-plasticized whey protein film has lower OP than glycerol-plasticized film. Sucrose-plasticized β-lactoglobulin film showed the lowest OP, followed by sorbitol and glycerol, while PEG 200- and 400-plasticized films exhibited the poorest oxygen barrier property (48). Glycerol-plasticized β-lactoglobulin

film had higher tensile strength (TS) and percent elongation than sorbitol-, PEG 200-, PEG 400-, and sucrose-plasticized films at equivalent amount of plasticizers (48). Chick and Ustunol (75) reported that sorbitol-plasticized lactic acid casein films were more effective in oxygen and water barrier properties than glycerol-plasticized film. Addition of plasticizers often decreases TS and increases elongation-at-break of caseinate films (76), MC films (62), peanut films (77), SPI films (68), gellan films (70), and whey protein films (69,71). Effects of using mixture of plasticizers on mechanical and WVP properties were investigated. Park, Bunn, Weller, Bergano, and Testin (78) reported that as the ratio of glycerol to PEG decreased, TS of wheat gluten film increased, while elongation and WVP decreased. A 50:50 mixture of PEG and glycerin plasticizers for protein edible films exhibited the highest TS (78). Environmental factors such as relative humidity and temperature of the room where films are made also affect the plasticizing effect of plasticizers. Gontard, Guilbert, and Cuq (52) reported that the plasticizing effect of water for wheat gluten film is highly temperature dependent.

C. SURFACTANT Surfactants are surface-active compounds that have the ability to reduce the interfacial tension between two interfaces since they have both hydrophilic and hydrophobic ends. They are usually incorporated into emulsion systems. Surfactants are chosen according to hydrophilic-lipophilic balance (HLB) and phase inversion temperature (PIT). HLB is a system of values of 1 to 40 according to the hydrophobic and hydrophilic portion of the surfactant. Surfactants with low HLB are used in water-in-oil (w/o) emulsification and surfactants with high HLB values were used in oil-in-water (o/w) emulsification. PIT is the value where emulsions reverse from o/w to w/o depending on the temperature. It is believed that o/w interfacial tension is the smallest at PIT. Naturally occurring emulsifiers, phospholipids, monoglycerides, soy lecithin, sodium stearoyl lactylate, sodium lauryl sulfate, propylene glycol alginate, and paraffin wax are some examples of food grade emulsifiers.

V. METHOD OF MAKING EFC Two broad categories of manufacturing EFC are: wet processing technologies (i.e., casting) and low-moisture processing technologies (i.e., compression molding and extrusion) (80).

A. CASTING Casting is one of the most common methods to make stand-alone films, which can be used to evaluate physical


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and chemical properties of films. Proteins or polysaccharides, in concentrated or naturally occurring form, are dispersed in a mixture of water (with addition of solvent for water insoluble proteins and polysaccharides) and plasticizer(s). After thoroughly mixing the components, pH of the film forming solution is adjusted to the desired range by titrating with acidic or basic solution. Subsequently, the film forming solution may undergo heat treatment for a period of time. Then a controlled amount of solution is filtered and poured into a casting plate. Then this film forming solution is allowed to dry to a stand-alone film at a specific condition (temperature and relative humidity) and for a specific time period (⬃12 to 24 hr). Finally, the dried film is peeled from the plate and evaluated for various basic film properties. Example of the casting procedure of making soy protein isolate film is showed in Figure 135.1. Drying temperature of the film should be taken into account since studies have shown that it had an effect on film properties. Thickness of wheat gluten films (81), and whey protein films (82) decreased by increasing the drying temperature. As temperature increases from 70°C to 80°C and 90°C, TS and elongation of peanut film increased while WVP and OP decreased. At 90°C, peanut films had the lowest WVP and OP and the highest TS (77). TS of wheat gluten film investigated by Kayserilioglu, Bakir, Yilmaz, and Akkas (81), on the other hand, decreased as drying temperature increased. Perez-Gago and Krochta (83) explained that WVP of an emulsion film of WPI-beeswax decreased as drying temperature increased, probably due to change in the lipid crystalline morphology and/or lipid distribution within the matrix.

which softens when it is heated, is placed on one half of a mold. Heat and pressure are applied to the mold once it is closed. Film material then fills the mold cavity and polymerization occurs. The film is, then, obtained by cooling the mold. One of the differences between compression molding and extrusion is that flowability of the film-forming material for compression molding can be low, while for extrusion, the material needs to have high flowability. Because compression molding has very limited production amount, it is economical for small production. Compression molding was used recently as one of the methods to make EFC. Foulk and Bunn (84) showed that SPI films could be produced by compression molding. Films were made according to the method developed by Poly-Med Inc. (84). Mechanical and barrier properties of the SPI films that have various solubility were significantly different. They concluded that compression molded acetylated SPI film could be used as commercial thermoplastic. Slightly yellow and transparent SPI films plasticized with ethylene glycol (EG) formed by compression molding under a pressure of 15 MPa at 150°C was developed by Wu and Zhang (85). Due to the physical cross-linking between protein chains induced by EG, water adsorption of SPI films was reduced. SPI films made by compression molding had improved TS, breaking elongation, water resistance and thermostability, and therefore, they suggested that the thermoplastic materials from SPI could be used commercially for food packaging. Other proteins such as whey proteins (86) and cottonseed proteins (80) were also used to successfully produce compression-molded films.

C. EXTRUSION B. COMPRESSION MOLDING Compression molding is one form of low-moisture processing method used to make EFC. Thermoplastic material,

Low-moisture process technologies take less energy and time. One of the advantages of extrusion is that it could be a continuous process in-line and could obtain larger

Dissolve 5 g of SPI in a constantly stirred mixture of 100 ml distilled water and 2.5 g plasticizer

Conditioned in 50% RH chamber for two days before being tested for WVP

pH of the solution adjusted to 10 ± 0.1 with 1 N NaOH

Dry at ambient conditions (25°C) for approximately 20 hr. It was then peeled from the plate

Heated for 20 min in a constant temperature water bath at 70°C

Poured (60 ml) onto a leveled Teflon-coated glass plate (22.5 cm × 30.5 cm)

Strained through eight-layered cheese cloth

FIGURE 135.1 Flowchart for making soy protein isolate film.

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production volume. Extrusion has been used to manufacture industrial polymers such as LDPE (87). A blend of polypropylene and thermotropic liquid crystalline polymer (TLCP), Rodrun LC5000, was fed to a twin-screw extruder and polymerized through a mini-extruder to form a film (88). Biodegradable films manufactured by melt blow extrusion were developed with polyvinyl alcohol (PVA) and collagen hydrolysate (CH) (89). A patent of a soy protein thermoplastic composition containing soy protein, carbohydrate filler, a reducing agent, a plasticizer, and water manufactured by extrusion was granted to Jane and Wang (90). They showed that this soy protein blend has the desirable flowability for processing by extrusion. Feasibility of using other edible film materials such as wheat gluten (91) and corn zein (92) in extrusion was also investigated. Redl, Morel, Bonicel, Vergnes, and Guilbert (93) suggested that extrusion of wheat gluten with plasticizers is feasible under steady-state conditions. Koh and Lim (94) concluded that protein cross-linking is an important characteristic of the leathery structure of extruded wheat gluten. They reported that extruded wheat gluten had higher water absorption capacity and lower protein solubility than unextruded wheat gluten. Plasticizers were also added to the polymer matrix to improve flexibility of the film for extrusion. Water and glycerol were added to extruded corn gluten meal (92) and extruded soy protein (90).

VI. FACTORS AFFECTING FILM FUNCTIONALITIES A. pH Formation of edible films only occurs at a certain pH range (46,95). In general, pH of the film solution should be away from the isoelectric point (pI) of the protein used as the film-forming macromolecule. SPI films formed well at pH range of 7.5 to11, while no films were formed at pH range of 3.5 to 5.5 due to coagulation of soy protein (95). When the pH of the film-forming solution of WPI-beeswax emulsion film was adjusted away from the pI of whey proteins, films had lower WVP (96). Also, if protein is one of the components of the film forming solution, the pH should be adjusted not to be extremely acidic or extremely alkaline, since intramolecular protein repulsive force develops under extremely acidic and alkaline conditions. Therefore, films formed in these conditions will be less dense and more permeable (51). Film opacity, solubility, WVP, and mechanical properties of wheat gluten films were affected by pH (52). They reported that the films made at pH 5 were the strongest, while films made at pH 6 had the lowest WVP. Jangchud and Chinnan (77) also found that as pH of the peanut film-forming solution increased, protein solubility increased, and the film was darker and more yellow.

Sian and Ishak (95) investigated the effect of pH on the composition of soybean protein-lipid films. They reported that films prepared at higher pH (pH ⬎ 7.5) had higher protein proportion and lower fat proportion than films that were prepared at lower pH.

B. HEAT TREATMENT AND IRRADIATION Heat treatment breaks intramolecular disulfide bonds in proteins and allows the proteins to unfold. Then the unfolded bonds interact and form intermolecular disulfide and hydrophobic bonds, which reduce mobility of protein solution. As temperature of the heat treatment is increased, solubility of SPI films decreased (47). Minimum requirement of heat treatment is different for various macromolecules. Whey protein films need to be heated at 75°C for 30 min in order to form intact water-insoluble films (69). Heat treatment also influences appearance. Heated films are smoother and more transparent than unheated films (97). Cuq, Boutrot, Redl, and Lullien-Pellerin (98) stated that thermal treatment induces inter- and intramolcular cross-linking of proteins which improve mechanical strength. Wheat gluten films developed by Micard, Belamri, Morel, and Guilbert (99) which underwent heat treatment (above 110°C for 15 min and above 90°C) were stronger and more flexible. When WPI-calcium caseinate and SPI-WPI films underwent γ-irradiation (100), puncture strength and water resistance significantly increased. It is due to protein crosslinkage which contributed to a more ordered and stable structure. Ouattara, Canh, Vachon, Mateescu, and Lacroix (101) also reported that covalent bonds formed between protein molecules under irradiation decreased WVP of caseinate-whey protein films.

C. CONCENTRATION AND MOLECULAR WEIGHT OF MACROMOLECULE An intact film cannot be obtained if the concentration of the film-forming macromolecule is too high or too low. If the concentration of macromolecules is too high, the filmforming solution will form a gel. If the concentration of macromolecules is too low, film will not form due to the lack of intermolecular interactions (71). Molecular weight (MW) of cellulose also had influence on film properties. As molecular weight of HPMC increased from 22,000 to 26,000 and 86,000, WVP of film decreased. This suggested that it may be due to decreased mobility of the molecules as MW increased (102).

VII. FUTURE RESEARCH DIRECTION Basic mechanical and barrier properties of stand-alone edible films have been extensively investigated. Various EFCs have shown high potential to be used commercially.


Composite and bilayer films, especially, received a lot of attention because of the improvement of water barrier property that they offer. Continuous research on understanding and modifying properties of composite/bilayer films are to be expected. Besides studying the basic properties of EFC alone, the latest interest of EFC research is to investigate the feasibility of applying EFC onto different food products. Physical structure, chemical reaction between the EFC and the product, and sensory properties (103,104) of the product should further be investigated.

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100. M Lacroix, TC Le, B Ouattara, H Yu, M Letendre, SF Sabato, MA Mateescu, G Patterson. Use of gammairradiation to produce films from whey, casein and soya proteins: structure and functional characteristics. Radiation Physics and Chemistry 63:827–832, 2002. 101. B Ouattara, LT Canh, C Vachon, MA Mateescu, M Lacroix. Use of gamma-irradiation cross-linking to improve the water vapor permeability and the chemical stability of milk protein films. Radiation Physics and Chemistry 63:821–825, 2002. 102. E Ayranci, BS Buyuktas, EE Cetin. The effect of molecular weight of constituents on properties of cellulosebased edible films. Lebensm Wiss u Technol 30: 101–104, 1997. 103. SY Lee, KL Dangaran, JX Guinard, JM Krochta. Consumer acceptance of whey-protein-coated as

104.

105.

106.

107.

compared with shellac-coated chocolate. J Food Sci 67(7):2764–2769, 2002. SY Lee, JM Krochta. Accelerated shelf life testing of whey-protein-coated peanuts analyzed by static headspace gas chromatography. J Agric Food Chem 50(7): 2022–2028, 2002. AH Brandenburg, CL Weller, RF Testin. Edible films and coatings from soy protein. J Food Sci 58(5): 1086–1089, 1993. G Cherian, A Gennadios, C Weller, P Chinachoti. Thermomechical behavior of wheat gluten films: effect of sucrose, glycerin and sorbitol. Cereal Chem 72(1): 1–6, 1995. HJ Park, MS Chinnan. Gas and water vapor barrier properties of edible films from protein and cellulosic materials. J Food Eng 25:497–507, 1995.

Part P Ingredients Technology

136

Seasonings and Spices

Zhang Lin

International Flavors & Fragrances (China) LTD

CONTENTS I. Seasonings ............................................................................................................................................................136-1 A. Basic Groups of Ingredients ........................................................................................................................136-2 1. Salt ......................................................................................................................................................136-2 2. Acid......................................................................................................................................................136-2 3. Flavour Enhancers ..............................................................................................................................136-2 4. Savoury ................................................................................................................................................136-3 II. Spices & Herbs ....................................................................................................................................................136-3 A. Classification of Spices by Sensory Characteristics ....................................................................................136-3 B. Main Spices in Asia ......................................................................................................................................136-3 1. Cinnamon ............................................................................................................................................136-3 2. Clove ..................................................................................................................................................136-3 3. Cumin ..................................................................................................................................................136-4 4. Fennel ..................................................................................................................................................136-4 5. Ginger ..................................................................................................................................................136-4 6. Red Pepper ..........................................................................................................................................136-4 7. Black Pepper........................................................................................................................................136-4 8. White Pepper ......................................................................................................................................136-4 9. Bay Leaves ........................................................................................................................................136-5 10. Chives ................................................................................................................................................136-5 11. Cilantro ..............................................................................................................................................136-5 12. Coriander ............................................................................................................................................136-5 13. Mint ....................................................................................................................................................136-5 14. Paprika ................................................................................................................................................136-5 15. Sesame Seed ......................................................................................................................................136-6 16. Turmeric ............................................................................................................................................136-6 References ....................................................................................................................................................................136-6

I. SEASONINGS [1]

Seasonings are compounds, containing one or more spices, or spice extractives, and other flavour ingredients such as salt, sugar, dairy products and flavour enhancers, which when added to a food, either during its manufacture or in its preparation, before it is served, enhances the natural flavour of the food and thereby increases it acceptance by the consumer.

The compounding of seasoning is considered a specialised, skilful art. The proper blending of such dissimilar components as spice extracts, essential oils, spices, salt,

sugars, monosodium glutamate, ribotides, dairy products, and the many other components that enter into complex seasoning mixtures require: ●

● ●

a high level of technical expertise and a long period of practical experience a list of government regulations and restrictions a sense of economics in the selection of the ingredients with the optimum use to which they can be used

A seasoning must be compounded in such a way that it increases the natural flavour of the product to be seasoned. 136-1

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It should not overpower or diminish the product’s flavour but add a balanced interest with an odour identity, a smoothly blended, rounded flavour with no perceptible undesirable aftertaste. In general the weaker the flavour of the product to be seasoned the lower the level of added seasoning required to achieve a satisfactory balance of flavour in the finished product.

A. BASIC GROUPS ● ● ●

● ● ● ● ● ● ● ● ●

●

OF INGREDIENTS

Salt Sugar/dextrose Food acids — citric/sodium diacetate/lactic/ malic/tartaric Flavour enhancers — MSG/I&G Herbs and spices Spice extracts Vegetable powders — tomato/onion/garlic Savoury — HVP/yeast extracts/reaction flavors Cheese/dairy powders Flavour top notes Colours Carriers — flour/starch/maltodextrin/lactose etc. Free flow agents

1. Salt Salt is an indispensable flavouring for all snacks, sweet or savoury. In many varieties of foods it is the predominant flavour note. Hundreds of chemical compounds are classified as salts, and most of the water-soluble ones exhibit what we recognise as a salty taste, but only pure sodium chloride gives this flavour in a form not modified by sour, bitter, or sweet tastes. In addition to having a pronounced and generally agreeable taste of its own, sodium chloride will modify other flavours. In most test situations, it has been found to enhance the sweetness of sugars and decrease the sourness of acids. In some liquid products the addition of small quantities of salt, even below the threshold level, will increase the apparent sweetness of dissolved sucrose. Wherever practical, salt should be applied to snacks as a topping. This ensures a quickly sensed saltiness which is a primary determinant of consumer acceptability. A sufficient level (super-threshold) should be applied to yield a distinct salty flavour, but gross overstating should be avoided because it can mask or depress desirable flavour notes such as the mild sweetness of potatoes, or accentuate undesirable flavours. 2. Acid The considerations which govern the choice of acid type and use concentration are extremely complicated. It has

been demonstrated that tartness or sour taste of the common food acidulants is directly related to the molar concentration of undissociated acid. Tartness values for various acids, as reported in the literature, vary considerably, probably reflecting differences in testing conditions. Citric acid and malic acid are quite close in organoleptic tartness value whereas tartaric acid is more tart. One part of citric or malic acid is reported to be equivalent to 0.8–0.7 parts of tartaric acid. The acids are reported to differ somewhat in their tartness character. Tartaric is slightly bitter, citric acid gives a sharper tartness peak than malic, which gives a smooth and long lasting tartness. Some general observations on the effect of organic food acidulants: ● ●

●

High viscosity reduces the organoleptic tartness. Organoleptic tartness drops when the free acid/sugar ratio is reduced. Organoleptic tartness is reduced by higher flavour level.

3. Flavour Enhancers Savoury or Umami is the descriptive term given to the glutamate found naturally in foods or as added monosodium glutamate. It is also used to describe combinations of glutamate and nucleotides. It usually works as a flavour enhancer, that is, bringing out the flavour of the food itself. Naturally occurring glutamate is found in foods as diverse as cheese such as parmesan, kelp, tomatoes, anchovies, and potatoes. Naturally occurring nucleotides — the main two being disodium-5⬘-guanylate (GMP) and disodium-5⬘-inosinate (IMP) — are mainly found in tuna fish, sardines, bonito, beef, prawns, chicken, and shitake mushrooms. Nowadays, monosodium glutamate is manufactured by fermentation commercially from molasses. Nucleotides are commercially produced by the natural fermentation of tapioca starch. Commercially, MSG is the more widely used — in various food products. More recently nucleotides have started to be used either in combination with naturally occurring glutamates, or with MSG. The three most common forms of nucleotides used are: IMP — Disodium inosinate GMP — Disodium guanylate Disodium ribotide (I ⫹ G) — which is IMP ⫹ GMP in a 50:50 mixture As mentioned before, glutamates can be used alone to impart the Umami flavour whereas nucleotides require a source of glutamate to function as a flavour enhancer. Nucleotides have a very marked synergistic effect with glutamate. The glutamate can be present naturally in the food product or can be added to the food. Even though


136-3

foods naturally contain nucleotides and glutamate, they are still used in seasoning as you would use salt. Very small amounts of nucleotides are added to replace some of the MSG to give a magnification of the flavour enhancement effect. Glutamates and nucleotides are completely watersoluble at use levels. IMP has a low moisture absorption which may be an important point when being used in dry mix products and favours. GMP has the strongest flavour enhancement and both nucleotides have high heat stability and tolerate a wide range of pH without large levels of decomposition. Nucleotides enable the manufacturer to control excessive saltiness in snack foods such as potato crisps and other snack items. MSG can be used to reduce salt levels and give the same amount of perceived saltiness to a product by bringing out the flavour.

Sour, astringent Sweet Sulfurous Warm, fruity Warm fragrant & cooling Warm heavy & aromatic Warm spicy and aromatic

Woody

B. MAIN SPICES

IN

Capers Anise, cardamom, fenugreek, star anise Garlic, onion Anise, bayleaf, caraway, cardamom, cumin, fennel, rosemary, savory Basil, oregano, peppermint, spearmint Cumin Allspice, basil, caraway, cardamom, cassia, celery, chervil, chilli, cinnamon, cloves, coriander, dillweed, ginger, mace, marjoram, nutmeg, pickling spice, sage, tarragon, thyme, saffron Cassia, cinnamon, cloves

ASIA[1] [2]

1. Cinnamon

4. Savoury We have many materials available to use which will enhance and/or add a savoury meaty flavour to seasonings. These products can be derived from yeasts and yeast autolysates, hydrolysed plant proteins, or meat products. Some materials add a savoury meaty flavour with no definite profile, some are specifically tailored to a profile (i.e., Chicken HVP, Beef yeast autolysate, and chicken processed flavours, etc). Available to us also are vegetable flavoured yeast autolysates, vegetable powders, many different sugars, starches, acids, and an entire range of natural identical flavours.

II. SPICES & HERBS

Cinnamon is the dried inner bark of various evergreen trees belonging to the genus Cinnamomum. At harvest, the bark is stripped off and put in the sun, where it curls into the familiar form called “quills.” Cinnamon in the ground form is used in baked dishes, with fruits, and in confections. Cassia is predominant in the spice blends of East and Southeast Asia. Cinnamon is used in moles, garam masala, and berbere.

[1]

Spices and herbs are aromatic natural products that are used to flavor food. Spices are the dried seeds, buds, fruit or flower parts, bark, or roots of plants, usually of tropical origin. Herbs are the leaves and sometimes the flowers of plants, usually grown in a climate similar to the Mediterranean.

A. CLASSIFICATION OF SPICES CHARACTERISTICS Flavour Characteristics Alliaceous Bitter

Fragrant & delicate Herbaceous Pungent & hot Pungent & sweet

BY

2.

Clove

SENSORY Spices

Onion, chives, shallots, garlic Celery seed, curry powder, fenugreek, hops, mace, marjoram, nutmeg, oregano, rosemary, saffron, savory, turmeric Bail, chives, shallots Dillweed, parsley, rosemary, saffron, sage, thyme Capsicum, ginger, horseradish, mustard, black and white pepper Cassia, cloves, cinnamon

Cloves are the dried, unopened, nail-shaped flower buds of the evergreen Syzygium aromaticum. They are reddishbrown in color and have a strong aroma. Cloves are an important ingredient in the spice blends of Sri Lanka and North India. They are used in garam masala, biryanis, and pickles. In the U.S., cloves are used in meats, salad dressings, and desserts. Clove is a key flavour contributor to ketchup and Worchestershire sauce seasoning blends. Chinese and German seasonings also depend on cloves to flavour meats and cookies.

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3. Cumin

Ginger is used in Indian curries, and Chinese, Japanese, and European spice blends. 6. Red Pepper

Cumin is the dried seed of the herb Cuminum cyminum, a member of the parsley family. The cumin plant grows to about 1 to 2 feet tall and is harvested by hand. Cumin is a key component in both chili powder and curry powder. The flavor of cumin plays a major role in Mexican, Thai, Vietnamese, and Indian cuisines. Cumin is a critical ingredient of chili powder, and is found in achiote blends, adobos, garam masala, curry powder, and baharat. 4. Fennel

Red pepper is the dried, ripened fruit pod of Capsicum frutescens, one of the most pungent capsicums. It is sometimes referred to as cayenne red pepper, having been named after the high heat chilies grown in the vicinity of the Cayenne River in French Guiana. Red pepper adds heat and bite to seasoning blends, meats, pickles, seafood, Italian, Indian, Mexican, and Caribbean cuisines. Red pepper is used in seasoned salt, chili powder, jerk, mole negro, and berbere seasoning blends. 7. Black Pepper

Fennel is the dried, ripe fruit of the perennial Foeniculum vulgare. Tall and hardy, this plant has finely divided, feathery, green foliage and golden yellow flowers. Oval seeds form in clusters after the flowers have died and are harvested when they harden. Fennel seeds are an important ingredient in seasoning blends of the Mediterranean, Italy, China, and Scandinavia. Fennel seeds may be roasted prior to incorporation into seasoning blends to intensify their flavor. Fennel is used in curry blends, Chinese five spice, mirepoix, and herbes de Provence. Fennel is also used to flavor fish, sausages, baked goods, and liquors. 5. Ginger

Ginger is the dried knobby shaped root of the perennial herb Zingiber officinale. The plant grows two to three feet tall. Once the leaves of the plant die, the thick roots, about 6 inches long, are dug up. Crystallized ginger is fresh gingerroot cooked in syrup and dried.

Black and white pepper are both obtained from the small dried berry of the vine Piper nigrum. For black pepper, the berries are picked while still green, allowed to ferment, and are then sun-dried until they shrivel and turn a brownish-black color. They have a hot, piney taste. Black pepper adds flavor to almost every food of every nation in the world. It is used in rubs, spice blends, salad dressings, and peppercorn blends. 8. White Pepper

Black and white pepper are both obtained from the small dried berry of the vine Piper nigrum. For white pepper, the berry is picked when fully ripe. The outer layer of shunken


136-5

skin is removed, leaving the dried, grayish-white kernel. It has a milder, more delicate flavor than black pepper. White pepper is used whole in pickling spices and marinades. Ground white pepper is used in light colored foods such as sauces and soups. It is especially popular in European cuisine.

that is otherwise identified as coriander, and from which coriander seed is obtained. Cilantro is used in salsas, chutneys, salads, dips, beans, and soups. Cilantro is used in Asian, Mexican, Indian, Tex Mex, Caribbean, and North African cuisines, and is used in seasoning blends such as masala, curry, salsa, and recados.

9. Bay Leaves

12. Coriander

Bay leaves or laurel, are the dried leaves of the evergreen tree, Laurus nobilis. The elliptically shaped leaves are light green in color and brittle when dried. They have a distinctively strong, aromatic, spicy flavor. Bay leaves is the approved term for this spice, but the name “laurel” is still seen frequently. It is used in soups, stews, stocks, pickles, marinades, tomato dishes, and meats. Mediterranean, French, Moroccan, and Turkish cuisines use bay leaves in spice blends such as bouquet garni and curry blends.

Coriander is the dried, ripe fruit of the herb Coriandum sativum. The tannish brown seeds have a sweetly aromatic flavor which is slightly lemony. A zesty combination of sage and citrus, coriander is actually thought to increase the appetite. Coriander is used in lentils, beans, onions, potatoes, hotdogs, chili, sausages, stews, and pastries. It is used in the cooking of North American, Mediterranean, North African, Mexican, Indian, and Southeast Asian cuisines, as well as spice blends, including curry powders, chili powders, garam masala, and berbere.

10. Chives 13. Mint

Chives, Allium schoenoprasum, are the reed-like stems of a perennial, bulbous plant of the lily family. The name “chives” is derived from the Latin cepa, meaning onion. Chives are a member of the onion family. It is used in cold soups, stir-fried items, cheese and cream sauces, dips, potatoes, and as a garnish. Chives are popular in European and Chinese cuisines and in seasoning blend fines herbes.

Mint leaves are dried spearmint leaves of the species Mentha spicata. The dark green leaves have a pleasant warm, fresh, aromatic, sweet flavor with a cool aftertaste. Mint leaves are use in teas, beverages, jellies, syrups, ice creams, confections, and lamb dishes. Mint is used in Afghanistani, Egyptian, Indian, and Mid-Eastern cuisines and spice blends such as chat masola, mint sauce, and green Thai curry.

11. Cilantro

14. Paprika

Cilantro is the dried leaves of the herb, Coriandrum sativum, an annual herb of the parsley family. Also known as Chinese parsley, cilantro has a distinctive green, waxy flavor. Cilantro is the usual name for the leaf of the plant

Paprika is the dried, ground pods of Capsicum annum, a sweet red pepper. It is mildly flavored and prized for its brilliant red color.

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Paprika is used in seasoning blends for barbeque, snack foods, goulash, chili, and the cuisines of India, Morocco, Europe, and the Middle East.

16. Turmeric

15. Sesame Seed

Sesame is the dried, oval-shaped seed of the herb Sesamum indicum. Sesame seed is harvested by hand. The seeds have a rich nut-like flavor when toasted. Sesame seed contains 25 percent protein. Sesame seeds are used to add texture and flavor to a variety of breads, rolls, crackers, and salad dressings. Middle Eastern, Muslim, and Asian seasoning blends use crushed, whole, and toasted sesame seeds for flavour and texture.

Turmeric is the dried root of the plant Curcuma longa. Noted for its bright yellow colour, it is related to and similar in size to ginger. Turmeric’s flavor resembles a combination of ginger and pepper. Turmeric is a powerful colouring agent. It is used to colour and flavour prepared mustard, pickles, relish, chutneys, and rice dishes as well as butter and cheese. It is also used in spice blends in the Caribbean, India, North Africa, the Middle East, and Indonesia such as curry powder and rendangs.

REFERENCES 1. Handbook of Spices seasonings and Flavorings by Susheela Raghavan Uhl. 2. Tiao Wei Pin Sheng Chan Gong Yi Yu Pei Fang by Zheng You Jun.

137

Sweet Flavor Application

Yuanchao Fang, Hangyu Jiang and Ming Cai International Flavors & Fragrances (China) LTD

CONTENTS I. Sweet Flavor Classification ................................................................................................................................137-1 A. Water-Soluble Flavors ................................................................................................................................137-1 B. Oil-Soluble Flavors ....................................................................................................................................137-1 C. Emulsion Flavors ........................................................................................................................................137-2 D. Powdered Flavors........................................................................................................................................137-2 II. Flavor Description ..............................................................................................................................................137-2 III. Food Flavor, Taste and Mouthfeeling Description and Analysis ......................................................................137-3 IV. Flavor Selection..................................................................................................................................................137-3 V. Sweet Flavor Application ..................................................................................................................................137-3 A. In Beverages ..............................................................................................................................................137-3 1. In Carbonated Drinks ........................................................................................................................137-4 2. In Juice Drink ....................................................................................................................................137-4 3. In Sports Drink and Isotonic Drink ....................................................................................................137-4 4. In Coffee Mix Drink............................................................................................................................137-5 5. In Powder Drink ................................................................................................................................137-5 B. In Dairy Products ........................................................................................................................................137-5 1. In Yogurt Drink....................................................................................................................................137-5 2. In Ice Cream ......................................................................................................................................137-6 C. In Confectionery ........................................................................................................................................137-6 1. In Hard Candy ....................................................................................................................................137-6 2. In Chewy Sweets ................................................................................................................................137-6 3. In Chewing/Bubble Gum ....................................................................................................................137-7 References ..................................................................................................................................................................137-7

Flavors are generally divided into two categories according to their end uses: sweet flavors and savory flavors. Sweet flavors include cola, orange, lemon, apple, strawberry flavors, etc. which are generally applied to sweet foods like beverage, dairy products, bakery products and confectionery. In some multinational companies, oral care flavors like peppermint and spearmint flavors are also divided into sweet flavors. Savory flavors include meat flavors, seafood flavors, mushroom, cheese flavors and so on, which are mainly used in savory foods such as spices, meat products, snack, seasoning, soup, etc.

A. WATER-SOLUBLE FLAVORS

I. SWEET FLAVOR CLASSIFICATION

Corn oil, triacetin and octyl and decyl glycerate (ODO) are used as solvent in this kind of flavors. This kind of flavor has higher concentration of chemicals, longer aroma retention ability and better stability against heating,

Sweet flavors can be divided into four categories according to their existence status:

Ethyl alcohol, propyl glycol and water are generally used as solvents for water-soluble flavors. Solvents account for 40%⬃99% of the composition in the formulae. These flavors are clear and have good topnote, but they are sensitive to heat. Water-soluble flavors are wildly used in beverage, ice cream, water ice, dairy products, pectin jelly, jam, etc.

B. OIL-SOLUBLE FLAVORS

137-1

137-2


but they are water-insoluble. Oil-soluble flavors are generally used in biscuit, confectionery, chocolate, chewing gum, etc.

C. EMULSION FLAVORS This kind of flavor is oil-in-water (o/w) flavor which contains two phases: water phase and oil phase. The oil phase is composed of flavor oils, weighting agents, vegetable oils, emulsifiers and antioxidants. The commonly used weighting agents include sucrose acetate isobutyrate (SAIB), ester gum, brominated vegetable oil (BVO), darmmar gum and elemi gum. There is strict legislation restrictions in different countries for each weighting agent. Because the specific gravity of flavor oils are generally between 0.84 and 0.87, and the specific gravity of beverage is above 1.00, weighting agents must be added to increase the specific gravity of oil phase. The specific gravity of several weighting agents is as follows: SAIB: 1.146 Ester gum: 1.085 BVO: 1.333 Darmmar gum: 1.065 Elemi gum: 1.03 The water phase consists of water, emulsifier (e.g. arabic gum), acid, preservative, thickening agent and antioxidant. Stokes’ Law plays an important part in the formulation of emulsion flavor. The law can be expressed with the equation as follows [1]: 2 gr2(d2 d1) V 9 η Where: V: g: r: d2: d1: η:

the velocity of creaming or sedimentation acceleration due to gravity the radius of the droplet the density of the dispersed phase the density of the continuous phase the viscosity of the continuous phase

According to Stokes’ Law, the following measures can be taken to decrease the velocity of creaming or sedimentation of droplets in the finished drinks: 1. Decrease the particle size at the premise of not influencing cloudiness 2. Decrease the difference of specific gravity between the two phases 3. Increase the viscosity of the continuous phase Emulsion flavors are wildly used in carbonated drinks, juice drinks and sports/isotonic drinks. It also can be used in ice cream, water ice, dairy products and bakery foods.

D. POWDERED FLAVORS This kind of flavor can be mainly divided into two categories. One is absorptive powdered flavor which is made by absorbing flavor base on carriers like maltose or maltodextrin. The other is encapsulation powdered flavor which is made by emulsifying and spray-drying the flavor base. This kind of flavor is mainly used in powder drinks.

II. FLAVOR DESCRIPTION It’s very important for food developers to give correct and precise description on flavor, which is the basic skills for food developer to create new food products. In flavor houses, primary, secondary and tertiary words are used to describe each flavor. For example, juicy, sweet, peely and other adjective words are used to describe orange flavor, so the primary word is orange, while juicy, sweet, peely and other adjective words are secondary words. If one orange flavor is very sweet with a little juicy note, it can be described as follows: Orange Sweet Juicy

(Primary) (Secondary) (Tertiary)

Generally, one can make the correct description on a variety of flavors after receiving at least one year’ training on description. In Table 137.1, a series of secondary/tertiary descriptive words are listed for commonly used flavors.

TABLE 137.1 Descriptive Words for Commonly Used Flavors Primary Words Orange Lemon Coffee Cola Apple Vanilla Banana Blueberry Grapefruit Grape Honey Mango Lime Peach Pineapple

Secondary/Tertiary Words Sweet, Juicy, Peely, Fresh, Oxidized, Aldehydic, Tangerine, Mandarin, Oily, Candy Juicy, Fresh, Peely, Oxidized, Oily, Candy Roasted, Brewed, Expresso, Sweet, Vanilla-like, Bitter, Fresh, Instant Spicy, Citrus, Woody, Oxidized, Vanilla-like Red, Green, Peely, Juicy, Delicious, Fresh, Ripe Vanillin, Hay-like, Creamy, French, Extracted Ripe, Green, Candy, Cooked, Spicy Juicy, Perfumed, Candy, Cooked, Ripe Juicy, Sweet, Bitter, Peely Concord, Muscat Floral, Herbal, Perfumed, Caramellized Juicy, Ripe, Green, Floral, Skinny Juicy, Peely, Oily, Candy, Fresh, Soapy Ripe, Skinny, White, Candy, Canned Juicy, Ripe, Canned, Candy


137-3

III. FOOD FLAVOR, TASTE AND MOUTHFEELING DESCRIPTION AND ANALYSIS In most cases, food developers tend to describe and analyze their competitors’ successful market sample. They are interested in the following information involved in the market sample: 1. Which kind of flavor is used in the product: orange, apple or lemon if the market sample contains only one type of flavor? 2. How many types of flavors are used in this product if the product contains several types of flavor: orange mango, orange lemon or orange mango peach? 3. Once the developer confirms that the product contains certain flavor (e.g. orange), he wants to know the flavor directions (e.g. juicy, sweet or sweet plus juicy). 4. Flavor solubility. For example, juice manufacturers add orange extracted flavors in juice product, but they may add orange topnote flavor or orange oil to improve the topnote of the juice product. The beverage developer has to gauge from his experience whether it contains orange topnote flavor or orange oil. 5. Food taste, mouthfeeling and texture. Flavors play an important part in food taste and mouthfeeling. Other factors affecting taste and mouthfeeling include sweeteners, acids, emulsifiers, thickening agents, mineral elements, vitamins, etc. The food developers must judge or guess from experience which specific function in the food product is caused by which specific kind of ingredient. Table 137.2 shows an example that describes a market coffee milk sample. TABLE 137.2 Flavor, Taste and Mouthfeeling Description of Market Coffee Milk Drink Coffee Milk Drink

Primary*

Secondary*

Tertiary*

Flavor

Coffee Milk Vanilla

Roasted Powdered Creamy, extracted

Burnt

Taste

Very sweet (judge from experience that sugar content is about 8%)

Mouthfeeling

Creamy, smooth, flavor lasting

* Only suitable for flavor description.

IV. FLAVOR SELECTION Flavor selection is the critical step for a food developer to create high-quality, popular and quality-consistent food. Much attention should be paid to the following factors during the selection of flavors: 1. Flavor supplier. 2. Legal status of flavors in different countries: natural, natural identical, artificial; Kosher or Halal; Does the flavor contain any chemical which is not allowed in the local country? 3. Flavor solubility: water-soluble or oil-soluble? 4. Flavor existence status: liquid, powder or emulsion? 5. Flavor flashpoint. Flavors with the flashpoint below 61°C are classified as dangerous goods, which are easy to fire or explode. These flavors should receive special care during the transportation, storage and usage. 6. Flavor price. 7. Flavor stability against heating, oxygen, light and storage. 8. Flavor dosage and specifications like specific gravity, refractive index, odor, color, etc. 9. Flavor combination. A food developer generally uses two or more flavors while creating a new food, especially matching a successful food sold well in the market. He has to use another flavor to complement the note which one flavor lacks; or use oil-soluble flavor to produce topnote in juice products although water-soluble flavor has been used. Flavor combination also causes the greatest difficulties for the competitors to match the market successful product.

V. SWEET FLAVOR APPLICATION In this chapter, we will concentrate on the application of sweet flavors to beverages, dairy products (e.g. yogurt, yogurt drinks, ice cream, water ice) and confectionery.

A. IN BEVERAGES The ingredients of beverage consist of water, sweetener, acidulant, colorant, flavor, stabilizer, thickener, antioxidant, concentrated juice, mineral element, preservative, etc. Flavors for beverage must be water-soluble. Generally, the ratio of flavor in the finished drink is between 0.01%⬃0.3%, but the needed dosage will depend on flavor category, beverage category and other factors. Although flavor only occupies a small ratio in the finished drink, it plays a critical role in the beverage flavor and acceptability.

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1. In Carbonated Drinks

a. Process flowchart of carbonated drinks There are two kinds of process for carbonated drinks: one time filling and two times filling. One time filling is much more popular than two times filling due to its consistent quality of the finished drink. Two times filling has been phased out by most beverage companies because the quality of the finished drink is inconsistent. The process flowchart of one time filling is as follows [2]: Water

Sugar

Acid, preservative, flavor, colorant, etc.

Water treatment

Heat to dissolve

Dissolve

Deaerate

Filter and decolor Blend

Absorb CO2

Cool

Mix quantificationally QC check

Clean

Bottle

Seal QC check Finished drink

b. Formula examples Example I: Carbonated cola drink The syrup: Sugar Phosphoric acid 85% Caffeine Color Cola flavor Water to make Throw: 1 5

650.0 g 3.60 g 0.60 g to suit as needed 1000 ml

a. Process flowchart of juice drink Treated water and sugar, acid, juice concentrate, etc. → Deaerate → Homogenize(170/40 bar) → Sterilize (105°C, 15S) → Fill (88°C) → Seal → Cool → QC check → Finished drink.

Sugar Citric acid Orange juice concentrate (8X) CMC(FH9, acid-proof) β-Carotene emulsion (1%) Orange emulsion Orange topnote Water to make

Example II: Carbonated orange flavored drink 778.0 g 1.50 g 9.00 g 0.175 g 0.040 g ⬃1.5 g 1000 ml

The syrup: brix 63.4°; acid% 0.90%; The finished drink: brix 13.1°; acid% 0.15%.

90.28 g 1.63 g 12.5 ml 1.0 g to suit ⬃1.0 g ⬃0.2 g 1000 ml

The finished drink: brix 9.50°; juice content (%) 10%.

Three kinds of orange flavors can be used in orange juice drink: orange emulsion, orange topnote flavor and washed orange flavor. Orange topnote flavor is used to improve the topnote, which dosage in the juice drink is 0.01%⬃0.02%; orange extract flavor improves body and bottom note, which dosage is 0.02%⬃0.08%. 3. In Sports Drink and Isotonic Drink

a. Process flowchart of sports drink and isotonic drink Sugar, glucose

Sweetener, acid, colorant, etc.

Heat to dissolve

Dissolve

Filter and decolor

The syrup: brix 53° The finished drink: brix 10.6°; volume of CO2 4.

The syrup: Sugar Sodium benzoate Citric acid Sunset yellow Tartarine Orange emulsion Water to make Throw: 1 5

2. In Juice Drink

b. Formula example: 10% orange juice drink

Cool

Fill

Two kinds of orange flavors can be used in carbonated drink: orange emulsion and water-soluble orange flavor, but the latter generally can’t be used solely in carbonated orange drink because it is a washed flavor and has relative weak flavor profile. The latter is generally used to improve body and bottom note. Orange emulsion can be solely used or combined with water-soluble orange flavor.

Mix Add flavors Add cloud Pasteurize (95C, 5S) Fill Seal Cool QC check Finished drink


137-5

b. Formula example: lemon flavored sports drink Sugar Glucose Acesulfame potassium Potassium citrate Sodium citrate Citric acid Malic acid Lemon washed flavor Cloud Water to make

50.0 g 15.0 g 0.14 g 0.30 g 0.30 g 1.60 g 0.80 g ⬃1.00 g ⬃1.00 g 1000 ml

The finished drink: brix 6.8°; total acid % 0.24%.

4. In Coffee Mix Drink

a. Process flowchart of coffee mix drink Sugar and emulsifier → Premix → Dissolve in hot water → Dissolve milk powder and starch → Add coffee extract → Dissolve sodium citrate, etc. → Add flavor → To make 1000 ml → homogenize @170/40 bar → Fill → Sterilize @121°C, 20 min → Cool → QC check → Finished drink. b. Formula example Sugar Whole milk powder Coffee extract (brix 51°) Sugar ester P1670 Sodium citrate Sodium ascorbate Instant textra (Nation starch) Vanilla flavor Milk flavor Coffee flavor Water to make

75.0 g 18.0 g 14.0 ml 0.40 g 0.30 g 0.30 g 2.0 g 0.40 g 0.33 g 0.85 g 1000 ml

The finished drink: pH 6.6; brix 11.0°.

5. In Powder Drink

a. Process flowchart of powder drink Sugar, acid, colorant, anti-caking agent, powder flavor, etc. → Blend → Sieve → Package → QC check → Finished powder drink. b. Example formula: mango flavored powder drink Sugar Citric acid Ascorbic acid Lake color #5 Lake color #6 Sodium citrate Anti-caking agent Malto dextrin Powdered cloud Powdered mango flavor Total

90.0 g 2.40 g 0.30 g 0.004 g 0.006 g 0.30 g 0.40 g 30.5 g ⬃0.30 g ⬃1.0 g 125.0 g

Use: 125 g dilute to 1000 ml for drink.

B. IN DAIRY PRODUCTS Yogurt, yogurt drinks, acidified milk drinks, ice cream and sherbets are classified as dairy products. Raw materials used in dairy products include the following: Fat: milk fat, cream, butter, butter oil, vegetable oil (sunflower oil, soybean oil, rapseed oil, etc.) Nonfat milk solids (NMS): proteins, lactose, mineral salts. Sweeteners: sucrose, glucose, HFCS, etc. Stabilizers: gelatin, pectin, xantham, carrageenan, agar-agar, CMC, guar, locust bean, sodium alginate, karaya, etc. Emulsifiers: monoglycerides, diglycerides, etc. Flavors: commonly used flavors in dairy products include coffee, milk, vanilla, nutty flavors (i.e. hazelnut), fruit flavors (i.e. orange, pineapple, strawberry), chocolate, cream, bean (i.e. green bean, red bean), ube yam, melon, etc. Water and air Juice preparations: orange, apple, strawberry, pineapple, banana, etc. Yogurt cultures Acids: citric acid, lactic acid, etc. Colorants Others

1. In Yogurt Drink

a. Process flowchart of yogurt drink Skimmed milk ↓ Pasteurize (e.g. 90⬃95°C for 3⬃5 minutes) ↓ Culturing to pH 3.8⬃4.2 ↓ Cool to about 20°C ←

Fruit juice Pectin dispersion Sugar, flavor, etc.

Slowly agitate for minimum 15 minutes ↓ Control pH 3.8⬃4.2 ↓ Pasteurize at 90⬃95°C for 10⬃15 seconds ↓ Homogenize at 150⬃200 kg/cm2 ↓ Cool to filling temperature ↓ Aseptic filling

137-6


candy, pectin jellies, chewy and bubble gums, chocolate, pressed tablet candies, jellies and marshmallows. Raw materials used in confectionery products involve:

b. Formula example: strawberry yogurt drink Skimmed milk yogurt Fruit juice (50% sugar) Pectin dispersion* Strawberry flavor Yogurt flavor Total

843 g 120 g 35 g ⬃1.0 g ⬃1.0 g 1000 g

* Pectin dispersion: 3.5 g pectin is dispersed in 31.5 g 65% sugar solution at high mixing speed. The finished yogurt drink: Fat: 7.6%; NMS: 8.0%; Sugar: 0.35%; Total solids: 15.95%.

2. In Ice Cream

a. Process flowchart of ice cream Mix at 50⬃60°C ↓ Pasteurize (e.g. 80⬃85°C for 20⬃25 seconds) ↓ Homogenize (e.g. 140/40 bar at 75⬃80°C) ↓ Cool to 4°C ↓ Age below the temperature 4°C

Sweetener: castor sugar, glucose syrup, invert syrup, synthetic and natural sweeteners Fat: butter, hydrogenated vegetable oil, cocoa butter Dairy products: fresh milk, butter cream, milk powder, condensed milk Gelling agents: starch, agar, gelatin, pectin, etc. Emulsifiers: lecithin, glycerol mono stearate, sucrose esters of fatty acids, span, tween, etc. Acid: citric acid Flavors: orange, lemon, strawberry, mint, blueberry, nutty flavors, etc. Nuts and fruits products Colorants Others 1. In Hard Candy

a. Process flowchart of hard candy Castor sugar, glucose syrup, water → Dissolve → Filter → Boil → Cool → Add flavor, acid and color → Blend → Cool → Desposit → Cool → Select → Pack b. Formula example: orange flavored hard candy

← Add flavor, color, etc. Freeze (e.g. 5.55°C at the outlet from the freezer) ← Incorporation of fruit, nut, cookie solids, ripple, etc. Harden to 30 ⬃ 35°C ↓ Store below the temperature 17°C

Castor sugar Glucose syrup Water Orange flavor Color Total

57.0% 29.7% 13.1% ⬃0.3% to suit 100%

2. In Chewy Sweets

b. Formula examples Table 137.3 displays typical formulas for ice cream in different regions.

C. IN CONFECTIONERY Market confectionery products can be mainly sorted as: hardboiled candy, chewy sweets (soft candy), gummy

a. Process flowchart of chewy sweets Gelatin, water

Glucose syrup, castor sugar, water

Hydrogenated palm oil

Dissolve at 60C

Dissolve

Melt

Filter

Filter

Gel

Boil (120C)

TABLE 137.3 Typical Formulas of Ice Cream in Different Regions [5] Ingredients Milk fat NMS Sucrose Glucose solids (36DE) Glucose solids (42DE) Stabilizer/emulsifier Flavor Total solids Overrun

U.S.A 10.0% 10.5% 12.0% 6.0% — 0.4% ⬃0.1% 38.9% 80–100%

Europe 8.0% 10.5% 12.0% — 3.0% 0.5% ⬃0.1% 34.0% 100–130%

Mix

Asia 6.0% 7.0% 14.0% 5.0% — 0.4% ⬃0.1% 32.4% 70–90%

Icing sugar

Blend Pull Shape Choose Pack

Flavor, acid, colorant


137-7

b. Formula example: peppermint flavored chewy sweets Castor sugar Water Glucose syrup Hydrogenated palm oil Emulsifier Gelatin 150 bloom Color Peppermint flavor Total

34.36% 18.38% 43.23% 3.06% 0.55% 0.22% to suit ⬃0.2% 100%

3. In Chewing/Bubble Gum

a. Process flowchart of chewing/bubble gum Gum base → Intenerate → Mix → Roll → Shape → Pack ↑ Icing sugar, flavors, other ingredients b. Formula example: blueberry flavored chewing/bubble gum Gum base Icing sugar

19.7% 59.7%

Glucose syrup Glycerin Blueberry flavor Total

19.8% 0.5% ⬃0.3% 100%

REFERENCES 1. X.S. Jiao. Natural food emulsifiers and emulsions (Chinese). Beijing: Science Press, 1999, pp. 100–150. 2. C.F. Shao, J.F. Zhao. Processing technology of soft drinks (Chinese). 7th ed. Beijing: China Light Industry Press, 1996, pp. 134–169. 3. Robert T. Marshall & W.S. Arbuckle. Ice Cream. 5th ed. New York: Chapman & Hall, 1997. 4. Y.S. Cai, W.Z. Zhang. Process technology and recipe of confectionary and chocolate (Chinese). Beijing: China Light Industry Press, 1999. 5. S.B.H. Guo. Process technology of dairy products. Beijing: China Light Industry Press, 2001.

138

Food Emulsions

John N. Coupland

Department of Food Science, The Pennsylvania State University

H. Sigfusson

Gorton’s Technology and Innovation Center

CONTENTS I. Emulsion Structure ..............................................................................................................................................138-1 A. Lipid Phase ..................................................................................................................................................138-3 B. Aqueous Phases ............................................................................................................................................138-4 C. Interface ........................................................................................................................................................138-4 D. Formation of Emulsions ..............................................................................................................................138-5 II. Properties of Food Emulsions..............................................................................................................................138-6 A. Texture ..........................................................................................................................................................138-6 B. Flavor ............................................................................................................................................................138-6 C. Color ............................................................................................................................................................138-7 III. Emulsion Stability................................................................................................................................................138-7 A. Creaming ......................................................................................................................................................138-8 B. Aggregation Kinetics....................................................................................................................................138-8 C. Types of Aggregation..................................................................................................................................138-10 IV. Effect of Process Conditions ............................................................................................................................138-11 Acknowledgements ....................................................................................................................................................138-11 References ..................................................................................................................................................................138-11

Only the simplest foods are present as one continuous phase. In practice, much of the texture and other complex behavior of food systems arises from phase heterogeneity. A common and relatively simple form of heterogeneity is when the food is present as a dispersed system — small particles of one material (or phase) in a second continuous phase [1]. We are concerned with that subset of dispersed systems where one of the phases is lipid and the other aqueous, i.e., emulsions. Some examples of water-in-oil food emulsions include milk, ice cream mix, mayonnaise, salad dressings, soups, beverage emulsions, and flavor emulsions. The classic definition of an emulsion requires both phases to be liquid but in foods this definition is frequently expanded to include systems where one or more phases are solid. It is possible to make an emulsion with either a lipid or an aqueous dispersed phase and this distinction largely governs the overall properties of the emulsions (e.g., a lipid-continuous system will disperse poorly in water, and have a low conductivity and a greasy mouthfeel). However,

in this work we will focus on aqueous-continuous emulsions. We will describe the structure of food emulsions and the effect this structure has on the properties of the food. We will consider the mechanisms of emulsion (de)stabilization and finally the effects of food processing on emulsion structure. Our goal in this work is to provide an accessible introduction to the main issues essential to the engineering of food emulsions, but clearly such a brief treatment cannot be comprehensive. In Table 138.1 we provide a selected bibliography of sources that may be helpful for further exploration of the subject.

I. EMULSION STRUCTURE Figure 138.1 shows an optical micrograph of a coarse food emulsion. The oil is present in spherical droplets in a continuous aqueous phase. The interfacial layer, although insignificant volumetrically, is essential to governing the 138-1

138-2


TABLE 138.1 Bibliography of Selected Further Reading in Food Emulsions Reference

Comments

“Colloids in Food” [54]

Although somewhat dated, the treatment of the fundamentals underlying the chemistry of food colloids remains unrivalled.

“An Introduction to Food Colloids” [55]

A simplified text covering many of the topics from #1.

“Foundations of Colloid Science” [27]

Highly technical and comprehensive on the fundamentals of dispersion technology. Particularly useful discussion of surfactants and self-assembled colloids.

“The Colloidal Domain” [16]

A good general reference particularly in the fields of the properties of surfaces and surfactants.

“Advances in Food Colloids” [56]

A useful update to #1.

“Food Emulsions: Particularly strong in the application of Principles, Practice and colloidal force arguments to food Techniques” [24] systems and analytical methods. “Physical Chemistry of Foods” [57]

Comprehensive treatment of all of the main aspects of food physical chemistry. Especially relevant are chapters 9–13 on aspects of dispersed systems.

FIGURE 138.2 Schematic diagram showing the effect of volume fraction on the structure and dynamics of an emulsion. (a) A dilute emulsion, the movement of the highlighted particle is not affected by other particles. This would be realistic up to a few percent volume fraction. (b) In a more concentrated emulsion (φ 10%) the movement of the highlighted droplet would be slowed by interactions with other droplets and the system would be more viscous and slower creaming than predicted from an extrapolation of the properties of the dilute system. (c) An ideally close packed emulsion where the particles are packed into a hexagonal array. The indicated droplet cannot readily move relative to the other droplets and the bulk material may develop elastic properties. Further concentration yields (d), a fluid foam where the droplets are distended into polyhedral shapes separated by narrow lamellae. Note ideal compression of (c) would yield a regular hexagonal structure but the polydisperse structure shown in (d) suggests some of the lamellae have ruptured to allow some coalescence.

First the amount of dispersed phase is typically expressed as a mass or volume fraction: mo φm ⫽ ᎏ mo ⫹ ma

FIGURE 138.1 Optical micrograph of a coarse food emulsion. The oil is present in spherical droplets several 10 s of micrometers in diameter (most food emulsions would be an order of scale finer) surrounded by an aqueous phase. The interfacial layer is seen as a thick line here, but that is an optical artifact. In reality the interfacial region discussed in the text would only be a few nanometers thick.

overall properties of the system. Although all emulsions must have an aqueous and a lipid phase and an interface between them, they may vary widely, and it is useful to develop a vocabulary to describe their microstructure.

vo φv ⫽ ᎏ vo ⫹ va

(138.1)

where φv and φm are the volume and mass fraction of dispersed phase and v and m are the volume and mass of the oil (subscript o) and aqueous (subscript a) phases. Mass and volume fractions are readily interconvertable knowing the density (ρ) of each phase. Droplet volume fraction can vary from zero to approaching 100% (Figure 138.2). As the volume fraction increases, the particles increasingly interact with one another until they are close packed (as shown in Figure 138.2). The maximum theoretical close packing of identical spheres is 0.7405, but in reality this type of highly organized structure does not occur and random close packing occurs at much lower volume fractions (0.64). Given an appropriate droplet size distribution (including particles approaching zero diameter) it would

Food Emulsions

138-3

Number

a c b

log size

FIGURE 138.3 Typical particle size distributions seen in emulsions. (a) has a larger median diameter than (b) but (b) is more polydisperse and so has more larger particles and may be less stable than (a). (c) is bimodal, very often the larger population of droplets is formed due to the flocculation of smaller droplets.

be possible to achieve a much higher volume fraction but Princen [2] points out that in practice most real polydisperse distributions do not exceed the perfect close packing of homogeneous spheres. Droplet volume fractions beyond close packing are only attainable by deforming the spherical droplets. Emulsion droplets are spherical (under all but the most extreme conditions) so can be characterized with a single length dimension. Real emulsions are polydisperse, i.e., contain droplets of varying sizes, and thus it may be more useful to represent their size as a distribution or in terms of a mean and polydispersity. For example a typical log-normal size distribution is shown in Figure 138.3a. Figure 138.3b has a lower median diameter, but as it has a broader distribution. It has more large droplets and may be effectively less stable. Figure 138.3c shows a bimodal distribution formed from the overlap of two polydisperse emulsions. Distributions such as Figure 138.3c are often seen in partly aggregated emulsions. Most stable emulsions have average diameters less than a few micrometers, and few are smaller than 100 nm.

A. LIPID PHASE The lipid phase is largely a mix of triacyglycerol molecules of animal or plant origin along with (typically) minor constituents of the lipids including free fatty acids and monoand di-glycerides. Food lipids typically crystallize over the range of temperatures found in food processing and storage so we may also be concerned with semi-crystalline droplets. In general the melting point of a fat decreases with decreasing chain length and increasing unsaturation. Fats cooled below their melting point may crystallize, but the crystallization process is often very slow. Fat crystallization is further complicated by the fact that there are often several stable solid forms under given conditions and their rate of

formation and interconversion may vary widely [3]. In the emulsified state the situation is further complicated as the fat is isolated into many self-contained droplets. In bulk fat it would require in principle only one nucleation event to crystallize all of the fat present but in the emulsified state each droplet must nucleate independently. In the most extreme case, each droplet is effectively pure and nucleation must occur homogeneously in the droplet [4]. Homogeneous nucleation is typically very slow and very large supercooling can occur when the number of droplets is much larger than the number of effective nucleating impurities. More commonly in foods there are sufficient impurities to allow most droplets to nucleate heterogeneously but this often still requires significant supercooling. Moreover, Walstra [5] pointed out that many real food emulsions contain several fat crystals, which implies multiple nucleation events per droplets. He argued this most likely occurs by secondary nucleation, the detachment of nuclei from the surface of a growing crystal. The pressure of the oil droplet surface also affects the development of crystal structures within the droplet. Lopez and co-workers [6, 7] showed that crystals are more disordered in the emulsion droplets than in bulk, presumably due to the physical constraints of the surface. The effect of the droplets was most strongly seen in the less stable crystal forms. These and other workers have also seen an emulsified fat crystallizes into an α-form more readily than the same fat in bulk [6–8]. A second major reaction we may be concerned with in emulsion lipids is oxidation [9]. Lipid oxidation can lead to rancid off-flavors and -odors that can spoil food when present at very low levels. Further oxidation can lead to the formation of potentially toxic compounds and can also degrade the nutritional value of foods. In general oxidation proceeds through a radical reaction between an unsaturated lipid and oxygen. Radicals are generated by interactions of metals, the action of light, or enzymatic activity and the reactivity of lipids increases greatly with the number of double bonds present. The reactivity of emulsified lipids is notably different from the bulk properties of the same lipid and offers new ways to control the reaction [10]. While oxygen is usually present in significant quantities in both the lipid and aqueous phases, some of the important oxidation catalysts are concentrated in one or either phase. Notable here are aqueous metal ions which are only effective catalysts of lipid oxidation when they can approach the lipid droplets. Consequently emulsion droplets with negatively charged surfaces (e.g., proteins pH ⬎ pI, sodium dodecyl sulfate) were much more prone to catalysis by iron cations than positively charged droplets. The actions of antioxidants are also different in dispersed systems. Frankel noted the “polar paradox” in which oil-soluble surfactants are more effective in stabilizing dispersed lipids while water-soluble surfactants are more effective in bulk [11]. He argued that in both cases

138-4


the antioxidant concentrates in the portion of the system most prone to oxidation (i.e., the emulsion droplets or the surface of the bulk respectively) where they can be most effective.

B.

AQUEOUS PHASE

The aqueous phase contains the water-soluble food ingredients including simple sugars and salts. Other ingredients may be largely water or oil soluble but partition to some extent between the two phases according to their partition coefficients: the ratio of the activity of the solutes in lipid and in water. Very often we are concerned with highly dilute additives such as flavors and antioxidants and it is reasonable to express a partition coefficient in terms of a ratio of concentrations. Lipids and sugars have partition coefficients close to infinity and zero, respectively but many other food ingredients have some solubility in either phase and while their equilibrium concentrations remain constant, individual molecules move dynamically between phases. The properties of the aqueous phase can also affect the interactions between the emulsion droplets. For example, the change on an ionizable group is a function of pH and so the amount of charge carried on a protein-stabilized emulsion droplets will decrease from a positive to a negative plateau value as the pH is increased (Figure 138.4). The ionic strength (⫽½ Σcz2, where c is the concentration of an ion of valence z) decreases the range of effective electrostatic interactions so they will be less important in salty systems. Some other components, particularly sugars, can act as co-solutes and stabilize proteins against thermal denaturation [12]. They achieve this by structuring the water around the hydrophobic groups on the protein and the same effect can reduce the hydrophobic drive to aggregation amongst denatured proteins.

Zeta potential (mV)

40 20 0 1.5 −20

3

4.5

6

7.5

9

10.5

pH

−40 −60 −80

FIGURE 138.4 Effective surface charge (ζ-potential) on fine hexadecane emulsion droplets stabilized with whey protein as a function of pH. The charge on the droplets is governed by the charge on the protein molecules and follows a typical sigmoidal shape with pH.

C. INTERFACE The fact that oil and water are immiscible and have a clear interface (Figure 138.1) stems from the strong waterwater attractive force. A water molecule is so strongly attracted to its peers that it will overcome the entropic drive for mixing with oil and remain as an isolated phase [13]. (In reality there is some small solubility of oil in water and although at the bulk scale an interface is a clear line separating the two phases, at the molecular level there is local mixing and an interfacial region where the concentration of both phases changes from its bulk value to its solubility in the second phase. It is possible to draw a Gibbs interface in this region so that the excess concentration on one side of the interface is equal to the deficit concentration on the other). The tendency of water and oil to demix also leads to a tendency to minimize the interfacial area because each surface water molecule is more exposed to the unfavorable oil phase than a corresponding molecule in bulk water [14]. The energy cost to increase the surface area of a system is manifested in the surface tension, a force opposing surface expansion: dG γ⫽ ᎏ dA

(138.2)

where γ is the interfacial tension, G is the surface excess free energy, and A is the surface area. The role of interfacial tension pushing against surface expansion also means droplet interiors are somewhat compressed (i.e., the Laplace pressure). Increased pressure can increase the solubility of lipid components and helps maintain the droplet’s spherical shape against applied forces. The unique molecular environment of a surface allows the accumulation of surface-active ingredients. Surfaceactive molecules have part of their structure water soluble and part oil soluble. Important examples in food emulsions are predominantly proteins [15] and to a lesser extent small molecule surfactants [16] and some hydrocolloids [17]. If a surface-active molecule is added to a two-phase system it can either remain free in one or both phases (enthalpic disadvantageous) or adsorb to the interface (entropically disadvantageous) [14]. The competition between terms means the amount of adsorbed surfactant will increase nonlinearly with added concentration. The maximum amount of sorbed surfactant is defined by packing concerns at the surface but typically for food proteins is in the order of a few mg m⫺2. Adsorbed surfactant shields the immiscible water and oil phases from one another and so decreases the interfacial tension from about 30 mN m⫺1 for a clean oil-water surface to a surfactant-dependent minimum at surface saturation [1]. The most important functional roles of the interfacial layer in controlling emulsion functionality are to: (i) lower the interfacial tension to ease the formation of the emulsions, (ii) self-repair incipient holes in the lamella separating two approaching droplets via the

Food Emulsions

Gibbs-Marangoni effect, and (iii) provide a basis for repulsive colloidal forces between droplets. This final point is crucial in limiting destabilization by aggregation (see below). Surface binding is not instantaneous. The potentially surface active molecule must first diffuse to the surface then “react” and bind. Diffusion is faster for smaller molecules so small molecules will usually develop an interfacial layer in a few seconds while a protein layer would take several minutes for the process to complete [14]. Although the binding of a single hydrophobic group to a surface is a spontaneously reversible event, the multiple binding of several sites on a polymer means that proteins will not typically spontaneously desorb from a surface. However if a more surface active material is added (i.e., small molecule surfactants) it may displace the protein from the interface by a competitive adsorption process. The surface protein concentration is not affected by small amounts of added surfactant, but will be completely desorbed following a small further increase [18]. Gunning and co-workers used atomic force microscopy to image protein displacement from a mica surface and saw that the initial portion of desorption the surfactant accumulated at the surface and pushed the protein into an increasingly thick and dense network surrounding the droplets [19]. At a critical level the surface protein network ruptured and detached from the surface. These workers termed this orogenic process as an analogy to the process of the formation of continents from tectonic plate activity. Oil soluble surfactants are less effective than aqueous surfactants at displacing proteins [20]. When globular proteins bind to a surface they often slightly rearrange their structures to better configure to the interfacial environment and as a result are often denatured. The interfacial protein concentration is relatively high even if the bulk concentration is relatively low and the properties of a concentrated protein solution are seen in two dimensions at the interface [21]. The proteins can cross react and form a viscoelastic two-dimensional gel that can improve the stability to coalescence. The same types of interprotein bonds responsible for bulk protein gelation (e.g., disulfide, hydrophobic) have been identified in the two dimensional surface analogue. Mixed protein films can phase separate

138-5

in two dimensions just as a thermodynamically incompatible polymer mixture can in bulk [22, 23].

D. FORMATION

OF

EMULSIONS

Emulsions can only be formed from bulk oil and aqueous phases through the application of amounts of energy. Some of the energy is used to create oil-water surface free energy but the bulk is “wasted” as heat losses in turbulent flow. Typically a coarse emulsion premix is made by blending or shaking the ingredients together. The particle size of the premix is very large, typically several (1–100) micrometers, and so they have a very short shelf life. However, the goal here is to produce a transiently homogeneous mixture that can be fed through a higher-energy secondary homogenization step for further particle size reduction to the desired final goal [24]. The second stage of homogenization can be achieved by a variety of technologies, e.g., colloid mill, ultrasonication, high pressure valve homogenizer, or microfluidizer, each of which enjoy various advantages and disadvantages but all serve to apply a critical mechanical stress to the droplets. As noted previously, emulsion droplets are spherical to minimize their surface area and are somewhat pressurized. To disrupt a droplet it must be stretched and eventually fractured and to achieve this the forces input from the homogenizer via the continuous phase must exceed the cohesive forces of the droplets (Figure 138.5) [25]. The cohesive strength of a droplet increases with surface tension and decreasing particle size so one of the main roles of a surfactant in facilitating homogenization is to reduce interfacial tension. The relationship between the applied forces and the operating conditions of the homogenizer depend on the type of flow occurring (i.e., laminar or turbulent) but generally particle diameter is believed to be inversely proportional to either shear rate in laminar flow or (power density) in turbulent flow [24]. It is possible for the homogenizer to very effectively fragment particles but there may be little or no net effect on the particle size because the newly formed droplets take a finite time to become coated in surfactant and may recoalesce before this can happen. Thus the second role of surfactant in homogenization is to rapidly diffuse to and adsorb to newly created

FIGURE 138.5 Schematic diagram showing the effect of applied force on a droplet. Initially the droplet is spherical but deforms into a spheroid under the applied force. Eventually a neck forms in the shape and fractures to form many smaller droplets. The small droplets formed can quickly recoalesce if they are not protected by surfactant or protein diffusion to the interface.

138-6


η⬘ ⫽ ki φi

surfaces to protect them before any droplet-droplet collisions occur.

II. PROPERTIES OF FOOD EMULSIONS We are rarely interested in food microstructure for its own sake but rather as a way to understand the sources of food quality [26]. So for food emulsions we are most concerned with how they affect perceived food texture, color, and flavor.

A. TEXTURE A dispersed system is always more viscous than the pure continuous phase due to the increased friction between the particle and the liquid layers causing greater energy dissipation. In a highly dilute, uncharged suspension containing non-interacting solid particles (volume fraction φ) the relative viscosity (η⬘, i.e., apparent viscosity normalized to that of the dispersed phase) is given by the Einstein relationship (Figure 138.6):

η⬘ ⫽ 1 ⫹ 2.5φ

(138.3)

The “hard-sphere” assumption may be acceptably valid when a thick surfactant layer limits the transmission of flow from the continuous to dispersed (liquid) flow but in most real foods there are significant particle-particle interactions that will further increase the effective viscosity (Figure 138.2b). Extensions to the Einstein equation are available to account for paired and multi-body interactions.

10

e

d

c

Relative viscosity

8 6

b

4 2

a

0 0.0

0.2

0.4 0.6 Volume fraction

0.8

1.0

FIGURE 138.6 Theoretical relative (i.e., normalized to the continuous phase) viscosity of an emulsion as predicted by (a) the Einstein equation, (b) a modified version of the Einstein equation including a second order term in volume fraction, and (c) the Krieger-Dougherty relation. (d) and (e) were calculated assuming the emulsion was flocculated. (e) and (f) were calculated assuming the emulsion was flocculated into flocs 10 times the primary particle radius with fractal dimensions 2.8 and 2.6 respectively.

(138.4)

i⫽0

where ki are constants (1, 2.5, 6, …). Using the first two powers Equation 138.4 reduces to Equation 138.3 and even using the third power it is only useful up to about 15 vol% [24]. At higher concentrations it is common to resort to empirical and semi-empirical expressions such as the Krieger-Dougherty relation:

φ η⬘ ⫽ 1 ⫺ ᎏ φmax

⫺2.5φmax

(138.5)

where φmax is a volume fraction approximating (0.6–0.7, Figure 138.2c) close-packing [27]. The φmax parameter can be allowed to relax between a high and low shear value to account for non-Newtonian behavior. The KriegerDougherty relation tends towards infinity at φ ⫽ φmax and gives no meaningful solution beyond that point. However highly concentrated emulsions have a real viscosity and also develop significant elastic properties (e.g., mayonnaise). In order to maintain the very high levels of dispersed phase (often ⬎80%) the droplets are distended and the microstructure approaches that of a foam with many polyhedral droplets in very close association (Figure 138.2d) [2].

B. FLAVOR Our perception of food flavor comes largely from volatiles released into the headspace in the mouth and detected by nerves at the back of the nose. As well as being volatile, many food flavors have a significant solubility in oil and aqueous phases so the presence of emulsified lipid can affect the kinetics and thermodynamics of their release and therefore our overall flavor perception. Considering the partitioning of a volatile between the oil, water, and headspace in a food emulsion we can quickly see the effects of fat content on the perceived flavor of foods (here presumed to be related to the absolute headspace concentration). Using the subscripts o, w, e, and g to refer to the oil, aqueous, overall emulsion, and headspace gas phases respectively we can define an effective partition coefficient between the emulsion and the headspace) [28]. Kgw Kge ⫽ ᎏᎏ 1 ⫹ (Kow ⫺ 1)φ

(138.6)

Figure 138.7a shows the effect of oil concentration on the headspace concentration of a number of different flavors with different partition coefficients (partition coefficient data from [24, 28]). The partitioning of the non-polar flavors (octanal, heptan-2-one) between the headspace and the emulsion decreases with oil concentration, the partition coefficient of the more volatile flavor (ethanol) increases. This is logical as oil provides a better reservoir for the non-polar flavors than the polar flavors. While volatile concentration does not necessarily correlate with sensory appreciation of flavor [29], this simple calculation shows the

Food Emulsions

138-7

magnitude of the changes that can be caused by changing the emulsion structure. For example, van Ruth and Roozen [30] studied the headspace concentration of volatiles above an oxidized sunflower oil in water emulsion and noted that while more volatiles partitioned into the headspace above the emulsion than the corresponding bulk fat, the relative change was different for different aromas. In another example fat content was shown to increase the amount of a non-polar flavor (linalool) released more than the polar flavor (diacetyl) [29]. Partition coefficients are thermodynamic constants but very often we are concerned with the dynamics of flavor release. Harrison and co-workers [28] proposed a mathematical model for the release kinetics of flavor from an emulsion. They assumed that as the droplets are relatively small, diffusion in the droplets and hence partitioning between oil and water occurs relatively fast, leaving the transport across the emulsion-headspace barrier as the ratelimiting step. Assuming the emulsion is well mixed they showed the concentration of gaseous volatile (cg) is given as:

cg(t) ve hD Age ᎏt ᎏ ⫽ 1 ⫺ exp ⫺ 1 ⫹ ᎏ Kgevg vg cg(∞)

(138.7)

0.005

where v is the phase volume, K is the partition coefficient, t is time, hD is the mass transfer coefficient in the emulsion, and A is the interfacial area of the emulsion surface (not the surface of the individual droplets). Emulsion particle size and volume fraction only entered into their formulation as terms controlling the viscosity of the emulsion (and hence hD). Neglecting this effect, this approach clearly shows the effect of Kge and hence oil content on the release kinetics. Some calculated rates of release of the aroma with partition coefficients calculated are shown in Figure 138.7b. While heptan-2-one is released more rapidly than ethanol in the higher water system (φ ⫽ 20%) the converse is true in the concentrated emulsion (φ ⫽ 80%). Harrison and co-workers obtained reasonable agreement with their model in a study of the release kinetics of diacetyl and heptan-2-one [28]. However other workers have challenged their assumptions and investigated the effect of particle size and interfacial barrier effects [31]. For example Miettinen and others [29] showed that decreasing droplet size had no affect on the amount of diacetyl flavor (i.e., polar) but increased the release of linalool (i.e., nonpolar) and the effects of surfactant type were quite minor (sucrose stearate vs. modified starch). The sensory perception of creaminess is associated with food emulsions and while it is related to the flavor present, rheology and particle size also play a complex role and vary by product [32].

0.004

C. COLOR

K ge

0.003 Heptan-2-one

Ethanol

0.002

Diacetyl

0.001

Octanal

0.000 0.0

0.2

0.4

(a)

1.0 0.8 Cg /Cg(inf)

0.6

0.8

1.0

Heptan-2-one ( = 80%) Ethanol ( = 80%)

Heptan-2one (

= 20%)

0.6 0.4

Ethanol ( = 20%)

0.2 0.0 (b)

Fine emulsion droplets have diameters approaching the wavelength of light and therefore scatter light efficiently. Consequently, emulsions containing oil concentrations above a few percent appear turbid and white. Finer emulsions scatter short wavelength light more strongly and therefore take on a bluish tinge. Theory has been developed to calculate the perceived color of an emulsion from droplet size and concentration. First the scattering effects of the droplets are calculated and from this reflectance spectrum and hence the tristimulus values [33].

Time

FIGURE 138.7 Calculated (a) partition coefficients and (b) flavor release kinetics for four volatiles as a function of emulsion volume fraction.

III. EMULSION STABILITY Emulsion destabilization is driven by the tendency to minimize interfacial area by lipid droplet coalescence, and by phase separation (under gravity) by creaming. These are both thermodynamic effects and so emulsions will inevitably break down eventually. The goal therefore is not absolute but adequate stability, i.e., we are as much concerned with kinetics as we are with thermodynamics. The product must last as long as expected for that type of food, be it seconds for vinaigrette dressing, days for a dairy product, or years for a beverage emulsion. Some of the main mechanisms are illustrated in Figure 138.8 and discussed further below.

138-8

(a) (a )


(b) (b )

(c) (c )

(d) (d )

(e) (e )

FIGURE 138.8 Schematic diagram illustrating the mechanisms of emulsion destabilization. The primary particles (a) flocculate (b) and rapidly cream (c) into a concentrated layer. The droplets may maintain their individual integrity in this state for a considerable period of time before (d) coalescing into a single phase. This is not the inevitable pathway, in some cases creaming (e) may occur without extensive flocculation. Note the two different creams shown (c and e) have different volumes. This is not the only route to destabilization.

A. CREAMING Oil is usually less dense than the aqueous phase of an emulsion, so it will tend to float to the surface in response to gravity or other applied forces. The movement of oil particles is impeded by viscous drag of the continuous phase and the net rate of particle movement (vs) can be approximated from a balance of these forces in Stokes’ equation: d 2(ρwater ⫺ ρoil )g vs ⫽ ᎏᎏ (138.8) 18ηc where d is oil droplet diameter, ρoil and ρwater are the densities of the respective phases, g is acceleration due to gravity, and ηc is the viscosity of the continuous phase. Equation 138.8 is based on a series of assumptions largely violated in real food emulsions, i.e., the droplets are isolated spheres moving in a Newtonian fluid (Figure 138.2a) but it provides some qualitative guidelines to limit creaming (e.g., minimize particle size, density difference, or increase the viscosity of the continuous phase to reduce the rate of creaming). For example homogenization reduces the size of milk fat droplets approximately tenfold so we would expect homogenized milk to be stable against creaming approximately 100 times longer. As the droplet volume fraction increases, particle-particle interactions become more important (Figure 138.2b), the rate of creaming slows and Equation 138.8 becomes less reliable. An analytical solution is available for moderate concentrations of spherical particles and in principle better fits could be achieved by incorporating higher powers of volume fraction. v ⫽ vs(1 ⫺ 6.5φ)

(138.9)

However a better description of the effect of volume fraction is achieved by assuming the effective viscosity (in

Equation 138.8) experienced by a creaming droplet in a concentrated emulsion is the viscosity of the emulsion (rather than the continuous phase) and calculating that using a Krieger-Dougherty type relation. In this formulation, the emulsion achieved solid-like character as the volume fraction approaches close packing and at this point creaming effectively stops (Figure 138.2c and d, [27]). Creaming can be readily and temporarily undone by gently shaking an emulsion. It can be measured by visually identifying the junction between a fat rich and fat poor region of a tube of emulsion, but only after creaming is well advanced. Earlier detection is possible using devices that can measure oil concentration as a function of position (e.g., ultrasonic velocity or optical reflectance), but it is often possible to identify systems likely to cream upon long term storage from particle size measurements or by using centrifugation to accelerate the process. In the remainder of this section we will discuss the aggregation mechanisms responsible for emulsion breakdown. We will first consider the kinetics of aggregation, the various types of aggregation and finally the effects of aggregation on emulsion properties.

B. AGGREGATION KINETICS Droplets in an emulsion move either by Brownian motion or under gravitational or other applied forces. The moving droplets collide and may (i) bounce off one another causing no change in the properties of the system, (ii) merge (coalesce) to form a single larger droplet, or (iii) stick together but maintain the shape of two conjoined droplets (i.e., flocculation). Either of the latter two cases will reduce the number of particles in the system and be a step towards destabilization. The rate of aggregation is given by the product of the rate of droplet-droplet collision (β) and the proportion of those collisions leading to reaction (α). Relatively simple terms for collision rates under static (i.e., perikinetic) and sheared (i.e., orthokinetic) conditions were derived by Smoluchowski and are given in Equations 138.10 and 138.11 respectively [34]. 2kT βperikinetic ⫽ ᎏ 3η

(138.10)

4G βorthokinetic ⫽ ᎏ d 3 3

(138.11)

where k is the Boltzmann constant, T is the temperature, η is the viscosity of the continuous phase, d is the droplet diameter, and G is the velocity gradient in the fluid. Smolokowski made several assumptions to derive these expression: (1) particles remain spherical after collision, i.e., complete coalescence, (2) no interparticle forces, (3) no particle or aggregate fracture, (3) fluid motion is exclusively either diffusional or laminar shear, (4) particles are of identical size, and (5) only two-body collisions occur. These

Food Emulsions

138-9

bases for a few of the more important interactions are described below:

Interaction energy

b Repulsive interaction

●

Electrostatic Repulsion. Most food emulsions are protein-stabilized and under most conditions those proteins have a significant net charge. Consequently protein-stabilized oil droplets act as charged particles. The magnitude of their charge depends on the pH (Figure 138.4) and the amount and type of interfacial protein. However in a given emulsion each droplet is likely to have a similar net charge and thus repel the other droplets. The magnitude of the electrostatic potential force is proportional to the square of surface potential and decreases exponentially moving away from the droplet. The exponential decay is faster in high ionic strength systems so the stability of charged emulsion often decreases in the presence of salt. (Note the ionic strength effect is theoretically independent of ion type. Some specific protein-ion interactions (notable casein and calcium) can also lead to strong droplet aggregation [36].) Demetriades and others [37, 38] studied the effect of salt and pH on the stability of thermally treated whey protein stabilized emulsions. They noted the emulsions thickened and creamed at pH pI and particularly at high salt concentrations. Presumably away from the isoelectric point the electrostatic repulsion was capable of preventing droplet aggregation.

●

Van der Waals Attraction. Transient dipoles in the bonds within matter lead to a weak colloidal attractive force. The force is relatively long-range, decaying with the reciprocal of distance, and is dependent on the dielectric properties of the component phases.

●

Hydrophobic Attraction. There is an energetic advantage to moving hydrophobic materials out of an aqueous environment. This is manifested in a strong attractive force between hydrophobic surfaces, which decays exponentially over quite long distances. While this force can be readily measured, until recently there has been relatively poor understanding of the physical basis of the relatively long range of the attraction [35]. However, Pashley [39] recently noted that oil was soluble (as a turbid dispersion) in water if the water was first rigorously degassed by freeze-thaw under vacuum. He proposed that the dissolved gasses were the cause of the longrange hydrophobic effect and once they were removed oil became effectively water-soluble. Emulsion droplets not adequately protected

c

a

Attractive interaction

Separation

FIGURE 138.9 Schematic illustration of some typical interaction potentials. (a) is attractive at all separation, (b) is repulsive, and (c) shows a barrier to droplet aggregation at intermediate separations.

are clearly violated in real systems so again the full analytical solution can only be used to (at best) give some qualitative guidance. More complete analytical and empirical solutions are available to deal with some of these but the simple equations are still widely used [34]. The collision efficiency parameter α is often used as a fitting parameter to describe flocculation kinetics, but should in principle be relatable to the interdroplet forces acting on the system. If two droplets approaching one another have a significant repulsive force between them they may not collide, whereas if the force is attractive they may veer towards one another and collide even if their original, unmodified trajectories would have caused them to miss. The collision efficiency parameter α would be respectively less than or greater than one in these cases. The interaction forces between droplets vary with range and tend towards zero at long separations. They can also be expressed as interaction potentials — the energy cost of bringing one particle to a given separation from a second particle. Some sample interaction potentials are given in Figure 138.9. The potential is negative at all separations for Figure 138.9a so an approaching particle will tend to be attracted to the second. At very low separations the potential becomes increasingly negative so it will be difficult for the reverse process to occur and the particles will irreversibly coalesce. Figure 138.9b is positive at all separations so the droplets will tend to be repelled from one another. Figure 138.9c is negative or zero at large separations so the particles may approach one another. There is then a positive barrier which can slow the aggregation of particles (if ⬎kT) but any particles which have sufficient potential energy to surmount the barrier will permanently coalesce into the potential energy pit at small separations. Interaction potentials can be measured directly or calculated as a sum of attractive and repulsive contributions from the physicochemical properties of the system [24, 35]. The physical

138-10


with surface active material have significant oilwater contact and will quickly aggregate as a result of the attractive hydrophobic forces. Surface protein usually shields the oil from the water and diminishes the hydrophobic attraction but surface denaturation can unfold the proteins and allow some protein-protein hydrophobic attraction to draw the droplets together. For example, Demetriades and others showed that thermally treating whey protein stabilized emulsions led to extensive aggregation [37, 38]. ●

Steric Repulsion. Adsorbed material acts as a barrier to coalescence at very short range (i.e., when the surface layers begin to overlap). The two main contributions to this mechanism are compression (volume exclusion) and mixing (osmotic) effects. Compression is always strongly repulsive but mixing can be attractive at slightly longer range depending on the solvent-polymer interactions [24]. Strong steric repulsion due to the protein layer at very short range is often responsible for the predominance of flocculation over coalescence in many food emulsions.

●

Depletion Interactions. Non-adsorbing particles are excluded from a region close to a surface equivalent to their hydrodynamic radius. In an emulsion, this sets up an osmotic pressure gradient between particle-rich and particle-free regions of the continuous phase that favors droplet aggregation to reduce the volume of this region. Depletion attraction is particularly important when a non-adsorbing polymer is added to a food emulsion (e.g., xanthan gum, protein aggregates) and can lead to extensive flocculation and creaming [40–42].

●

Hydrodynamic Repulsion. In sheared systems we can add hydrodynamic interactions to this list. Hydrodynamic forces represent the energy required to make the fluid between the approaching droplets “get out of the way” and so produces a repulsive force. The relative movement of the fluid will also tend to cause approaching particles to rotate and take on a curved trajectory and miss one another. Consequently the presence of hydrodynamic forces will always reduce the collision frequency, sometimes by up to 5 orders of magnitude, compared to rectilinear trajectories [34].

C. TYPES

OF

AGGREGATION

Flocculation is the aggregation of oil droplets without mixing of their contents whereas during coalescence the

membrane separating the approaching droplets is breached and allows the dispersed phases of the droplet to mix. Once oil is allowed to flow between droplets they quickly revert to a (larger) spherical shape. Coalescence can take place either following droplet collision (see above) or after periods of prolonged storage in a concentrated emulsion (often in a cream layer). The resistance of the interdroplet membrane to rupture depends on the strength of the protein films and the ability of the interdroplet repulsive forces to maintain a thick aqueous layer between the droplets. Surfactant layers can also favor film rupture if their optimal surface curvature favors hole formation [43] and oppose it through the Gibbs-Marangoni effect [44]. Extensive coalescence increases the particle size and therefore the tendency of an emulsion to cream. In practice, coalescence is rarely the initial cause of food emulsion failure. More common is flocculation whereby droplets collide and stick together but the membrane separating them does not rupture. There is no mixing of oil so the individual droplets retain their spherical shape and a large, porous, and frequently fractal floc develops. A floc immobilizes a mixture of dispersed and continuous phase so the effective volume fraction of a flocculated emulsion, and hence the viscosity (Equation 138.3) is much greater than the corresponding unflocculated emulsion. Coalescence decreases the number of droplets but causes no change in the volume fraction of the emulsion and hence no change in product rheology. Both flocculated and coalesced droplets have larger particle sizes so often cream more readily than the primary emulsion. Flocs can be characterized in terms of a size and density (fraction of the floc taken up by particles) but in many cases it is possible to ascribe a fractal dimension to the flocculated structure. A fractal object shows some level of self-similarity over several orders of scale. All objects show some relation between length and mass, for Euclidean solids, planes, and lines it is cubic, quadratic, or linear respectively, but for fractal objects it is non-integer. To describe a fractal floc the closer fractal dimension of the floc to three, the more the particles pack together to form a “perfect” Euclidean 3D object, i.e., complete coalescence. The lower the fractal dimension, the more open the structure. Fractal dimension can be used to calculate an effective volume fraction φeff: 3⫺D

R φeff ⫽ φ ᎏ r

(138.12)

where R is the floc radius, r is the primary particle radius, φ is the particle volume fraction, and D is the fractal dimension (⫽ 1–3). The effective volume fraction can be used in the Krieger-Dougherty type relation to link microstructure and viscosity. Plots of viscosity as a function of volume fraction are shown in Figure 138.6d and e for R/r ⫽ 10 and D ⫽ 2.8 and 2.6 respectively. The looser floc (Figure 138.6e) reaches a critical volume fraction at

Food Emulsions

lower concentrations. Fully coalesced droplets (D ⫽ 3) behave the same as unflocculated emulsion (Figure 138.6c). Emulsions may flocculate when too little protein is used in their formation. In this case a single protein strand may encompass several droplets and cause them to stick together). Factors which increase attractive colloidal forces or decrease repulsive forces (see above) such as adjusting pH to pI or thermal denaturation of the protein layer will also tend to induce flocculation. This can also be seen as factors that make the aqueous phase a poorer solvent for the protein will tend to favor protein precipitation and hence emulsion destabilization [45]. Related to both flocculation and coalescence is partial coalescence which is a novel mechanism of emulsion destabilization which can occur when the droplets are semi-crystalline (especially in sheared, refrigerated dairy products [44]). The fat crystals penetrate the aqueous layer separating the colliding droplets and then liquid oil flows out to wet the crystal surface and to reinforce the link between the droplets. The solid fat network in each droplet provides a skeleton to prevent the oil flowing completely between droplets and thus prevents full coalescence and maintains the characteristic shape of individual droplets. However if a partially coalesced emulsion is heated so the fat crystals melt they will merge and can lead to significant oiling off. Partially coalesced fat droplets play a role in supporting the foam of whipped cream and ice cream [46]. In conclusion, the typical mode of emulsion destabilization in real food emulsions is flocculation with associated increases in viscosity and often complete or partial phase separation. Later the membranes separating the flocculated droplets may break allowing coalescence and eventual oiling off. (When semi-crystalline droplets are sheared partial coalescence with associated formation of visible clumps or even phase-inversion may dominate.) It is notable that the distinct mechanisms of emulsion destabilization introduced above do not in practice occur in isolation but instead catalyze one another.

IV. EFFECT OF PROCESS CONDITIONS Foods are processed to improve their safety and quality and sometimes these affect the colloidal forces responsible for the stabilization of emulsions. Thermal Treatments. Most cooking operations involve some thermal denaturation of proteins. The extent of denaturation depends on the type of protein present at the surface, the temperature and time to which it is exposed, and to a lesser extent the pH and other co-solutes present. Thermal denaturation leads to some protein unfolding and the increased exposure of hydrophobic amino acids to the aqueous phase. The increased hydrophobicity leads to protein aggregation between the surface proteins and either (i) unadsorbed protein, or (ii) adsorbed protein on the same droplets (intradroplet bonding), or (iii) adsorbed

138-11

protein on other droplets (interdroplet bonding). Of these, (iii) is the most significant as it leads to droplet flocculation and consequent changes in emulsion properties. The prevalence of each case depends on the relative concentration of oil droplets and non-adsorbed protein, and the inter-particle forces acting. Chilling is important as it induces oil crystallization within the droplets. Droplet oil tends to crystallize more slowly than bulk oil due to the isolation of active catalysts but once there are crystals present there is an increased likelihood of partial coalescence and the formation of visible clumps in the product [44]. Freezing can quickly destabilize many emulsions via a variety of mechanisms. Firstly droplets are forced into very high concentrations in the unfrozen channels between the ice crystals [47]. This can first lead to the conventional forms of droplet aggregation found in concentrated unfrozen emulsions [48], in particular partial coalescence [49]. In addition the dehydrating effect of freezing on the surfactant head groups can alter spontaneous curvature and favor the formation of pores between droplets and eventual full coalescence [47]. In some emulsions the freezing will induce polymerpolymer interactions and droplet flocculation and the formation of a cryo-gel [50, 51]. Other Operations. High pressure treatment can have a denaturing effect on proteins and thereby affect the functional properties of emulsions [52]. The effect of the pressure treatment depends on the nature of the protein and on the solvent conditions, for example, 700 kPa had no effects on the surface pressure of casein but significant effects on whey. Pulsed electric fields have an antimicrobial effect often exploited to reduce the microbial loads on minimally processed foods but have little effect on the properties of food emulsions, for example, disrupting flocs and rupturing larger droplets [53]. Irradiation inactivates food spoilage organisms by damaging their nuclear material. Extremely high levels of irradiation could in principle trigger more rapid lipid oxidation or damage other ingredients present but it is unlikely the levels used in food processing would have any detectable effect on the physical microstructure.

ACKNOWLEDGEMENTS We are grateful to Dr. Julian McClements for generously providing a pre-print of parts of the forthcoming second edition of his book and to Ms. Andrea Docking for providing Figure 138.1.

REFERENCES 1. Walstra, P., Dispersed Systems: Basic Considerations, in Food Chemistry, O.R. Fennema, Editor. 1996, Marcel Dekker: New York. p. 95–156.

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Food Emulsions

35. 36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

K. Larsson, and J. Sjoblom, Editors, 4th ed. 2004, Marcel Dekker: New York. Israelachvili, J., Intermolecular and Surface Forces. 2nd ed. 1992, London: Academic Press. Dickinson, E. and E. Davies, Influence of ionic calcium on stability of sodium caseinate emulsions. Colloids and Surfaces B-Biointerfaces, 1999. 12(3–6): p. 203–212. Demetriades, K., J.N. Coupland, and D.J. McClements, Physical properties of whey protein stabilized emulsions as related to pH and NaCl. J. Food Sci., 1997. 62: p. 1–6. Demetriades, K., J.N. Coupland, and D.J. McClements, The effect of temperature on the stability of whey protein stabilized emulsions. Journal of Food Science, 1997. 62: p. 462–467. Pashley, R.M., Effect of degassing on the formation and stability of surfactant-free emulsions and fine teflon dispersions. Journal of Physical Chemistry B, 2003. 107(7): p. 1714–1720. Dickinson, E. and M. Golding, Depletion flocculation of emulsions containing unadsorbed sodium caseinate. Food Hydrocolloids, 1997. 11(1): p. 13–18. Chanamai, R. and D.J. McClements, Depletion flocculation of beverage emulsions by gum arabic and modified starch. Journal of Food Science, 2001. 66(3): p. 457–463. McClements, D.J., Ultrasonic determination of depletion flocculation in oil-in-water emulsions containing a non-ionic surfactant. Colloids and Surfaces A., 1994. 90: p. 25–35. Kabalnov, A. and H. Wennerstrom, Macroemulsion stability: The oriented wedge theory revisited. Langmuir, 1996. 12(2): p. 276–292. Walstra, P., Emulsion stability, in Encyclopedia of Emulsion Technology, P. Becher, Editor. 1996, Marcel Dekker: New York. p. 1–62. Dickinson, E., Structure, stability and rheology of flocculated emulsions. Current Opinion in Colloid & Interface Science, 1998. 3(6): p. 633–638.

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46. Goff, H.D., Colloidal aspects of ice cream — a review. International Dairy Journal, 1997. 7(6–7): p. 363–373. 47. Saito, H., et al., Coalescence of lipid emulsions in floating and freeze-thawing processes: Examination of the coalescence transition state theory. Journal of Colloid and Interface Science, 1999. 219(1): p. 129–134. 48. Bibette, J., Stability of thin-films in concentrated emulsions. Langmuir, 1992. 8(12): p. 3178–3182. 49. Harada, T. and K. Yokomizo, Demulsification of oil-inwater emulsion under freezing conditions: Effect of crystal structure modifier. Journal of the American Oil Chemists Society, 2000. 77(8): p. 859–863. 50. Patmore, J.V., H.D. Goff, and S. Fernandes, Cryogelation of galactomannans in ice cream model systems. Food Hydrocolloids, 2003. 17(2): p. 161–169. 51. Lozinsky, V.I., Cryogels on the basis of natural and synthetic polymers: Preparation, properties and application. Uspekhi Khimii, 2002. 71(6): p. 559–585. 52. Galazka, V.B., E. Dickinson, and D.A. Ledward, Influence of high pressure processing on protein solutions and emulsions. Current Opinion in Colloid & Interface Science, 2000. 5(3–4): p. 182–187. 53. Barsotti, L., E. Dumay, T.H. Mu, M.D.F. Diaz, and J.C. Cheftel, Effects of high voltage electric pulses on protein-based food constituents and structures. Trends in Food Science & Technology, 2001. 12(3–4): p. 136–144. 54. Dickinson, E. and G. Stainsby, Colloids in Food. 1982, London: Applied Science Publishers. 55. Dickinson, E., An Introduction to Food Colloids. 1992, Oxford: Oxford University Press. 56. Dickinson, E. and D.J. McClements, Advances in food colloids. 1995, Glasgow: Blackie Academic & Professional. 57. Walstra, P., Physical Chemistry of Foods. 2003, New York: Marcel Dekker.

139

Food Gums: Functional Properties and Applications

Florian M. Ward and William H. Hanway TIC Gums, Inc.

Richard B. Ward RBW Consulting

CONTENTS I.

II.

III. IV. V. VI.

Hydrocolloids: Overview of Chemistry and Functionality ............................................................................139-2 A. Hydration Rate, Functional and Rheological Properties ........................................................................139-2 B. Proximate Composition: High Soluble Dietary Fiber Content ..............................................................139-3 C. Plant Exudates as Emulsifying Agents ..................................................................................................139-3 1. Gum Acacia or Gum Arabic ............................................................................................................139-3 2. Modified Gum Acacia: New Emulsifying Systems ..........................................................................139-4 D. Seed Polysaccharides ..............................................................................................................................139-4 1. Guar Specialty Products: Deodorized and Hydrolyzed Guar Gums ..............................................139-4 2. Locust Bean (Carob) Gum ..............................................................................................................139-4 E. Seaweed Polysaccharides ........................................................................................................................139-5 1. Agar and Agaroid Series as Gelling Agents ....................................................................................139-5 2. Carrageenans: Protein-Reactive Gelling and Stabilizing Agents ....................................................139-6 3. Alginic Acid Derivatives ..................................................................................................................139-6 4. Propylene Glycol Alginate: An Emulsifying Gum ..........................................................................139-6 F. Microbial Polysaccharides ......................................................................................................................139-7 1. Gellan Gum ......................................................................................................................................139-7 2. Xanthan Gum ..................................................................................................................................139-7 G. Pectins ....................................................................................................................................................139-7 H. Inulin: A Fructooligosaccharide ..............................................................................................................139-8 I. Cellulose Gum and Methylcellulose ......................................................................................................139-8 General Applications in the Food and Beverage Industries ..........................................................................139-8 A. Dairy Foods and Beverages ....................................................................................................................139-8 1. Ice Cream and Other Frozen Dairy Products ..................................................................................139-9 2. Cream Cheese and Sour Cream ......................................................................................................139-9 3. Acidified Milk Beverage ..................................................................................................................139-9 B. Bakery Products, Cereals, and Snack Foods ........................................................................................139-10 C. Salad Dressings and Sauces ..................................................................................................................139-11 D. Beverage Emulsions ..............................................................................................................................139-11 E. Confections and Candies ......................................................................................................................139-12 1. Gelatin Substitutes: Hydrocolloid Gelling Agents ........................................................................139-12 F. Meat and Poultry Products ....................................................................................................................139-12 Fat Mimetics and Functional Foods ..............................................................................................................139-13 Gum Systems: Synergy and Interaction ........................................................................................................139-13 Maximum Usage Levels and Quality Specifications ..................................................................................139-14 Prehydrated or Agglomerated Gums and Gum Systems ..............................................................................139-14 139-1

139-2


VII. Analytical Methods for Evaluation of Gums ................................................................................................139-14 VIII. Summary and Recommendations ..................................................................................................................139-15 Acknowledgements ..................................................................................................................................................139-15 References ................................................................................................................................................................139-15

I. HYDROCOLLOIDS: OVERVIEW OF CHEMISTRY AND FUNCTIONALITY Hydrocolloids, commonly known as water-soluble gums, are high molecular weight plant polysaccharides, usually with some inorganic or mineral content and low levels of protein. Gums are naturally occurring, water-soluble polymers with thickening, film-forming, and/or gelling properties based on their chemical nature, given specific conditions. Gums as complex carbohydrates exhibit properties that are affected by many factors including the following: active functional groups as substituents, molecular size, orientation, molecular association, water-binding and swelling, concentration, particle size, degree of dispersion, temperature, pH, processing conditions, etc. (1, 2, 3). Soluble dietary fiber is defined as being resistant to degradation by human digestive enzymes and can help decrease high serum cholesterol levels. Water-soluble gums are good sources of soluble dietary fiber (about 85% on a dry basis) when used as an ingredient in processed foods. Low viscosity gums, such as gum acacia, inulin, hydrolyzed guar, and polydextrose, may be used as fiber source at higher levels in nutraceutical products. Gums can also be used as one of the ingredients in fat mimetic systems due to their ability to bind as much as 100-fold their weight of water. This chapter aims to highlight the functional properties of various commercially available hydrocolloids for applications in food and beverage products. A list of the various categories, examples, and botanical sources of naturally occurring hydrocolloids is shown in Table 139.1A. Examples of the most important chemically modified gums are shown in Table 139.1B.

A. HYDRATION RATE, FUNCTIONAL AND RHEOLOGICAL PROPERTIES Gums are highly functional ingredients in beverages, salad dressings and sauces, snack foods, cereal products, and other food systems (1, 2, 3). In food applications, they function in a variety of ways (Tables 139.2A and 139.2B), an attribute that may be related to their viscosity characteristics or to their water binding, gelling, and other specific properties. These characteristics ensure the production of high quality food products with extended shelf life. As is evident from Table 139.3, viscosity ranges of the various hydrocolloids can vary greatly due to their chemical nature and degrees of branching and polymerization. Depending on their chemical nature, type of branching, molecular weight, residual ionic charge, and ability to

TABLE 139.1A Types of Natural Hydrocolloids Type (Function)

Examples

Botanical Source

Plant Exudates (Emulsifying & Film-Forming Agents)

Gum Arabic Karaya Tragacanth

Acacia sp. Sterculia sp. Astragalus sp.

Seed Gums (Thickeners & Water Binders)

Guar Locust Bean

Cyamopsis tetragonolobus Ceratonia siliqua

Seaweed Extracts (Gelling Agents & Film Formers)

Carrageenan Agar Alginate

Chondrus, Eucheuma spp. Gracilaria, Gelidium spp. Laminaria, Macrocystis spp.

Microbial Gums (Thickener/Gelling)

Xanthan Gum Gellan Gum

Xanthomonas campestris Pseudomonas elodea

Plant Extracts (Gelling/Thickener)

Pectins Inulin Konjac Flour

Apple & citrus fruits Chicory & Artichokes Amorphophallus sp.

TABLE 139.1B Types of Chemically-Modified Water-Soluble Gums Type Cellulose Derivatives (Thickening & Suspending Agents)

Examples

Synonym

Cellulose Gum

Sodium carboxymethyl cellulose Methyl Cellulose Cellulose ether Cellulose Gel Microcrystalline cellulose HPMC Hydroxypropylmethyl cellulose EHEC Ethylhydroxyethyl cellulose Modified Gums Modified Esterified Gum Acacia (Emulsifying Agents) Gum Acacia

undergo intermolecular or intermolecular associations, the gums exhibit a variety of rheological properties. Shearthinning behavior or pseudoplascticity is shown by xanthan gum while gum acacia, a branched, globular arabinogalactan is Newtonian (i.e., does not decrease in viscosity with increasing shear rate) up to 40% concentration. Thixotropic behavior has been observed in some cellulose gum solutions, depending on degree and uniformity of substitution. To optimize the functional properties of gums, it is essential that the product is completely dispersed and fully


TABLE 139.2A Functions of Gums in Food Products (1) Function Adhesive Binding agent Bulking agent Clouding agent Crystallization inhibitor Coating agent Emulsifier Encapsulating agent Fat mimetic Film former

139-3

TABLE 139.4 Proximate % Composition of Selected Gums

Application Glazes & cereal clusters Granola bars & sausages Sugar-free foods Fruit beverages Ice cream, syrup, & candy Confectionary Beverage emulsions & salad dressings Spray-dried flavors Low-fat cookies & dressings Edible films & coatings

Gum Agar Arabic Carrageenan Guar Locust Bean Tragacanth Xanthan a b

Complex Carbohydratesa

Protein

Ash

Othersb

Sodium mg/100 g

85.0 85.0 60.0 85.0 80.0 80.0 85.0

⬍1 2–3 ⬍1 3–6 6.0 2.5 0–2

6.5 3.8 35.0 1.5 1.2 3.0 10.0

8.5 9.3 5.0 9.9 12.8 14.5 5.0

⬍10 100 3,000 250 ⬍10 10 5,000

Soluble dietary fiber. Primarily moisture.

TABLE 139.2B Functions of Gums in Food Products (2)

B. PROXIMATE COMPOSITION: HIGH SOLUBLE DIETARY FIBER CONTENT

Function

As mentioned earlier, gums have minute quantities of lipids and contain low levels of protein, depending on their source. They consist primarily of complex carbohydrates derived from plants or from the biosynthesis of end products by pure microorganisms (e.g., xanthan gum and gellan gum). They act as soluble dietary fiber (Table 139.4), which has been reported to lower serum cholesterol and improve gastro-intestinal function as well as improve glucose tolerance. Seaweed extracts also contain an appreciable level of ash, which may naturally occur with the gum or may be the consequence of manufacturing conditions.

Foam stabilizer Gelling agent Protein reactive colloid Reduce cooking loss Suspending agent Syneresis inhibitor Thickening agent Whipping agent

Application Whipped toppings, mousse Pie fillings, custard, gummy bears Whey beverage, acidified milk Meat & poultry injection Marinades & dressings Cheese, frozen foods Sauces, gravies, smoothies Whipped yogurt & cream

TABLE 139.3 Relative Viscosities of Gums Hydrocolloid CMC Guar Gum Locust Bean* Xanthan Carrageenan* Karaya Tragacanth Sodium Alginate Gum Acacia

Viscosity Rangea 4,000–6,000 3,000–5,000 2,500–3,500 1,000–2,000 100–1,500 300–1,000 500–750 200–400 2–10

a

Centipoise (cP) after 24 hours at 25°C (77°F) with 1% gum solns. (Brookfield Viscometer), RV4 at 20 rpm, except for gum acacia, LV2 at 60 rpm. * Requires heating to 180°F to hydrate completely.

hydrated. Factors affecting hydration rate and functionality include the following: (a) chemical nature of the hydrocolloid, (b) particle size distribution, (c) method of incorporation of ingredients, (d) temperature, (e) pH and the presence of other ionic salts, (f) shear rate, (g) duration of mixing, (h) synergy between gums, (i) potential incompatibility between food components, (j) factors required for gel formation including cations and percent solids, and (k) processing conditions and equipment design.

C. PLANT EXUDATES AS EMULSIFYING AGENTS 1. Gum Acacia or Gum Arabic Gum arabic is derived from the plant exudate of Acacia senegal or related Acacia species grown mainly in African regions. The general properties of this plant exudate are affected by the age of the tree, the amount of rainfall, type of storage conditions, and other factors. Salts and other electrolytes as well as temperature can affect the viscosity of acacia, which is typically not more than 300 cps at 30% gum level. The highly branched, compact structure may account for its low viscosity. The emulsifying properties are attributed to the protein moieties covalently linked to the polysaccharide (2). Gum arabic is an anionic heteropolysaccharide consisting of an arabinogalactan complex (about 88%), an arabinogalactan-protein complex (10.4%), and a glycoprotein fraction (about 1.2%). It consists of rhamnose and glucuronic acid, in addition to arabinose and galactose (Figure 139.1). Gum arabic exhibits emulsifying properties and may be used at high concentrations up to 30% in spray dried flavors, due to its unusually low viscosity. When the protein moiety in gum acacia is damaged by high temperature and other specific processing parameters, the emulsifying capacity of the gum may be adversely


2. Modified Gum Acacia: New Emulsifying Systems The emulsifying properties of gum acacia are significantly enhanced by introducing a covalently linked lipophilic group through reaction with compounds such as octenylsuccinic anhydride (5). Modified gum acacia may be used to replace gum tragacanth and emulsifying starches used in bakery emulsions. Modified guar gum has also been used as a replacement for propylene glycol alginate in emulsion stability studies (5). These new products are members of the series of modified, emulsifying hydrocolloids covered by U.S. Patent No. 6,455,512 (6). A petition for GRAS certification has been approved after a thorough review by a panel of experts. The new products are available for initial evaluation in various formulations including beverage emulsions, cosmetics, and salad dressings.

D. SEED POLYSACCHARIDES

limit of 10%. It swells in cold water and is one of the highly efficient water thickening agents used in the food industry. Solutions of guar are non-Newtonian and pseudoplastic or shear-thinning in nature (Figure 139.3). More recent specialty guar types have been developed to reduce the grassy odor and flavor, using a proprietary manufacturing process (7). Reduced odor guar gum is recommended for various food and beverage products with delicate flavor and odor. Hydrolyzed guar gum with lower viscosity (60 to 150 cps at 2%) may also be used to increase the fiber content of the finished product. The viscosities of two special types of deodorized guar as a function of concentration after hydration for 2 and 24 hours are shown in Figure 139.4. Viscosity loss is reversible when heat is applied and subsequently removed. Guar gum can vary in its viscosity, rate of hydration, and dispersion properties depending on the conditions under which it is manufactured. Being the same as other gums, it has a high dietary fiber content of 80 to 85%. 2. Locust Bean (Carob) Gum Locust bean gum is a non-ionic polysaccharide obtained from Ceratonia siliqua, a tree that belongs to the Family Leguminoseae, and consists of mannose and galactose sugar units in a ratio of 4:1 (Figure 139.5). Unlike guar gum, which hydrates rapidly in cold water, locust bean gum has to be heated to 80°C (176°F) for full hydration. The distribution

1. Guar Specialty Products: Deodorized and Hydrolyzed Guar Gums Guar gum is a non-ionic galactomannan isolated from the seeds of the shrub that belongs to the species Cyamopsis tetragonolobus, Fam. Leguminoseae. It is grown in Pakistan and India and may also be cultivated in Texas, Arizona, and other arid regions of the U.S. The structural building blocks of guar are the sugars mannose and galactose at a ratio of 2:1 (Figure 139.2). The protein content ranges from 3 to 6% but the Food Chemicals Codex allows a maximum

CH2OH O HO

CH2OH

O

OH

OH HO

O

O

HO CH2

CH2 O

O

O

O

O

OH HO

OH HO

O

OH HO

CH2OH

OH HO

CH2OH

O

x

FIGURE 139.2 Structure of guar gum. O 1,6 linkage 3000

O O 1,4 linkage

CH2

O O

Gum Acacia is highly branched and has C-1,4 and C-1,6 glycosidic linkages as shown above in addition to C-1,3 linkages (not shown).

FIGURE 139.1 Glycosidic linkages in disaccharide repeating units in gum acacia.

Viscosity, cPs

HOH2C

O

HO

O

CH2OH

MW = 1− 2 × 106

Mannose/Galactose ~ 2/1

O

affected. Gum arabic is widely used in the food industry due to its emulsifying properties, low viscosity, high fiber content, water-binding capacity, and adhesive and filmforming properties (4). Gum arabic is reported to be incompatible with vanillin, pyrogallol, vanillin, phenol, thymol, cresols, tannin, etc. However, it is compatible with most other plant hydrocolloids, proteins, carbohydrates, and starches.

O

139-4

Temperature 26°C, Brookfield programmable rheometer

2500 2000 1500 1000 500 0 0

20

40 60 Shear rate (1/sec)

80

FIGURE 139.3 1% Deodorized guar: shear rate vs. viscosity.


139-5

of D-galactosyl groups in guar and locust bean gum has been described by Baker and Whistler (8). Food grade locust bean gum should have a protein content not exceeding 8% as specified in the Code of Federal Regulations. Solutions of locust bean gum are non-Newtonian with zero yield value and, thus, flow as soon as slight shear is applied. When combined with xanthan, locust bean gum yields heat-reversible, pliable gels. It also acts synergistically with kappa carrageenan to form strong, somewhat elastic gels. Locust bean gum is classified as a direct food additive under FDA regulations. Dilute solution properties of guar and locust bean gum in sucrose have been characterized by Richardson et al. (9).

mixed with water, and treated with alkali to facilitate the extraction of the polysaccharide. Alcohol is sometimes used to precipitate the concentrated extract. The general principles involved in the purification and isolation of these gums from the seaweeds are essentially similar, but the specific procedures are proprietary in nature and differ slightly for each manufacturer. The structure of the repeating unit of the gelling components from agar and carrageenan are shown in Figures 139.6 and 139.7, illustrating the presence of the ester sulfate groups that account for the anionic character of agar and carrageenan.

E. SEAWEED POLYSACCHARIDES

Agar and its agarose constituents have been characterized by Armisen (10). Agar consists of a repeating unit of betaD-galactose attached to 3,6-anhydro-alpha-L-galactose. It is isolated from seaweeds that mainly belong to the Gelidium, Gelidiella, or Gracilaria species. Traditional agar can bind about 100 times its weight of water, and, when boiled to 212°F and cooled, forms a strong gel. It is one of the most potent gel-forming gums known and, unlike most other gelling gums, has a gelation temperature that is far below the gel-melting temperature. A solution of agar (1.5%) congeals in the range of 32 to 39°C (89.6 to 102.2°F) but does not melt below 85°C (185°F). This property is important in many of its applications in the food industry. A more recently developed type of agar (Agar RS-100) from TIC Gums, Inc. does not require boiling as does the traditional agar (11). The seaweed sources are subjected to a series of manufacturing procedures that yield a product that may be hydrated at 170 to 180°F instead of 212°F. This is a desirable feature, considering the expense involved in boiler operations in the industry. A series of synergistic systems, the Agaroid Series, that make use of non-boiling agar and other hydrocolloids

1. Agar and Agaroid Series as Gelling Agents

Some seaweed extracts from the Family Rhodophyceae (agar), Phaeophyceae (algin), and Gigartinaceae (carrageenan) are used as gelling and stabilizing agents in various food products and beverages, based on the optimum gum levels and conditions required for functionality. The seaweeds are typically dried by sun-drying or mechanical means, washed to remove sand, salt, and other debris,

Viscosity, cPs

2500 2000 1500 1000 500 0 0.0%

0.2%

0.4% 0.6% 0.8% Guar concentration (%)

Guar bland A 2 hr Guar bland A 24 hr

1.0%

1.2%

Guar bland B 2 hr Guar bland B 24 hr

FIGURE 139.4 Deodorized guar levels vs. viscosity.

CH2OH O

Mannose/Galactose ~ 4/1

OH HO

O

CH2OH

CH2

O

O O OH HO

O O

O

CH2OH

FIGURE 139.5 Structure of locust bean gum.

O

OH HO

OH HO

OH HO O

HO

CH2OH

x

139-6


have been developed (11) to replace gelatin as gelling agent. Gelatin is not acceptable to some religious groups and has been recently associated with the incidence of mad cow disease, which has increased the demand for gelatin substitutes from the food industry. 2. Carrageenans: Protein-Reactive Gelling and Stabilizing Agents Carrageenan, a water-soluble gum, is a sulfated, linear, anionic polysaccharide composed of D-galactose and 3, 6-anhydro-D-galactose derived from red seaweeds including Eucheuma, Gigartina, or Chondrus species. Carrageenans act as strongly anionic polyelectrolytes, a property that accounts for their high protein reactivity. Due to the presence of the ester sulfate groups, an interaction occurs with charged amino acid groups in proteins above the isoelectric point. The three common types of carrageenans –– kappa, iota, and lambda –– differ in degree and location of sulfated ester groups and the linkage of the repeating units (Figure 139.7). Bixler (12) con-Carrageenan Moieties -D-galactose CH2OH O OH

CH2 O O

O

O

O OH

OH

3,6-anhydro--D-galactose

Agarose Moieties -D-galactose OH

O

CH2OH O O

CH2 OH

O

3,6-anhydro--L-galactose

FIGURE 139.6 Repeating chemical units of gelling agents.

−

O3SO

CH2OH O

CH2 O O

Kappa

O O

−

O3SO

CH2

O O

4. Propylene Glycol Alginate: An Emulsifying Gum An ester derivative of alginic acid, propylene glycol alginate is widely used as an emulsifier in salad dressings and other types of oil-in-water emulsions. Propylene oxide is reacted with alginic acid to esterify partially the hydrocolloid’s mannuronic or guluronic acid units. The use of food colloids and polymers in emulsifying systems has HOOC

OSO3−

CH2OH HO

O

HOOC

O OH

O O

CH2OSO3− O

O O

OH

H (30%) O SO − (70%) 3

O

O

O

Iota

Lambda

Alginic acid is a high molecular weight linear polysaccharide derived from Laminaria, Macrocystis, Lessonia, and other related seaweed species. It consists of homo- and heteropolymic sequences composed of mannuronic and guluronic acid units (Figure 139.8). The guluronic and mannuronic acid content of the alginate affects the nature of the gel that is formed. Sodium alginate in the presence of calcium ions yields gels that are not thermally reversible. The method of addition and type of calcium salt added affects the properties of the final gel. A calcium sequestrant may be used to weaken the gel or delay its setting time. Gel systems may also be prepared using alginates by controlling pH. Neiser (14) characterized the gel formation in heat treated bovine serum albumin-sodium alginate systems.

OH

OH CH2OH

3. Alginic Acid Derivatives

O

O OH

ducted studies and reviewed the properties of refined and semi-refined carrageenans. In terms of solubility in water, both kappa and iota hydrate at above 70°C, while the lambda type is soluble in cold water. In cold milk, lambda carrageenan disperses and thickens while both the iota and kappa carrageenans are insoluble. Kappa carrageenan requires potassium ions to gel, while iota carrageenan requires calcium ions to form a heat reversible and flexible gel at 1.5% gum. The non-gelling type, lambda carrageenan is usually used to thicken milk, an action enhanced by tetrasodium pyrophosphate. Thomas (13) described the applications of carrageenans as thickening and gelling agents for food.

O OSO 3−

FIGURE 139.7 Repeating chemical units of carrageenans.

-D-Mannuronic acid moiety

O

-L-Guluronic acid moiety

The mannuronic to guluronic acid ratio varies with the seaweed source

FIGURE 139.8 Glycosidic linkage repeating units in alginates.


139-7

grown in a special nutrient medium under controlled conditions. It is approved for food use in many countries, including the United States and Canada. Food grade xanthan is also a good source of dietary fiber and should have an ash content that does not exceed 10.0%. Xanthan gum solutions are extremely pseudoplastic and exceed most common gums in this respect. Viscosity is reduced with increasing shear; viscosity is regained after shear is released. This property is an advantage when pumping gum-thickened liquids. Xanthan gum is an excellent emulsion stabilizer, although it is not an emulsifier by definition. It is typically used in salad dressing emulsions. Xanthan gum is stable over a wide range of pH (2 to 10) and temperature, which makes it an ideal stabilizer in a variety of applications (3).

been investigated extensively by Dickinson and Walstra (15).

F. MICROBIAL POLYSACCHARIDES 1. Gellan Gum Gellan gum (16) is a fermentation product of Pseudomonas elodea grown under aerobic, submerged condition. The active, high-acyl gellan gum consists of a linear sequence of tetrasaccharide repeating units. Cations such as potassium ions, in addition to interchain reactions and hydrogen bonding with water, help stabilize the structure that gives rise to gel formation. The influence of calcium ions, acetate, and L-glycerate groups in the gellan double helix has been investigated by Chandrasekaran and Thilambali (17). By varying the degree of acylation, a range of gel textures can be generated. The viscosity of gellan gum solutions decrease markedly with increasing temperature, but functionality is retained upon cooling. Ratios of gellan gum and gum arabic at 1:1 exhibit an increase in gel strength by about 60% at 0.5% gum level. Gellan gum is used in food formulations that require gelling properties including jams, jellies, dessert gels, pie fillings, puddings, frostings, and dairy products.

G. PECTINS Citrus and apple byproducts have been widely used as sources of pectin. Other sources include tropical fruits such as guava, papaya, mango, etc. Under appropriate conditions, pectins (polygalacturonans) are gel forming. The main component of pectin is D-galacturonic acid partly esterified with methoxyl groups. The degree of esterification is initially high, but the pectin methylesterase enzyme present in most tissues can cause demethoxylation. Pectins can be classified into high methoxy, low methoxy, and amidated. High methoxy pectins require more than 60% solids and low pH (less than 3.5) to form a heat-irreversible gel. Low-methoxy pectins (between pH 1 to 5) require calcium to gel and may yield a heatreversible gel at 25 to 35% solids given the proper gelling

2. Xanthan Gum Xanthan gum is a highly branched polysaccharide consisting of repeating units of D-glucose, D-mannose, and D-glucuronic acid (Figure 139.9). It is a biosynthetic product of a pure bacterial culture of Xanthomonas campestris

MW = 3 to 7.5 × 106 CH2OH O O

CH2OH O O

OH

CH2OH O O

O

OH

OH

O

OH

OH O CH2 OCCH3 O

O

OH

OH

FIGURE 139.9 Structure of xanthan gum.

M + − OOC

+O

OH

OH

O

OH

OH

O

OH

O O

M+ COO − OCH2 O O C CH3 O

OH

OH

COO M O

O

OH

O H3CCOCH2

O

OH

OH −

O

CH2OH O O

O

OH

OH

HOCH2

CH2OH O

OH OH M+ = Na+ , K + , or 1/2 Ca++ , Comb-like, double helix

x

139-8


conditions. Schols et al. (18) describe the structural features of native and commercially extracted pectins.

II. GENERAL APPLICATIONS IN THE FOOD AND BEVERAGE INDUSTRIES

H. INULIN: A FRUCTOOLIGOSACCHARIDE

Hydrocolloids, alone or in combination with other gums, other ingredients, and emulsifiers, are widely used in the dairy, bakery, confection, beverage, and snack food industries. They are also used in flavor emulsions, cereal products, candies, and confections. As indicated in the preceding section, their functionality is affected by the chemical nature of the gum as well other factors including pH, temperature, % total solids, presence of cations, and synergy or incompatibility with other ingredients. Before actual production, planning equipment design, and scaling-up manufacturing procedures, the specific requirements of the gum or gum system should be taken into consideration by the engineer and food technologist. For example, if the filling viscosity is a limiting factor in production, gums that do not attain full viscosity in cold water are required. Shear forces at high temperature should be taken into consideration with some specific gums and starches to avoid excessive degradation. Similarly, high acid and high temperature can cause gum hydrolysis in the food system and should therefore be avoided where possible. For each food or beverage product, all the basic requirements to maximize functionality of the gum and the other ingredients should be adopted to achieve the desired sensory qualities and shelf-life stability. The oral behavior of food hydrocolloids and emulsions, including the relationship of zero shear viscosity and the maximum aroma intensity of guar, xanthan, and sodium alginate solutions, has been extensively investigated by Malone et al. (19).

A relatively new food ingredient, inulin is isolated from the root of the chicory plant Cichorium intybus and other plant sources. It has gained wide use over the past several years due to the following features: readily soluble fiber, “neutral” taste, odor, and color, gelling properties, and ability to stabilize foams and emulsions. Inulin is a combination of fructose chains, the chain length varying from 2 to 60 fructose units. It has also been reported to provide only 1.6 Kcal/g as compared to 4.0 Kcal/g for starch and other carbohydrates (2). In addition, it has a glycemic index of zero, does not stimulate insulin excretion, has been claimed to be 100% soluble fiber, and is recommended in the modern diet for diabetics.

I. CELLULOSE GUM AND METHYLCELLULOSE Cellulose is a linear polymer consisting of beta-D-anhydroglucose units (Figure 139.10). Sodium carboxymethylcellulose (cellulose gum), derived from cellulose, is universally known as CMC. Purified CMC is a tasteless, odorless, and free-flowing powder and is widely used in the food industry. There are various types of CMC manufactured throughout the world. These differ in degree of substitution and viscosity. CMC is recommended in products where clarity is an essential feature. The manufacturer should specify the limits for % insolubles as well as turbidity values and degree of substitution, to ensure that the CMC grade meets the requirement for clarity in the finished product. Other derivatives of cellulose include methylcellulose and hydroxypropylmethylcellulose (HPMC). Methylcellulose, at suitable levels, gels upon heating, a property that makes it useful as a binder for products subjected to heating. Microcrystalline cellulose (MCC) is unmodified, insoluble cellulose that has been reduced to a small particle size and has been used as a fat mimetic in combination with CMC (11).

CH2OH O O

CH2OH O O

OH OH

OH

The conventional, older method of heat processing, batch heating at 145°F for 30 minutes, favored hydration of the gums. With the advent of HTST (high temperature-short time, 171°F for 16 seconds), hydration of gums requiring heat to unfold has become problematic. Ultra-high temperatures, 285 to 302°F for 2–3 seconds, further shorten

CH2OH O O

OH

A. DAIRY FOODS AND BEVERAGES

CH2OH O O

OH

O

OH

OH

CH2OH O

OH

O

OH OH

→ 4)--D-Glup-(1→4)--D-Glup-(1 x

FIGURE 139.10 Structure of cellulose.


the time available for hydration as well as pose stability risks in acidic beverages. The extremely high heat of UHT processing denatures proteins that were not affected by the lower heat treatments. This unfolding can be utilized for additional viscosity, although precipitation problems may arise depending on the isoelectric point of the protein substrate. 1. Ice Cream and Other Frozen Dairy Products Ice cream (20) is composed of milk ingredients, sweeteners, stabilizing agents, and flavoring. The milk ingredients used may include cream, milk, condensed milk, powdered milk, butter, and frozen cream. Fat is an important component in ice cream due to its mouthfeel and richness of texture. Whey solids are used in lower quality ice creams due to cost considerations. Whey consists of whey protein and other components that are not precipitated by high acidity. A by-product of the cheese industry, whey is now being used in beverages and nutritional sports supplements. Certain types of whey can impart a cheesy or salty flavor to the ice cream. Seventy-five percent of whey solids consists of milk sugar, or lactose. Too high a level of whey in the cream causes a defect, called “sandiness,” due to specific structural forms developing. The “sand” or lactose crystals do not melt in the mouth. Plain ice cream must contain at least 10% milkfat and 20% total milk solids. Air is whipped into the ice cream mix and the structure is quickly frozen. An ice cream with 100% overrun contains 50% air. Too much air causes rapid meltdown and is regulated by the Code of Federal Regulations. Stabilizers in ice cream, particularly gums, provide smoothness in body and texture, retard ice and lactose crystal growth, and increase freeze-thaw stability. In addition, they contribute to creamier meltdown, lowered sensation of coldness, improved body, overrun control (product uniformity), and resistance to melting. The stabilizer, which increases viscosity and adsorbs to the air lamellae, also aids in the suspension of flavoring particles. Gums stabilize the product by restricting the movement of water and solutes. They hold free water as water of hydration or by immobilization within a gel structure. In a sucrose-lactose solution simulating the colloidfree phase of an ice cream mix, the effects of CMC and guar gum on ice crystal formation were studied. Freezing profiles, obtained over short-term intervals, showed that the addition of guar gum significantly retarded ice crystal propagation in the sugar solution, whereas addition of CMC showed no effect (21). Stabilizers are reported to have no significant effect on the amount of freezable water or enthalpy of melting. Although they exhibit no effect on the freezing point depression of ice cream, the gums do limit the growth of crystals during recrystallization (22). The size of ice crystals in ice

139-9

cream determines the perception of iciness. Properly processed ice cream leaves the filler with small ice crystals. After transport, storage, and home freezing, ice crystal size grows. The consumer considers the ice cream defective, and sensory studies describe the defect as being coarse and icy. Defects in body and texture in ice cream are typically described by the following sensory properties: coarse or icy, gummy, weak, churned or greasy, soggy, and sandy. The use of polysaccharide gum helps inhibit the development of several of these defects. Carrageenan is an excellent stabilizer and is used in chocolate milk and ice cream. It forms a lattice structure that holds water and keeps it from migrating. The kappa carrageenan component exhibits gel-forming properties. Used with other stabilizers to prevent wheying off, kappa carrageenan reacts with kappa-casein micelles in milk to form a weak pourable gel. It is used in ice cream, evaporated milk, infant formulas, freeze-thaw stable whipped cream, and emulsions in which milk fat is replaced with vegetable oil. Guar gum is an excellent, cost-effective thickener that is cold-water soluble and is typically combined with locust bean gum and carrageenan at recommended ratios to maximize its functionality in the ice cream product. Some gum systems form microscopic spherical particles that mimic the rheology and mouthfeel properties of emulsified fat. These systems may include locust bean gum, guar gum, carrageenan, xanthan gum, cellulose gum, and microcrystalline cellulose. Other frozen products in which gums are used include frozen custard or French ice cream, sugar-free ice cream, mellorine, frozen yogurt, fruit sherbets, and water ices. Mellorine is similar to ice cream but the milkfat is fully or partially replaced by vegetable or animal fat. It should contain not less than 6% fat. 2. Cream Cheese and Sour Cream Gums, particularly combinations of carrageenan, guar gum, and locust bean gum, help minimize or retard syneresis or weeping in cream cheese and sour cream. Xanthan gum is also usually combined with guar gum and locust bean gum to form a synergistic component for cream cheese and similar formulations. The gum is added after the curd is formed by the inoculum, or, as in the case of imitation sour cream, after the protein is curdled with a lactic acid and citric acid mixture. 3. Acidified Milk Beverage Acidified milk beverages that are subjected to UHT pasteurization may be stabilized by the use of pectin or a combination of pectin and a deodorized guar gum at 0.4 to 0.5%. A prototype formulation is shown in Tables 139.5A and 139.5B. The gum system is dry blended with sugar and added to the milk, then allowed to mix and hydrate. The orange juice and the remaining balance of the milk

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TABLE 139.5A Acidified Milk Beverage (1) (Prototype Formulation, pH 4.2 & 22°Brix) Ingredient Orange Juice Whole Milk Sucrose Gum System* Flavor (as required)

% 64.6 25.0 10.0 0.4

* Pectin, deodorized guar gum, and propylene glycol alginate.

TABLE 139.5B Acidified Milk Beverage (2) (Prototype Formulation) Suggested Procedure 1. Pour milk into a mixing tank. 2. Dry blend sucrose and gum system. 3. Start blending at low speed, add the sucrose-gum blend to the mixer with the milk. Increase speed of the blender by 20%. 4. Let the gum hydrate for fifteen minutes in the mixing tank. 5. Add orange juice slowly and let mix for five minutes, adjust pH to 4.1–4.2 with citric acid, if necessary. 6. Pass through a thermal processing unit at 220°F (104°C) for 20 sec. Cool with chilled water to 40°F (4°C). 7. Package in sterile containers.

are then added to complete the formula. The UHT treatment involves heating at 220°F for 20 sec, after which the mixture is homogenized at 2500 psi and then cooled down with chilled water to 40°F and packaged in sterile containers. The product should show no significant wheying off or separation over a 6-week period. Pectin can also be used to stabilize UHT-treated fruit smoothies, buttermilk, fruit juice, milk drinks, and aseptic yogurt drinks. Pectin, which is anionic, stabilizes the milk protein and protects it from denaturation, thus keeping it suspended. The effect of carrageenan on sensory properties of milk beverage model systems is described by Yanes et al. (23).

B. BAKERY PRODUCTS, CEREALS, AND SNACK FOODS Hydrocolloids are used in bakery and snack foods (24, 25) due to their ability to bind water and improve the texture of the products as well as act as gelling agents. Gum arabic, used at 0.08 to 0.20 mg/kg of the flour, has been reported to improve the baking properties of rye and wheat flour (3). The anti-staling property of gum arabic and other hydrocolloids, when used in bread and cookies, has also been reported (3). Gum arabic is a main component in some glazing agents due to its adhesive properties. It also yields pliable and stable icing bases. The emulsifying ability of gum arabic is used in baker’s emulsions, in combination with other gums such as gum tragacanth. As a surfactant

and foam stabilizer, it may be used in whipped cream or toppings. Since gum acacia is high in dietary fiber, it may be used as a texturizer and bulking agent in powdered bakery mixes. Cellulose gum prolongs shelf life in bread and increases water retention as well as volume of the dough. Comparison of the moisture content of reduced fat oatmeal cookies and cakes prepared with and without gums showed higher moisture levels in gum-treated samples. CMC may also be used as a film-forming coat and adhesive in doughnut glazes (24, 25). Cellulose gum at 0.10% use level reduces ice crystal growth in frozen dough products and improves freeze-thaw stability. Fruit fillings with CMC in combination with gelforming systems help reduce syneresis or weeping. Methylcellulose is another derivative of cellulose that has been reported to increase moisture content and improve the sensory ratings of doughnuts. Microwaveable cakes showed uniformity of moisture distribution attributed to thermal gelation in methylcellulose-treated cake samples. Carrageenan is used to strengthen and extend the protein ingredients in bread or cake mixes. It is also used as an additive in various dough products to help improve the loaf volume, loaf shape, and texture. The freeze-thaw stability of pasta products is improved by the addition of 0.05 to 0.10% of carrageenan. Kappa carrageenan is used in breading and batter mixes due to its protein reactivity. Lambda carrageenan, a non-gelling type, is used to bind or retain moisture. It also contributes viscosity to sweet dough products. Iota carrageenan, which requires calcium ions to form a heat-reversible and flexible gel, may be used in fruit applications. Cake and doughnut mixes with 0.1% carrageenan show better moisture retention and softer texture in the final product than mixes without the protein-reactive hydrocolloid. Another important property of kappa carrageenan is its ability to form gels in the presence of potassium ions, and also to form rigid gels with locust bean gum. This gel-forming ability may be used in preparing piping gels, bakery jellies, and similar products. The ice crystal formation in frozen dough products is retarded by the addition of 0.1% carrageenan, thus improving the texture in frozen dough. Alginates in combination with xanthan may be used to increase batter viscosity and increase cake volume. They also act as a cold-water gel base for instant bakery jellies and instant lemon pie fillings. Freeze-thaw stability of the fillings has been reported to improve in samples treated with alginates. In icings, alginates reduce stickiness and cracking. Alginates stabilize fat dispersion in whipped toppings and stabilize meringue products. Some of the uses of agar important to the baking industry include its ability to stabilize icings or glazes by preventing water migration. It has also been used to reduce tackiness and to prevent adhesion of the sugar coating to the wrapper. Other applications include its use as a stabilizer


in pie fillings, piping gels, meringues, cookies, and similar products. Bakery jellies that are heat reversible may be prepared with the use of 1 to 2% amidated pectins at 40 to 65% solids. Weeping or syneresis of pie fillings and glazes is also retarded or inhibited by the use of pectins, in combination with other gums. Guar gum is used in cake mixtures to improve moisture retention in the finished product. It is a thickening agent and stabilizer for baked goods. Guar also helps to increase volume in yellow cake, probably by aiding in air entrapment. The whipping properties of toppings and icings are enhanced by the addition of 0.1 to 0.2% of guar based on the weight of the finished product. Guar gum in combination with other hydrocolloids has been used to increase soluble dietary fiber content in bread. In a study, panelists evaluated “internal” scores for bread formulated with guar gum and carrageenan. The internal scores evaluated include grain, mouthfeel, crumb body, and taste aroma. Results of the study show high acceptability of the high-fiber bread with gums (24). Guar is shown to have water-binding properties when used in bread doughs at 0.15%. When used at 0.1 to 0.2% in fruit pie fillings, it prevents the water from boiling out. Syneresis or weeping is retarded by the use of locust bean gum in gel desserts. In bakery fillings, guar gum prevents water migration from the filling to the pastry due to its water-binding property. Freeze-thaw stability in frozen doughs is improved by use of xanthan gum. In baked goods, xanthan appears to inhibit starch retrogradation and improves shelf life of the finished product.

C. SALAD DRESSINGS AND SAUCES “Full fat” salad dressings usually contain 30 to 60% oil and mayonnaise about 70 to 80%. Gums as thickeners, stabilizers, and emulsifiers are widely employed in these types of products industry. The most common emulsifying gum in high-oil salad dressings and sauces is propylene glycol alginate, an esterified form of alginate. Gum acacia may also be used to emulsify the oil but is used in combination with thickeners such as guar gum and xanthan gum. The synergy between xanthan gum and guar gum is well known (3) and utilized in stabilizing salad dressings. Xanthan gum adds acid stability and guar gum provides viscosity at lower cost than other thickeners. In products that contain milk protein such as creamy Italian or ranch dressing, a low concentration of carrageenan (0.05 to 0.10%) may also be used for its protein reactivity. The formulation for a pourable creamy Italian salad dressing with 10% oil is shown in Table 139.6. The stabilizer used may include xanthan gum, guar gum, propylene glycol alginate, and starch. The sensory evaluation results of salad dressing bases using a 35% oil salad dressing and

139-11

TABLE 139.6 Creamy Italian 10% Oil Dressing Formulation Ingredients

%

Water Vinegar (100 gr.) Non fat dry milk Sugar Maltodextrin Soybean oil Salt Xanthan/guar/gum acacia/starch system

66.15 7.00 5.00 4.50 4.50 10.00 1.50 1.35

100.00 Procedure: 1. Add 10 parts to 1 part gum system to form a slurry. 2. Add oil/gum slurry to water while mixing. Hydrate for 10–15 min. with good agitation. 3. Add the rest of the ingredients. 4. Run through a colloid mill at medium setting to form a stable emulsion with small particle size.

Opacity 4

Control Gum system

3 2 1

Mouthfeel

Cling

0

Acidity

Creaminess

FIGURE 139.11 Sensory analysis of salad dressings (fat mimetic system vs. control).

a 3% oil dressing using a fat mimetic gum system are shown in Figure 139.11. The attributes, opacity, cling, acidity, creaminess, and mouthfeel are comparable using a hedonic rating scale.

D. BEVERAGE EMULSIONS Oil-in-water emulsions are employed widely in the food, beverage and pharmaceutical industries. Flavor components in beverage can be mainly oil-soluble, water-soluble, or a combination of both, and flavor retention in the beverage requires a good emulsifying system. Beverages mainly consist of water, sweeteners, acidulants (phosphoric acid or citric acid), flavors, and other additives such as coloring agents, weighting agents, vitamins, and minerals. The total solids may vary from 9 to 14% and the beverage may contain alcohol. Clear beverages require that any insoluble components of the citrus oils be removed by special treatment.

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Typical beverage emulsion formulations, using a weighted or unweighted emulsion with gum acacia, an emulsifier with an HLB (hydrophile-lipophile balance) value of 10 to 11, are shown in Tables 139.7A, 139.7B, and 139.8, respectively). Weighted emulsions may contain SAIB (sucrose acetate isobutyrate), ester gum (glyceryl abietate) or BVO (brominated vegetable oil) as weighting agents to adjust the specific gravity of the flavor oil. The emulsification procedure involves homogenization at about 3500/500 psi in a two-stage homogenizer. Homogenizers are special devices that disperse a mixture by forcing it through a tiny orifice under very high pressure. Microfluidizing equipment may also be used to prepare emulsions with very fine particle size (about 90% less than 2 microns). TABLE 139.7A Prototype Formulation: Weighted Beverage Emulsion (1) Ingredients

%

Gum acacia, spray-dried* Citrus oil/ester gum/SAIB** Sodium benzoate Citric acid Water (added to make 100 mL)

15.0 10.0 0.1 0.1

* May be replaced by 5% modified gum acacia. ** Sucrose acetate isobutyrate.

TABLE 139.7B Prototype Formulation: Weighted Beverage Emulsion (2) Procedure: 1. Add the preservatives to the water and mix thoroughly. 2. While mixing add the gum acacia gradually to the vortex of the solution. 3. Allow the gum to hydrate by mixing for 1 hour. 4. Dissolve the ester gum in the oil thoroughly by mixing for 2 hours. 5. Add the weighted oil from #4 to the gum solution. 6. Mix using a Ross mixer at medium speed for 10 min. 7. Homogenize at 3500/500 psi. 8. Pack into sterile containers.

TABLE 139.8 Prototype Formulation: Emulsion

Unweighted

Ingredients Gum acacia, spray-dried Citrus oil Sodium benzoate Citric acid Water (added to make 100 mL) Note: Procedure as in weighted emulsion.

E. CONFECTIONS AND CANDIES Confectionery products include a spectrum of sweet goods, specifically candies and similar products. Sugar confectioneries include nougats, fondant, caramels, toffees, and jellies (e.g., gum drops and orange slices). Chocolate confections include chocolate and assorted chocolate-covered fruits, nuts, and cremes. Gum acacia has the ability to retard or inhibit sugar crystallization and is used in the manufacture of pastilles and soft candy where sugar content is very high. In caramels, it will also help emulsify the fat to distribute it more uniformly and prevent oil from forming a rancid oily film. Gum acacia coacervates with gelatin are also used as chewy candy centers in many popular products flavored with peppermint or spearmint. 1. Gelatin Substitutes: Hydrocolloid Gelling Agents Gelatin substitutes in gummy bears and similar products have been developed in recent years because gelatin is not acceptable to some religious groups and is sometimes associated with “mad cow disease.” However, the properties of gelatin, including melting point, flexibility, and mouthfeel, are difficult to simulate with other hydrocolloids and textural differences between products prepared with gelatin and gelatin substitutes can be detected by the consumer. Gummy candies, using gelling agents such as pectin, agar, carrageenan, or combinations of these with modified starches have been introduced in the market, but with limited success. A formulation for a gummy candy without gelatin is shown in Table 139.9. A comparison of the texture profile analyzer (TPA) curves of gummy candy with gelatin and those prepared with some other gum systems containing agar and pectin as gelatin substitutes is shown in Figure 139.12.

F. MEAT AND POULTRY PRODUCTS For poultry and meat injection, fine-mesh carrageenan incorporated with the brine significantly reduces cooking loss, thus increasing yield. Marinades may also be thickened and stabilized with guar gum and xanthan gum, a

Beverage % 15.0 10.0 0.1 0.1

TABLE 139.9 Gummy Candy with Pectin and Agar System Ingredients Sucrose Glucose syrup Agaroid gum system Trisodium citrate Citric acid (50%) Water (to make 100%)

% 46.0 25.0 2.0 0.25 0.15


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heat-stable gum. In meat analogs or minced meat products, carrageenan at 0.5% to 1.0% level has been shown to reduce syneresis and act as moisture binder and adhesive (11).

III. FAT MIMETICS AND FUNCTIONAL FOODS The typical North American diet provides a continuing challenge to the food industry to formulate highly acceptable low-fat products, particularly those low in trans-fatty acids, for the consumer. Unless an integrated approach is used, fat or oil mimetics may yield products that are inferior to the full-fat, high-calorie counterparts. Initially the role of the fat in the specific product has to be determined and then steps taken to simulate or mimic the functionality and sensory qualities of fat or oil being reduced. Some of the important factors to consider include the following: mouthfeel and texture characteristics, impact on threshold value or perception of flavor, functionality of the fat in the product, processing conditions during manufacturing scale-up, shelf life, microbial stability, and water activity. Hydrocolloid systems consisting of gums, starches, and other components may be used as fat mimetics (26). However, it is not recommended that all the fat or oil is replaced in the formulation, since the lipids have special functional properties that the gum systems may not be able to replace. Partial reduction of fat or oil up to 50% replacement may be feasible so as to prevent the significant changes in the threshold value of flavor compounds. The flavor components are significantly affected by the fat or oil content of the finished product as shown by significant changes in threshold value. A combination of gum acacia, a food starch, and alginates has been successfully marketed for low-fat muffin and other bakery mixes (26). The main component of the system, gum acacia, is an unusual gum that offers advantages over other gums as a fat mimetic for the following reasons.

Gel strength (g/cm2)

1400 1200 1000 800

Unlike other gums, it has low viscosity about 100 cps at 25% solids and contains 2 to 3% protein (which provides its good emulsifying properties). It imparts smooth mouthfeel and acts as a lubricant, inhibits ice and sugar crystallization, is high in dietary fiber, has adhesive properties, and may be used for spice adhesion instead of oil. Fat mimetic systems using gums and starches incorporated in a low-fat cookie formulation have been shown to yield a product with longer shelf life, based on higher moisture content retention and lower hardness values measured using a Steven’s Texture Analyzer. Gelling agents such as carrageenan and agar may also be used in fat mimetic systems to simulate the texture of fat in icings and glazes. In developing low-fat systems, the fat is usually replaced with water and other solids. It is imperative that the formulator minimize the water activity gradient between the substrate and the filling or glaze, to reduce water migration. The high soluble dietary fiber content of hydrocolloids makes them essential components of functional/ nutraceutical foods and beverages. For example, the use of low viscosity guar gum and gum acacia in high-fiber beverages such as smoothies or fruit concentrates is increasing. In granola bars and dry beverage mixes, the health benefits are being augmented by adding gums as sources of soluble fiber including the following: inulin, gum acacia, polydextrose, and hydrolyzed guar.

IV. GUM SYSTEMS: SYNERGY AND INTERACTION When certain specific gums are used in combination, the functional properties are significantly enhanced or modified due to synergistic action. For example, a combination of xanthan gum and locust bean gum at 1.0% gum level will form a heat-reversible flexible gel whereas the individual gums are not gel forming (27). The interaction of locust bean gum with kappa carrageenan, to yield

Candy with 3.5% Gelatin Candy with 2% Pectin/Agar system 6% Gelatin alone

600 400 200 0 −200 −400

Time (0 − 2 min.) *Stevens texture analyser QTS 25

FIGURE 139.12 TPA of gummy candy with pectin/agar system vs. gelatin.

139-14


7000

600

Viscosity

Viscosity (cP)

Gel strength

5000

500 400

4000

300

3000 200 2000

Gel strength (g/cm2)

6000

100 1000 0

0

r ua

/G

n ha

r ua

G

C

BG

M

r/L

C

nt

Xa

ua

r ga

A

G

n/

Xa

Initial

Retorted*

*Retorted 30 min. at 250°F

FIGURE 139.13 Effect of heat processing on various gums.

heat-reversible gels with lower degree of syneresis than gels made with kappa carrageenan alone, has also been used in baked goods. Synergy between alginate, gum acacia, and starches has been utilized to formulate fat-mimetic systems as discussed earlier. Guar/xanthan provide strong viscosity synergism when used in combination (1, 2). A comparison of the effects of heat processing (retort temperature at 250°F for 30 minutes) on some gums and gum blends is shown in Figure 139.13. Some gums, due to their synergy, are more resistant to heat degradation when used in combination with each other.

V. MAXIMUM USAGE LEVELS AND QUALITY SPECIFICATIONS The maximum permitted usage levels of gums in foods and beverages vary according to the Code of Federal Regulations. The Food Chemicals Codex of the Food and Drug Administration also establishes the specifications for each gum, and a summary of the FCC standards for guar gum, as an example, is shown in Table 139.10. In addition, the food manufacturer should specify for each gum or gum system specific microbial limits, gel strength, viscosity, particle size distribution, and other relevant parameters, in order to avoid unwarranted variations in the quality of the finished formulation.

VI. PREHYDRATED OR AGGLOMERATED GUMS AND GUM SYSTEMS Proprietary processes of agglomeration or “prehydration” of gums and gum systems have been developed by a number of ingredient manufacturers (11). The end products hydrate faster, have no lumping problems and are virtually

TABLE 139.10 FCC & USP/NF Standards: Guar Gum Tests Required Identification Acid* insoluble matter Arsenic Total ash Galactomannans Heavy metals Lead Loss on drying Protein Starch

FCC

NF (USP)

A. Opalescent, viscous solution B. No appreciable increase in visc. ⭐7.0% Same ⭐3 ppm Same ⭐1.5% Same ⭓70% Same ⭐0.002% Same ⭐5 ppm ⭐0.001% ⭐15% Same ⭐10% Same No blue color Same produced

* H2SO4.

dust-free, resulting in fewer incorporation problems, reduced dusting, mote efficient use of equipment, and production and labor costs. The agglomerated gums such as CMC and gum acacia have been shown to reduce motor load in a twin-screw extruder when incorporated at low levels with the initial dry mix (28). They also improve the mouthfeel and texture of corn cereal extrudates, based on sensory evaluation. These so-called prehydrated gum stabilizers are also preferred for use in low-oil salad dressings and in low-moisture systems.

VII. ANALYTICAL METHODS FOR EVALUATION OF GUMS A laboratory designed to analyze and evaluate individual hydrocolloids and gum systems requires a number of essential instruments and equipment. For the study of viscosity and other rheological properties of gum solutions, a programmable rheometer (e.g., a Brookfield™ model DVIII)


is useful in characterizing the effect of shear rate, temperature, time, and stress. A Bostwick™ flow meter or consistency meter can be used to measure flow of a viscous fluid vs. time. For the proximate analyses of gums and their basic composition, the laboratory should be equipped with instruments for measuring pH, ash, protein, fat, and moisture. HPLC (high pressure liquid chromatography) equipment with appropriate columns, refractive index measurement capabilities, and UV detectors may be used to analyze monosaccharide ratios of gums and help detect cross-contamination, adulteration, and the presence of bulking agents. To analyze molecular weight changes and effects of various factors such as pH and temperature on the stability, hydrolysis, or degradation of gums, gel permeation chromatography using various known standards is useful. To save instrumentation costs, independent analytical laboratories may of course be utilized at any time whenever deemed necessary. For gel strength measurements, programmable texture analyzers that can determine parameters such as hardness, cohesiveness, adhesiveness, and gumminess help evaluate the texture of the gel or the finished gelled product. This may help the sensory evaluation specialist by providing an objective comparison of the texture profiles of products prepared with gelatin with those containing polysaccharide gelling agents. A useful instrument for evaluating emulsifying systems such as gums for beverage emulsions, is a particle size analyzer (e.g., a Coulter Counter). Preparation of stable emulsions requires a homogenizer that can operate at 2500 up to 6000 psi. A microscope equipped with a digital camera can be used to analyze the morphology of starches and the emulsion particle size of salad dressings, creams, and similar products. A differential scanning calorimeter may be used to analyze melting point and gelling temperatures and other endothermic and exothermic properties such as glass transition temperatures of various hydrocolloid systems.

VIII. SUMMARY AND RECOMMENDATIONS Hydrocolloids are highly functional ingredients in foods and beverages. Preferably, they should be incorporated with some of the dry ingredients such as flour or sugar to avoid lumping or incomplete hydration. The gum manufacturer should specify the various requirements for optimum functionality of the gum (pH, % solids, temperature of hydration, salts, co-factors, etc.). The impact of large-scale processing operations and the possible need for special equipment design should be jointly evaluated by the food technologist and the chemical engineer. This can reduce production problems during scale-up from the R & D or applications laboratory to the manufacturing plant.

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In developing a fat mimetic system in food products, a systems approach should be used in which a variety of synergistic components, including gums, are used to duplicate the functional and sensory characteristics of the specific full-fat product. The development of a zero fat formulation is generally not recommended since significant changes in threshold value of flavor components in going from a high-oil to oil-free medium make it difficult to simulate the flavor of regular fat products. However, fat or oil reduction and increase in soluble fiber in the average American diet are essential, requiring the food manufacturer to make healthy alternative products and functional foods available to the consumer. Using an individual gum for a certain application may not be adequate to achieve the desired quality attributes in the finished product. Instead, a combination of the optimum ratio of specific gums that exhibits synergistic properties may be required. The food technologist and the gum supplier should be familiar with the chemical and functional properties of each component in the food system and the resulting interactions between various ingredients and at varying processing conditions.

ACKNOWLEDGEMENTS The authors would like to thank Steve Andon and Chris Andon, of TIC Gums, Inc., for administrative and financial support; and Ken Kuschwara & Jim Caulfield of the R & D Group of TIC Gums, Inc., for their technical assistance.

REFERENCES 1. Whistler, R. L. and J. BeMiller. Industrial Gums. Academic Press, Inc., New York. 1993. (642 pages). 2. Philips, G. O. and P. A. Williams. Handbook of Hydrocolloids. CRC, Woodhead Publishing Ltd., England. 2000. (450 pages). 3. Glicksman, M. Food Hydrocolloids. CRC Press, Inc. Boca Raton, FL. Volumes I to III, 1982. 4. Ward, F. M. Uses of Gum Acacia in the Food and Pharmaceutical Industries. Nothnagel et al. Kluwer Academic/Plenum Publishers, pp. 231–239, 2000. 5. Ward, F. M. Modified hydrocolloids with enhanced emulsifying properties. In: Gums and Stabilizers for the Food Industry 11. P. A. Williams and G. O. Philips, Eds. Royal Society of Chemistry, U.K., pp. 218–322, 2002. 6. Ward, F. M. 2002. U.S. Patent No. 6,455,512. Water-Soluble Esterified Hydrocolloids. September 4, 2002. 7. Ward, F. M. Hydrolysed and deodorized guar gum. In: Gums and Stabilisers for the Food Industry 10. P. A. Williams and G. O. Philips, Eds. Royal Society of Chemistry, U.K., pp. 429–438, 2000.

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8. Baker, C. W. and Whistler, R. L., 1975. Distribution of D-Galctosyl groups in guar and locust bean gum. Carbohydr. Res. 45, 237–243. 9. Richardson, P. H., et al., 1998. Dilute solution properties of guar and locust bean gum in sucrose solutions, Food Hydrocolloids 12, 339–348. 10. Armisen, R. 1991. Agar and agarose constituent of agaragar. Hydrobiology, 221, pp. 159–166. 11. Tic Gums, Inc., 1993–2003. Product Data Bulletins, Belcamp, Maryland. 21017, USA. 12. Bixler, H. J. 1996. Refined and semi-refined carrageenan. Food Hydrocolloids 96. San Diego, March 1996. 13. Thomas, W. R. 1997. Carrageenan in Thickening and Gelling Agents for Food, 2nd edn. A. P. Imeson, Ed. Blackie, London, pp. 45–59. 14. Neiser, S., Draget, K. and Smidsrod, O. 1998. Gel formation in heat-treated bovine serum albumin-sodium alginate systems. Food Hydrocolloids 12, 127–32. 15. Dickinson, E. and Walstra, P. 1993. Food Colloids and Polymers. Stability and Mechanical Properties. Royal Society of Chemistry. 16. Sworn, G., Gellan Gum. In Philips, G. O. and P. A. Williams. Handbook of Hydrocolloids. CRC, Woodhead Publishing Ltd. England, pp. 117–134, 2000. 17. Chandrasekaran, R., and Thilambali, V. G. 1990. The influence of calcium ions, acetate and L-glycerate groups on the gellan double helix. Carbohydr. Polym. 12, 431–432. 18. Schols, H. A., Ros, J. M., Dass, P. J. H., Bakx, E. J., and Voragen, A. G. J., 1998. ‘Structural Features of Native and Commercially Extracted Pectins, Gums, and Stabilizers for the Food Industry,’ 9, Wrexham, The Royal Society of Chemistry.

19. M.E.Malone, I.A.M. Appelqvist, I.T. Norton. 2003. Oral behaviour of food hydrocolloids and emulsions. Taste and aroma release. Food Hydrocolloids 17, 775–784. 20. Arbuckle, W. S. Ice Cream. Van Nostrand Reinhold Co. New York, pp. 49–94, 1986. 21. Wang, S. T., S. A. Ringer, P. M. T. Hansen. 1998. Effects of carboxymethyl cellulose and guar gum on ice crystal propagation in a sucrose-lactose solution. Food Hydrocolloids 12, 211–215. 22. Marshall, R. T. and D. Goff. 2003. Ice cream. Food Technology 57(5), May, 32–45. 23. Yanes, M. L., Duran, & E. Costell. 2002. Effect of hydrocolloid type and concentration on flow behaviour and sensory properties of milk beverages model systems. Food Hydrocolloids 16, 605–611. 24. Ward, F. M. and S. Andon. 1993. The use of gums in bakery foods. Ranhotra, Gur. Ed. Technical Bulletin, American Institute of Baking. Manhattan, KS, USA, pp. 1–8, 1993. 25. Ward, F. M. and S. Andon. 1993. Water-soluble gums used in snack foods and cereal products. Cereal Foods World 38, 748–742. 26. Ward, F. M. 1997. Hydrocolloid systems as fat mimetics in bakery products: icings, glazes and fillings. Cereal Foods World 42(5), 386–390. 27. Nussinovitch, A., Ed. 1997. Hydrocolloid Applications: Gum Technology in the Food and Other Industries. Blackie Academic and Professional, London. 28. Mulvaney, S., Rizvi, S. S. H and G. Ryu. The use of cellulose gum and gum acacia in corn extrudates. Technical Report. Joint study: TIC Gums and Dept. of Food Science, Cornell University. New York, 1993, 15 pages.

140

Pectins

Hans-Ulrich Endress, Frank Mattes, and Karl Norz Herbstreith & Fox KG, Pektin-Fabrik Neuenbürg

CONTENTS I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

Introduction ....................................................................................................................................................140-2 Occurrence of Pectins ....................................................................................................................................140-2 Chemical Structure of Pectins........................................................................................................................140-2 Biochemistry of Pectins ................................................................................................................................140-4 Pectins in Cell Wall Architecture ..................................................................................................................140-5 Determination of the Pectin Content and Kind of Pectic Substances ..........................................................140-5 Chemical, Enzymatic, and Mechanical Modifications of Pectins ................................................................140-5 Pectins as Polyelectrolytes ............................................................................................................................140-7 Rheology of Pectins ......................................................................................................................................140-7 The Molecular Weight of Isolated Pectins ....................................................................................................140-8 Pectin Manufacturing ....................................................................................................................................140-9 Properties of Isolated Pectins ......................................................................................................................140-11 A. High Methylester Pectin ......................................................................................................................140-12 B. Low Methylester Pectin ......................................................................................................................140-13 C. Nutritional Aspects ..............................................................................................................................140-14 XIII. Food Legislative Aspects ............................................................................................................................140-14 XIV. Dissolving Pectin ........................................................................................................................................140-15 XV. Jams, Jellies, and Marmalades ....................................................................................................................140-15 A. Traditional Jams and Jellies ................................................................................................................140-15 B. Low Sugar Jams and Jellies ................................................................................................................140-18 C. Gelling Powder and Gelling Sugar ......................................................................................................140-20 XVI. Fruit Preparations ........................................................................................................................................140-20 A. Baking Stable Fruit Preparations ........................................................................................................140-21 B. Cake Glazing ........................................................................................................................................140-22 C. Dairy Fruit Preparations ......................................................................................................................140-23 XVII. Confectionery ..............................................................................................................................................140-24 XVIII. Beverages ....................................................................................................................................................140-25 A. Juices and Soft Drinks..........................................................................................................................140-25 B. Dairy Beverages and Soy Drinks ........................................................................................................140-26 XIX. Ice Cream and Sorbets ................................................................................................................................140-27 XX. Other Food Applications ..............................................................................................................................140-28 A. Savory Products....................................................................................................................................140-28 B. Desserts and Other Dairy Products ......................................................................................................140-29 C. Bakery Products ..................................................................................................................................140-30 XXI. Summary ......................................................................................................................................................140-30 References ..................................................................................................................................................................140-30

140-1

140-2


I. INTRODUCTION Pectins belong to a group of closely related polysaccharides or pectic substances, located in the middle lamella and primary cell walls of higher plants (dicotyledons). The general term protopectin is often used to designate the native, insoluble pectins in the cell walls that cannot be extracted by methods that are non-destructive or non-degradative. The dominant feature of pectins is a linear chain of α-(1,4)-linked D-galacturonic acid units in which varying proportions of the carboxyl groups are esterified with methanol to methyl esters. This group of pectic substances covers the following: ●

● ●

different methyl esters (pectins with different degree of esterification — DE), their salts (pectinates) unesterified pectic acid, its salts (pectates) different neutral polysaccharides linked to the polygalacturonan backbone such as arabinans, arabinogalactans, arabinoxylans, and galactans

Portions of these neutral polysaccharides are a part of the isolated, commercial pectins divided into HM pectins and LM pectins with correspondingly high and low methyl ester content. Amidated pectins are obtained by saponification of HM pectins with ammonia under alkaline conditions.

II. OCCURRENCE OF PECTINS Beside cellulose, hemicelluloses, glycoproteins, and lignin pectins form a major part of the cell wall of all higher plants. The concentration of pectins is highest in the middle lamella, a tissue responsible for the adhesiveness of cells (1), and decreases from primary to secondary walls, where pectins are almost absent. Pectins participate in plant physiology: water retention, ion transport, porosity, growth, and the size and shape of cells. Pectins are involved in defense mechanisms against infections by plant pathogenic

micro organisms, generating by their enzymatic attack (mainly using a polygalacturonase PG) oligogalacturonides with a degree of polymerisation between 10 and 15, which can be recognized by the plant (so called elicitors). This results in an activated metabolism (2). This reaction may be used to activate plant cell cultures for a higher productivity. The specific functions of pectins in distinct parts of the cell walls or plant tissues are influenced by the amount and nature of specific molecules present (3).

III. CHEMICAL STRUCTURE OF PECTINS Studies on pectins from many sources have illustrated that pectin is a heteropolysaccharide (see Table 140.1) (4). Pectins consist of a linear zigzag shaped structure of axial-axial linked α-(1,4)-D-galacturonic acid units, a result of the equatorial position of the carboxyl group. The poly-galacturonic acid chain is interrupted by “inserted” α-(1,2)-linked L-rhamnopyranosyl units resulting in a kink that determines the linear portion of the corresponding pectin segment (see Fig. 140.1) (5, 6). Results from X-ray diffraction analyses fail to confirm: ●

●

if the pectins are right- or left-handed double or single helix what the number of repeating units is

Pectin segments or fractions with low content of L-rhamnose are described as smooth regions of pectins or homogalacturonans. Those with high L-rhamnose content are known as hairy regions or rhamnogalacturonans. The neutral sugar side chains consisting of mainly L-arabinose and D-galactose are bound by covalent linkages to the L-rhamnose units. L-fucose is found as the terminal end of these side chains. As minor sugars also D-xylose, D-glucose, D-mannose, and D-apiose are found next to further rare sugars. The minor sugars can occur as single unit side chain such as D-Xylose or as short side

Galactan

GUS

RHA

GUS

Galactan

GUS

RHA

GUS

GUS

GUS

GUS

GUS

RHA

n GUS GUS = Galacturonic acid RHA = Rhamnose

FIGURE 140.1 L-rhamnose in the galacturonan chain.

RHA

GUS

GUS

Pectins

140-3

TABLE 140.1 Galacturonic Acid and Neutral Sugars in Some Pectic Materials (4) Applea

Sugar Beetb

Carrotc

Plumd

Potatoe

Yield

14f

11.1f

13.5f

28.6f

13.1g

GalA Rha Fuc Ara Xyl Man Gal Glc

58.0 3.0 23.0 1.0 1.0 5.0 3.0

54.9 3.2 12.5 0.2 — 8.1 0.3

54.7 3.8 11.7 0.2 0.7 8.3 1.2

43.0 1.5 5.9 0.4 1.1 15.2 3.8

43.6 1.3 7.0 0.4 0.5 5.5 4.5

a

Pectins extracted with hot water (7). Pectins extracted by 0.05 M NaOH, 4°C after extraction with water, oxalate, acid (8). c Pectins extracted by 0.05 M HCl at 85°C (9). d Pectins extracted by water at room temperature (10). e Pectins extracted with CDTA, pH 6.8, 20°C (11). f Yield calculated from an alcohol-insoluble residue. g Yield calculated from cell-wall material after SDS and DMSO treatments. b

chains whereas L-arabinose and D-galactose form complex structures. The arabinans are branched polysaccharides with a backbone of α-(1,5)-linked arabinofuranosyl residues with α-(1,2)- and α-(1,3)-linked arabinofuranosyl side chains. Pectins with attached arabinans can be isolated from many fruits and vegetables like apples, sugar beet, apricots, carrots, cabbage, onion, and pears. Citrus fruits, potato, soy beans, grapes, apples, onions, tomatoes, and others contain arabinogalactans, described as two structurally different forms. Type I consists of a β-(1,4)-linked linear chain of D-galactopyranosyl residues with short chains of linear α-(1,5) arabinans connected to O-3. Type II is a highly branched polysaccharide with ramified chains of β-(1,3)- and β-(1,6)-linked D-galactopyranosyl residues terminated by L-arabinofuranosyl and to a smaller extent by L-arabinopyranosyl residues. Albersheim and co-workers (12–14) studied the structure of suspension cultured sycomore cells and described rhamnogalacturonan I and II. Rhamnogalacturonan I was analyzed to have a linear structure of alternating α-(1,4)linked D-galacturonosyl and α-(1,2)-linked L-rhamnosyl residues with a bunch of different neutral sugar side chains. Rhamnogalacturonan II is a very minor and complex heteropolysaccharide consisting of about 30 glycosyl residues. Using chemical (β-elimination) or enzymatic (endoPG or endo-PL) degradation techniques to split the polygalacturonan backbone, the following is observed. The L-rhamnosyl residues and the neutral sugar side chains are not homogeneously distributed over the pectin chain. Sequences rich in neutral sugars are interspersed with almost pure poly-D-galacturonosyl blocks. This finding distinguishes pectic polysaccharide as smooth or hairy

regions (15–18) which are also homogalacturonans and rhamnogalacturonan I (13, 19, 20). The figures in literature on the length of homogalacturonan blocks vary from 25 (21) to 40–60 (22), and 72–100 galacturonic acid units (17). But also non-sugar substituents (beside the methyl ester of the carboxyl groups or the amide groups at C-6) bound to C-2 or C-3 of the galacturonic acid like acetic acid and phenolic acids can be found with some pectic substances. The degree of esterification (DE) or methylation (DM) is described as the percentage of esterified galacturonic acid units. If more than 50% of the carboxyl groups are esterified, the pectin is called high methyl ester (HM) pectin; if less than 50%, low methyl ester (LM) pectin. Pectic acid is defined to have a DE of 10%. The carboxyl groups (free and esterified) can be distributed statistically (random) or blockwise along the pectin molecules. The distribution pattern influences the reactivity of the pectins with bivalent cations and positively charged proteins. Acid treatment and most microbial pectin esterases (PE) deesterify HM pectins statistically. Plant pectin esterases work blockwise creating high reactive zones of negatively charged sequences. This property is ambiguous when used in commercial pectin applications. Also, this property distinguishes between intra- and intermolecular distribution of carboxyl groups. “Intermolecular” refers to an inhomogeneous pectin preparation. The degree of acetylation (DAc) is defined as mol% of acetic acid calculated on the content of galacturonic acid. This may result in a DAc higher than 100% because C-2 and C-3 of the galacturonic acid and other sugars of the pectin can be acetylated. Apple and citrus pectins have a negligible DAc whereas beet pectin is highly acetylated. A DAc of about 25% can be found in some commercial beet pectins, e.g., those by Herbstreith & Fox. Galacturonic acid content (GalA), degree of esterification (DE), and degree of acetylation (DAc; mol/mol) of some pectins extracted from different plants are summarized in Table 140.2 (4). Fry (30), Rombouts and Thibault (8), and the research group of Ralet and Thibault (31, 32) described ester-linked ferulic acid units in beet and spinach pectin, linked to arabinosyl and galactosyl residues. Fry (33) was investigating the possible role of phenolic compounds of the primary cell wall in the hormonal regulation of growth. By these ferulic esters pectin chains can be cross-linked by phenolic coupling. In plant tissue these phenolic bonds are of big importance and can be used for firming fruits for food application. Also, gels from beet pectin can be formed by oxidative coupling, using phenoloxidase and peroxidase. Isolated commercial pectins seem to belong to the group of homogalacturonans. However, they are obtained from apples, citrus fruits, sugar beet, or sunflower heads by extraction conditions cleaving covalent bonds to the hairy part of pectic substances firmer bound into plant tissue by neutral sugar side chains.

140-4


TABLE 140.2 Galacturonic Acid Content (GalA), Degree of Methylation (DM) and Degree of Acetylation (DAc; mol/mol)a of Some Pectins (4) Origin

Extraction

GalA (%)

DM (%)

DAc (%)

● ● ●

Ref.

Mango Acidic 54 68 4 (23) Sunflower Acidic 81 17 3 (23) Sugar-beet 0.05 M HCl; 85°C 65 62 35 (8) Carrot 0.05 M HCl; 85°C 61 63 13 (24) Grape Oxalate pH 4.5; 20°C 63 69 2 (25) Sunflower Oxalate 83 27 10 (26) Peach HCl to pH 2; 80°C 90 79 4 (27) Siberian apricot EDTA 64 57 8 (28) Cythere plum 0.05 M HCl; 85°C 65 65 16 (29) Potato HCl, pH 2; 80°C 40 53 15 (4) a Calculated on the assumption that all acetyl groups are bound to galacturonic acid.

IV.

Other observations confirm that pectin synthesis may differ:

●

The precursors of the nucleotide-sugars for pectin synthesis are hexose-phosphates from photosynthesis and stored starch or sucrose. The main sugars of pectic substances are D-GalA, L-Ara, D-Gal, D-Man, L-Rha, D-Xyl, L-Fuc. They are synthesized accordingly: 1. All can be synthesized enzymatically via D-Glc1-phosphate, uridine-diphosphate-D-glucose (UDP-D-Glc), and their corresponding uridinediphosphates by different nucleotide-sugar transformation pathways. 2. All can be recycled from walls by the salvage pathway. 3. D-GalA, D-Xyl, and L-Ara can also be synthesised enzymatically by the myoinositol pathway from UDP-D-GlcA and the subsequent and corresponding UDP-sugars/UDP-sugar acids.

BIOCHEMISTRY OF PECTINS

A most recent and excellent description of the biosynthesis of pectins is by Mohnen (34). Pectin is said to be the most complicated polysaccharide in plant cell wall. Since we do not know its exact composition and the synthetic process of various fractions of pectic polysaccharides, we may benefit from a summary of what information is available: At least 12 activated sugar substrates (nucleotidesugars), 14 distinct enzyme activities (for their production), and 58 glycosyl-, methyl-, and acetyltransferases are required for pectin synthesis. Mohnen (34) cites comprehensive reviews on: ●

● ● ●

pectin structure, pectin and cell wall synthesis (35–40) nucleotide-sugar interconversion pathways (41) wall biosynthetic genes (38, 42) glycosyltransferases (43)

The synthetic process of pectin is often described as follows: ●

● ● ●

●

synthesized as homogalacturonans in the cis-Golgi branched in the trans-Golgi cisternae highly esterified in the medial and trans-Golgi transported as high esterified, branched pectin to the plasma membrane in vesicles, which move along actin filaments via myosin motors, and subsequently inserted into the wall or the cell plate followed by deesterification to create calciumreactive sequences

in different cell types in different species at different points during development, or even at different locations in the same wall (cited in 34)

Examples of well investigated enzymes in these pathways are: ●

●

●

UDP-glucose-6-dehydrogenase (EC 1.1.1.22), oxidating UDP-D-glucose to UDP-D-glucuronic acid UDP-glucuronate-4-epimerase (EC 5.1.3.6), transforming UDP-D-glucuronic acid into UDPD-Galacturonic acid UDP-xylose-4-epimerase, transforming UDPD-xylose into UDP-L-arabinose.

UDP-xylose is synthesized from UDP-D-glucuronic acid by UDP-GlcA-decarboxylase (EC 4.1.1.35). L-rhamnose is proposed to be produced from UDP-D-glucose via UDP-4-keto-6-deoxy-glucose (by UDP-Glc-4,6dehydrogenase) and UDP-4-keto-6-deoxy-L-mannose (by UDP-4-keto-L-rhamnose-3,5-epimerase), with a final conversion via UDP-4-ketorhamnose-reductase. The nucleotide sugars are transported into the Golgi and used as substrate by a pectin biosynthetic glycosyltransferase that transfers the glycosyl residue onto a growing polymer (34).

V.

PECTINS IN CELL WALL ARCHITECTURE

Are pectins and their fractions including neutral sugar side chains covalently linked to other polysaccharides or

Pectins

glycoproteins in cell walls? Some observations from complex early models suggest such linkages, the existence of which has never been proven (44, 45). Current accumulated reports suggest the following: 1. The existence of several woven but independent networks in the cell walls and pectin network is one of them, situated next to cellulosexyloglucan-cellulose bridges (46, 47) 2. The existence of independent protein-protein structures (48) instead of the hydroxyprolinrich protein extension structure crosslinked to pectin Pectins are linked with each other through Ca2bridges (21), via borate esters between apiose residues (40), diferulic acid bridges between arabinoxylans side chains (16), and finally esters between the carboxyl groups of the galacturonic acids (49). In growing plants, cell wall structures undergo a continuous change. Crosslinkages are made and broken, especially when a cell divides, expands, or matures. An in-depth discussion on interactions between pectins and other polymers is reported by Mort (50).

VI. DETERMINATION OF THE PECTIN CONTENT AND KIND OF PECTIC SUBSTANCES To characterize pectic substances and their change during growth, ripening, storage, and processing in plant tissue a sequence of events is noted. Pectins are enriched and purified from interfering plant components by washing with hot alcohol to remove alcohol soluble components from the tissue or by fractional extraction techniques. Native enzymatic activities in the plant material are inactivated instantaneously. The repeated washing with hot alcohol results in an AiR (alcohol insoluble residue) composed mainly of cell wall materials, protein, and starch (51). The latter have to be removed by degradation with pure enzymes under conditions not affecting the pectic substances. Starch can also be removed with 90% dimethyl sulfoxide. The cell wall material undergoes further characterization and fractional extraction techniques (chemically and/or enzymatically). Substances, soluble or solubilized under the respective extraction conditions, are yielded as watery extract to be precipitated in alcohol. The alcohol insoluble substance (AiS) is dried, grinded, and analyzed. Note that all extraction and fractionation conditions modify the pectic substances to a certain extent. The evaluation of the different fractions and the knowledge of the enzymatic activity provide us with the ambiguous image of the native pectic substances which we have today.

140-5

The chemical fractionation techniques exploit the different properties of pectic substances. A common procedure is the subsequent use of: 1. cold and/or hot water or buffer solutions for already soluble pectins, mainly HM 2. monovalent buffer solutions to solubilize pectins fixed by bivalent cations 3. chelating agents like EDTA, CDTA, oxalate, or hexametaphosphate for HM and LM pectins crosslinked and bound in cell wall by bivalent cations 4. cold and/or hot acid with different pH, temperature, and time regimes for HM pectins crosslinked with cellulose by neutral sugar side chains 5. cold alkali to prevent β-elimination such as sodium carbonate or sodium hydroxide, often in combination with sodium borohydride to protect the reducing end of polysaccharides, suitable for hemicellulose extraction crosslinked with pectins Other chemicals for extraction are also used (4, 52). Voragen et al. (4) give an overview on enzymatic extraction and fractionating procedures. One approach is based on the degradation of the rhamnogalacturonan backbone, using endo-polygalacturonase, pectin esterase/endo-polygalacturonase, endo-pectinlyase, or endo-pectatelyase, and recently rhamnogalacturonases. In general two fractions of degradation products are obtained, oligogalacturonides and rhamnogalacturonides, that are rich in neutral sugars. These fragments can be further characterized. Degree of polymerization depends on enzyme combination, sugars, and glycosidic linkages which clarify the structure of the heteropolysaccharides present. The second approach is to use non-pectolytic enzymes for the extraction of unaltered pectins. This approach is often discussed as an alternative to industrial acidic pectin extraction. According to available data, it is not possible to yield comparable pectin quantities using combinations of cellulases and hemicellulases (53, 54). However, the use of an endo-glucanase in combination with an endopectinlyase can result in a significant pectin yield in the laboratory. But the gel strength of this pectin is poor.

VII. CHEMICAL, ENZYMATIC, AND MECHANICAL MODIFICATIONS OF PECTINS Pectic substances can be attacked at the polygalacturonic main chain, the neutral sugar side chains, or at the methyl, acetyl, and phenolic ester groups. Glycosidic bonds between uronic acids are relatively stable against hydrolysis; arabinofuranosyl bondings are weak in acidic conditions. This difference is used for pectin extraction by

140-6


acidic hydrolysis to split the neutral sugar side chains, which connect the pectins with the cellulosic fibrils fixing pectin in the cell walls. This way the insoluble protopectin is converted to soluble pectin. The methyl and acetyl ester groups are partially hydrolyzed by the extraction procedure, which is sometimes used to extract pectins with a smaller degree of esterification. Pectin solutions are stable at pH 2.5–4.5. At a lower pH the a.m. hydrolysis occurs. This is used to produce pectins with a lower degree of esterification, changing, within the region of HM pectins, their setting behavior from rapid set (DE 70%) to slow set (DE ca. 60%). Further deesterification results in low ester pectins. At low pH values also acetyl groups from sugar beet pectin are removed and jellifying beet pectins with poor gelling strength are formed. At low pH, e.g., boiling in 12 M HCl, the galacturonic acid releases CO2. This reaction quantitates the pectin content (55). At pH 5 and elevated temperature, the glycosidic bonds of esterified polygalacturonic acid main chains are split at the non-reducing side next to a methyl esterified galacturonic acid unit. This is done by β-elimination, resulting in an unsaturated galacturonic acid molecule with double bonding between C-4 and C-5 (56). Because this reaction can occur wherever there is an esterified acid group in the molecular chain, a small amount of degradation can cause a large loss in viscosity, gelling power, and other functional properties. This reaction is slow at low temperatures. Increasing the pH accelerates saponification of ester groups, which competes with β-elimination. Pectic acid is stable in alkaline conditions. We can take advantage of β-elimination to decrease water binding capacity of pectins by soaking legumes in neutral mono-valent buffer solutions and by liming of fruit press cakes after juice production. Such treated residues can be further pressed to increase solids before drying. Pectins can also be degraded by oxidants. The most common chemical modification is the alkaline amidation of pectins in alcoholic suspension, using ammonia. By this reaction methyl ester groups are converted to acid amid groups (–CONH2). Free carboxyl groups cannot be amidated. Pectins can also be esterified with methanol at a low pH in methanolic suspension, using sulphuric acid. As a by-product, sodium methyl sulphate can be produced with a limit of 0.1% in commercial pectins per FDA and FCC. Crosslinking of pectins by epichlorhydrin is successfully used to prepare affinity chromatography columns to separate pectinases. Crosslinking by phenolic coupling was mentioned earlier. There are several enzymes involved in pectin degradation. 1. glycosidases catalyzing the degradation of neutral sugar side chains (group of arabinanases, galactanases, xylanases, etc.) 2. esterases, pectin esterase (PE) (EC 3.1.1.11), and pectin acetyl esterase

3. enzymes splitting linkages between the galacturonosyl residues of the pectin main chain like endo- and exo-polygalacturonase (PG) (EC 3.2.1.15), endo- and exo-pectate-lyases (PAL) (EC4.2.2.2 and 4.2.2.9), and pectin-lyase (PL) (EC 4.2.2.10) The reaction products of the hydrolases are identical with those from chemical hydrolysis. The lyases work by a trans-elimination mechanism. PG and PAL split low methyl ester pectins and pectates at an unesterified galacturonic acid unit. PL splits a site next to an esterified group, catalyzing the β-elimination reaction. More recently a group of rhamnogalacturonases (hydrolases and lyases from Asp. aculeatus) were described, acting on highly branched regions of pectins, liberating oligosaccharides consisting of rhamnose linked to galacturonic acid and to galactose (optional) (18, 57). Commercial pectinases are often mixtures of enzymes mentioned above and several hemi-cellulases, cellulases, and proteases. Producers may use one or both enzymatic systems: 1. a system based on pectin lyase, degrading pectin alone with the disadvantage of creating unsaturated oligogalacturonides that are sensitive to browning 2. a system based on polygalacturonase in combination with pectin esterase, converting HM pectins to LM pectins, the substrate for the polygalacturonase Bock et al. (58) characterized pectins ground by a vibration mill as follows: ●

●

The molecular weight was decreased by “mechanolysis.” The degree of esterification, the neutral sugar content, and the reactivity against calcium ions, assuming the pectins could form a gel, was almost unchanged.

Ralet et al. (59, 60) and Ralet and Thibault (61) suggested extrusion cooking as a method for pectin production. The extrusion process converted the pectic substances of the cell walls of apple, citrus, and sugar beet into soluble pectins. The softening of plant tissue after extrusion cooking is also associated with this degradation. Bondar und Golubev (62) announced a new desintegration and extraction technique for pectins, using cavitation.

VIII. PECTINS AS POLYELECTROLYTES Pectic substances are negatively charged polyelectrolytes. Their charge density and their apparent dissociation

Pectins

140-7

constant pKa calculated from pH measurement depend on the degree of dissociation. The pK0 determined for pectins is independent of pectin concentration and of their degrees of polymerization, esterification, amidation, and acetylation. The values obtained are in the range of 3.0 to 3.3, close to that (3.5) of monomeric galacturonic acid (cited in (4)). At pH 3.0, the dissociation of pectins is almost repelled; they are almost undissociated and uncharged. This is an important premise for pectin chain association in the case of jellification of HM pectins. Monovalent cations (Na, K, etc. and NH4 ) are bound to pectins electrostatically only. These ions can weaken pectin association in tissues as in thickened and jelled products. Magnesium ions do not support pectin jellification and follow also only electrostatical theories. The other alkaline earth cations cause pectin chain associations resulting in: ● ● ● ●

tissue hardening increased viscosity gel formation of LM pectins precipitation of their pectinates

Such observations or effects are dependent on: ● ● ●

cation concentration, ionic strength, and pH degree of esterification distribution of free carboxyl groups (random or blockwise) [for HM pectins]

When HM citrus pectins or HM pectins are slightly deesterified, using a plant, blockwise acting pectin esterase (creating blocks of free carboxyl groups), there is a significant increase in affinity and viscosity with calcium ions. This effect improves protein stabilization and increases the tendency towards syneresis which impairs jellification. Ca2, Sr2, Ba2, Cd2, Ni2, and Pb2 ions influence the circular dichroism behavior of pectins in the same way. That is, associate two pectin chains if blocks of free carboxyl groups with adequate length are present, i.e., 7, 10, and 20 units (21, 63). Practically LM pectins with a degree of esterification of about 40% and less show gelling properties by this mechanism. This chain association is described as “egg-box” gelling mechanism. Two pectin chains arrange parallel and symmetrically and form together (due to the axial-axial glycosidic linkages of the galacturonic acid polymer) negatively charged cavities in which the cations fit as eggs in a box. Thom et al. (64) and Debongnie et al. (65) describe Cu2 ions to behave different, and circular dichroism spectra are also different. In food processing usually calcium salts of different solubility (like calcium chloride, calcium lactate, calcium citrate, and calcium mono- and di-phosphates together with monovalent buffer salts to influence velocity of chain association) are used together with LM pectins (DE about

44%) to form gels at reduced soluble solids (sugar content). LM pectins are also described to bind heavy metals as an antidote for heavy metal poisoning and as an ion exchanger to remove heavy metals from effluents. Calcium pectinate is used as fat replacer forming soft, creamy insoluble particles. Pectins can be precipitated as their aluminium salts (ancient pectin production method) or copper salts (ancient quantitative pectin determination method — Cuprizon method).

IX. RHEOLOGY OF PECTINS Using pectins for technological reasons means that in general the producer has some means of influencing flow behavior and texture. The ability to form gels under acidic conditions is one such method under the disposal of the producer. The rheological properties of native pectins are used in food industry. If these properties are negative pectins have to be degraded in the manufacturing process. Pectin degrading enzymes are used to extract and concentrate juices. To achieve a high yield or capacity for the pressing systems, pectinases with other cell wall degrading enzyme activities are used in mash treatment (liquifaction). Stored apples will respond best to treatment with these enzymes for their content of soft tissue and high soluble pectin facilitates the binding of juice in the mash. Berries like black and red currant, grapes, etc. are also treated with pectinases for juice production to increase yield and color intensity. Some of the anthocyanes are bound to pectic substances and will be released into the juice if the pectin is degraded. Tomato is handled in two stages in the cold break process: degrading the pectin and subsequent concentration to tomato concentrate. In the case of manufacturing tomato ketchup, the hot break process is used to maintain pectin quality and flow behavior by inactivating native pectinases immediately after squeezing the tomato juice. Axelos et al. (66) reported the dividing line between: ●

●

dilute and semi dilute pectin solution in 1 M NaCl: a concentration C* between 0.7% and 1.0% semi dilute and concentrated regime: a concentration C** between 8% and 10%

Up to C* the specific viscosity corresponds to C[η]1.2 and beyond C** to C[η]3.3. Values of the Huggins coefficient kh, which characterizes the effect of molecular interaction on the viscosity, vary with the degree of esterification, ranging between 0.37 and 0.6. Pectins are insoluble in organic solvents and soluble in water to give a viscous solution. Like other viscous gums, it needs care in dispersing the powder rapidly into the water. Lumps of powder easily become coated with a gel layer

140-8


which makes further solubility slow and difficult. Appropriate techniques to prepare pectin solutions: ● ●

●

a high-shear mixer separating pectin particles by dilution with soluble powders like sugar (10 fold quantity of pectin) dispersing the pectin in high concentrated sugar syrups (at least 50% soluble solids to prevent significant swelling of the pectin) with subsequent dilution to less than 30% soluble solids. Under heating and moderate agitation the pectin will dissolve rapidly.

Pectin will not dissolve completely at high sugar concentrations. Thoroughly dissolved pectin will have no “fish-eyes” on a glass plate and will not feel gritty when rubbed between the fingers. Solutions with 3 to 4% pure pectin can be prepared easily. Industrial processing with more than 10% pectin in hot water will exceed the limit of complete pectin dissolution. Pectins with a low degree of esterification dissolve better in the form of monovalent metal salts. Bi- and trivalent cations decrease the solubility of all pectins. Pectic acid is only soluble in water as ammonium, sodium, or potassium pectates at pH 6. Pectins are also soluble in DMSO, formamide, dimethylformamide, and warm glycerol (67). Industrial pectin solutions with unchanged molecular weight exhibit a pseudoplastic flow behavior. Depending on raw material and extraction conditions, the viscosity of pectin solutions varies. Often the viscosity of pectins is standardized with neutral sugars like dextrose and sucrose to achieve constant quality for every batch. Solutions of high esterified pectins show little elastic shares. The viscosity of a pectin solution decreases with increasing pH and ionic strength. In combination with calcium salts and some sugar, a solution of low esterified pectins will form a soft network with measurable elastic properties. The viscous properties of pectin solutions are used in fruit juices, fruit based products, and soft drinks to increase viscosity, resulting in a better mouth feel and a higher impression of fruitiness and sweetness. The increased viscosity also improves cloud stability and reduces coalescence of oil droplets in emulsions (68). Cloud is also stabilized by bigger hydration if the cloud is positively charged and enveloped by a negatively charged pectin layer with high hydration (69). Fruit preparations produced with low methyl ester pectins also shows pseudoplastic flow with shear thinning during mechanical stress like pumping and dosing. This lowers the forces acting on the fruits during these processing steps and reduces their destruction. To prevent fruit floating these fruit preparations are produced with pectins giving a significant yield point. In contrast a

formulation using only viscosity for thickening will only delay floating. To prevent floating the yield point has to be higher than the lifting force of the fruit pieces. The yield point can be adjusted by the calcium reactivity of the pectin in combination with calcium concentration, soluble solids, and ionic strength. Fruit preparations produced with pectins show also little thixotropy and a high ability to regenerate after removal of the stress. These factors are very important for fruit preparations for yogurt where fruit preparations are produced and filled aseptically in big containers which are shipped to the dairy plant. There the fruit preparation is pumped out of the container and an even distribution of fruit pieces is a quality criterion. After pumping the fruit preparation has to be mixed with the white mass of the yogurt or metered into the yogurt beaker with subsequent over-layering with yogurt without the reaction between low methyl ester pectin and free calcium ions of the yogurt. Jams, jellies, and marmalades produced with pectins are visco-elastic solids. The ratio of viscous to elastic shares varies, depending on: ● ● ●

degree of esterification of the pectins pH conditions kind of pectin used

Viscous shares increase with increasing pH and decreasing DE of high methyl ester pectins. Apple pectin gels show higher viscous shares with good spreadability whereas citrus pectins have less viscous and more elastic shares. The elastic shares make the products more brittle and less spreadable, with a higher tendency to syneresis. This also can be shown by the smaller linear viscoelastic range of citrus pectin gels compared to apple pectin gels. These figures can be obtained using rheometers with oscillating techniques. The phase displacement angle δ between stress and strain indicates the proportion of elastic (G) to viscous shares (G) whereas ● ● ●

pure viscous liquid, δ 90° pure elastic solid, δ 0° viscoelastic solid, 0° δ 90°.

X. THE MOLECULAR WEIGHT OF ISOLATED PECTINS The figures on molecular weight of pectin published in the literature vary significantly from about 40.000 to more than 4 million Daltons. This variation is not only influenced by source of raw material, kind of plant tissue, and extraction conditions of the analyzed pectins but also by the analytical method itself. Owens et al. (70) first established that values, determined by viscosity measurements of pectin solutions with different concentrations and extrapolation to zero concentration, depend on the

Pectins

constants (K) and (a) used in the Mark-HouwinkSakurada equation [η] K Mηa. Values for different pectins can be found in Voragen et al. (4) and literature cited. For the exponent (a) values between 0.8 and 0.9 seem to be confirmed, representing a slightly stiff conformation of pectins. Polydispersity and aggregation phenomena are reasons for big discrepancies between weight-average (determined by laser light scattering) and number-average (determined by membrane osmometry or end group analysis) molecular weight values. Fishman et al. (71) published a critical re-examination of molecular weight and dimensions for citrus pectin. More detailed information on molecular weight distribution of pectins can be obtained by combinations of size exclusion chromatography (HPSEC) with multi-angle laser light scattering (MALLS) (72). The molecular weight for isolated pectins for food application as thickener or gelling agent is estimated to be 60.000 to 120.000 Dalton.

XI. PECTIN MANUFACTURING In 1825 Braconnot was the first who isolated a substance from plants, which showed gelling properties. The term pectin has been derived by him from the Greek “πηχτos,” meaning to congeal or solidify. It took up to 1917 until Ehrlich described pectic substances being composed of galacturonic acid. The high water binding and the ability to form gels of pectin quickly attracted the interest of the food industry. Before a defined pectin became available, the liquid pectin obtained from concentrated apple juice was used to decrease the influence of the natural content of fruit pectin in the raw material of the fruit processor. The first commercial application of pectin was as a gelling agent for jams, jellies, and marmalades, to balance the different pectin contents of the processed fruit. Kertesz (73) is giving the most comprehensive review of the early work on isolated pectins. Despite the wide occurrence of pectins in nature only a few raw materials were established as sources for commercial pectins. Reasons for this are the facts that only a few plants contain pectins with suitable properties for food applications, still the main use for pectins, and that the raw material has to be available in a sufficient quantity and constant and storage stable quality. Still pectins are produced mainly from by-products of fruit juice industry, namely apples and citrus fruits whose fresh or carefully dried pomace or peels after washing to remove citric acid and sugars are used. In Central Europe, pectin is produced from dried apple pomace and dried lemon and lime peels, whereas in Central and South America, pectin used to be obtained also from fresh orange, lemon, and lime peels. At present, North America is no longer a supplier of pectin. Instead, it is a major consumer.

140-9

Sugar beet chips are of minor importance due to their lower pectin quality. Sunflower heads fail as a pectin source because pectin quality declines rapidly before harvest time. The careful handling of raw material is essential in the manufacturing process of pectin. For example, in the production of apple pectin, the dried apple pomace from the manufacture of apple juice is the raw material. After the apple mash has been de-juiced, the pomace is dried immediately to prevent the degrading enzymes in the plant tissues from reducing the quality of the pectin. Drying also stabilizes the pomace by reducing micro-organisms to a minimum, permitting the product to be used to produce pectin year-round. Some producers use pectin degrading enzymes to increase fruit juice output and facilitate the de-juicing. The pomace from this operation cannot be used to produce pectin. Apple pectins, especially from unripe apples are often associated with native starch, which can be removed by enzymatic treatment. The degree of esterification in unripe apples is nearly 100%. Industrial extraction yields 10 to 15% of pectin out of dried apple pomace (or about 0.5% calculated on fresh apples) with a degree of esterification of up to 80%. The distribution of free carboxyl groups is very even. Apple pectins show a higher proportion of neutral sugars than citrus pectins resulting in summary in gels with high viscous shares, low tendency to syneresis, and high internal gel strength. Citrus pectin production uses the peels of de-juiced and de-oiled fruit. Washing the peels prior to processing is essential in the removal of remnants such as aromatic oils and bitter components. If such remnants cling to the peels and if these peels are used to produce pectin, they may cause an off-flavor, resembling a bitter, rancid taste in the end product. Thus for the production of a product that is stable when shipped and stored, the peels must be dried. If a producer wants to extract pectin from wet peels, he should do it in the vicinity of a citrus processing plant. Also, enzymes in the plant tissues will start to degrade the available pectin if bottlenecks develop in the extraction process or an excess of raw material prevails. The yield of citrus pectins from dried lemon or lime peels is about 30 to 35%. Orange and mandarin peels are seldom used due to their lower pectin content and pectin quality. Grapefruit pectin is produced mainly for nutritional applications to lower cholesterol (74). Citrus pectins usually have a lower content of neutral sugars and a higher concentration of D-galacturonic acid as apple pectins. Due to a higher pectin esterase activity in citrus fruits, acting block wise as all plant pectin esterases do, citrus pectins often show blocks of free carboxyl groups which are high calcium reactive. The citrus pectin gels are more elastic and brittle, and have a higher tendency to syneresis and pregelification with calcium ions present in the cooking. The advantage of the higher reactivity with calcium ions is the better protein stabilizing

140-10


property especially of heat sensitive casein in acidified milk drinks. Sugar beet chips are of minor importance. Compared to apple or citrus pectin, pectins from sugar beet have higher neutral sugar content and are partially esterified with acetic acid and ferulic acid. The acetylation is preventing gelation, but even acidic or enzymatic deacetylation does not create proper gelling pectins due to the low molecular weight of beet pectin. However studies have been carried out that show improved gelation of sugar beet pectin through oxidative coupling (75). It could be shown that beet pectins have emulsifying properties due to their hydrophobic (acetyl and methyl groups) and as well hydrophilic (hydroxyl and carboxyl groups) nature (68). Sunflower heads contain low ester pectins which are also acetylated. Interesting properties can be found in sunflower pectin as long as the sunflower is blossoming until an early mature state of the kernels showing a white and fluffy inflorescence. But when harvesting sun flowers for oil production the heads are dried out, sometimes after a killing frost, turning sunflower pectin almost to pectic acid with a very low solubility. Several other plant raw material like cabbage, onion peels, mango, cotton, etc. have been investigated as sources for commercial pectins. Up to now all efforts failed to introduce a new source of pectin. Additionally several international pectin specifications limit the sources of pectins to apple, citrus, and beet. Citrus fruits and apples are not processed with the main purpose of pectin production in mind. One of the main tasks of pectin producers is to secure the supply of high quality raw material by keeping in constant contact with fruit juice producers. The essential know-how of pectin producers is the manufacture and maintenance of pectins with a constant/consistent good quality despite adverse factors, which include variable contents and molecular weights. Such factors, as indicated above, result from differences within the raw materials and production parameters in the fruit juice processing plant. At first the pectin containing extract is produced in an acidic medium. Here, the insoluble protopectin is converted into its soluble form. Additionally water-soluble ingredients like sugars and phenolic compounds will also be dissolved. The insoluble residue, which mainly contains cellulose and other water-insoluble matter, will be separated and dried and may be used as animal feed. The extract containing pectin is further purified to remove suspended particles. It is then added to an alcohol solution in which the insoluble pectin will precipitate. After the alcohol is removed using standard procedures, the pectin is dried prior to grinding and sieved. The ground and sieved pectin represents the base material for “standardization,” i.e., the adjustment of pectin with sugar to ensure constant/consistent functional properties.

Citrus pectin is produced in much the same way as that for apple pectin. In contrast, the use of wet peels is possible for the production of citrus pectin. This reduces one energy intensive working cycle, i.e., drying of the citrus peels. However, this procedure depends on a close proximity between a pectin producer and a fruit juice producer for countries in Central or South America. Differences in the pectin quality within the raw material depend on fruit maturity and enzymatic activity and must be normalized or accounted for in pectin production. In contrast, dried citrus peels are available year round. Important suppliers are countries in Central and South America, Spain, and Italy. Depending on the extraction conditions and quality of the raw material, large differences in the molecular weights of pectin exist. Nonetheless, the molecular weight of the manufactured pectin is an essential factor for its gelling properties and determines its commercial quality. The degree of esterification has decisive influence on the gelling behavior and the application of the pectin. Pectin directly precipitated after extraction has, due to its natural properties, a very high degree of esterification. It is also known as rapid set pectin. Other types of pectin, like slow set pectins or low methylester pectins, which have a low degree of esterification, can be produced by adjusting some parameters of the extraction process, e.g., pH and temperature. It is possible to carry out de-esterification during or after the extraction. Pectins are amidated under alkaline conditions, in the presence of ammonia. In this process methylester groups will be transferred to amid groups. These pectins contain three functional groups: methylester, carboxyl, and amid groups. The term pectin is generally used for a poly-galacturonan, which has been extracted from cell walls. Pectins with a defined structure can be produced via additional processing steps. In order to differentiate the large number of pectins, they can be classified according to their chemical nature (Table 140.3). Because of the presence of free carboxylic acid groups, pectins have weak acid properties, which can affect and change the pH-value of a food system. Added pectins may TABLE 140.3 Classification and Definition of Pectins Description Protopectin Pectic acid Pectate Pectin Pectinate Pectic substances

Chemical Characterization Water-insoluble native pectin network Polygalacturonic acid (degree of esterification  > E S Product; d(AA) k(AAT) dt

k2 AH2.AH  > Product

k1 AH2 AH >

k3 AH2  > Product

k1 AH  > Product;

AA loss is first order in relation to AA AA f(T) Arrhenius relation Describes 3 reactions involved in the overall destruction of AA:

n.a.

Zero-order

Aer./ First-order Anaer. Two distinct Arrhenius profiles [4.4, 21.1] and [29.4, 46.1]°C Employment of orthogonal polynomials in the analysis of variance indicates that the mechanism of AA degradation was not the same at all temperatures

Aer.

(Continued )

Good Rates of AA degradation are dependent (66) R2 0.97 upon O2 availability, which is in turn dependent upon temperature and moisture content The equation derived to predict the amount of AA lost during an unsteady state heating process was successful when tested under conditions approximating a linear T rise (error for the final predicted value 5%)

(57)

(74)

57.5

The rate of AA destruction was influenced by pH, reaching a maximum near pK of AA The rate of copper-catalysed destruction of AA increased with copper conc. and was affected by pH Ea changes with pH, with a minimum at pH 4.06 (3.3 kcal/mol) A mathematical model was developed for the rate of AA loss as function of T, pH and Cu A computer simulation program was developed to predict AA stability in tomato juice Predictions were in good agreement with results of the shelf-life tests

Good Values of U.S. RDA of vitamin C are given 0.97 R2 0.99 At each specific temperature all 14 juices, regardless of plant or processing season, showed essentially similar percent vitamin C retention The Arrhenius plot showed two distinct temperatures regions, with a critical transition region between 22 and 26.7°C

Good*

92–6

60

TDT cans 5 T: 110, 115, 121, 126, 132°C 6 hours

°brix: concentrated during the study from 11 to 62 °brix

Initial AA content: 34.8, 112.5, 204.8 mg/100 g

60 min

3 aw: 0.9, 0.8, 0.69

Temperature: 60 to 110°C

Intermediate Jacketed, stirred moisture reactor with air food material space above reactants

Peas

Grapefruit Juice

Food/Model Package/Storage System Conditions


Ascorbic acid

Ascorbic acid

Ascorbic acid

O2 monitored

Compounds Other Under Study Compounds

Oxygen mass transfer aw

Temperature

°brix

Added AA concentration

Compounds/Factors Affecting Degradation

Aer./ Anaer.

Aer./ Anaer.

Anaer.

Atm. Cond.

Retention (%)

Second-order

First-order

n.a.

30–96

85 First-order Dependent on T by Arrhenius Eq. Ea 5 kcal/mol (11°brix) Ea 11.3 kcal/mol (62°brix) Arrhenius coeff. dependent on °brix Polynomial curve fitting, empirical kinetic equation correlating rate of reaction with temperature and degree of concentration

Applied Kinetic

Observations The same equation yielded less accurate predictions of AA losses during extrusion (error for the final predicted value 10%)

(58)

Ref.

n.a.

The effect of oxygen transport on degradation rate was determined by comparing the experimental observations with theoretical predictions for a series of four regimes (a regime is characterised by different relative rates of O2 mass transfer and chemical reaction) The most likely explanation for the experimental data is regime III — the chemical reaction rate is sufficiently fast that all the oxygen reacts in a thin film near the interface between the food and the gas phase, which enhances the mass transfer rate due to chemical reaction

(68)

Reasonable Discussion on the values of Ea for (59) good* this study and others, both for aerobic and anaerobic conditions, reaching the conclusion that the differences on Ea indicate that kinetic studies should be conducted for different food systems

Good Initial AA conc. has no significant effect either on rate of deterioration 0.972 R2 0.999 or mechanism A model combining kinetic data with process variables was developed and proved useful in predicting and optimising vit. C retention processes where grapefruit juice is subjected to any combination of thermal and concentration treatments

Fit Quality

Can

Grapefruit

Closed system with headspace and control of head-space gases 4 T: 30 to 55°C Initial AA conc 30, 35, 40, 50, 65 g/L 3 O2 levels 10, 15, 21%

Can (6 oz) 18 months 6 T (°C) 18, 5, 12, 17, 25, 37 4°brix 11°, 34°, 44°, 58

Buffered model system (pH 6.1)

SSOJ (var. Valencia) and OJ concentrate

Lime

Lemon

Tangerine

Whole Fruit

Orange

Ascorbic acid

Ascorbic acid

Ascorbic acid

Browning Furfural Sensorial changes

°brix

Temperature

Oxygen

Ascorbic acid addition

Temperature

Production factors Climate Position of fruit on the tree Maturation Rootstock effects Citrus variety (and fruit parts) Processed products: seasonal variability, processing effects, storage time and temperature - Vitamin C destruction: reaction order and reaction rates (aerobic and anaerobic mechanisms) - Effects of container - Influence of juice constituents

-

Aer./ Anaer.

Aer.

T 37°C different from first-order

T 25° first-order

Second-order

First-order

95–10

75

Good*

Good 0.96 R2 0.99

AA loss depends on the degree of concentration of the O. J. (increase with °brix)

(70)

(67)

(Continued )

Oxygen concentration was maintained throughout the experiment Ascorbic acid autoxidation is dependent on dissolved oxygen concentration It appears that the rate of oxygen dissolution into the AA solution is dependent on both temperature and headspace oxygen levels

The variability of vitamin C in fresh fruit (60) is due to variety, climate, horticultural practice, maturity stage and storage conditions Processing fruit into juice products results in minimal loss of vitamin C potency but subsequent storage finished of the product at higher temperatures results in considerable loss From the point of view of the consumer, numerous investigations have shown that fresh processed single strength and reconstituted citrus juices may be kept in a refrigerator for a reasonable length of time (4 weeks) without serious loss of vit C; even when juice is stored at room temperature, storage time is limited more by loss of palatability than by loss of vit C Aerobic and anaerobic mechanisms are mainly responsible for loss of vit C in processed products The mode of breakdown of vitamin C can best be explained by a 1st-order reaction but a significant quadratic time effect has been determined by polynomial regression calculations Plots of log rate (vit C loss) vs. 1/T for canned orange juice showed two distinct Arrhenius profiles, whereas canned grapefruit showed only one

Aqueous solution with AA

Lemon Juice

Aqueous solution with AA

Package system: n.a. 11 hours 2 T: 30 and 71°C Oxygen levels:

T: 36°C

Initial dissolved oxygen content: 0.41, 1.44 and 3.74 mg/L

Glass flasks, 250 mL, covered with AL foil Temperature

Browning

Prod k2 ↑k5

k1

k2

Ascorbic acid

Dissolved oxygen content

AA oxidation has a firstorder kinetics at one time and zero-order at another time:

Furfural: zero-order reaction

Furfural

Browning: Zero-order with a lag period

AA: First or second-order

First-order (reversible) for DHA and diketogulonic acid First-order reaction for AA loss

Prod ↑k4

AA DHAA DKA

Prod ↑k3 k1

Mechanism of degradation:

HMF: first-order reaction

Aer.

Aer./ Anaer.

Aer.

Applied Kinetic

HMF

Temperature

Initial dissolved oxygen content

Continuous aeration.

Temperature

Compounds/Factors Atm. Affecting Degradation Cond.

Ascorbic acid

β-carotene

Compounds Other Under Study Compounds

Erlenmeyer Ascorbic flasks (open) acid 4 T 25, 62, 75, 86°C DehydroInitial AA ascorbic conc 500 mg/L acid 90 minutes



17.5–28.2

AA: 47.1

72.2–50

Retention (%)

The AA-to-DHA-to-DKA mechanism fits the data reasonably well An irreversible path from AA to products appears to exist; the rate constant k3 appears to be large enough that it may be possible to degrade measurable amounts of AA without any DHA being formed

Observations

Good*

Rate constants are independent of initial AA content

AA: 1st AA: Initial oxygen content did not order: 0.85 affect significantly the rate of AA R2 degradation and furfural formation 0.87 Correlation between AA and the other compounds was between 0.8 and 0.9 2ndBrowning: The lag period before order: n.a. browning increased depended on the initial dissolved oxygen Browning: concentration, being greater for the 0.90 R2 lower initial concentration 0.95 Highly significant correlations were obtained between browning index, HMF and furfural (0.96), HMF suggesting that all 3 would be 0.96 R2 suitable as chemical indices of 0.98 storage temperature abuse in Furfural lemon juices; initial oxygen content did not affect significantly the rate 0.98 R2 0.99 of furfural formation Highly significant correlations were obtained between browning index, HMF and furfural (0.96), suggesting that all 3 would be suitable as chemical indices of storage temperature abuse in lemon juices

Good*

Fit Quality

(72)

(61)

(73)

Ref.

5T (°C): 10, 20, 30, 40, 50

18 weeks

Can, bottle

SSOJ TB cartons (made from 64 days concentrate) Initial dissolved O2 content 4.45 ppm T (°C): 4, 20, 37, 76, 75 L-AA addition (supplemented with 0.34 M, 1 mL)

Grapefruit juice

Ascorbic acid

Valencia Glass bottles O.J. (200 mL) (Pasteurised at 92°C for T: 4, 22.5, 35 30 sec) and 45°C

Ascorbic acid

Browning

Furfural

Ascorbic acid

Packaging system: n.a.

Buffer solution

saturation with air, oxygen, or 10% O2-90% N2 B-carotene: 80°C 2 initial AA conc. 114, 266 mg/L

Temperature

Oxygen

Ascorbic acid

Temperature

Temperature Total solids pH, acidity Formol no. Reducing Sugars

Catalytic metals: Fe(III) Cu(II)

AA addition Continuous aeration

Aer./ Anaer.

n.a.

Anaer.

Aer.

Impossible to say which best fits the data

Zero, first and secondorder were applied (4 to 37°C)

Reaction order should be between zero and one, for temperatures 30°C

Furfural: Zero-order

AA: Zero-order

First-order

kb ka A  > B  >C First order: ka kb (A)o Zero order: ka kb (A)o First order reaction in respect to -carotene

60.4–2

Furf: n.a.

AA: 97–47

n.a.

Zero-order: 0.97 R2 0.985 1st-order: 0.93 R2 0.99 2nd-order: 0.97 R2 0.97

n.a.

Good*

n.a.

Initial sudden drop of oxygen, intensified at higher temperatures Initial drop correlates with AA degradation during initial stage of storage After the dissolved oxygen reaches the equilibrium, L-ascorbic acid decomposition occurs independently of oxygen

(Continued )

(79)

Browning in citrus juices involves a (92) complex group of reactants that produce an assortment of brown pigments of highly unstable characteristics; based on these reasons and results of other researchers, the authors believe that it is inaccurate to define browning by a simple zero or first-order reaction No simplistic models should be applied to define the complex series of events leading to brown discoloration of citrus juices, especially within the temperature region of 30–50°C

(65) At high temperature (45°C) furfural production relates very well to AA degradation (r 0.96), but for lower T this relation is not so obvious

(62) The first order rate constant in an air saturated catalytic metal free solution is less than 6 107 s1 at pH 7 Ascorbate can be used in a quick and easy test to determine if the near-buffer solutions are indeed “catalytic metal free”

Ascorbic acid

Vials (10 mL) HMF 4 T (°C): 90, 100, 110, 120 5 experimental times: 20, 40, 60, 80, 100 min

4 pH levels: 2.5, 2.5, 4.5, 6

Constant O2 content

T: 25°C

700 min (11.7 h) pH: 3.0 to 5.0 3 T: 26.5, 30, and 33°C

Sweet Glass flasks: Ascorbic acid aqueous 60 mL (no Browning model headspace) 210 days at 24°C, system: aw 0.94 125 days at 33°C, and pH 3.5 105 days at 45°C, 140 h at 70°C, 90 h at 80 and 90°C. Initial AA conc 330 mg/kg Stored in the dark

Grapefruit juice

Buffer solution

Ascorbic acid

Sucrose solution

BOD bottle (300 mL) Initial AA conc 6.67 g/L

Compounds Under Study



Oxygen

Additives: potassium sorbate, sodium bissulfite

Humectants: glucose, sucrose, sorbitol

Temperature

Temperature

pH

Cu (II) — citrate complexes

pH

Sucrose

Temperature

Compounds/Factors Other Compounds Affecting Degradation

Reaction order, according to empirical kinetics of formation was 0.31

First-order

Dependent on T by Arrhenius equation

Second-order in relation to dissolved oxygen

Applied Kinetic

Aer./ AA: First-order Anaer. Browning: Zero-order

n.a.

Aer.

Aer.

Atm. Cond.

AA: 20

n.a.

78.5–87

Retention (%)

n.a.

Good*

n.a.

Good 0.98 R2 0.99

Fit Quality

At lower temperatures (24, 33, 45°C) the humectants protected L-AA from destruction (sugars being the most effective due to the structure forming effect they have) At higher temperatures characteristic of processing (70, 80, 90°C) humectants with active carbonyls (glucose, sucrose) promoted AA destruction and nonenzymatic browning reactions AA destruction occurred mainly

There was a lag phase in kinetics of HMF formation Ea 130 7 kJ/mol, kr 0.0105 0.00040(mg/L)0.69 s1 (the used reference temperature is not mentioned)

The rate of cupric ion-catalysed oxidation was found to be first order with respect to ascorbic acid. The effects of cupric ion conc. and pH suggests a mechanism involving the formation of a transition complex between monoascorbate ion and Cu(II)-citrate chelate

Molar ratio between O2 and AA: 1 mol of O2 per mol of AA Ea(AA) was higher in presence of 10% sucrose than in non sucrose controls, and the addition of sucrose reduced the rate of reaction at the temperature tested Effects of sucrose on AA: pH independent physical effect retards AA oxidation; pH dependent catalytic effect accelerates AA oxidation

Above 2 ppm of dissolved oxygen content there is one mechanism of reaction and below 2 ppm there is another

Observations

(91)

(99)

(63)

(78)

Ref.

pH Initial oxygen

2-hexanal α-terpineol

240 minutes

6 T: 60, 70, 75, 80, 90, 99°C

Dehydroascorbic acid

Ascorbic acid

Temperature

TDT tubes (with headspace)

Cupuaçu nectar

p-vinyl guaiacol

Temperature

Furfural

Temperature

Culture tubes (15 1.5 cm) Abuse time/ /temperature protocol T (°C): 75, 85, 95 Time: 15, 30, 60 min pH: 3.1, 3.8, 4.5 Initial O2: 6.2, 0.6 ppm

Fresh Polyethylene Ascorbic acid squeezed bottle O.J. 24 months storage Unpasteurised T(°C): 23 Frozen Initial AA 406 mg/L pH 3.7 °brix: 11.4

Orange juice

Aer./ Anaer.

n.a.

Aer./ Anaer.

CDHAA CiDHAA

C*AA Ci*AA

k2 k1

k1 k2 C*AA >CDHAA >CDKGA i k t C*AA C AA * e 1

After a transformation of variables (C*AA CAA CiAA) the reaction was treated as two consecutive irreversible reactions:

k1 k2 >DKGA >DHAA20-carbon) omega-6 fatty acids Total long chain (>20-carbon) omega-3 fatty acids a

Breast Milk Egg Lipids

Commercial Formula

38.3 45.0

34.2 38.4

52.8 19.6

1.1

1.4

0.0

1.1

3.3

0.0

Adapted from Reference 67.

through the placenta as in mammals. This biologically self-contained model allows a close relationship between nutritive substances and their physiological utilization. Thus, the laying hen and the chick (avian model) can be useful models for studying the net transfer of nutrients from maternal sources and its effect on the progeny (70). In this model, chicks with severe deficiency or excess of a nutrient could be obtained through diet manipulation. Upon hatching, the newly hatched chick can be used to study the effect of maternal (yolk/laying hen) or neonatal (chick) dietary nutrients (excess or deficient) on metabolic effects and/or other behavioral changes in the progeny. Furthermore, as incubation takes only 21 days, the time span involved in raising multi-generation progeny with severe deficiency of a particular nutrient (e.g., essential fatty acids) can easily be reduced. The hatched chick, like newborn infants, must obtain the bulk of its nutrients from dietary supply.

A. FERTILIZED EGGS AND HATCHED CHICKS FOR INFANT FATTY ACID NUTRITION RESEARCH Because fat composition can be manipulated by diet, the egg is a unique research tool in studying the role of maternal dietary fatty acids on the lipid metabolism in the progeny. In mammals, the nervous system is the organ with the greatest concentration of lipids after adipose tissue. These lipids are structural and are high in long chain PUFA of the n-6 and n-3 series. DHA is the predominant n-3 fatty acid, and arachidonic acid is the major n-6 fatty acid in the central nervous system of mammals and avians. Deficiency of long chain PUFA has been reported to cause impaired visual acuity, abnormal electroretinogram, and reduction in intellectual performance. In the human brain, the last intrauterine trimester is the most active period of brain tissue growth and DHA accumulation. During prenatal life, the accretion of long chain PUFA in the human brain is of quadratic type, the increase being most rapid towards the end of gestation and continuing into early life, plateauing

by two years of age, by which time 90% of human brain growth is completed (71). Considerable similarities exist between mammalian and avian species in the accretion of LCPUFA during embryonic development (72). The effect of maternal diets high or low in n-3 essential fatty acids on the PUFA metabolism of the brain tissue of developing progeny was investigated using laying hens, eggs, and hatched chicks (73). Regardless of dietary supply, an intense transfer of lipids, DHA, and arachidonic acid from maternal supply (yolk sac) resulted in a preferential incorporation of DHA and arachidonic acid in the chick (73). The changes in maternal dietary source of fat during development of brain tissue can affect the fatty acid composition of the progeny in mammals (74). Thus, despite the obvious developmental difference between mammals and avians, the subsequent usage and metabolism of PUFA is similar. This is evidenced by accretion and preferential uptake of long chain PUFA by the chick brain during the last week of incubation, suggesting that the egg and the hatched chick are unique research models in studying the effect of maternal diet on the metabolism of PUFA in the brain. Considering the uniqueness of egg in offering several nutrients and other biologically active components that contribute to human health and the health and development of a new life during its 21-day incubation, eggs are nature’s first and original functional food. Much work remains to be done before the nutritional significance of this biological and chemical entity will be fully known.

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154

Ice Cream and Frozen Desserts

H.D. Goff

Department of Food Science, University of Guelph

R.W. Hartel

Department of Food Science, University of Wisconsin-Madison

CONTENTS I. Formulations and Ingredients ..............................................................................................................................154-2 A. Product Definitions and Formulations ........................................................................................................154-2 1. Ice Cream..............................................................................................................................................154-2 2. Reduced Fat Products ..........................................................................................................................154-3 3. Sherbet ..................................................................................................................................................154-3 4. Frozen Yogurt........................................................................................................................................154-4 5. Fruit Ices and Sorbets ..........................................................................................................................154-4 B. Sources and Functional Roles of Ingredients ..............................................................................................154-4 1. Fat ........................................................................................................................................................154-4 2. Milk Solids-not-Fat ..............................................................................................................................154-5 3. Sweeteners ............................................................................................................................................154-8 4. Stabilizers..............................................................................................................................................154-9 5. Emulsifiers ..........................................................................................................................................154-10 II. Manufacturing and Structure of Frozen Dessert Products ................................................................................154-10 A. Mix Manufacture ......................................................................................................................................154-10 1. Blending..............................................................................................................................................154-10 2. Mix Calculations ................................................................................................................................154-10 3. Pasteurization and Food Safety Issues................................................................................................154-14 4. Homogenization..................................................................................................................................154-16 5. Aging ..................................................................................................................................................154-16 B. Dynamic Freezing......................................................................................................................................154-16 1. Principles of Ice Crystallization ........................................................................................................154-17 2. Operation of the Freezer Barrel ..........................................................................................................154-24 3. Overrun Calculations ..........................................................................................................................154-27 4. Fat Destabilization and Foam Stabilization........................................................................................154-28 C. Flavors and Flavor Addition ......................................................................................................................154-31 D. Packaging and Static Freezing ..................................................................................................................154-34 E. Novelty/Impulse Product Manufacture......................................................................................................154-36 F. Storage and Distribution ............................................................................................................................154-38 III. Product Quality and Shelf-Life ........................................................................................................................154-39 A. Flavor Defects ............................................................................................................................................154-39 B. Texture Defects ..........................................................................................................................................154-40 1. Recrystallization ................................................................................................................................154-40 2. Lactose Crystallization ......................................................................................................................154-43 3. Shrinkage ............................................................................................................................................154-44 IV. Conclusions ......................................................................................................................................................154-44 Acknowledgment........................................................................................................................................................154-44 References ..................................................................................................................................................................154-45 154-1

154-2


This chapter is focused on frozen desserts, dairy or non-dairy, that are characterized by being concomitantly whipped and frozen in a scraped surface freezer, and subsequently consumed in the frozen state. There are many product variations on this category, ice cream and lower fat versions being the most common, but also including sherbets and sorbets, frozen yogurt, soy-based frozen desserts, etc. Thus we begin with definitions and formulations of the major products within this category. However, there are many features of these products that are similar, hence many other aspects can be treated collectively. We will review the sources and functional roles of ingredients, mix manufacturing, including formulation calculations, the dynamic freezing process, including structure and structure formation, the static freezing (hardening) process, product storage and distribution, and finally, a review of shelf-life and quality aspects. Although we use “ice cream” in the generic sense throughout this chapter, all of these topics are relevant to all products within this category. It is not possible to provide a complete coverage of all aspects of ice cream and frozen desserts in one chapter. However, various aspects are covered in numerous books (1,2), book chapters (3–8), and review papers (9–11).

I. FORMULATIONS AND INGREDIENTS A. PRODUCT DEFINITIONS AND FORMULATIONS 1.

Ice Cream

The most common product within the category of frozen desserts is ice cream. The legal definition of ice cream is controlled by regulations and varies with jurisdiction, but it is generally a sweetened product containing milkfat and milk solids-not-fat (msnf), and is frozen while being whipped. The general composition of most ice cream products is shown in Table 154.1. Some of the factors affecting the choice of composition include legal requirements, which must be met, the quality desired in the finished product (increasing fat and total solids are usually associated with increasing quality), and the cost to be borne by the consumer. Premium products usually command a higher price. There are no specific definitions of common industry-accepted terms, such as premium or super-premium ice cream, but a relationship between fat content, total solids content, air content, and cost (also affected by quality and proportion of inclusions and marketing issues) exists, as illustrated in Table 154.2. Suggested formulations for a range of ice cream products are presented in Table 154.3. Several trends are evident. There is usually an inverse relationship between fat and total solids compared to msnf. As discussed in Section I.B.2, the lactose component of the msnf is quite insoluble and above its saturation level in ice cream, so with increasing lactose content in a decreasing quantity of

TABLE 154.1 The General Composition of an Ice Cream Mix Component

Range of Concentration ⬎10–16%

Milkfat Milk solids-not-fat Proteins, lactose, minerals

9–12%

Sweeteners Sucrose Corn syrup solids

10–14% 3–5%

Stabilizers Guar, locust bean gum (carob), carrageenan, carboxymethyl cellulose (cellulose gum), micro-crystalline cellulose (cellulose gel), sodium alginate, xanthan, gelatin

0–0.25%

Emulsifiers Mono- and di-glycerides, Polysorbate 80

0–0.25%

Water

55–64%

water, the risk of lactose crystallization increases. There is also generally an inverse relationship between corn syrup solids (starch hydrolysate sweetener, sometimes referred to as “glucose solids”) levels and total solids. The corn syrup solids will contribute to a firmer, chewier texture, which is more desirable when there are less solids present. Likewise, as total solids increases, there is less requirement for stabilizer. This is generally due to the fact that increasing stabilizer-in-water ratios lead to enhanced gumminess, which becomes undesirable at high levels. Also, a reduction in the water content means there are diminished problems associated with ice recrystallization. Additionally, as fat levels in a mix increase, there is generally less need for emulsifier in order to optimize the extent of partial coalescence of the fat. Further discussion on many of these aspects of formulations can be found in the appropriate sections of the chapter. Soft-serve ice cream is very similar to its hard-frozen counterpart in composition, but is sold at a different point

TABLE 154.2 Average Values for Fat and Total Solids Content, Overrun, and Cost among the Categories of Ice Cream Component Fat content Total solids Overrun Cost

Economy

Standard

Premium

SuperPremium

Legal minimum, usually 10% Legal minimum, usually 36% Legal maximum Low

10–12%

12–15%

15–18%

36–38%

38–40%

⬎40%

~100% Average

60–90% Higher than average

25–50% High


154-3

TABLE 154.3 Suggested Mixes for Hard-Frozen Ice Cream Products Percent (%) Milk Fat Milk Solids-not-fat Sucrose Corn Syrup Solids Stabilizer * Emulsifier * Total Solids

10.0 11.0 10.0 5.0 0.35 0.15 36.5

11.0 11.0 10.0 5.0 0.35 0.15 37.5

12.0 10.5 12.0 4.0 0.30 0.15 38.95

13.0 10.5 14.0 3.0 0.30 0.14 40.94

14.0 10.0 14.0 3.0 0.25 0.13 41.38

15.0 10.0 15.0 — 0.20 0.12 40.32

16.0 9.5 15.0 — 0.15 0.10 40.75

*Highly variable depending on type; manufacturers’ recommendations are usually followed.

in its production stage, and usually with a much lower overrun content. Suggested formulations are shown in Table 154.4 for soft-serve ice cream, but it should also be recognized that much of the soft-serve on the market today falls into the low-fat, or ice milk category, with fat contents typically around 4%. Generally, while the fat content is kept lower, the msnf content is generally higher than for hard-frozen products. Lactose crystallization is not a problem in these products, as they are consumed immediately after freezing. Corn syrup solids are often used, but can lead to an enhanced sensation of gumminess. Stabilizers are also generally used for viscosity enhancement and mouthfeel, but their function in ice recrystallization is no longer needed. Dryness and shape retention, however, are a big concern in soft-serve products, hence the emulsifier content is generally kept high.

suited for typical acidic fruit flavors, e.g., citrus. The sugar and acid levels in fruits or fruit purees have to be considered in the final formulation, and are included in the numbers suggested above. Acidity is usually added in the form of citric or tartaric acid, and this level of acidity modifies the perception of sweetness that would otherwise be created by the high level of sugars. Acid should not be added to ice and sherbet mixes until just before freezing. Heating of some stabilizers in the presence of acid will reduce their effectiveness. Adding acid to a sherbet mix in which the milk solids have been included, may result in aggregation/precipitation

2.

Milk Fat Milk Solids-not-fat Sucrose Corn Syrup Solids Stabilizer * Emulsifier * Total Solids

Reduced Fat Products

Ice milk was the traditional lower fat ice cream product for many years; but this category has been reclassified by many regulatory jurisdictions to include three reduced fat categories: light ice cream, lowfat ice cream (the traditional ice milk), and non-fat ice cream. Light or “reduced fat” ice creams are usually in the range of 5–7.5% fat. Lower fat versions are usually in the range of 3–5% fat. It has generally been possible to produce lower fat products, as low as 4% fat, with traditional ingredients, but further fat reductions have generally involved the incorporation of fat-replacers. These are discussed further in Section I.B.1. Suggested formulations for light and low-fat ice creams are presented in Table 154.5. 3.

Sherbet

Sherbet is usually taken to be a frozen dairy dessert made from a milk product, but containing a low (usually legallydefined maximum, e.g., 5%) level of milk solids, including milk fat, a high level of sweeteners (sugar and corn syrup solids, 30–35%), and added acidity (usually to greater than a legally defined minimum, e.g., 0.35%, expressed as lactic acid). Suggested formulations are given in Table 154.6. Because of the acidified nature of sherbets, they are most

TABLE 154.4 Suggested Mixes for Soft-Frozen Ice Cream Products Percent (%) 10.0 12.6 13.0 — 0.15 0.20 36.0

10.0 12.0 10.0 4.0 0.15 0.20 36.3


TABLE 154.5 Suggested Mixes for Low-Fat Ice Cream or Ice Milk Products (3–5% Fat) and Light Ice Cream Products (6–8% Fat) Percent (%) Milk Fat Milk Solids-not-fat Sucrose Corn Syrup Solids Stabilizer * Emulsifier * Total Solids

3.0 13.0 11.0 6.0 0.35 0.10 33.65

4.0 12.5 11.0 5.5 0.35 0.10 33.45

5.0 12.5 11.0 5.5 0.35 0.10 34.45

6.0 12.0 13.0 4.0 0.35 0.15 35.5

8.0 11.5 12.0 4.0 0.35 0.15 36.0


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TABLE 154.6 Suggested Sherbet Components


Mixes

Showing

Typical

Percent (%) Milk fat Milk Solids-not-fat Sucrose Corn Syrup Solids Stabilizer/emulsifier Citric acid (50% sol.) Water Total

0.5 2.0 24.0 9.0 0.3 0.7 63.5 100.0

1.5 3.5 24.0 6.0 0.3 0.7 64.0 100.0

of the protein. Sherbet generally requires the addition of milk solids, and at least some fat (~0.5%) is desirable, as it tends to lubricate the dynamic freezer and provides a slightly more pleasant mouthfeel than non-fat products. In many multi-product manufacturing settings, ice cream mix is widely used as a source of milk solids, and the amount added will depend upon the level of milk solids desired. Overrun should be kept much lower in sherbet than that in ice cream, usually 30–35%. 4.

Frozen Yogurt

Yogurt is a well-established dairy product, is generally perceived to be characterized by developed acidity (lactic acid) from fermentation of lactose by bacterial culture, and may or may not include live culture. The acidity destabilizes the casein micelles in the milk, and they, in turn, establish the typical acid gel. Frozen yogurt, therefore, should be much like the unfrozen version, and be also characterized by developed acidity from fermentation. The example formulation in Table 154.7 is typical of a more traditional frozen yogurt. However, in most legal jurisdictions, frozen yogurt is not standardized, so a wide range of products exists, including those in which the acidity is not developed by bacterial culture, but has been added in the form of citric acid. To make a traditional frozen yogurt, as in Table 154.7, the processing occurs in two steps: the manufacture of a fermented yogurt-like ingredient, and the blending of this

TABLE 154.7 Suggested Frozen Yogurt Formulation Percent (%) Milk Fat Milk Solids-not-fat Sugar Stabilizer Water Total

2.0 14.0 15.0 0.35 68.65 100.0

product with the rest of the ingredients. For example, 20% of the mix in Table 154.7, consisting of skim milk and skim milk powder, blended to give 12.5% msnf, is pasteurized at 85–90°C, cooled to 40 to 43°C, inoculated with a yogurt culture (typical of yogurt processing), and incubated as the yogurt portion. When the fermentation is complete (to the desired acidity), the “yogurt” is cooled. To make the “sweet” mix, the cream, sugar, stabilizer, and the balance of the skim milk powder and skim milk are combined, pasteurized, homogenized, cooled (typical for ice cream processing), and then blended with the “yogurt.” The completed frozen yogurt mix is then aged and prepared for flavoring and freezing. 5.

Fruit Ices and Sorbets

“Ice” or “sorbet” is likewise typically not defined in legal regulations, but is generally taken to be much the same as sherbet, except that milk solids are not included. Sorbets are generally frozen in a swept surface freezer, while ices are generally frozen quiescently in molds. Both sorbets and ices are usually fruit-based, and ingredients include combinations of fruit and/or fruit juices, sugar, stabilizer, and water. Overrun is very low, as aeration is difficult to achieve without protein or emulsifier. To make water ice or sorbet mixes from the above suggested sherbet formulae, delete the fat and msnf. B.

SOURCES AND FUNCTIONAL ROLES INGREDIENTS

1.

Fat

OF

The fat component of frozen dairy dessert mixes increases the richness of flavor, produces a characteristic smooth texture by lubricating the palate, helps to give body, and aids in producing desirable melting properties (1,6). The fat content of a mix also aids in lubricating the freezer barrel while the ice cream is being manufactured. Limitations on excessive use of fat in a mix include cost, a hindered whipping ability, decreased consumption due to excessive richness, and high caloric value. Fat contributes 9 kCal/g to the diet, regardless of its source. During freezing of ice cream, the fat emulsion that exists in the mix will partially coalesce (destabilize) or churn as a result of emulsifier action, air incorporation, ice crystallization, and high shear forces of the blades (6,12). This partial churning is necessary to set up the structure and texture in ice cream, which is very similar to the structure in whipped cream (13). This process will be discussed in Section II.B.4. The fat content is an indicator of the perceived quality and/or value of the ice cream. Ice cream must have a minimum fat content of 10% in most legal jurisdictions. Premium ice creams generally have fat contents of 14 to 18%. It has become desirable, however, to create light ice creams, ⬍10% fat, with the same perceived quality. In addition to


structure formation, fat contributes a considerable amount of flavor to ice cream, which is difficult to reproduce in lowfat ice creams. Fat content must be altered by at least 1% before any noticeable difference appears in the taste or texture (1). Several recent papers have examined the effect of source and quantity of milk fat on sensory and textural characteristics of ice cream (14–20). Milkfat as a fat source for ice cream formulations is in widespread use in North America, Australia and New Zealand, and parts of Europe. The triglycerides in milkfat have a wide melting range, ⫹40° to ⫺40°C. The crystallization patterns of milkfat are also very complex, due in part to the large variation in fatty acids and large numbers of different triglycerides present (21). Consequently, there is always a combination of liquid and crystalline fat at refrigeration and subzero temperatures. Alteration of this solid: liquid ratio at freezer barrel temperatures, through natural variation or fat fractionation, may affect the ice cream structure formed. The best source of milkfat in ice cream for high quality flavor is fresh sweet cream, from fresh sweet milk (1). Other sources of milkfat include sweet (unsalted) butter, frozen cream, or condensed milk blends. Whey creams have also been used, but may lead to flavor or texture problems. Vegetable fats are used extensively as fat sources in ice cream in the United Kingdom and parts of Europe, but only to a very limited extent in North America. Three factors of great interest in selection of fat source are the way in which the fat crystallizes, the temperature-dependent melting profile of the fat, especially at refrigerator and freezer temperatures, and the flavor and purity of the oil (6). For optimal partial coalescence during freezing, it is important that the fat droplet contain an intermediate ratio of liquid:solid fat at the time of freezing. Crystallization of fats occurs in three steps: subcooling of the oil (below the equilibrium crystallization temperature) to induce nucleation, heterogeneous or homogeneous nucleation (or both), and crystal propagation. In bulk fat, nucleation is predominantly heterogeneous, with crystals themselves acting as nucleating agents for further crystallization, and subcooling is usually minimal. However, in an emulsion, each droplet must crystallize independently of the next. For heterogeneous nucleation to predominate, there must be a nucleating agent available in every droplet, which is often not the case. Thus in emulsions, homogeneous nucleation and extensive subcooling are expected (6). Blends of oils are often used in ice cream manufacture, selected to take into account physical characteristics, flavor, availability, and cost. Hydrogenation is often necessary to achieve the appropriate melting characteristics. Palm kernel oil, coconut oil, palm oil, sunflower oil, peanut oil, and fractions thereof with varying degrees of hydrogenation are all used to some extent. Tong and co-workers (22) substituted a portion of milkfat in ice cream with safflower oil, a highly unsaturated oil, in an attempt to lower the saturated

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fatty acid content of the final product. They reported that increasing concentration of safflower oil decreased overrun, but had little effect on the extent of fat destabilization at lower substitution levels. There has been a great interest in the marketplace for the development of lower fat alternatives to traditional ice cream products. As a result, a large amount of product development time has been used in searching for a combination of ingredients that will replace the textural and flavor characteristics of fat in ice cream (17,18,23). These often involve the use of fat substitutes. Such products may be formulated with starch or other polysaccharides, proteins, or lipids, but their main requirement is to provide less calories to the product than traditional fat sources in the diet. A great deal of technical literature exists on the various properties of the products being marketed by a number of commercial firms. Schmidt and co-workers (24) studied the rheological, freezing, and melting properties of ice milks manufactured with protein-based or maltodextrinbased fat alternatives. They concluded that the carbohydrate-based alternatives resulted in greater affects on mix rheology, while the protein-based alternatives were more similar to ice cream, due in part to the functional contributions of proteins to food systems, especially in the area of emulsification and air incorporation. Ice cream products are very complex systems, both in structure and flavor. In creating products that are meant to deliver to the consumer the same attributes but with less fat or calories, it is imperative that the structural element of fat be considered to the same extent as flavor in order to deliver high quality products and develop market share for these products. 2.

Milk Solids-not-Fat

The milk solids-not-fat (msnf) or serum solids improve the texture of ice cream, aid in giving body and chew resistance to the finished product, are capable of allowing a higher overrun without the characteristic snowy or flaky textures associated with high overruns, and may be a cheap source of total solids (25). The msnf contain the lactose, caseins, whey proteins, minerals (ash), vitamins, acids, enzymes, and gases of the milk or milk products from which they were derived. The content of msnf used in a mix can vary from 10 to 14% or more. Whole milk protein blends contain both caseins and whey proteins, and this category includes most of the traditional sources of milk msnf, fresh concentrated skimmed milk, or spray dried low-heat skim milk powder. However, most ice cream formulations now use another source or sources of msnf or milk protein to replace all or a portion of skim milk solids, for both functional and economical reasons (26). When assessing replacements for skim milk solids, an important consideration is the levels of protein, lactose, and ash in the ingredients being assessed (27). Lactose is not very sweet and not very soluble, and therefore, during

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freezing of ice cream, it is freeze-concentrated beyond maximum solubility (supersaturated) and thus potentially prone to crystallization. Lactose crystals are very undesirable in ice cream, causing the defect known as sandiness. Lactose, being a disaccharide, also contributes to freezing point depression in the mix, so its concentration must be closely controlled. In addition, the milk salts affect both the flavor and texture of ice cream. Also, when replacing skim milk solids, sufficient total solids must be added to limit the water content of the mix and meet legal minimum total solids requirements. For these reasons, it is often desirable to replace skim milk solids with a product(s) with similar concentrations of lactose and protein. Lactose can be reduced through ultrafiltration, or modified by limited hydrolysis to its constituent monosaccharides; either change will affect the concentration of the ingredient that can be used and the subsequent protein level achieved in the ice cream. Buttermilk solids have often been cited as a useful substitute for skim milk solids. Buttermilk contains a higher concentration of fat globule membrane phospholipids than skim milk. Thus, it can be used for its emulsifying properties to reduce the need for emulsifiers, or in formulations where it is undesirable to add emulsifiers (1). It is possible to produce concentrated protein products from the casein portion of milk proteins, the most common for use as a food ingredient being sodium caseinate. The use of sodium caseinate in ice cream has been investigated, and a small percentage may be useful in contributing to functional properties, particularly aeration and emulsification (28,29). However, the functionality of sodium caseinate is different than that of micellar casein, the form in which it is found in milk ingredients, and this needs to be considered when proposing its use. It can contribute positively to aeration, but may lead to an emulsion that is too stable to undergo the required degree of partial coalescence. It is therefore most desirable in the serum phase, rather than at the fat interface. There has been a great deal of attention to the use of whey products in ice cream. Whey contains fat, lactose, whey proteins, and water, but very little, if any, casein. While skim milk powder contains 54.5% lactose and 36% protein, whey powder contains 72–73% lactose and only about 10–12% protein. Thus, it can aggravate some of the problems associated with high lactose. However, an increasing number of whey products are available that have higher protein and lower lactose contents, mostly processed by membrane technology. Many of these can provide much higher quality than the traditional whey ingredients (26,29). Whey protein concentrates with similar protein and lactose contents to skim milk solids can be produced. Protein content can vary from low values of 20–25% to 75% or more. In addition, the level of lactose can be modified by hydrolysis, although the freezing point depression effect of the higher monosaccharide content

must be considered. Ash content can be reduced by demineralization. Whey protein isolates, which contain no lactose, are also available for blending with other ingredients to form the msnf content of ice cream formulations. Proteins contribute much to the development of structure in ice cream, including emulsification, whipping, and water holding capacity (8,30). The interfacial behavior of milk protein in emulsions is well documented, as is the competitive displacement of proteins by small molecule surfactants (31–35). In ice cream, the emulsion must be stable to withstand mechanical action in the mix state, but must undergo sufficient partial coalescence to establish desirable structural attributes when frozen. These include dryness at extrusion for fancy molding, slowness of melting, some degree of shape retention during melting, and smoothness during consumption. This implies the use of small molecule surfactants (emulsifiers) to reduce protein adsorption and produce a weak fat membrane that is sensitive to shear action (7,11,12,29,36–41). Bolliger and coworkers (42) showed that protein adsorbed to the fat droplets (mg m⫺2) in ice cream mix correlated with major characteristic analyses describing the fat structure in ice cream (fat agglomerate size, fat agglomeration index, solvent extractable fat) (Figure 154.1). The loss of steric stability from the globule, which was contributed from micellar adsorption, accounts for its greater propensity for partial coalescence during shear. Partial coalescence is responsible for establishing a three-dimensional aggregation of fat globules that provide structural integrity (see Section II.B.4). This is especially important if such integrity is needed when the structural contribution from ice is weaker (i.e., before hardening or during melting). Variables that affect the destabilization of fat in ice cream have been well studied (43–46). With respect to protein contribution to fat globule integrity, it is obvious from the studies to date that a weak surface layer is most desirable (8). Segall and Goff (47) examined the susceptibility of ice cream emulsions to partial coalescence during shear when the emulsion was prepared with varying concentrations and type of protein, while still retaining sufficient quiescent emulsion stability. The membranes of fat globules stabilized by whey protein isolate were more susceptible than those made from sodium caseinate or casein micelles, while those made from partially hydrolyzed whey proteins did not show sufficient quiescent emulsion stability. However, when casein was added to the whey protein-stabilized emulsion, after homogenization, further casein adsorption to the whey protein membrane was rapid. Nevertheless, an understanding of protein structures and protein:surfactant interactions at the fat interface may lead to better control over the extent of partial coalescence desirable in the finished product. Milk proteins are well known for their foaming properties, and during the manufacture of ice cream, air is


154-7

80

100 Solvent extractable fat (%)

Fat destabilization index

r 2 = 0.92 60

40

20

0

75

50

25

0 6

8

10

12

6

8

10

12

80

30 2=

r 2 = 0.81

0.95 Fat globule % > 3 µm

r Fat globule size (d4,3, µm)

r 2 = 0.90

20

10

0

60

40

20

0 6

8

10

12

6

Adsorbed protein (mg

8

10

12

m–2)

FIGURE 154.1 The effect of protein adsorbed to the fat globules in the mix on the fat destabilization index, solvent extractable fat, and fat agglomerate size in ice cream (42).

incorporated to about 50% phase volume. Thus, it should not be surprising that milk proteins contribute to stabilizing the air interface in ice cream. This air interface is very important for overall structure and structural stability (48). Loss of air can lead to a defect known as shrinkage (see Section III.B.3), the occurrence of which is fairly common and very significant for quality loss and unacceptability of the product (49). The process of whipping heavy cream includes an initial protein adsorption to the air interface, and a subsequent adsorption of fat globules and their associated membrane to the existing protein air bubble membrane (13). Globular fat adsorption to air interfaces is known to stabilize air bubbles from rapid collapse (50). Proteins at the fat interface have also been shown to play an important role during the aeration of emulsions (51). However, the actual contribution of protein to ice cream aeration and its interaction with both the added emulsifying agents (which are also surface active) and partially coalescing fat at the air interface has been less well studied. Incorporation of air into ice cream is rapid, within seconds, and at the same time, viscosity of the surrounding matrix is increasing exponentially due to freezing, such that air bubbles after formation become physically trapped into a semi-solid matrix, making their collapse quite difficult.

Similar to the role of milk protein in aeration, the role of this protein in the unfrozen aqueous phase is recognized but less well studied than the role at the fat interface. Milk proteins interact with water, and the subsequent hydration is responsible for a variety of functional properties, including rheological behavior. Thus, freeze-concentration of proteins in ice cream must lead to a sufficient concentration to have a large impact on the viscosity of the unfrozen phase and its subsequent effect on ice crystallization, ice crystal stability, and solute mobility (52). Jonkman and co-workers (53) studied the effect of ice cream manufacture on the structure of casein micelles and found that the micelles per se were not affected by the process. Although the stability of the micelle was expected to be affected by low temperature, this was offset by an increasing concentration of milk salts in solution during freeze-concentration, such that the micelle remained intact in a similar state to that found in mix. Polysaccharides are also added to ice cream mix to enhance solution viscosity and to impact ice crystallization behavior. Commonly used polysaccharides can be incompatible in solution with milk proteins, leading to a microscopic or macroscopic phase separation (54), a phenomenon that has been studied in milk and ice cream-type systems (55–57). Goff and co-workers (58) examined the

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interaction between milk proteins and polysaccharides in frozen systems using labeled polysaccharides and fluorescence microscopy, and demonstrated a clear phase separation between the two, leading to discernable networks created by freezing from both locust bean gum and milk proteins. It has also been shown in ice cream that when in solution with polysaccharides, the casein aggregates into distinct networks (58). Flores and Goff (59) demonstrated that milk proteins had a large impact on ice crystal size and stability. It thus appears that microscopic phase separation of the milk protein induced by polysaccharides, and “aggregation” of casein into a weak gel-like network, promoted also by freeze-concentration, may be at least partly responsible for ice crystal stability, and thus improvement of texture during consumption. Lactose, or milk sugar, is a disaccharide of glucose and galactose that does not contribute much to sweetness of ice cream, since it is only 1/5 to 1/6 as sweet as sucrose (21). Lactose is relatively insoluble and crystallizes in two main forms, α monohydrate and β anhydrous, depending on conditions. The α monohydrate crystals, which take on a characteristic tomahawk shape, lead to the defect known as sandiness when they are allowed to grow sufficiently large (about 15 µm). Lactose content of ice cream mix is about 6% if no whey powder has been used in the formulation. Levels of lactose in ice cream mix in excess of this leads to reduced freezing point, causing a softening of the ice cream and the potential for development of iciness, a greater potential for lactose crystallization or sandiness, and salty flavors (60). The lactose solubility in water at room temperature is about 11% (21). During freezing, this concentration is exceeded as a result of freeze concentration (water removal in the form of ice). When 75% of the water is frozen in a mix consisting originally of 11% msnf (6% lactose), the lactose content in the unfrozen water corresponds to ~40%. Probably much of the lactose in ice cream exists in a supersaturated, amorphous (non-crystalline) state, however, due to extreme viscosity (61). Stabilizers help to hold lactose in a supersaturated state due to viscosity enhancement. 3.

Sweeteners

Sweet ice cream is usually desired by the consumer. As a result, sweetening agents are added to ice cream mix at a rate of usually 12–17% by weight. Sweeteners improve the texture and palatability of ice cream, enhance flavors, and are usually the most economical source of total solids (1). Their ability to lower the freezing point of a solution imparts a measure of control over the temperature-hardness relationship (see Section II.B.1). In determining the proper blend of sweeteners for an ice cream mix, the total solids required from the sweeteners, the sweetness factor of each sugar, and the combined freezing point depression of all sugars in solution must be calculated to achieve the

proper solids content, the appropriate sweetness level, and a satisfactory degree of hardness (5,6,62). The most common sweetening agent used is sucrose, alone or in combination with other sugars. Sucrose, like lactose, is most commonly present in ice cream in the supersaturated or glassy state, so that no sucrose crystals are present (6,61). It has become common practice in the industry to substitute sweeteners derived from corn starch or other starch sources such as rice, for all or a portion of the sucrose (1,4). A typical sweetener blend for an ice cream mix usually includes 10–12% sucrose and 4–5% corn syrup solids (corn starch hydrolysate syrup, commonly referred to as “glucose solids”) (1,4). The use of corn syrup solids in ice cream is generally perceived to provide enhanced smoothness by contributing to a firmer and more chewy texture, providing better meltdown characteristics, bringing out and accentuating fruit flavors, reducing heat shock potential which improves the shelf-life of the finished product, and providing an economical source of solids (62,63). During the hydrolysis process, starch, a high molecular weight polymer of the monosaccharide glucose (dextrose), is continually and systematically cleaved by enzymes (α amylase, glucoamylase, and β amylase) to produce mixtures of medium and low molecular weight units (Figure 154.2). The medium molecular weight saccharides (dextrins) are effective stabilizers and provide maximum prevention against coarse ice crystal formation, which is reflected in improved meltdown and heat shock resistance. They also improve cohesive and adhesive textural properties. The smaller molecular weight sugars provide smoothness, sweetness, and flavor enhancement. With the appropriate use of enzyme technology, corn syrup manufacturers have the ability to control the ratios of high to low molecular weight components, and the ratios of maltose, the disaccharide, to glucose, the monosaccharide. Glucose monosaccharide offers sweetness synergism with sucrose, but, being half the molecular weight, has greater freezing point depression than either sucrose or maltose. The ratio of higher to lower molecular weight fractions can be estimated from the dextrose equivalent (DE) of the syrup. Figure 154.2 shows that as the DE decreases, the sweetness increases, but the freezing point decreases (more freezing point depression) and the contribution to viscosity and chewiness in the mouth also decreases. Thus, optimization of DE and concentration of corn sweeteners are required for the most beneficial effects. Ice cream manufacturers usually use a 28 to 42DE syrup, either liquid or dry (1,62). High maltose syrups modify the effect of dextrose on the freezing point (62,63). With further enzyme processing (glucose isomerase), glucose can be converted to fructose (Figure 154.2), as in the production of high fructose corn sweeteners. The resultant syrup is much sweeter than sucrose, although it has half the molecular weight, and thus contributes more to freezing point depression than sucrose. These modifications to properties would also


Corn starch

154-9

Dextrose equivalent Maltose 28 DE • Disaccharide 42 DE • Same freezing pt. 55 DE depression of 64 DE

• Not sweet • Very little freezing pt. depression (high freezing pt.) • High molecular wt.

• Sweetness increases • Molecular wt. decreases • Freezing pt. decreases • Viscosity decreases

sucrose

Glucose (dextrose) • Monosaccharide • 80% as sweet as sucrose • Twice the freezing pt. depression of sucrose

Fructose • Monosaccharide • 1.8x sweeter than sucrose • Twice the freezing pt. depression of sucrose

FIGURE 154.2 An illustration of the products that result from the hydrolysis of corn starch and their properties relevant to ice cream manufacture.

require optimization of all sugars for appropriate use of HFCS, although it has been shown that blends of high fructose syrup, high maltose syrup, and low DE syrup can be utilized to provide appropriate sweetness, freezing point depression and total solids in the absence of sucrose (62,63). 4. Stabilizers Ice cream stabilizers are a group of ingredients (usually polysaccharides) commonly used in ice cream formulations. The primary purposes for using stabilizers in ice cream are to produce smoothness in body and texture, retard or reduce ice and lactose crystal growth during storage, especially during periods of temperature fluctuation, known as heat shock (64), and to provide uniformity to the product and resistance to melting (1,4). They also increase mix viscosity, stabilize the mix to prevent wheying-off (e.g., carrageenan), aid in suspension of flavoring particles, produce a stable foam with easy cut-off and stiffness at the barrel freezer for packaging, slow down moisture migration from the product to the package or the air, and help to prevent shrinkage of the product volume during storage (65). Stabilizers must also have a clean, neutral flavor, not bind to other ice cream flavors, contribute to acceptable meltdown of the ice cream, and provide desirable texture upon consumption (65). Limitations on their use include production of undesirable melting characteristics, excessive mix viscosity, and contribution to a heavy, soggy body. Although stabilizers increase mix viscosity, they have little or no impact on freezing point depression. Gelatin, a protein of animal origin, was used almost exclusively in the ice cream industry as a stabilizer, but has gradually been replaced with polysaccharides of plant origin due to their increased effectiveness and reduced cost (1). Stabilizers currently in use include: a) carboxymethyl cellulose, derived from the bulky components

or soluble fibre of plant material; b) locust bean gum (carob bean gum) which is derived from the beans of the tree Ceratonia siliqua, grown mostly in the Mediterranean; c) guar gum, from the guar bush, Cyamoposis tetragonolba, a member of the legume family grown in India and Pakistan for centuries, and now grown to a limited extent also in the USA; d) xanthan, a bacterial exopolysaccharide produced by the growth of Xanthomonas campestris in culture; e) sodium alginate, an extract of seaweed, kelp, or brown algae; or, f) carrageenan, an extract of Chondus crispis (Irish Moss), a red algae, originally harvested from the coast of Ireland, near the village of Carragheen. Each stabilizer has its own characteristics, and often two or more of these stabilizers are used in combination to lend synergistic properties to each other and improve their overall effectiveness. Guar, for example, is more soluble than locust bean gum at cold temperatures, thus it finds more application in HTST pasteurization systems. Carrageenan is a secondary colloid used to prevent wheying-off of mix, which is usually promoted by one of the other stabilizers (1,6); hence it is included in most blended stabilizer formulations. The mechanisms by which ice cream stabilizers affect freezing properties or limit recrystallization (see Section III.B.1) have been extensively studied, but are as yet not fully understood. Ice recrystallization in ice cream has recently been reviewed (10,66). It appears from the literature available to date that stabilizers have little (67) or no (68,69) impact on the initial ice crystal size distribution in ice cream at the time of draw from the scraped surface heat exchanger. They also has little or no impact on initial ice growth during quiescent freezing and hardening (52,70,71), but do limit the rate of growth of ice crystals during recrystallization (59,67–69,72–78). They have no effect on the freezing properties of an ice cream mix, e.g., freezing point depression (79,80), amount of freezable water or enthalpy of melting (71,81,82), or heterogeneous nucleation (83), and thus may not have been expected to

154-10


affect initial ice crystallization processes. With respect to recrystallization, there has not been a demonstrable correlation between viscosity of the unfrozen mix and recrystallization rate (74,79,80,84). The protective effect of stabilizers also appears not to be related to a modification of the glass transition (74,82,84,85). However, it has been suggested that they modify the ice crystal serum interface, either through surface adsorption to the crystal itself (68,69,76,78); by modifying the rate at which water can diffuse to the surface of a growing crystal during temperature fluctuation, or the rate at which solutes and macromolecules can diffuse away from the surface of a growing ice crystal (67,85); or by some other modification of the ice serum interface (86). It must be remembered that freeze-concentration of the unfrozen phase results in a polysaccharide concentration several times higher than what was present in the original mix. Most polysaccharides are also incompatible in solution with milk proteins, which leads to further localized concentrations. Recent research by Goff and co-workers (58) has focused on the ability of at least some stabilizers to form a cryo-gel and entrap ice crystals within this gel. Phase separation of polysaccharides and proteins also appears to be related. Control of ice recrystallization may then relate to microstructural differences in solute concentration at the surface of the crystal.

are of two main types: the mono- and diglycerides, and the sorbitan esters. Mono- and diglycerides are derived from the partial hydrolysis of fats of animal or vegetable origin. The sorbitan esters are similar to monoglycerides, in that the sorbitan esters have a fatty acid molecule, such as stearate or oleate, attached to a sorbitol molecule; whereas the monoglycerides have a fatty acid molecule attached to a glycerol molecule. To make the sorbitan esters water soluble, polyoxyethylene groups are attached to the sorbitol molecule. Polysorbate 80, polyoxyethylene sorbitan monooleate, is the most common of these sorbitan esters. Polysorbate 80 is a very active drying agent in ice cream (12), and is used in many commercial stabilizer/emulsifier blends.

5.

1. Blending

Emulsifiers

Emulsifiers have been used in ice cream mix manufacture for many years (87,88). They are usually integrated with the stabilizers in proprietary blends, but their function and action is very different than that of the stabilizers. They are used for: improvement of the whipping quality of the mix; production of a drier ice cream to facilitate molding, fancy extrusion, and sandwich manufacture; smoother body and texture in the finished product; superior drawing qualities at the freezer to produce a product with good stand-up properties and melt resistance; and more exact control of the product during freezing and packaging operations (1,87–89). Their mechanism of action can be summarized as follows: They lower the fat/water interfacial tension in the mix, resulting in protein displacement from the fat globule surface, which in turn reduces the stability of the fat globule to partial coalescence that occurs during the whipping and freezing process, leading to the formation of a fat structure in the frozen product that contributes greatly to texture and meltdown properties (12). The extent of protein displacement from the membrane, and hence the extent of dryness achieved, is a function of the emulsifier concentration (6,90). Their role in structure formation will be described further in Section II.B.4. Egg yolk was formerly commonly used as an ice cream emulsifier. Emulsifiers used in ice cream manufacture today

II. MANUFACTURING AND STRUCTURE OF FROZEN DESSERT PRODUCTS A. MIX MANUFACTURE Ice cream processing operations can be divided into two distinct stages: mix manufacture and freezing operations (Figure 154.3). Ice cream mix manufacture consists of the following unit operations: combination and blending of ingredients, batch or continuous pasteurization, homogenization, and mix aging.

Ingredients are usually preblended prior to pasteurization, regardless of the type of pasteurization system used. Blending of ingredients is relatively simple, if all ingredients are in the liquid form, as automated metering pumps or tanks on load cells can be used for measurement, and tanks, usually conical-bottom and agitated, are used for mixing. When dry ingredients are used, powders are added through either a pumping system under high velocity, or through a liquifier, a large centrifugal pump with rotating knife blades that chop all ingredients as they are mixed with the liquid (3). 2. Mix Calculations The general object in calculating ice cream mixes is to turn the formula, which is based on the desired components, into a recipe, which is based on the actual ingredients to be used to supply the components and the amount of mix desired. The formula is given as percentages of fat, msnf, sugar, corn syrup solids, stabilizers, and emulsifiers. The ingredients to supply these components are chosen on the basis of availability, quality, and cost. Table 154.8 illustrates the relationship between the major components, the main ingredients that supply the major components, and the minor components that are supplied with the major ones for each ingredient.


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Formulation -Sweetener -Emulsifier -Fat -Milk SNF -Stabilizer -Water Batch pasteurization

Homogenization

Cooling

Blending Continuous Pasteurization/Homogenization/Cooling Liquid ingredients

Dry ingredients

Air incorporation

Continuous freezing Aging

Packaging Batch freezing/Whipping

Flavor/Color addition

Particulate addition Hardening

Storage/Distribution

FIGURE 154.3 A schematic illustration of the processing steps in ice cream manufacture.

The first step in a mix calculation is to identify the composition of each ingredient. In some cases the percentage of solids contained in a product is taken as constant or provided by an ingredient supplier, while in others, the composition must be obtained by analysis TABLE 154.8 Sources of the Major Components in Ice Cream Mix, as well as the Minor Components Supplied by these Ingredients Component

Ingredients to Supply (but also supplies)

Milkfat

Cream (msnf, water) Butter (msnf, water)

Milk solids-not-fat (msnf)

Skim powder (water) Condensed skim (water) Condensed milk (water, fat) Sweetened condensed (water, sugar) Whey powder (water)

Water

Skim milk (msnf) Milk (fat, msnf) Water

Sweetener

Sucrose Corn syrup solids Liquid sugars (water)

Stabilizers/ emulsifiers

(e.g., the fat content in milk or cream). If there is only one source of the component needed for the formula, for example, the stabilizer or the sugar, it is determined directly by multiplying the percentage needed by the amount needed, e.g., 100 kg of mix @ 10% sugar would require 10 kg sugar. If there are two or more sources, for example 10% fat coming from both cream and milk, then an algebraic method may be utilized. Computer programs developed for mix calculations generally solve simultaneous equations based on mass and component balances. For manual calculations, a method known as the “Serum Point” method has been derived (1,4). This method has solved the simultaneous equations in a general way so that only the equations need to be known and not resolved each time. In the Serum Point method, 9% msnf is assumed in the aqueous (serum), non-fat portion of all milk ingredients. Thus, the msnf content of milk or cream is calculated as (100 ⫺ percent fat) ⫻ 0.09. This section will illustrate mix calculation solutions using algebraic techniques and the Serum Point method. EXAMPLE PROBLEM 1 - Mix using cream, skim milk, and skim powder (three sources of msnf, three sources of water), solution shown by both the Algebraic and Serum Point Methods. Desired : 100 kg mix @ 13% fat, 11% msnf, 15% sucrose, 0.5% stabilizer, 0.15% emulsifier. On hand: Cream @ 40% fat; skim milk; skim milk powder @ 97% msnf; sugar; stabilizer; emulsifier.

154-12


Solution via an algebraic method: Solution (Note: only one source of fat, sugar, stabilizer, and emulsifier, but two sources of msnf): Cream 13 kg fat 100 kg cream 100 kg mix ⫻ ᎏᎏ ⫻ ᎏᎏ 100 kg mix 40 kg fat ⫽ 32.5 kg cream Sucrose 15 kg sucrose 100 kg mix ⫻ ᎏᎏ ⫽ 15 kg sucrose 100 kg mix Stabilizer

Point method, and the solution of the above example by that method, along with the derivation of the equations, follows. The Serum Point calculation assumes 9% msnf in skim milk and the skim portion of all dairy ingredients. It then solves the calculation beginning with the most concentrated source of msnf first. It is advisable to solve a problem with the Serum Point method on the basis of 100 kg, and then scale it up to the desired mix quantity by multiplying by the appropriate factor, e.g., solution for each component for 100 kg ⫻ 50 ⫽ solution for 5000 kg.

Solution of Problem 1 via the Serum Point method: 1. Amount of skim milk powder needed is found by the following formula: msnf needed ⫺ (serum of mix ⫻ 0.09) ᎏᎏᎏᎏ ⫻ 100 % msnf in powder ⫺ 9

0.5 kg stabilizer 100 kg mix ⫻ ᎏᎏ ⫽ 0.5 kg stabilizer 100 kg mix

⫽ kg skim powder

Emulsifier 0.15 kg emulsifier 100 kg mix ⫻ ᎏᎏ ⫽ 0.15 kg emulsifier 100 kg mix Skim milk and skim powder, Note: two sources of the msnf Now, let x ⫽ skim powder, y ⫽ skim milk. MASS BALANCE (All the components add up to 100 kg, so skim powder ⫹ skim milk ⫽ 100 ⫺ mass of other ingredients)

The derivation of Equation 154.1 is shown at the end of the problem. For now, just assume that this equation will work! The serum of the mix is found by adding the desired percentages of fat, sucrose, stabilizer, and emulsifier together and subtracting from 100 [i.e., “serum” ⫽ msnf (or “serum solids”) ⫹ water]. In the present problem then, 100 ⫺ (13 ⫹ 15 ⫹ 0.5 ⫹ 0.15) ⫽ 71.35 kg serum. Substituting in Equation 154.1 we have:

x ⫹ y ⫽ 100 ⫺ (32.5 ⫹ 15 ⫹ 0.5 ⫹ 0.15) MSNF BALANCE (Equal to 11% of the mix and coming from the skim milk, the skim powder, and the cream, so the portion from the skim powder and skim milk ⫽ 11 kg ⫺ the contribution from the cream). The msnf portion of the skim milk and cream are taken as 9% of the non-fat portion, i.e., 9% in the case of the skim milk and (100⫺40) ⫻ 0.09 ⫽ 5.4% in the case of the cream. 0.97 x ⫹ 0.09y ⫽ 0.11(100) ⫺ (0.054 ⫻ 32.5) Once the appropriate equations have been written, they need to be solved algebraically. x ⫹ y ⫽ 51.85 so y ⫽ 51.85 ⫺ x 0.97 x ⫹ 0.09 y ⫽ 9.245 0.97 x ⫹ 0.09 (51.85 ⫺ x) ⫽ 9.245 0.97 x ⫺ 0.09 x ⫹ 4.67 ⫽ 9.245 0.88 x ⫽ 4.58

from the mass balance from the msnf balance substituting

x ⫽ 5.20 kg skim powder y ⫽ 46.65 kg skim milk The above shows the simultaneous solution of 2 equations with 2 unknowns. Likewise, if there were 3 unknowns, e.g., fat, msnf, and the total weight, then three equations could be written, one for each of fat, msnf, and weight. However, the above problem could also be solved with the Serum

(154.1)

(71.35 ⫻ 0.09) 4.58 11 ⫺ ᎏᎏ ⫻ 100 ⫽ ᎏ ⫻ 100 97⫺ 9 88 ⫽ 5.20 kg skim powder 2.

The weight of cream (since there is only one source of fat) will be 100 kg cream 13 kg ⫻ ᎏᎏ ⫽ 32.5 kg cream 40 kg fat

3. 4. 5. 6.

The sucrose will be 15 kg/100 kg mix. The stabilizer will be 0.5 kg/100 kg mix. The emulsifier will be 0.15 kg/100 kg mix. The weight of mix supplied so far is, Cream Skim powder Sucrose Stabilizer Emulsifier

32.50 kg 5.20 kg 15.00 kg 0.50 kg 0.15 kg 53.35 kg

The skim milk needed therefore is 100 ⫺ 53.35 ⫽ 46.65 kg. It is always important to check your solutions to ensure they give the desired result. Such a proof is shown below, where the quantities of each ingredient and the quantities


154-13

of each component in each ingredient are laid out in a table and summed. The total mass of each component divided by the total mass of mix should yield the desired percentage.

MASS BALANCE X ⫹ Y ⫽ Total mix ⫺ components already added X ⫹ Y ⫽ 100 ⫺ (13 ⫹ 15 ⫹ 0.5 ⫹ 0.15) (the “Serum of the Mix”) X ⫹ Y ⫽ 71.35

Proof: Ingredients

Kilograms

Cream Skim milk Skim powder Sucrose Stabilizer Emulsifier Totals

32.50 46.65 5.20 15.00 0.50 0.15 100.0

Kgs. Fat

Kgs. msnf

Kgs. T.S.

13.0 — — — — — 13.0

1.75 4.20 5.05 — — — 11.0

14.75 4.20 5.05 15.00 0.50 0.15 39.65

Derivation of the Serum Point equations: Problem 1 is resolved again using simultaneous equations in a general way to show where the serum point equations come from. On hand:

cream @ 40% fat (supplies fat, water, and msnf, therefore can be thought of as a mixture of fat and skim milk) skim milk @ 9% msnf (supplies water and msnf) skim milk powder @ 97% msnf (supplies water and msnf) sucrose stabilizer emulsifier

Solution - Only one source of fat, sucrose, stabilizer, and emulsifier kg fat ⫽ 100 kg mix ⫻ 13 kg fat/100 kg mix ⫽ 13 kg fat (The explanation for this assumption becomes clearer in a moment!) kg sucrose ⫽ 100 kg mix ⫻ 15 kg sucrose/100 kg mix ⫽ 15 kg sucrose kg stabilizer ⫽ 100 kg mix ⫻ 0.5 kg stab./100 kg mix ⫽ 0.5 kg stabilizer kg emulsifier ⫽ 100 kg mix ⫻ 0.15 kg emul./100 kg mix ⫽ 0.15 kg emulsifier - Two sources of msnf Let X ⫽ skim powder (kg) Let Y ⫽ skim milk (kg) ⫹ skim milk in cream (kg)

(so Y ⫽ 71.35 ⫺ X) MSNF BALANCE ⫹

0.97X “Serum solids fraction in powder”

0.09Y

⫽

(0.11 ⫻ 100)

“Serum solids “Serum solids fraction fraction in mix” in skim”

0.97 X ⫹ 0.09 (71.35 ⫺ X) ⫽ 11 0.97 X ⫹ (0.09 ⫻ 71.35) ⫺ 0.09 X ⫽ 11 0.97 X ⫺ 0.09 X ⫽ 11 ⫺ (0.09 ⫻ 71.35) 11 ⫺ (0.09 ⫻ 71.35) X ⫽ ᎏᎏᎏ 0.97 ⫺ 0.09 Which is equal to: kg skim powder msnf needed ⫺ (0.09 ⫻ serum of mix) ⫽ ᎏᎏᎏᎏ ⫻ 100 % msnf in powder ⫺ 9 (which is Equation 154.1!) 4.58 X ⫽ ᎏ ⫽ 5.20 kg powder 0.88 kg cream ⫽ 13 kg fat ⫻ 100 kg cream/40 kg fat ⫽ 32.5 kg cream kg skim ⫽ 100 ⫺ 32.5 ⫺ 15 ⫺ 0.5 ⫺ 0.15 ⫺ 5.2 ⫽ 46.65 kg EXAMPLE PROBLEM 2 - Mix using cream, milk, and skim powder (three sources of msnf, three sources of water, and two source of fat); solved by both the Algebraic and Serum Point Methods. Desired: 100 kg mix containing 14% fat, 9.5% msnf, 15% sucrose, 0.4% stabilizer, 1% frozen egg yolk. On hand: Cream 30% fat, milk 3.5% fat, skim milk powder 97% solids, sucrose, stabilizer, and egg yolk (50% solids). The solution to this problem will be shown by the simultaneous solution of 3 equations, since there are three sources of msnf, three sources of water, and two source of fat, and by the Serum Point method. Both produce the same results. Follow whichever method you prefer. Computer programs exist that solve simultaneous equations; writing

154-14


the equations, however, requires an understanding of the objectives of the problem.

Substituting we have, 3.236 ⫻ 100 9.5 ⫺ ( 69.6 ⫻ 0.09) ᎏᎏᎏ ⫻ 100 ⫽ ᎏᎏ 88 97⫺9

Solution via the algebraic method: 15 kg sucrose Sucrose: 100 kg mix ⫻ ᎏᎏ ⫽ 15 kg sucrose 100 kg mix 0.4 kg stabilizer Stabilizer: 100 kg mix ⫻ ᎏᎏ 100 kg mix ⫽ 0.4 kg stabilizer 1 kg egg yolk Egg yolk: 100 kg mix ⫻ ᎏᎏ 100 kg mix ⫽ 1 kg egg yolk Now, let x ⫽ skim powder, y ⫽ milk, and z ⫽ cream.

⫽ 3.68 kg powder 2. Amount of sucrose required is 15.0 kg. 3. Amount of stabilizer required is 0.4 kg. 4. Amount of egg required is 1.0 kg. 5. Determine amount of milk and cream needed. Materials supplied so far are 3.68 kg powder, 15 kg sucrose, 0.4 kg stabilizer, and 1 kg egg yolk, a total of 20.08 kg. 100 ⫺ 20.08 ⫽ 79.92 kg milk and cream needed. 6. Determine the amount of cream by following formula: % fat in milk)

kg fat needed ⫺ (kg cream and milk needed ⫻ ᎏᎏ

100

MASS BALANCE All the components add up to 100 kg, so the sum of the three unknowns ⫽ 100 ⫺ the sum of the known mass of the other components. x ⫹ y ⫹ z ⫽ 100 ⫺ (15 ⫹ 0.4 ⫹ 1)

% fat in cream ⫺ % fat in milk

(154.2)

Note: Equation 154.2 is derived from a generalized fat balance, in much the same way that Equation 154.1 was derived. Substituting we have,

MSNF BALANCE Equal to 9.5% of the mix and coming from the milk, the skim powder, and the cream; assume 9% in the skim portion of the milk and cream so that the msnf of the milk ⫽ 0.09 ⫻ (100 ⫺ 3.5) and of the cream ⫽ 0.09 ⫻ (100⫺30)

冢

3.5 14 ⫺ 79.92 ⫻ ᎏ 100

0.035y ⫹ 0.3z ⫽ 0.14 (100) These equations could now be solved to produce the final outcome: x ⫽ 3.7 kg skim powder y ⫽ 37.7 kg milk z ⫽ 42.3 kg cream

Solution via the Serum Point method: 1.

冣

30 ⫺ 3.5 11.20 ⫻ 100 ⫽ ᎏ ⫻ 100 ⫽ 42.26 kg cream. 26.5

0.97x ⫹ 0.08685y ⫹ 0.063z ⫽ 0.095 (100) FAT BALANCE Equal to 18% of the mix and coming from the milk and cream

⫻ 100

7.

Amount of milk needed ⫽ 79.92 ⫺ 42.26 ⫽ 37.66 kg of milk.

Proof: Ingredients

Kilograms

Kgs. Fat

Kgs. msnf

Kgs. T.S.

Cream Milk Skim powder Sucrose Stabilizer Egg yolk Totals

42.26 37.66 3.68 15.00 .40 1.00 100.00

12.68 1.32 — — — — 14.00

2.66 3.27 3.57 — — — 9.50

15.34 4.59 3.57 15.00 .40 .50 39.40

Determine the amount of skim milk powder required by using Equation 154.1:

With Equations 154.1 and 154.2, most complex mix problems can be solved. There are additional complications for the use of condensed skim or whole milk, and for liquid sugars. See Ref. 1 for further details.

msnf needed ⫺ (serum of mix ⫻ 0.09) ᎏᎏᎏᎏ ⫻ 100 % msnf in powder ⫺ 9

3. Pasteurization and Food Safety Issues

⫽ skim powder Serum of mix ⫽ 100 ⫺ (14 ⫹ 15 ⫹ 0.4 ⫹ 1.0) ⫽ 69.6.

Pasteurization is the biological control point in the system, designed for the destruction of pathogenic bacteria. If raw milk or cream are used as ingredients, it could be


154-15

that these are contaminated with a human pathogen from the dairy farm. Therefore, it is essential that pasteurization be carefully designed and closely monitored. If raw dairy ingredients are not used, contamination from a human source could also occur, and thus the use of pasteurization conditions that eliminate pathogens is mandated by most legal jurisdictions. In addition, it serves a useful role in reducing the total bacterial load, and in solubilization of some of the components (proteins and stabilizers). Both batch and continuous (high temperature short time or HTST) systems are in common use (3). In a batch pasteurization system, blending of the proper ingredient amounts is done in large jacketed vats equipped with some means of heating, usually saturated steam or hot water. The product is then heated in the vat to at least 69°C (155°F) and held for 30 min to satisfy legal requirements for pasteurization, or equivalent times and temperatures as determined by the local legal jurisdiction. The heat treatment must be severe enough to ensure destruction of pathogens and to reduce the bacterial count to a maximum (e.g., 10,000 per gram), depending on the legal jurisdiction. Following pasteurization, the mix is homogenized using high pressures and then is passed across some type of heat exchanger (plate heat exchanger or double or triple tube heat exchanger) for the purpose of cooling the mix to refrigerated temperatures (4°C).

Continuous pasteurization is usually performed in an HTST heat exchanger following the blending of ingredients in a large, insulated feed tank. Some preheating, to 30 to 40°C, may be necessary for solubilization of the components. The HTST system is equipped with a heating section, a cooling section, and a regeneration section (Figure 154.4). Mix first enters the raw regeneration section, where cold or preheated mix is heated to as warm as possible on one side of a plate heat exchanger, while the pasteurized hot mix is cooled to as low as possible running countercurrent on the opposite sides of the plates. Following the raw regeneration section, the mix enters the heating section where a minimum temperature of 80°C is obtained. The mix is held at this temperature for 25 sec by flowing either through a series of holding tubes or through an additional set of plates in the HTST unit. Holding times much longer than the minimum can be accomplished with longer holding tubes. Holding times of 2 or 3 min may produce superior mixes that retain many of the advantages of batch pasteurization (4,6). After leaving the holding tube, the mix enters the homogenizer, depending upon the particular configuration, then flows back through the pasteurized side of the regeneration section and enters the cooling plates, where a chilled brine solution or chilled water bring the mix down to around 4°C.

HTST continuous plate pasteurizer

Cold water*

9 Cold, past. mix

6

2

Hot, Warm, past. raw mix mix

3 Warm, raw Warm mix water

Frame Plates 5 External holding tube

Screw press

Warmer water*

Cool, past. mix 8

Cooling section

Cool, Cold, past. raw mix mix 7

1

Regenerator

Hot, Hot raw water mix 4 Heating section

*or brine, or glycol

FIGURE 154.4 A schematic illustration of the side view of a plate heat exchanger used for HTST pasteurization of frozen dairy dessert mixes. Numbers indicate the sequence of flow of mix. Italics are used to differentiate the material on one side of a section from the material on the other.

154-16


4. Homogenization Homogenization is responsible for the formation of the fat emulsion by forcing the hot mix through a small orifice under pressures of 15.5 to 18.9 MPa (2000–3000 psig), depending on the mix composition. The actual mechanism of fat disruption within the homogenizer is thought to result from turbulence, cavitation, and velocity gradients (energy density) within the valve body (91). The 4–8 fold increase in the surface area of the fat globules is responsible in part for the formation of the fat globule membrane, comprised of adsorbing materials attempting to lower the interfacial free energy of the fat globules (92,93). Koxholt and co-workers (94) have recently shown that sufficient fat structure in the mix for optimal ice cream meltdown was created by homogenization pressures on the first stage of 10 MPa, in mixes with up to 10% fat content, and that higher pressures were not required. With single stage homogenizers, fat globules tend to cluster as bare fat surfaces come together or adsorbed molecules are shared. Therefore, a second homogenizing valve is commonly placed immediately after the first, with applied back pressures of 3.4 MPa (500 psig) (3), allowing more time for surface adsorption to occur. However, Koxholt and co-workers (94) have recently shown that two-stage homogenization is not necessary for ice cream mixes up to 10% fat content in order to achieve optimal fat structuring and ice cream meltdown. The net effects of homogenization are in the production of a smoother, more uniform product with a greater apparent richness and palatability, and better whipping ability (1). Homogenization also decreases the danger of churning the fat in the freezer, and makes it possible to use products that could not otherwise be used, such as butter and frozen cream. 5.

Aging

An aging time of four hours or greater is recommended following mix processing prior to freezing. This allows for hydration of milk proteins and stabilizers (some viscosity increase occurs during the aging period), crystallization of the fat globules, and a membrane rearrangement, to produce a smoother texture and better quality product (6,11). Non-aged mix is very wet at extrusion and exhibits variable whipping abilities and faster meltdown (1,6). The appropriate ratio of solid:liquid fat must be attained at this stage. This is a function of temperature and the triglyceride composition of the fat used; as a partially crystalline emulsion is needed for partial coalescence in the whipping and freezing step, as discussed in Section II.B.4. Emulsifiers generally displace milk proteins from the fat surface during the aging period (12,36,95), and this is also discussed in detail in Section II.B.4. The whipping qualities of the mix are usually improved with aging. Aging is performed in insulated or

refrigerated storage tanks, silos, etc. Mix temperature should be maintained as low as possible (at or below 4°C) without freezing.

B. DYNAMIC FREEZING In a continuous, scraped surface freezer, numerous processes take place that ultimately influence the overall quality of the ice cream. One of the most important steps, of course, is freezing water into ice. At the same time as ice is being formed, there is also air incorporation, leading to development of air cells and the desired overrun. In addition, destabilization of the fat emulsion (partial coalescence, see Section II.B.4) takes place during freezing, which promotes incorporation and stabilization of the air cells. All of these processes take place simultaneously in the minute or less that ice cream spends in the dynamic freezing step. Following this initial phase of ice formation in a dynamic freezer, where about half of the water is turned into ice, there is a static freezing step, often called hardening (see Section II.D). The mechanisms that lead to ice formation in an ice cream freezer are quite complex. Ultimately, the product exiting the freezer contains numerous, small ice crystals. As seen in Figure 154.5 (96), the ice crystals in ice cream at the exit of the freezer are somewhat block-shaped and vary in size from a few microns to over 100 µm. A typical size distribution for hardened ice cream is shown in Figure 154.6 (6). The large number of very small ice crystals, estimated to be 4 ⫻ 109 crystals per liter (97), gives ice cream its smooth, cool character. The ice crystals must remain below some threshold detection size, often given as about 50 µm mean size (1), for the ice cream to remain smooth. When crystals become larger than this, the ice cream may be considered coarse. Control of ice crystallization to produce the desired number and size of crystals is critical to producing high quality ice cream.

80 µm

0 min –14.0°C

FIGURE 154.5 Ice crystals in ice cream, as observed using light microscopy (96).


154-17

18 16

Mean diameter (hardened): 45–55 µm Range of sizes: 8 to 170 µm Number: 4 × 109 crystals/litre

14 12

%

10 08 06 04 02 0

20

40

60

80 100 120 140 160 180 Size (µm)

FIGURE 154.6 Typical ice crystal size distribution for hardened ice cream (6).

1.

Principles of Ice Crystallization

When ice freezes or crystallizes from any solution, several steps must take place. First, the solution must be cooled below the freezing (melting) point of the solution. The temperature difference between the actual temperature and the freezing point temperature of the mix is the driving force for freezing. Once an appropriate driving force has been attained, formation of the solid ice phase from the liquid molecules must occur. This step is called nucleation, where tiny bits of crystalline ice have just started to form. Once these nuclei begin to form, they continue to grow until some phase equilibrium has been obtained. In freezing, ice continues to form until a thermal equilibrium between the freezing product and its environment has been reached. The total amount of ice that forms (at any storage temperature) depends on the system. For pure water, all of the water is converted to ice as long as the temperature is below 0°C. In ice cream, however, the

Air

other ingredients influence the freezing process and determine how much water turns to ice (the ice phase volume) at any temperature. Both the total amount of ice as well as the nature of the ice dispersion (size, shape, etc.) influence the physical properties of the final ice cream product. After the product is frozen, the ice phase continues to undergo recrystallization. Recrystallization is a term used for a combination of several events, including melting, growth and ripening, that occur after the initial ice crystal phase has been developed. Recrystallization leads to changes in the distribution of ice crystals within the system based on the thermodynamic difference in melting point between large crystals and small ones. Typically, recrystallization occurs with no change in ice phase volume. In continuous ice cream manufacture, mix is pumped into the freezer and flows along the length of the barrel. As the ice cream moves from the inlet to the outlet, ice is frozen, fat is destabilized and air is injected, as shown in Figure 154.7. The mix enters the freezer barrel at a temperature between 0 and 4°C and begins to freeze as it contacts the metal wall cooled by expanding refrigerant (ammonia or Freon). Ice forms at the barrel wall since this is where the driving force for freezing is the highest. However, the ice layer that forms is rapidly scraped off of the wall and dispersed into the center of the freezer barrel where the ice changes form depending on temperature conditions and mixing parameters. As the mix moves axially along the freezer barrel, the amount of ice formed increases as the bulk average temperature of the slurry decreases. At the draw (exit) of the freezer, approximately half of the initial water in the mix is frozen into ice and the product is a pumpable slurry of partially frozen ice cream. The change in temperature along the length of the freezer for a typical ice cream operation is shown in Figure 154.8 (98). The final temperature and the amount of ice formed depends on the rate of freezing within the barrel of the freezer. This is controlled by the flow of refrigerant (ammonia or Freon) on the outside of the barrel, the throughput rate of ice cream through the freezer and the type of mixing device used within the barrel of the freezer. In general, conditions in a scraped surface freezer

Refrigerant vapor

Mix 2 to 4°C Liquid refrigerant

Ice cream –5 to –6°C • 50 to 100% overrun • 40 to 50% water frozen to ice • 20 to 60% fat destabilization

FIGURE 154.7 A schematic diagram to represent inputs and outputs during the continuous freezing of ice cream.

154-18


10

Temperature (°C)

5

0

100 RPM –5 250 RPM 500 RPM –10 0

0.2

0.4

0.6

0.8

1.0

Axial distance (fraction of barrel length)

FIGURE 154.8 Axial profile of ice cream temperature as a function of dasher speed within the barrel of a scraped-surface ice cream freezer (98).

Firmness

a. Phase/state behavior Freezing Point Depression. In order for ice to freeze, the temperature of the solution has to be lowered below its freezing point. The temperature at which a solution freezes, or the freezing point, is determined by the concentration and type of solutes present in the mix. The presence of dissolved salts and sugars causes the freezing point of water to be lowered. This freezing point depression occurs because the solute molecules interact with water and inhibit the ability of the water molecules to come together and form an ice crystal lattice (or freeze). The extent of freezing point depression is based on the number of solute molecules and their size. Small molecules have the greatest effect; the higher the concentration of these small molecules, the lower the freezing point. Thus, ice cream mixes made with a high concentration of milk salts and lactose, with high sugar content, or with high content of low-molecular weight sweeteners, have lower freezing points. For example, use of high fructose corn syrup as a sweetener gives a lower freezing point (compared to the use of sucrose) due to the addition of lower molecular weight sugars. Mixes made with high

levels of msnf have a low freezing point due to the addition of milk salts and lactose. The freezing point of the ice cream mix is an important quality control parameter since it governs the amount of ice that can form at a given temperature, which affects the quality and textural attributes of the ice cream. As seen in Figure 154.9 (99), melting rate increases and firmness decreases with increasing freezing point (as indicated by osmolality) (99,100). As the freezing point of the mix goes down (osmolality increases), ice cream contains less ice and more unfrozen water at any given temperature, which leads to ice cream that is less firm and melts at a faster rate.

Melting rate

are controlled to give a compromise between the draw temperature (amount of ice frozen) and the stiffness of the ice cream exiting the freezer. The ice cream should be as frozen as possible (since here is where control of ice formation occurs), yet be sufficiently fluid to incorporate inclusions and/or fill the containers without leaving air gaps. This compromise depends to some extent on the type of product being produced and its final form.

Freezing point temperature

FIGURE 154.9 Effects of freezing point of ice cream mix on melting rate and firmness of final product (based on data from Ref. 99).


154-19

Freezing point depression also can be calculated based on principles of thermodynamics (96), assuming ideal solutions and dilute concentrations. At the point where the two phases (solid ice and liquid water) are in equilibrium, the chemical potentials of the two phases are equal and the following equation can be developed.

冤

冥

1 ∆H 1 ⫽ ln(Xw) ᎏ ᎏ⫺ᎏ T R T0

(154.3)

Here, ⌬H is the latent heat of fusion, R is the ideal gas constant, T0 is the freezing point of pure water, T is the freezing point of solution with mole fraction of water of Xw. For aqueous foods, equation (154.3) may be modified to give: C (Tf ⫺ T0 ) ⫽ K ᎏ MW

(154.4)

where, Tf is freezing point (°C) of a solution with concentration C (in g/100 g water), MW is the molecular weight of the dissolved solute, and K is a conversion factor (equal to 1.86 for water). In simple systems, Equation 154.4 gives a good estimate of freezing point and can be used to show the relationship between freezing point and solute content. For example, the freezing point depression curves for several sugars are shown in Figure 154.10 (96). Note that fructose has a lower freezing point than sucrose at any equal concentration (wt %), because it has lower molecular weight and there are more molecules of fructose added (at equivalent mass of sugar). Conventional corn syrup solids (42DE), which contain numerous longer-chain saccharides, have a higher freezing point than sucrose. In more complex food formulations, the sum of each of the components that

0

20

impact freezing point depression is needed. In ice cream mix, it is the combination of sweeteners and milk ingredients used in the formulation that leads to the specific freezing point depression curve for any mix. Sugars (from sweetener and msnf) and salts (from msnf) are the main components that impact freezing point depression of an ice cream mix. Typically, freezing point depression of an ice cream mix is calculated from Equation 154.4 by taking the sucrose equivalents of all the important components that influence freezing point. Sucrose equivalency values for common sweeteners have been developed (101) for use in ice cream formulations. The contributions of both sweeteners and salts on freezing point are then summed (102) to obtain the initial freezing point of the mix. Equation 154.4 can also be used to calculate the amount of water frozen into ice for a given ice cream at any temperature by varying the concentration since freeze concentration of the unfrozen phase occurs during freezing. Based on the approximate freezing point depression curve and the assumption of slow freezing, the amount of water converted to ice at any temperature can be calculated by a mass balance. For a typical ice cream, a relationship between temperature and the amount of water frozen into ice is obtained, as shown in Figure 154.11 (103). Since Equation 154.4 technically only works for dilute, ideal solutions, it does not apply very accurately at higher concentrations found in the unfrozen phase of ice cream. Thus, correction factors have been developed based on experimental data for frozen sucrose solutions (104). To calculate the freezing point of a given mix, the effects of sweeteners and salts must be summed. The effects of sweeteners are obtained by summing the

40

60

80

0

Freezing temperature (°C)

–5

–10 Fructose Sucrose

–15

42 DE corn syrup 42 DE high maltose

–20

–25 Concentration (wt%)

FIGURE 154.10 Freezing point depression curves (freezing temperature as a function of concentration) for several sugars (96).

154-20


100

Water frozen into ice (%)

80

60

40

20

0 –30

–20

0

–10 Temperature (°C)

FIGURE 154.11 Examples of the approximate amount of water frozen into ice for ice cream of standard formulation at given temperatures, based on an equilibrium freezing curve for that formulation (based on Ref. 103).

contributions of sucrose, lactose (from msnf) and any corn syrups added. For an ice cream mix containing only sucrose, Equation 154.5 is used (1). [(msnf ⫻ 0.545) ⫹ S]100 SEsw ⫽ ᎏᎏᎏ W

Freezing Points

(154.5)

Here, SEsw is the sucrose equivalence from sugars, S is sucrose content, W is water content (100 – total solids, %) and 0.545 is the percentage of lactose typically found in msnf. To obtain the freezing point depression associated with this level of sugars, FPDsw, Table 154.9 is used (1). The contribution to freezing point depression from salts in msnf is found from Equation 154.6. msnf ⫻ 2.37 FPDsa ⫽ ᎏᎏ W

(154.6)

Here, FPDsa is the sucrose equivalence for salts contained in msnf, and the constant 2.37 is based on the average molecular weight of the salts present in msnf. To obtain the freezing point depression of the ice cream mix, FPDt, the two contributions are summed. FPDt ⫽ FPDsw ⫹ FPDsa

Sucrose Equivalent (%) 0 5 10 15 20 25 30 35 40 45 50

(°C)

(°F)

0.00 ⫺0.42 ⫺0.83 ⫺1.17 ⫺1.50 ⫺2.08 ⫺2.67 ⫺3.58 ⫺4.39 ⫺5.69 ⫺7.00

32.00 31.25 30.50 29.90 29.30 28.25 27.20 25.55 24.10 21.75 19.40

Now, find the freezing point depression for this level of sucrose equivalent from Table 154.9. By interpolation, FPDsw ⫽ 2.31°C

(154.7)

EXAMPLE PROBLEM 3: Calculate the initial freezing point of an ice cream mix containing 16% sucrose, 12% msnf, and 60% water (40% total solids). First, calculate the sucrose equivalents from Equation 154.5: [12 ⫻ (0.545) ⫹ 16]100 SEsw ⫽ ᎏᎏᎏ ⫽ 37.57 60

TABLE 154.9 Freezing Point Depression in Sucrose Equivalents (1)

For salts, from Equation 154.6: 12 ⫻ (2.37) FPDsa ⫽ ᎏᎏ ⫽ 0.47°C 60 Find the total freezing point depression for the mix from Equation 154.7: FPDt ⫽ FPDsw ⫹ FPDsa ⫽ 2.31 ⫹ 0.47 ⫽ 2.78°C


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Thus, the initial freezing point temperature for this ice cream mix is ⫺2.78°C. Freezing Curve. In order for ice to form, the temperature of the system (T) must be below the freezing point (Tf) of the mix. The extent of subcooling (∆T ⫽ Tf ⫺ T) determines the rate of freezing, as discussed in the next section. Once freezing occurs, though, several things take place. The change in phase due to formation of ice causes a release of heat (latent heat of fusion), which increases the temperature in the vicinity of the phase change; this heat is removed by the refrigerant. At the same time, removal of water from the mix in the form of ice causes an increase in concentration of the remaining unfrozen phase, which has a lower freezing point due to the higher concentration. Thus, in the vicinity of the ice crystals, the temperature increases and the freezing point decreases. This leads to a freezing profile (Figure 154.12) dependent on the rate of freezing (96). For slow freezing, once nucleation starts, the temperature increases to approximately the melting point, due to fast release of latent heat, and then begins to decrease as further heat is removed and the concentration increases. Slow freezing results in a freezing profile that essentially follows the freezing point depression curve. As freezing continues, the unfrozen phase becomes more and more concentrated and temperature continues to decrease. This leads to an increase in viscosity of the unfrozen phase until ultimately, the viscosity is sufficiently high that the freeze-concentrated unfrozen phase becomes glassy. That is, at some low temperature (the glass transition temperature, Tg), the unfrozen phase solidifies into a glassy state. Note that this is not a true solid (in the sense

of a crystalline solid), but rather it is a high viscosity fluid that acts like a solid for as long as the temperature remains low. The point where the glassy state is formed during slow freezing is called the maximally freeze-concentrated temperature (Tgⴕ), as seen in Figure 154.12. For various ice cream mixes, Tgⴕ has been found to be around ⫺30 to ⫺35°C (85,105). For slow freezing, the amount of ice formed at any temperature is obtained as described in the previous section since the system follows the freezing point depression curve. If freezing is very rapid, the temperature and concentration of the solution falls somewhere below the freezing point depression curve, as shown in Figure 154.12. In this case, Figure 154.11 no longer applies and the amount of ice formed at any temperature is less than that shown in Figure 154.11 and is dependent on the rate of freezing.

b. Nucleation The driving force for freezing is the temperature difference between the actual temperature of the system and the freezing (melting) point (T⫺Tf). At higher subcooling, freezing occurs more rapidly; that is, the rate of ice formation is a strong function of the thermal driving force (∆T). The onset of nuclei formation is the point when the water molecules convert into molecules in an ice crystal lattice. When the temperature driving force is sufficiently high (temperature sufficiently below the freezing point), there is sufficient energy for the water molecules to overcome the energy barrier needed to form an ice crystal surface (the interface between crystal and liquid). Typically, ice formation begins on a surface that catalyzes the formation of ice crystals. This surface may

Solution Initial

Temperature

Ice + solution

Final Rapid freezing

Slow freezing

Tg′

Glass

Solution concentration

C g′

FIGURE 154.12 A phase diagram for solutions (e.g., ice cream mix) showing the path of freezing (temperature and solution concentration) for freezing at different rates. Figure shows schematic representation of freezing point depression and glass transition curves (adapted from Ref. 96). Tgⴕ and Cgⴕ represent point of maximally freeze-concentrated solution.


be that of the vessel that contains the solution or particles distributed throughout the solution that provide sufficient energy to order the water molecules in solution and promote nuclei formation. In commercial ice cream manufacture, it is likely that nucleation initially occurs by formation on the metal surface (inner barrel wall) exposed to the refrigerant, since that is where the driving force (∆T) is highest. The rate of nucleation (number of nuclei formed per unit volume per unit time) for melt systems has been described by Equation 154.8 (96,106).

冦

BT f2 ⌬G⬘ J ⫽ Aexp ⫺ ᎏᎏ ⫹ ᎏv (⌬Hf)2(Tf ⫺ T)2 kT

冧

Nucleation

Rate

154-22

Growth

Tg

B

(154.8)

Here, J is nucleation rate, A is a frequency factor (or preexponential term), B is a constant depending on the solutes present, Tf is freezing (melting) point, k is Boltzman’s constant, ∆Hf is latent heat of fusion, T is system temperature and ∆Gⴕv is a diffusion-limited term that describes the mobility of water molecules. Equation 154.8 clearly shows the dependence of nucleation rate on operating parameters, particularly the temperature driving force. When the system temperature, T, is close to the freezing point temperature, Tf, the temperature driving force (∆T) and nucleation rate are low. In fact, at temperatures close to Tf, nucleation is so slow that the system may effectively remain unfrozen for long times, even though the temperature is below the freezing point of the solution. However, when ∆T is sufficiently high, or when system temperature falls sufficiently below Tf, Equation 154.8 predicts a sudden onset of nuclei formation. As the driving force (∆T) increases, the rate of nuclei formation increases precipitously, giving rise to the spontaneous nature of freezing once it has initiated. When ∆T increases to too high a value, nucleation rate once again decreases due to the limited mobility of water molecules. As temperature goes down, the viscosity increases substantially, until eventually the system becomes glasslike. At this point, the ∆Gⴕv term overwhelms the ∆T term in Equation 154.8, and nucleation rate again goes to zero. Thus, there is a maximum in the nucleation rate curve as shown schematically in Figure 154.13 (9). In a commercial scraped-surface freezer, the primary temperature driving force for nucleation occurs at the barrel wall. On the jacket side of this metal wall, liquid refrigerant (either ammonia or Freon) is vaporizing to provide the cooling effect. Vaporizing refrigerant removes heat from the ice cream mix nearest to the barrel wall and creates a high degree of subcooling in the mix at that region (9), as seen in Figure 154.14. Ice forms on the metal surface of the barrel wall where the temperature driving force is highest and catalytic nucleation sites exist (microscopic imperfections in the wall itself). Without agitation and scraping, this ice layer would continue to grow and increase in thickness until a thermal equilibrium

A

Tf

Temperature

FIGURE 154.13 Rates of nucleation and growth of ice over the temperature range from glass transition temperature (Tg) to melting point (Tf). A and B represent temperatures where nucleation rate is low and high, respectively (9).

Bulk ice cream mix –2 to –6°C Heat transfer ~ –26°C

Ice layer Metal wall

~ –30°C

Ammonia

FIGURE 154.14 Approximate driving force (∆T) for freezing of ice cream in a continuous freezer with vaporizing ammonia as refrigerant.

was attained between unfrozen mix and the coolant. In commercial freezers, the rotating scraper blades repeatedly clean off the metal surface of the barrel wall. Based on an agitator speed of 200 RPM and a six-bladed agitator, it can be calculated that the metal surface is scraped every 0.05 s. Thus, ice has very little chance to build up on the barrel wall. Recent studies (107,108) using videomicroscopy to observe ice formation on a cooled surface suggest that the scraper blade effectively cleans most of the ice off the metal wall at each scraping. Small pockets or shards of ice left on the wall serve as seeds for subsequent growth of the ice layer between scrapings. These studies suggest that the ice layer initially grows out along the surface to fill an ice layer on the metal wall rather than initially growing out into the solution. Most likely, the scraper blade removes the regrown ice layer before substantial growth into the solution (away from the wall) has occurred. The ice layer that is scraped off the metal wall is dispersed into the bulk mix circulating around the agitators.


The nature of the ice layer scraped off the metal wall in a commercial freezer has been the subject of much discussion in the past decades. Based on work by Schwartzberg and co-workers (109,110), it has been suggested that the ice layer in a scraped-surface freezer forms as dendritic (or needle-shaped) ice crystals extending into the solution (9). The scraper blade then removes these dendrites from the surface and disperses them into the center of the barrel, where subsequent recrystallization and ripening occur. Recent experiments suggest a different form of ice crystals at the barrel wall. Rather than dendrites extending into the solution, it appears that ice initially grows horizontally along the metal surface since this is the most favorable direction for heat transfer (107,108). The ice crystals in this layer are most likely needle-like although this has not been shown conclusively. Because growth is extremely rapid at the low temperatures of the metal wall, this ice layer is comprised of multiple ice crystals surrounded by concentrated mix. Before this layer has a chance to form more perfect crystals and exclude solvent molecules from the mix, it is scraped off by the blades and dispersed into the bulk of the freezer. In “slushie” machines that produce iced fruit drinks, the first evidence of ice formation when the refrigeration unit is turned on is thin “flakes” of ice removed from the refrigerated metal surface. Apparently, the scraper blade removes a layer of slush composed of ice and concentrated mix that temporarily maintains its integrity in the bulk, appearing as a thin layer or flake of ice approximately 0.5 to 1 cm in diameter. A submersed microscope in a batch scraped-surface freezer initially catches large (about 250 µm across) sheets of ice that take a hexagonal form (111). Similar forms have been seen growing horizontally along a cooled metal surface (108). These polycrystalline ice flakes are distributed into the bulk of the freezer by the action of the scraper blades. What happens next depends to some extent on the nature of the bulk phase within the barrel of the freezer. For freezers with open dashers and internal mixers, the ice layer is mixed well with the warmer mix farther away from the refrigerated barrel wall. Here, the blades of the internal dasher can break the ice “flakes” into smaller shreds or pieces. In addition, melting, growth and ripening take place due to fluctuations in temperature that arise from the heat being removed by the barrel wall and the latent heat associated with melting and growth. A complex heat and mass transfer environment exists in which the ice crystals ultimately grow to product size and shape. Ice crystals exiting the scraped-surface freezer are typically disk-shaped with sizes ranging from a few microns to over 50 µm. In a closed dasher (one with high displacement of barrel volume), where the ice cream essentially flows in an annular space between the two cylinders (barrel and dasher), there is much less internal mixing and less opportunity for melting, growth and ripening.

154-23

Nevertheless, enough of these processes take place that the ice crystals exit the freezer as disk-shaped crystals (as seen in Figure 154.5).

c. Growth, ripening, and equilibration Within the barrel of the scraped-surface freezer, several complex processes related to freezing take place simultaneously. Furthermore, each process affects the nature of the other processes, primarily through influences on heat transfer. The thin layer of polycrystalline ice and slush that is scraped off the barrel wall is colder than the fluid at the center of the barrel. Thus, the first thing that happens is that the colder slush flake cools the surrounding environment as it, in turn, is warmed up. This warming, coupled with mechanical agitation, causes the flake to be broken down into smaller shreds, as has been observed by submersible microscope in a batch freezing apparatus (111). The polycrystalline ice crystals contained within the slush flakes are dispersed into the bulk solution where they melt, grow or ripen according to the conditions in their immediate environment. In regions where temperature is slightly higher than the slush from the wall, the ice crystals begin to melt. However, melting takes heat out of the solution as latent heat, which subsequently cools the surrounding environment. The direction of heat transfer determines which regions get the most cooling effect. In the regions where temperature is a little lower than the slush from the wall, ice crystals grow due to the temperature driving force. However, growth causes a release of latent heat, which warms the surrounding environment. The rate of ice crystal growth is primarily influenced by two mechanisms. Ice crystal growth depends on the rate of counter-diffusion of solute molecules away from the growing interface and on the rate of heat transfer removal from the environment through either the solution or the ice crystal itself (112). The solute molecules present in the ice cream mix (i.e., sugars, salts, proteins, hydrocolloids, etc.) must diffuse away from the growing surface to allow incorporation of water molecules into the existing crystal lattice structure. The rate of diffusion of these solutes depends on the molecular size (larger molecules diffuse more slowly) and the concentration gradients existing during growth. Once water molecules are incorporated into the crystal lattice, there is a release of the latent heat of fusion, which must be removed by conduction and/or convection mechanisms. In an agitated environment, heat transfer generally occurs most rapidly by convective processes with fluid movement carrying away the heat from the growing crystal surface. Further complicating these dynamics of melting and growth within the freezer barrel is the thermodynamic mechanism of ripening (112). Ripening is based on the slight difference in equilibrium (e.g., freezing temperature) between crystals of different size. It is well known that very small crystals (less than about 5 µm for ice) have

154-24


a slightly lower freezing point than large crystals (96). Thus, very small crystals may actually melt at the same time (in the same environment) that larger ice crystals continue to grow. In fact, it is this principle of ripening that leads to changes in ice crystals due to recrystallization in storage.

d. Controlling freezing The principles of freezing discussed in the previous section are applied in commercial ice cream manufacture to make products with the desired number and size distribution of ice crystals for the highest quality. In the continuous commercial freezer described above, conditions are controlled to maximize production of numerous, small ice crystals. A low-temperature refrigerant (vaporizing ammonia or Freon) is used to lower the temperature of the mix quickly to about –25°C at the surface of the freezer barrel. This low temperature (high temperature driving force for nucleation) causes nucleation to occur rapidly, and results in formation of many small nuclei. Even though these nuclei ripen and grow as they make their way to the exit of the continuous freezer, they remain quite small (20 to 25 µm). Compare the commercial situation above to that in a small batch home freezer. In both cases, ice forms on a cold metal surface in contact with a refrigerant, with a scraper blade periodically removing the ice layer formed

at the wall. In the batch freezer, an ice-brine solution is made to lower the temperature of the ice cream mix. However, this brine reaches temperatures of perhaps only ⫺10° to ⫺12°C. This warmer temperature means that nucleation occurs at a significantly lower driving force than in the commercial freezer (liquid ammonia at about ⫺30°C). According to Figure 154.13, the rate of nucleation is significantly lower in the batch freezer, due to the lower ∆T, than in the continuous freezer and thus, fewer ice crystals are formed. When the final ice cream products are hardened to the same temperature, the product from the batch freezer, which contains fewer crystals, ends up with significantly larger ice crystals (and potentially coarser ice cream) than the product from the continuous freezer, which has many more smaller crystals. This principle is described schematically in Figure 154.15 (113). 2.

Operation of the Freezer Barrel

In larger ice cream manufacturing plants, ice cream mix is initially frozen into a semi-frozen slurry in continuous freezers. These units are scraped-surface freezers designed to carefully control ice formation, air incorporation and fat destabilization. Small-scale operations may utilize a batch freezer, where a single batch of ice cream is frozen at a time. In small soft-serve ice cream and custard stands, batch freezers are sometimes used that involve

Few nuclei formed

Many nuclei formed

FIGURE 154.15 Schematic depiction of ice crystal size distributions obtained from batch (top) and continuous (bottom) ice cream freezers, based on nucleation rate (113).


discontinuous freezing, where ice cream is produced on an as-needed basis.

a. Continuous, scraped-surface freezer A schematic of a commercial, continuous freezer is shown in Figure 154.16. Ice cream mix at a temperature of 0 to 4°C is pumped into the main barrel of the scraped-surface freezer under a pressure of 4–5 atmospheres (3) where it is frozen and aerated at the same time. The pressure inside the barrel is applied to reduce air phase volume and hence increase heat transfer. Refrigerant is introduced to the outside wall of the annular space between the two concentric cylinders, where vaporization of the refrigerant occurs to provide the refrigeration effect. Heat is removed from the ice cream as it freezes inside the barrel through the walls. Typically, either ammonia or Freon, kept at high pressure to maintain the liquid state, is pumped into the freezer where a lower pressure allows it to expand and vaporize to provide the refrigeration effect. Vaporized refrigerant is removed from the freezer and recompressed in a mechanical refrigeration system. Refrigerant pressure is controlled to maintain the desired temperature (about –30°C) and driving force for heat transfer removal. The rotating dasher, operating at 150 to 300 RPM within the freezer, holds scraper blades that contact the metal wall and scrape away the slush freezing on the inside of the barrel wall. As the mix enters the freezer barrel, several things take place at the same time: water freezes in the mix, air is incorporated and the fat emulsion becomes partially coalesced. Control of these multiple factors is necessary to make ice cream with the desired physical and sensory characteristics. As discussed in the previous section, freezing of water occurs in the barrel and control of ice crystal formation is critical to product quality and shelflife. Since the mix enters the freezer slightly above its

The continuous ice cream (barrel) freezer

Stainless steel cover Insulating layer Refrigerant Ice cream annulus Scraper blades Dasher (hollow, with solid beater)

FIGURE 154.16 Schematic of the main components of the heat exchanger in a typical continuous ice cream freezer.

154-25

freezing point, sensible heat must be removed to lower the temperature to the point where nucleation occurs. This occurs first at the barrel wall with vaporizing refrigerant separated from the ice cream mix by only a thin layer of metal. At the wall, the mix is quickly cooled below the freezing point and nucleation occurs. It has been estimated that the temperature just on the inside of the barrel wall is about ⫺26°C, based on heat transfer resistances of the metal wall and perhaps a thin layer of ice present on the inside of the barrel wall (9). Since the initial freezing point of the mix is about ⫺2°C, there is a significant driving force {(⫺2) ⫺ (⫺26) ⫽ 24°C} for nucleation at the wall and freezing occurs rapidly. Since the refrigerant temperature is maintained along the length of the freezer, the temperature at the barrel wall along the length of the freezer does not change significantly. That is, temperature just at the inside of the barrel wall is likely to be close to ⫺26°C along the length of the entire freezer barrel. In the center of the barrel, however, the mix temperature is quite different from at the wall and a temperature gradient in the radial direction exists. Temperature in the center of the barrel may remain above the freezing point for some time as the mix works its way from the inlet to the outlet of the freezer. Eventually, as more and more ice scraped from the wall is mixed in with the warmer mix at the center of the barrel, the temperature in the center gradually decreases. It is at the center of the barrel where melting, growth and ripening occur, as discussed in the previous section. Thus, temperature at the center is essentially adiabatically controlled, based on the complex interactions (melting, growth, ripening, etc.) that take place. It is thought that the decrease in temperature along the length of the barrel at the center of the freezer follows approximately the freezing point depression curve as more and more water is removed in the form of ice (9). Russell and co-workers (98) measured the temperature profile along the length of an experimental freezer and found that temperature decreased rapidly initially (near the inlet), decreased more slowly in the middle section and then increased slightly towards the outlet of the freezer, as seen in Figure 154.8 (98). At higher (500) dasher RPM, the temperature decreased to a greater extent than at lower (100) dasher RPM, which suggests that convective mixing from the colder environment near the wall is better with higher agitation rates. However, the mechanical energy input at the wall of the freezer with a higher agitation rate decreases the efficiency of nucleation and leads to ice cream with larger mean ice crystal size (98). There was slight increase in temperature of the ice cream just prior to the end of the barrel where the ammonia jacket ended and no longer provided a cooling effect. This indicates that the ice cream within the freezer barrel was slightly subcooled below the freezing point and the

154-26


release of latent heat at the end of the freezer caused the temperature to go up slightly. Once the ice cream was removed from the freezer, however, no temperature changes were observed when the ice cream was held adiabatically. This indicates that no additional crystallization took place once the ice cream was removed from the freezer and suggests that ice cream as it exits the freezer is at a point nearly on the freezing point depression curve for that temperature. Thus, estimates of the amount of water frozen into ice at any temperature that are based on freezing along the freezing point depression curve are essentially correct. The importance of surface nucleation of ice at the barrel wall was shown by attempts to promote nucleation of ice through addition of ice-nucleating bacteria in a commercial continuous ice cream freezer (114). Ice-nucleating bacteria (Pseudomonas syringae) were added to an ice cream mix, and the mix was frozen under typical operating conditions in a pilot plant freezer. Ice crystal size of the ice cream exiting the freezer was identical for the control mix and the mix containing the ice nucleator. Since these nucleators promote nucleation in the bulk solution, this result suggests that the rate of ice nucleation at the wall of the freezer barrel was so high that the presence of ice nucleators had no effect on the total number of crystals formed in the freezer. At the same time as freezing is taking place within the barrel, changes are also occurring to the lipid phase and air component. In commercial scraped-surface freezers, filtered compressed air is injected under pressure through a diffuser at the end of the barrel where the mix is input (3). The fine air bubbles formed in the diffuser are incorporated within the mix as the dasher rotates within the barrel. The air cells are broken down into smaller and smaller bubbles based on the shear forces within the freezer as the ice cream is formed (115). Dispersion of air into fine bubbles (about 20 µm in size after draw) requires that freezing occur at the same time to increase the shear forces within the freezer. Whipping air into ice cream mix without freezing results in lower amounts of overrun incorporated and larger air bubble sizes (115). The fat emulsion also undergoes important changes in the barrel of the scraped-surface freezer (see Section II.B.4). Emulsifiers are added to the ice cream mix to decrease the stability of the emulsion droplets and allow partial destabilization during freezing. The shear forces within the freezer result in breakdown of the fine (⬍ 3 µm) emulsion droplets and lead to partial coalescence of the fat globules. In this case, partial coalescence of the emulsion results in clusters of fat globules that are attracted to the air-serum interface. These partially-coalesced fat globules provide stabilization to prevent coalescence of the air cells so that many small air bubbles remain intact within the ice cream. It is this network of clusters of fat globules that provides meltdown resistance to the finished ice cream. The refrigeration effect needed for ice cream freezing has been estimated by treating the distinct phases of the

freezing process (116). The total energy required may be estimated as the sum of the energy required to cool the mix from the initial temperature to the freezing point, the energy associated with the latent heat needed to convert a certain amount of water into ice and the energy needed to cool the slush to the draw temperature (1). Although this approach gives only an approximation of the true refrigerant requirements for freezing ice cream, based on the simplifying assumptions, the values obtained give a starting point for calculating refrigeration load in an ice cream facility.

b. Batch freezer Operation of a batch freezer proceeds in much the same manner as for a continuous freezer with several notable differences. That is, similar events take place in batch freezing as just described for continuous freezing, with the ice cream remaining in one place rather than moving along the length of the freezer barrel as in a continuous freezer. One notable difference in batch freezing is that there is typically a lower ratio of heat transfer surface to volume of ice cream. Thus, heat transfer is generally not as efficient in batch freezers compared to continuous freezers. Another typical difference between continuous and batch freezing is the nature of the refrigerant used. In commercial batch freezers, as found for soft-serve or custard-type freezers, vaporizing Freon may be used to provide the refrigeration effect. In this case, the temperature differential at the wall of the freezing cylinder may be as low as those found in continuous freezers. Hence, very small ice crystals are formed at the wall, scraped off by the mixing blades and then dispersed into the mix at the center of the cylinder. The temperature profiles at the wall and center of the freezing cylinder are very similar to those found in continuous freezers, except the temperature changes with time during freezing. When the temperature of the bulk of the ice cream reaches the desired draw temperature, or when the consistency of the ice cream within the barrel reaches some preset or desired value, the ice cream is drawn from the freezer. Typically, draw temperatures from batch freezers are similar to those in continuous freezers. However, due to the quantity of mix to freeze, the residence time required to achieve this draw temperature is much longer than in the continuous freezer, typically 15–30 minutes compared to approximately 1 to 2 min, and the resulting slower rates of freezing result in more recrystallization events in the barrel, larger crystal sizes, and slightly coarser texture when first frozen. Another significant difference between batch and continuous freezing involves the nature of air incorporation. In batch freezers, the mix is allowed to whip at atmospheric pressure. Hence, whipping properties of the mix are very important and overrun is more variable, being controlled simply by the headspace remaining after the mix charge is put into the barrel. In the continuous freezer, air is injected through controlled valves, so whipping properties of the mix are perhaps less important and overrun control is


154-27

exact. Air distribution occurs under pressure in the continuous freezer, and it is the rapid expansion of the air bubbles at draw that establishes the air bubble interface. Soft-serve ice cream freezers contain a swept-surface barrel freezer similar to the batch freezer, but they also contain a mix hopper that permits entry of a charge of mix each time a portion of the semi-frozen ice cream is removed. Thus, the complete barrel is only emptied on shut-down. The air handling systems of some large installation soft-serve ice cream freezers are a hybrid between batch and continuous freezers, in that the air inlet and barrel itself are pressurized to allow more exact control of overrun. 3. Overrun Calculations Overrun is the industrial calculation of the air added to frozen dessert products, and it is calculated as the percentage increase in volume that occurred as a result of the air addition. The following examples will show calculations of overrun by volume and by weight, without and with the addition of particulates, and will also show calculations of target package weights. When packages are being filled on a processing line, package weights should be closely monitored. Deviations can be attributed to variations in the fill level of the package (packaging machine adjustment), variations in the ratio of ice cream to particulate addition (ingredient feeder or ripple pump adjustment), or variations in the overrun of the ice cream (freezer barrel adjustment). Determining manufacturing overrun by volume, no particulates: The equation for overrun determination of a production run, based on the definition of overrun as above, is as follows:

Determining manufacturing overrun by volume, with particulates: Example. 40 L mix plus 28 L pecans gives 110 L butter pecan ice cream, using Equation 154.9: 110 ⫺ 28 ⫽ 82 L actual ice cream surrounding the nuts. Vol. of ice cream ⫺ Vol. of mix used % Overrun ⫽ ᎏᎏᎏᎏ Vol. of mix used 82 ⫺ 40 ⫽ ᎏ ⫻ 100% ⫽ 105% 40 The pecans do not incorporate air. This type of a determination might be useful if, for example, defects in a given mix were known to show up at ⬎115% overrun. Otherwise, in a calculation of total output, a calculation similar to the one above, with no particulates, may be more useful. Determining package overrun by weight, no particulates : % Overrun ⫽ Wt. of mix ⫺ Wt. of same vol. of ice cream ᎏᎏᎏᎏᎏ Wt. of same vol. of ice cream ⫻ 100%

Must know density of mix (wt. of 1 L), usually 1.09 ⫺ 1.1 kg /L (see example below). Example. If 1 L of ice cream weighs 560 g net weight (exclusive of package), assuming a density of 1.09 kg/L, using Equation 154.10: 1090 ⫺ 560 % Overrun ⫽ ᎏᎏ ⫻ 100% 560

% Overrun ⫽

⫽ 94.6% Overrun

Vol. of ice cream produced ⫺ Vol. of mix used ᎏᎏᎏᎏᎏ Vol. of mix used ⫻ 100%

(154.10)

(154.9)

Determining package overrun by weight, if the ice cream has particulates in it, gives very little information because both the ratio of ice cream to particulates and the air content of the ice cream affect the final weight.

Example. 500 L mix gives 980 L ice cream, using Equation 154.9: 980 ⫺ 500 ᎏᎏ ⫻ 100% ⫽ 96% Overrun 500

Determining mix density: The density of mix can be calculated as follows: 100 ⫻ 1.07527 冫冤 ᎏ % total solids % Fat % Water ⫹ 冢 ᎏᎏ ⫺ ᎏ 冣 ⫻ 0.6329 ⫹ ᎏ 冥 100 100 100 % fat

Any flavors added, such as chocolate syrup in the next example, that become homogeneous with the mix can incorporate air and are thus accounted for in the following way. Example. 80 L mix plus 10 L chocolate syrup gives 170 L chocolate ice cream, using Equation 154.9: 170 ⫺ (80 ⫹ 10) ᎏᎏ ⫻100% ⫽ 88.8% Overrun (80 ⫹ 10)

(Wt. per litre of water)

⫽ Wt./L mix

(154.11)

Example. Calculate the weight per litre of mix containing 12% fat, 11% msnf, 10% sugar, 5% corn syrup solids,

154-28


0.30% stabilizer, and 38.3% total solids, using Equation 154.11: 1.0 kg/L ᎏᎏᎏᎏᎏᎏ 0.12 ⫻ 1.07527 ⫹ (0.383 ⫺ 0.12) ⫻ 0.6329 ⫹ 0.617

冣

100 ⫹ 1冣冢ᎏ 100 So, 100 kg gives a yield of 12 ⫹ 165.4 ⫽ 177.4 L

⫽ 1.0959 kg/L of mix Determining target package weights, no particulates: Use the following formula: Weight of given vol. of ice cream Wt. of same vol. of mix ᎏᎏᎏ ⫽ (Desired overrun ⫹ 1) 100

冢

91 kg 91 kg of ice cream or ᎏ ⫽ 165.4 L 1.1 kg/L

(154.12)

100 kg 1 L weighs ᎏ ⫽ 564 grams 177.4 L In many cases, ice creams of different flavors are manufactured to provide the same weight per package for the consumer. As a result, overrun of the actual ice cream in the product varies from flavor to flavor, depending on the density and addition ratio of the particulate ingredients. 4. Fat Destabilization and Foam Stabilization

Example. Desired 90% overrun, mix density 1.09 kg/L, using Equation 154.12 1.09 kg ᎏ net wt. of 1 L ⫽ (90 ⫹ 1) ⫽ 573.7 g 100 Also, the density of ice cream can be calculated in a similar manner from Equation 154.12, density of mix ᎏᎏ Density of ice cream ⫽ (Overrun ⫹ 1) 100 Example: Density of mix 1100 g/L, 1100 g/L ᎏᎏ @100% Overrun, density of ice cream ⫽ (100 ⫹ 1) 100 ⫽ 550 g/L

Figuring target package weights, with particulates: Example. Ice cream with candy inclusion; density of mix 1.1 kg/L; overrun in ice cream 100%; density of candy 0.748 kg/L*; candy added at 9% by weight, (i.e. 9 kg to 100 kg final product). In 100 kg final product, we have: 9 kg 9 kg of candy (or ᎏᎏ ⫽ 12.0 L) 0.748 kg/L

* Note: density of particulate pieces containing void spaces must be determined by first crushing the material to eliminate void spaces, given that ice cream will fill in the voids after incorporation.

The texture of ice cream is perhaps one of its most important quality attributes. It is the sensory manifestation of structure; thus, establishment of optimal ice cream structure is critical to maximal textural quality. While the dynamic freezing process is generally associated with the formation of the ice phase, aeration and agitation during this process are also responsible for the formation of colloidal aspects of structure, viz., the formation of air bubbles and the partial coalescence of the fat into a major structural element (Figure 154.17). The colloidal structure of ice cream begins with the mix as a simple emulsion, with a discrete phase of partially crystalline fat globules surrounded by an interfacial layer comprised of proteins and surfactants (Figure 154.18) The continuous, serum phase consists of the unadsorbed casein micelles in suspension in a solution of sugars, unadsorbed whey proteins, salts, and high molecular weight polysaccharides. During the “freezing” stage of manufacture, the mix emulsion is foamed, creating a dispersed phase of air bubbles, and is frozen, forming another dispersed phase of ice crystals (Figure 154.19). Air bubbles and ice crystals are usually in the range of 20 to 50 µm and are surrounded by a temperature-dependent unfrozen phase (60). In addition, the partially-crystalline fat phase in the mix at refrigerated temperatures undergoes partial coalescence during the concomitant whipping and freezing process, resulting in a network of agglomerated fat, which partially surrounds the air bubbles and gives rise to a solidlike structure (Figure 154.18) (12,40,43,44,117). The development of structure and texture in ice cream is sequential, basically following the manufacturing steps. To properly describe the role of fat in the structure, it is necessary to begin with the formation of the emulsion and the role of the ingredients present at the time of homogenization, with particular reference to the fat, proteins, and emulsifiers. After preheating or pasteurization, the mix is at a temperature sufficient to have melted all the fat present, and the fat passes through one or two homogenizing valves. Immediately following homogenization, the newly


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Ice cream mix (×10,000)

Ice cream (×1000) 3-D fat network

Mixed membrane of protein and emulsifier

Air

Ice

Casein micelles Partially-crystalline fat emulsion

Solution of dissolved solutes Freeze-concentrated unfrozen phase

FIGURE 154.17 A schematic representation of the structure of ice cream mix and of ice cream.

formed fat globule is practically devoid of any membranous material and readily adsorbs amphiphilic molecules from solution (93). The immediate environment supplies the surfactant molecules, which include caseins, undenatured whey proteins, phospholipids, lipoprotein molecules, components of the original milkfat globule membrane, and any added chemical surfactants (6,93). These all compete for space at the fat surface. By controlling the adsorbing material present at the time of homogenization, it may be possible to predetermine the adsorbing substances and thus create a membrane with more favorable functional attributes, utilizing natural proteins rather than relying on the chemical surfactants (47). The membrane formed during homogenization continues to develop during the aging step and rearrangement occurs until the lowest possible energy state is reached (95). The transit time through a homogenization valve is in the order of 10⫺5 to 10⫺6 seconds (91). Protein adsorption or unfolding at the interface may take minutes or even hours to be complete (21). It is clear, therefore, that the immediate membrane formed upon homogenization is a function of the microenvironment at the time of its creation, and that the recombined membrane of the fat globule in the aged mix is not fully developed until well into the aging process (12). Emulsifiers are not needed in an ice cream mix to stabilize the fat emulsion, due to an excess of protein and other amphiphilic molecules in solution (87,88). If a mix is homogenized without any emulsifier, both the whey proteins and the caseins will form this new fat globule membrane, with the caseins contributing much more to the bulk of the adsorbed protein. However, if added emulsifiers are present, they have the ability to lower the interfacial tension between the fat and the water phases lower than the proteins. Thus they become preferentially adsorbed to the surface of the fat (12,32,95). As the interfacial tension is lowered and proteins are eliminated from the surface of the fat, the surface excess (quantity of adsorbed material, mg/m2) is reduced (42) and the actual membrane becomes weaker to subsequent

destabilization. This is due to the fact that the protein molecules, and particularly the caseins, are considerably larger than the emulsifier molecules, such that a membrane made up entirely of emulsifier is very thin (Figure 154.18). This results lower surface excess, although the emulsion is thermodynamically favored due to the lowering of the interfacial tension and net free energy of the system. Crystallization of fat also occurs during aging, creating a highly intricate structure of needle-like crystals within the globule (Figure 154.18). The high melting point triglycerides crystallize first, and continue to be surrounded by liquid oil of the lower melting point triglycerides. It has been reported that fat crystallization of emulsified milkfat at refrigerated temperature reaches equilibrium within 1.5 hours (6). A partially crystalline fat droplet is necessary for clumping to occur. van Boekel and Walstra (118) found emulsion stability of a paraffin oil in water emulsion to be reduced by six orders of magnitude when crystals were present in the dispersed phase. This has been attributed to the protrusion of crystals into the aqueous phase causing a surface distortion of the globule (118). The crystal protrusions can then pierce the film between two globules upon close approach. As the crystals are preferentially wetted by the lipid phase, clumping is thus inevitable. This phenomenon may account for partial clumping of globules under a shear force. The clusters thus formed actually hold the ice cream serum in their interstices resulting in the observed dryness. These fat globule chains may also envelope the air cells thus improving overrun (36), however, fat crystals are also known to impair overrun development in whipped cream (21). The next stage of structure development occurs during the concomitant whipping and freezing step. Air is incorporated either through a lengthy whipping process (batch freezers), drawn into the mix by vacuum (older continuous freezers) or injected under pressure (modern continuous freezers) (1). This process causes the emulsion to undergo partial coalescence or fat destabilization, during which clumps and clusters of the fat globules form and build an

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c f f (A)

(B)

f

a

a

f (C)

(D) a

c

a

f

fc

f (E)

(F)

fn

f c (G)

(H)

FIGURE 154.18 The effect of added emulsifier/adsorbed protein on structure of ice cream mix, ice cream, and melted ice cream. A-B, ice cream mix with no emulsifier and with added Polysorbate 80, respectively, as viewed by thin section transmission electron microscopy; f⫽fat globule, c⫽casein micelle, arrow (in B)⫽crystalline fat, bar⫽ 0.5 µm. See Ref. 36 for methodology. C-D, ice cream with no emulsifier and with added polysorbate 80, respectively, as viewed by low temperature scanning electron microscopy; a⫽air bubble, f⫽fat globule, bar⫽ 4 µm. See Ref. 61 for methodology. E-F, ice cream with no emulsifier and with added Polysorbate 80, respectively, as viewed by thin section transmission electron microscopy with freeze substitution and low temperature embedding; a⫽air bubble, f⫽fat globule, c⫽casein micelle, fc⫽fat cluster, bar⫽ 1 µm. See Ref. 121 for methodology. G-H, melted ice cream with no emulsifier and with added Polysorbate 80, respectively, as viewed by thin section transmission electron microscopy; f⫽fat globule, c⫽casein micelle, fn⫽coalesced fat network, bar⫽ 1 µm in G and 5 µm in H. See Ref. 36 for methodology.

internal fat structure or network into the frozen product (1,6) in a very analogous manner to the whipping of heavy cream (13). During the initial stages of whipping of cream, air bubbles have been shown to be stabilized primarily by

beta casein and whey proteins with little involvement of fat (13). Adsorption of fat to air bubbles occurred when the fat globule membrane coalesced with the air water interface. Only rarely did fat spread at the air water interface. The final cream is stabilized by a cross-linking of fat globules surrounding each air cell to adjacent air cells, thus building an infrastructure in the foam (119). In skim milk foams, the initial air water interface is also formed by the serum proteins and soluble β-casein, with little involvement of micellar casein. Micelles become attached as a discontinuous layer, but are not deformed or spread (21). It can be postulated that air cell incorporation into ice cream mix follows a similar mechanism. Cross-linking of fat globules from one air cell to the next, thus forming an infrastructure, is less likely due to the reduction in dispersed phase volume from the heavy cream system to the ice cream mix system. However, it must also be borne in mind that the air bubbles, fat globules, and aqueous phase are being freezeconcentrated at the same time. The fat globule clusters formed during the process of partial coalescence are responsible for surrounding and stabilizing the air cells and creating a semi-continuous network or matrix of fat throughout the product, resulting in the beneficial properties of dryness upon extrusion during the manufacturing stages (aids in packaging and novelty molding, for example), a smooth-eating texture in the frozen dessert, and resistance to meltdown or good standup properties (necessary for soft serve operations) (6,120). Fat destabilization is enhanced by the emulsifiers in common use (12,88). When the emulsion is subjected to the tremendous shear forces in the barrel freezer, the thin membrane created by the addition of surfactant is not sufficient to prevent the fat globules from colliding and coalescing, thus setting up the internal fat matrix (36). If an ice cream mix is subjected to excessive shearing action or contains too much emulsifier, the formation of objectionable butter particles can occur as the emulsion is churned beyond the optimum level. Polysorbate 80, having a small molecular weight and producing the lowest interfacial tension compared to mono- and diglycerides displaces more protein, resulting in a very thin membrane, and thus produces the maximum amount of fat destabilization (36). The extent of fat destabilization can be quantified in several ways. It is sometimes presented as a % change in turbidity as measured by a spectrophotometer on diluted samples of mix and ice cream (12). It can also be determined based on a solvent extraction technique using a mild solvent, since coalesced fat becomes increasingly susceptible to extraction, whereas emulsified fat does not (95). As well, it can be presented as a change in size distribution of fat globules as measured by laser light scattering techniques (e.g., %>3 µm, since 0% of the mix emulsion was greater than 3µm) (42). Gelin and co-workers (37) demonstrated through light scattering measurements of fat globule size distribution


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s

a

f

i

i

a s s (A)

(B)

(C)

FIGURE 154.19 Low temperature scanning electron micrographs of the overall structure of ice cream. A) General overview of spatial distribution of ice crystals (i) within the unfrozen phase (s). Bar (in C) ⫽ 100 µm. B) Higher magnification showing air bubbles (a) and ice crystals (i) embedded into the unfrozen serum (s) as discrete phases. Bar (in C) ⫽ 40 µm. C) High magnification picture of an air bubble, showing fat globules (f) adsorbed at the air interface and also dispersed in the unfrozen phase (s). Bar ⫽ 20 µm.

and aggregation that the freezing step is responsible for considerable fat aggregation. This aggregation is initially reversible through dissociation with SDS, but not after fat crystal sintering has occurred. They have also shown the changes occurring to the protein distribution between the aqueous and adsorbed states. It was obvious from their study that the homogenization step accounted for a large amount of adsorbed protein, and that casein was preferentially adsorbed over the whey proteins. The aging and freezing-hardening-thawing steps each accounted for subsequent protein desorption, again mostly of the caseins. The sequential process of partial coalescence during ice cream freezing has also been examined (12). The incorporation of air alone, or the shearing action alone, independent of freezing, are not sufficient to cause the same degree of fat destabilization as when ice crystallization occurs concomitantly. The freezing process causes an increase in concentration of the mix components, such as proteins and mineral salts, in the unfrozen water phase. It is believed that the ice crystals contribute to the shearing action on the fat globules, due to their physical shape, and that the concentration of components also leads to enhanced destabilization. However, to create the desired extent of fat destabilization, whipping and freezing must occur simultaneously (87). Goff and co-workers (121) examined air interfaces in ice cream and fat:air interactions using transmission electron microscopy with freeze-substitution. The structures created by increasing levels of fat destabilization in ice cream (achieved through increased emulsifier concentration in the mix in both batch and continuous freezing) were observed as an increasing concentration of discrete fat globules at the air interface (Figure 154.18), and increasing coalescence and clustering of fat globules, both at the air interface and within the serum phase (Figure 154.18). Air interfaces at the highest levels of fat destabilization were not completely covered by fat globules. It has been suggested that the air interface in ice cream may

be covered by a thin layer of non-globular liquid fat (6). However, there was no evidence of a surface layer of free fat in the work of Goff and co-workers (121). Further, air interfaces in a fat-free ice cream formulation showed a very similar, continuous membrane as those from a formulation containing fat. This further suggests that the air bubble membrane itself is comprised of protein, with discrete and partially-coalesced fat globules subsequently adsorbed.

C. FLAVORS AND FLAVOR ADDITION Ice cream and frozen dessert manufacturers offer a wide variety of flavors and particulate ingredients to consumers, which are often the basis upon which consumers make selection choices. Some of the major flavors and flavor categories, based on consumption in North America, are shown in Table 154.10. Ingredients are added to ice cream in three ways during the manufacturing process: in the mix tank prior to freezing (for liquid flavors, colors, fruit purees, flavored syrup bases, or anything else that will become homogeneous within the ice cream); through a variegating pump (for ribbons, swirls, ripples, revels, etc.); or through an ingredient feeder (for

TABLE 154.10 Ice Cream Consumption by Flavor, 2002 Annual, Canada and the U.S. Flavor Vanilla Nut Flavors Chocolate Fruit flavors Neapolitan Bakery Flavors

Percentage of Production Volume 28.4 10.4 8.0 7.6 7.4 5.8

Data from the International Dairy Foods Association.

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particulates — fruits, nuts, candy pieces, marshmallows, cookies and bakery pieces, etc.). In the case of the latter two, this equipment is added in series after the continuous freezer, when the ice cream is already semi-frozen. Often, these may be placed in sequence for complex flavors involving multiple components, e.g., a variegating pump and an ingredient feeder or two ingredient feeders. Ingredients added into the semi-frozen ice cream should be as cold as possible, either refrigerated or stored at subzero temperatures, so as not to cause any melting and recrystallization of the ice crystals at this point in the process. Vanilla. Vanilla is the most popular flavor for ice cream in North America. Vanilla ice cream is used to make milkshakes, sundaes, floats and other types of desserts at the retail level, and is often an a accompaniment to other desserts, such as cakes or pies. Vanilla is also used in many other flavors as a flavor enhancer, e.g., chocolate flavor is improved by the presence of a small amount of vanilla. Vanilla comes from a plant belonging to the orchid family called Vanilla planifolia, grown typically in Mexico, the islands off the east coast of Africa (particularly Madagascar), Tahiti, South America (Guadeloupe, Dominica, Martinique), and Indonesia (Java). Bourbon beans from Madagascar are often considered the finest and account for over 75% of world production. From each blossom of the vine that is successfully fertilized comes a pod that reaches 15–25 cm in length, picked at 6–9 months. It requires temperatures of 24–29°C day and night throughout the season, as well as frequent rains with a dry season near the end for development of flavor. Pods are immersed in hot water to stop biological activity of the seed (which also serves to increase enzyme activity), then fermented for 3–6 months by repeated wrapping in straw to “sweat” and then uncovering to sun dry. 5–6 kg green pods produce 1 kg cured pods. Beans are then aged 1–2 years. Enzymatic reactions during aging produce many compounds, of which vanillin is the principal flavor compound. However, there is no free vanillin in the beans when they are harvested. It develops gradually during the curing period from glucosides, which break down during the fermentation and “sweating” of the beans. Extraction takes place as the beans are chopped (not ground) and placed in a stainless steel percolator. Cold alcohol (no heat involved) and water are pumped over and through the beans until all flavoring matter is extracted. Vacuum distillation takes place for a large part of the solvent. The desired concentration is specified as twofold, fourfold, etc. Each multiple must be derived from an original 10g beans/100 mL of alcoholic extract. Vanillin can be and is produced synthetically to a large extent. Vanillin is contained in many types of woods and thus is a by-product of the pulp industry. Compound flavors are produced from combinations of vanilla extract

and vanillin. Vanillin may be added at one ounce to the fold for compound flavors. The number of folds plus number oz. of vanillin equals the total strength, e.g., 2 fold ⫹ 2 oz. ⫽ 4 fold vanilla-vanillin. However, more than 1 oz to the fold is deemed imitation. Usage level in the mix is a function of purity and concentration. Typically a single fold natural vanilla is recommended at 3–6 mL/L mix, a two fold vanilla-vanillin at 2–3 mL/L mix. Some vanillin may improve flavor over pure vanilla extract, so often natural and artificial compound flavors are more desirable than pure natural flavors; however, too much vanillin results in harsh flavors. Chocolate and Cocoa. The cacao bean is the fruit of the tree Theobroma cacao (“Cacao, food of the gods”), which grows in tropical regions such as Mexico, Central America, South America, West Indies, and the African West Coast. The beans are embedded in pods on the tree, 20–30 beans per pod. When ripe, the pods are cut from the trees, and after drying, the beans are removed from the pods and allowed to ferment for 10 days (microbiological and enzymatic fermentation). Beans then are washed, dried, sorted, graded and shipped for processing. Figure 154.20 shows a flow diagram for the processing of chocolate and manufacture of cocoa. At the processing plant, beans are roasted, the seed coat is removed and the interior of the bean, called the nib, is ground. Friction melts the fat and the nibs flow from the grinding as a liquid, known as chocolate liquor. The composition of chocolate liquor is about 55% fat, 17% carbohydrate, 11% protein, 6% tannins and many other compounds. After the cocoa butter is pressed from the chocolate liquor, the remaining press cake is now the material for cocoa manufacture. The amount of fat remaining determines the cocoa grade: medium fat cocoa, 20–24% fat; low fat 10–12% fat. There are many types of chocolate that differ in the amounts of chocolate liquor, cocoa butter, sugar, milk, other ingredients, and vanilla. Imitation chocolate is made by replacing some or all of the cocoa fat with other vegetable fats. For ice cream, this provides improved coating properties and enhanced resistance to melting. White chocolate is made with cocoa butter, milk msnf, sugar, but no cocoa or chocolate liquor. There are two types of cocoa available, namely, American (domestic) and Dutch (alkalized). The latter is treated with an alkali (sodium hydroxide, etc.) to increase solubility, darken the color, and modify the flavor. The Dutch type is usually preferred in ice cream because it gives a darker, less red color but the choice depends upon consumer preference, desired color (Blackshire cocoa may also be used to darken color), strength of flavor, and fat content of the ice cream (19). For chocolate ice cream manufacture, cocoa is more concentrated for flavoring than chocolate liquor (55% fat) because cocoa butter has relatively low flavor. Hence, low fat cocoa powders are usually utilized at 2–3% (w/w) in the mix. Cocoa is usually added with other dry ingredients at the blending


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Fermented and dried cocoa beans

Cleaning and roasting

Breaking and winnowing Shells Nib Germ separation Milling Chocolate liquor

Cocoa manufacture Alkalization (optional)

Chocolate manufacture Addition of sugar, flavour, milk, cocoa butter, etc.

Fat pressing Mixing and refining Presscake

Conching

Cocoa butter

Breaking, grinding, and sifting

Tempering

Molding

Enrobing

Plain or milk chocolate

Chocolate-coated products

Cocoa powder

FIGURE 154.20 The processing of cocoa into ingredients typically used in chocolate ice cream.

stage, and pasteurized and homogenized with the rest of the mix. Blends of cocoa (2–3%) and chocolate liquor (2%) or chocolate liquor alone (5%) can also be used to produce a chocolate ice cream with enhanced smoothness and with the typical full-fat flavor of chocolate products. Chocolate mixes have a tendency to become excessively viscous, so stabilizer and corn syrup solids content and homogenizing pressure need to be slightly lowered to account for the enhanced viscosity. Sucrose content is generally increased by 2–4% (w/w) in the mix to offset the slight bitterness from the cocoa. One frequent defect with chocolate ice cream, particularly soft-serve, is chocolate specking. Cocoa becomes entrapped in partially coalesced fat, which then darkens. Alleviation of excessive fat destabilization usually alleviates this problem. Fruit Ice Cream. Fruit flavors are quite popular in ice cream. Fruit for ice cream can be utilized as fresh fruit, raw frozen fruit, “open kettle” processed fruit, or aseptically processed fruit cooked in swept-surface heat exchangers. Fruit additions should use sufficient fruit (15–25% w/w) of choice quality for best fruit ice cream. The more highly flavored the fruit, the less required in ice

cream. Fruit should be kept in large pieces in the ice cream where possible, and that is usually a function of the incorporation method. Ingredient feeders are used with continuous freezers to add the fruit pieces or sugared fruit preparations, while a portion or all of the fruit juice, as appropriate when straining of fruit is employed, is added directly to the mix. In the batch freezer, fruit juice is added with the mix at the start of the batch, and the fruit pieces are added when the mix has been partially frozen or at draw. Some small-scale ice cream processors may find it desirable, for a variety of reasons, to use fresh fruit. Such use involves all of the preparation steps of washing, sorting, peeling, destoning, etc. If fresh fruit is being added to ice cream, it should be prepared with sugar in such a way as to allow the sugar to penetrate the fruit. Otherwise, it will freeze to form solid lumps in the ice cream. Sugar draws out juice by osmotic dehydration. If fruits are to be pureed, this will not be necessary, although sugar does help to bring out flavor. With strawberries it is advisable to slice in half and treat with sugar at the rate of at least 20–30% sugar, allowing the berries to stand in a cool temperature until sufficient sugar has been absorbed. Sugared fruit can

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either be strained to separate juice from pulp, or can be cold-stabilized with the use of pectin or starch prior to adding to the ice cream. In this way, the juice and pulp can be added at the same time through the ingredient feeder. Frozen fruit for ice cream is usually frozen with the addition of a suitable content of sugar, usually 25–30%. Frozen packs must be thawed before use. Forced thawing with heat will cause rupture of the fruit with resulting poor appearance. Where discrete fruit pieces are not desired in the ice cream, forced thawing may be used. Thawing usually results in juice separation, unless the product has been cold-stabilized with starch or pectin, and if so, this juice should be strained and added to the mix before freezing. Polysorbate 80 (see Section I.B.5) is sometimes added to the mix prior to the freezing of fruit ice cream, particularly if the fruit is “wet.” This aids in producing a dry ice cream to help incorporate the fruit addition. Depending on the strength of flavor of the fruit preparation and the concentration utilized, it may be necessary to augment fruit flavors with the addition of natural or artificial flavors. Also, sometimes the addition of citric acid to the mix is desirable. Fruit can be processed by cooking in a syrup with added sugar to a total sugar content (°Brix) of 50–60%, and is often stabilized with pectin or starch. This processed fruit moves the problems of procurement, variability, and quality from the ice cream manufacturer to the fruit manufacturer/supplier. The fruit manufacturer can source fruit from around the world and blend it from a variety of sources to achieve year-round supply and consistency. Fruit preparation ensures removal of debris, stones, pits, skins, etc., and cooking ensures microbial safety. By cooking in sugar, the fruit will not freeze as a solid in the ice cream, which provides a more pleasant texture. For the ice cream manufacturer, this product is available in a ready-touse form, with no need for thawing, straining, etc., so it involves no product loss. Fruit processed by open kettle methods, however, often provides a cooked flavor that detracts from the natural fruit flavor desired by the ice cream manufacturer and consumer. The processing of such fruit aseptically in scraped surface heat exchangers provides the opportunity to offer an improved flavor and color, a more consistent product, no preservatives, and a longer shelf-life. Variagates. Variagates are injected through a positive pump connected to a small-diameter nozzle or nozzles within the stream of ice cream from the continuous freezer. They are available as a prepared base, e.g., chocolate, butterscotch, marshmallow, strawberry, cheese cake concentrate, etc., and are usually incorporated at 10% (w/w) of ice cream. Almost any flavor can be variegated into ice cream in a variety of contrasting ice cream flavors and colors. A good variegating syrup should not settle out or run into pools in the ice cream. It must not become icy during storage.

Nuts in Ice Cream. Nut-flavored ice creams are also very popular, although concern for consumers with nut allergies has meant strict segregation of nuts from non-nut products and declaration of possible cross-contamination with nuts, and has limited the use of nut flavors in recent years. Nuts should be used in generous amounts, usually around 10% (w/w), and kept in large pieces. Commonly used are walnuts, pecans, filberts, almonds, and pistachios. Brazil nuts and cashews have been tried without much success. Pecans are usually roasted with butter and incorporated into a butter pecan ice cream. Pistachios may be treated in somewhat the same manner as pecans, or may be used in the characteristic pistachio ice cream, which is usually colored green and is flavored with bitter almond. Raw walnuts may be preferred to roasted for flavor, but some form of heat (oven) treatment should be given to walnuts to eliminate surface microbial contamination. Walnuts are often used with a maple flavoring. Almonds are commonly dry roasted to a point just before burning, and are added to the mix flavored with vanilla or almond flavoring. Filberts are roasted dry to a light brown color. The skins are removed (blanched), and the nuts reduced in size by chopping. They are added to a mix mildly flavored with vanilla. Due to potential contamination with extraneous (e.g., shells) and foreign matter, nuts require extensive cleaning and screening. Nuts must be processed in a clean sanitary premise following good manufacturing practices. Nuts should be either oil roasted or heat treated to reduce any bacteria. Microbiological testing for Standard plate count, coliforms, E. coli, yeasts, molds, and Salmonella sp. is carried out randomly but routinely, and testing for aflatoxin (mold toxin from Aspergillus flavus) is performed on peanuts. Nutmeats should be stored either at subzero temperatures in a freezer, or at least at 2–4°C to maintain freshness and reduce problems with lipid oxidation in the nuts. Color in Ice Cream. Ice cream should have a delicate, attractive color that is closely associated with the type of flavoring material that has been added. In some instances, ice cream mix may be slightly colored to give it the shade of the natural product, e.g., 15% (w/w) fruit produces only a slight effect on color and may need to be augmented. Some fruit solid packs may already be colored by the fruit manufacturer, for convenience to the ice cream manufacturer. Most colors are of synthetic origin and can be purchased in liquid or dry form. Color solutions can easily become contaminated, and therefore must be fresh.

D. PACKAGING AND STATIC FREEZING Once the ice cream exits the freezer as a partially-frozen slush, particulate flavors can be added, and then it is pumped into a package, sealed, and hardened. When the semi-solid ice cream exits the continuous freezer, it should have the correct stiffness, or ability to flow, for its


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intended use. For ice cream intended for direct packaging, about half of the water is frozen to ice when the ice cream exits the freezer, and it should still be sufficiently fluid to flow and completely fill a package without leaving void spaces. If the draw temperature of the freezer is too low, or the mix is otherwise frozen too much, the ice cream exiting the freezer will be too stiff for proper packaging. In some cases, as for frozen novelties, this high degree of stiffness may be desired so that the ice cream maintains its shape prior to hardening. Packages of ice cream are sent to a hardening room or tunnel for further freezing. The aim of hardening is to remove heat so that the ice cream cools quickly to temperatures below –18°C. The time required for hardening primarily depends on the size of the package entering the hardening facility, and the nature of the refrigeration process within the hardening facility. Very small containers, as in 0.5L or smaller cups, may take as little as 30 minutes to harden properly; whereas larger bulk-sized containers may take 24 hours. If cartons of ice cream are collected on a pallet prior to hardening, the time for the center-most container to reach hardening temperatures may be substantially longer than 24 hours. Most commercial facilities allow between 12 and 24 hours in the hardening facility to ensure proper freezing. As the ice cream cools, additional ice freezes in accordance with the freezing point depression curve. It is important to note that, typically, no new ice crystals (nuclei) are formed during hardening, since the thermal driving forces are generally too small to promote nuclei formation. Thus, the increase in ice content (ice phase volume) comes about

through a general increase in the size of all existing ice crystals. Clearly, the number of ice crystals formed in the initial freezing step will have a big impact on the ice crystal size of the final hardened ice cream. Typically, ice crystals increase in size about 10 to 15 µm during hardening. That is, the mean ice crystal size after drawing from the continuous freezer may be about 25 to 30 µm, but the mean size after hardening is more likely to be between 40 and 45 µm. The speed of cooling has a significant impact on the ice crystal size, and this may vary through the container. The ice cream near the outside of the package cools the fastest. The ice cream near the center is insulated by the rest of the ice cream and cools much more slowly. For example, Donhowe (122) followed the temperature decrease at different locations in a half-liter cylindrical container of ice cream during hardening, as shown in Figure 154.21 (10). The surface cooled most rapidly, with the center taking nearly 10 minutes to even start cooling. During that 10 minute delay, the ice crystals at the center of the package were undergoing recrystallization at a rapid rate due to the high temperature. The result is that the ice crystals in the ice cream at the center of the container had substantially larger mean size than the ice crystals in the product near the surface, as seen in Figure 154.22 (10). This effect becomes even more dramatic when larger-sized containers are hardened. For example, the ice cream at the center of a pallet of containers may remain at elevated temperatures for substantially longer than the 10 minutes in this example, and the mean size can get considerably larger. Proper hardening is critical to maintaining the highest quality of the ice cream.

–5 r/R = 0 (center) r/R = 13

Temperature (°C)

–10

–15 r/R = 1 (surface)

r/R = 2/3 –20

–25 0

5

10

15

20

25

Time (min)

FIGURE 154.21 Temperature profiles as a function of time at different distances from the center (relative radial dimension, r/R) during hardening of a half-liter cylindrical container of ice cream at –30°C (10).

154-36


40 35 Draw

Percent of total

30

Hardened: surface Hardened: center

25 20 15 10 5 0 0

20

40

60

80

100

120

Crystal size (µm)

FIGURE 154.22 Ice crystal size distributions for ice cream at different points within a half-liter container after hardening to –30°C. Points correspond to container positions in Fig. 154.21 (10).

The speed of cooling in the hardening facility also depends on the type of refrigeration system chosen. There are numerous options for hardening ice cream. The choice of hardening facility depends on many factors, including the size of the operation, the types of ice cream products being frozen, as well as other economic factors. In some cases, as in small operations, the packages of ice cream may simply be transported to an air blast freezer for hardening. In this case, cold air blowing across the packages removes heat from the ice cream as it freezes further. Typically, air at –30°C, cooled by a mechanical refrigeration system, blows past the packages. Good air flow across each individual package is necessary to obtain the fastest rate of cooling. In larger operations, packages of ice cream are placed on a conveyor (e.g., spiral configuration) and transported through a hardening tunnel to provide rapid convective cooling. The tunnel is maintained at ⫺35° to ⫺40°C and with very high air velocity. The residence time of a package on the conveyor may be between 40 and 160 minutes, which is sufficient to lower the temperature to about –18° to ⫺25°C (1). Again, cold air (⫺30 to –40°C) blowing across the individual packages provides a rapid rate of cooling in the hardening tunnel. Product exiting the tunnel is then transported to a storage freezer for further distribution. Another type of hardening system is the plate freezer, which works well for products in containers with flat sides. In the plate freezer, the containers come in contact with a metal surface (the plates) on both sides (top and bottom). The plates are cooled internally with circulating

refrigerant so conductive heat transfer is excellent between plates and ice cream. Hardening in a plate freezer can be accomplished in as little as 2 hours (1). The choice of packaging material is based on many considerations. From a heat transfer standpoint, the package should have a sufficiently high heat transfer rate that the ice cream cools rapidly in the hardening facility, so that ice crystals are maintained as small as possible. However, during storage and distribution of the ice cream, a good insulating package is desired to minimize thermal fluctuations (and minimize recrystallization during storage). Thus, a compromise on the type of packaging material used is necessary, and often the choice comes down to marketing considerations and the price of the packaging material, with heat transfer and product concerns essentially ignored.

E. NOVELTY/IMPULSE PRODUCT MANUFACTURE Ice cream products designed for single servings are widely available, and are often purchased to be handheld items, eaten immediately after purchase. Many of these items are designed specifically for the children’s market, so a vast array of shapes exist, and new introductions and variations occur frequently. As a result, this category of products is often referred to either as novelty or impulse products, and account for a larger share of the ice cream and frozen dessert market in many countries of Europe and Asia than do packaged items designed for home consumption. Examples include stick or stickless bars, cups, and cones. They can be made of many types


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of frozen desserts, including ice cream with its various fat contents, frozen yogurt, sherbet, puddings, tofu, sorbet, gelatin, and fruit ices. To these are frequently added chocolate, baked items such as wafers and cakes, and numerous kinds of fruit. Recent advances in novelty manufacture equipment have greatly increased the number of products available. This equipment is usually high-speed for mass production, but at high capital cost, so production of such items is a specialty market. Strict portion control is a common attribute of modern equipment. Marketing of these items is a large factor in their success. Novelties can be formed in any of several ways. Most novelty freezing equipment uses ice cream direct from a continuous freezer, at various draw temperatures, in order to get the appropriate consistency for the next step. Different configurations of novelty items include direct filling into a preformed single-service cup or edible cone, layering ice cream between biscuits, as in ice cream sandwiches, filling into molds and then quiescently freezing the molds, or extruding ice cream through various shapes or dyes (1). In the molding method (Figure 154.23), unfrozen mix, such as juice or fruit ice formulations, or ice cream from the continuous freezer, usually at higher than normal draw temperature so it is not too stiff, is transferred to molds that are immersed in or sprayed with chilled brine or glycol. After the product has been partially

Clean, empty molds

Filling

frozen, sticks are inserted and freezing is completed in the molds. The molds then progress to a section where they are lifted from the secondary refrigerant and briefly exposed to heat (warm brine or water) to loosen the bar. An extractor there picks up the novelty by the stick and passes it to the next station. This station can be an enrober, decorator, or packaging apparatus. Individual packaged items are typically placed in bags or boxes, which may be packed in cartons. Because they typically are very hard when packaged, it is unnecessary to transfer them through a hardening tunnel before sending them to cold storage. Some flexibility with external shapes is possible, however with the use of metal molds, the mold shape must allow for the product to be extracted. Some machines are equipped with flexible molds that peel off the surface of the frozen product during extraction, allowing for more surface features. It is also possible to produce “splits,” products with multiple layers from exterior to inner core, on molding machines by filling the mold with the first layer (e.g., fruit ice), allowing for partial freezing of this layer, then sucking the remaining unfrozen material from the inner core and refilling with another material (e.g., ice cream). In belt-type molding equipment, as in Figure 154.23, the molds are then cleaned prior to refilling. Mold freezing equipment is also available in a rotary table-type configuration. The extrusion method (Figure 154.24) involves extraction of ice cream from a continuous freezer at

Optional suck-out Partial freezing of core and refilling

Stick insertion

Further freezing, optional sauce insertion

Extraction: bars lifted and carried on to enrobing/ coating, packaging

Refrigerated CaCl 2 brine

Sanitizing Rinsing Washing Rinsing

FIGURE 154.23 A schematic illustration of molded novelty freezing equipment used in the production of molded ice cream novelties.

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Ice cream from freezer barrel

Reciprocating "hot-wire" or knife blade

Layering of syrups, nuts, etc.

Ice cream "bars" Conveyor belt running to enrobing or hardening operations

Horizontal extrusion

Ice cream from continuous freezer

Reciprocating "hot-wire" or knife blade

Extrusion nozzle in various shapes

Horizontal stick insertion

Ice cream "bars" Conveyor belt running to enrobing, hardening, packaging, etc.

Vertical extrusion

FIGURE 154.24 A schematic illustration of horizontal and vertical extrusion and continuous belt-type freezing equipment used in the production of extruded ice cream novelties.

lower-than-average draw temperatures, about ⫺6° to ⫺8°C. The ice cream is then pumped through an extruder nozzle and sliced into portions by an electrically heated wire cutter. The extruder may take a horizontal or vertical form (Figure 154.24). The external contour of the slice may be almost any desired shape, as is dictated by the shape of the extruder nozzle. By placing different extrusion nozzles inside each other, intricate designs can be formed. Complex extrusions in which multiple flavors or colors are extruded require the use of multiple continuous freezers. Cold-forming or pressing of the extruded item is also possible, allowing complex shapes, designs, patterns, words, etc., to be embossed into the frozen item. If a stick item is desired, the stick is inserted in the extruded ice cream. The pieces are formed on or dropped onto carrier plates and pass through a freezing chamber at ⫺40°C, with rapid air circulation for fast freezing. Each piece is removed from the carrier plate as it emerges from the freezing chamber. Alternatively, a liquid nitrogen dip can be utilized for rapid setting of surface layers. Portions to be coated with chocolate or other coating are then transferred to an enrober, then through a chill tunnel to set the coating.

F. STORAGE AND DISTRIBUTION Once ice cream has been frozen and hardened, it then goes through a storage and distribution system designed to get the product to the point of commercial use. This may be a retailer’s freezer cabinet, and ultimately, in the case of take-home packaging, the consumer’s freezer; or it may be another retail outlet like a scooping shop. Whatever the case, the steps and sequence of storage and distribution are critical to maintaining the highest possible quality of the ice cream. Once the ice cream comes out of the hardening facility, it is typically stored in a low-temperature (⫺25 to ⫺30°C) freezer within the plant itself until it is shipped to its next destination. It is difficult to generalize the series of distribution points for ice cream, since this depends on many factors, including the size of the ice cream manufacturer, the radius of distribution, and the facilities available. Some companies have their own distribution resources, including refrigerated trucks, whereas other companies must rely on contractors for distribution. In some cases, the ice cream goes first to a central warehouse; whereas in other cases the product may go directly


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to retail outlets. Everington (123) shows a typical timetemperature history for distribution of ice cream. Keeney (124) reported on a survey of ice cream manufacturers and presented some typical time scales for storage at several points in the distribution chain. The time ice cream spent in the factory before shipping varied from 1 to 4⫹ weeks, with 2 weeks being most common (36%). The next stage in distribution was a warehouse or distribution center, where most companies (64%) reported that the ice cream spent over 4 weeks before being shipped to the point of purchase. The majority of ice cream (68% of respondents) was purchased within 2 weeks at the retail outlet and used within 2 weeks of the consumers getting the product to their homes. However, in both the retail and consumer stages, some respondents (21%) reported that the ice cream was kept for greater than 4 weeks. Since temperatures are typically more variable in retail outlets and the consumer’s freezer than in the factory or warehouse freezers, ice cream that spends a long time at warmer temperatures is more prone to becoming coarse, as the ice crystals continue to get larger by recrystallization. Ben-Yoseph and Hartel (125) report some typical conditions and storage times at various stages in ice cream distribution, as shown in Table 154.11. These numbers were obtained from anecdotal reports from various sources, and are only meant to indicate the range of conditions that might be found (122). Ben-Yoseph and Hartel (125) used data on recrystallization of ice cream, coupled with rates of heat transfer into a half-gallon container of ice cream, to predict the increase in size of ice crystals at various locations within the container (center to surface) as it progressed through the distribution system presented in Table 154.11. Not surprisingly, the retailer’s outlet and the consumer’s freezer were two of the most significant sources of quality loss. However, any point of transport from one center to another is cause for concern, as temperature spikes (heat shock) due to lack of control can cause significant product damage in a short time. TABLE 154.11 Approximate Distribution Sequence for Ice Cream (125) Mean Air Fluctuationa Temperature Amplitude

Storage Site

Storage Time

Manufacturing plant Distribution vehicle from plant Central warehouse Distribution vehicle from warehouse Supermarket storage Consumer vehicle from supermarket Home freezer

2 weeks 6 hours

⫺22.0°C ⫺19.0°C

2.0°C 2.8°C

4 weeks 3 hours

⫺24.0°C ⫺19.0°C

6.0°C 2.8°C

1 week 0.5 hour

⫺15.6°C 21.0°C

2.8°C 0°C

1 week

⫺12.0°C

2.8°C

a

Approximate amplitude of temperature fluctuations.

III. PRODUCT QUALITY AND SHELF-LIFE A. FLAVOR DEFECTS There can be numerous flavor and textural defects associated with ice cream. Excellent reviews on ice cream defects can be found in Refs. 1 and 126. Flavor defects are classified according to origin, and include those associated with the flavoring system (lacks fine flavor, lacks flavor, too high flavor, unnatural), the sweetening system (lacks sweetness, too sweet, syrup flavor), the dairy ingredients (acidic, salty, lacks freshness, old ingredient, oxidized/metallic, rancid, whey), processing (cooked), and others (absorbed from storage, stabilizer, neutralizer, foreign). The dairy ingredients give rise to many of the common flavor defects in frozen dairy dessert products. Acid flavors may develop due to microbial growth in the dairy ingredients used in the manufacture of mix or in mix before freezing. However, off-flavor development due to microbial growth is dependent on the type of organisms present. Acidity is developed by lactic-acid organisms, but the organisms that grow at refrigerated temperatures are mostly psychrotrophs, and off-flavors associated with their growth are usually fruity and/or bitter in nature, due to peptides derived from proteolysis. Salty flavors may arise from formulations that are too high in msnf, especially if whey powder is used. Whey powder tends to be higher in natural milk salts than does skim milk powder. However, it should also be recognized that salt is often an ingredient in mix formulations, for flavor enhancement, and too much salt may have been used. Another source of high salt flavor may be salted butter, used in error rather than sweet butter. Defects in ice cream flavor associated with the fat phase are usually related to either lipolysis of free fatty acids from triglycerides by the action of lipases (known in the dairy industry as rancidity), or autoxidation of the fat resulting in oxidized flavors (oxidative rancidity as distinct from lipolytic rancidity). These defects tend to be present in the raw ingredients used in ice cream manufacture, rather than promoted by the manufacturing process itself. However, similar precautions to the processing of milk must be taken to ensure that these flavor defects are not present. Oxidation of milk and other fats proceeds by the wellknown autoxidation reaction in three stages: initiation, propagation, and termination. In milk, the initiation reactions involve phospholipids present in the fat globule membrane. Free radicals formed from phospholipids are then able to initiate oxidation of triglycerides, especially in the presence of copper and proteins (21). During propagation, antioxidant compounds, such as tocopherols and ascorbic acid, are depleted, while peroxide derivatives of fatty acids accumulate. Peroxides, which have little flavor, undergo further reactions to form a variety of carbonyls, some of which are potent flavor compounds, especially

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some ketones and aldehydes. Most methods available to monitor lipid oxidation are unsuitable as an early index of oxidized flavor development in milk: measurement of peroxides is not useful because peroxides are unstable intermediates; tests based on colorimetric reaction of thiobarbituric acid with malonaldehyde show some correlation to sensory values, but are rather insensitive; and direct measurement of oxygen uptake is only suitable for controlled experimental conditions. Milk may oxidize as a result of either factors extrinsic or intrinsic to the milk (21,127). Important extrinsic factors include contamination with metals, temperature of storage, oxygen tension, heat treatment, agitation, light, and acidity. Both copper and iron may catalyze lipid oxidation, but probably only copper is significant in milk. Added copper is much more potent than natural copper, because a significant portion of added copper goes directly to the fat globule (21). Significant intrinsic factors affecting milk fat oxidation include metallo-proteins, such as milk peroxidase and xanthine oxidase, endogenous ascorbic acid which acts as a co-catalyst with copper to promote oxidation, endogenous copper content, and endogenous antioxidants, mainly tocopherols. Fresh forage is well known to control spontaneous oxidation, as indicated by obvious seasonal effects on the incidence of oxidized flavor. This effect is probably due to increased levels of endogenous antioxidants. Hydrolysis of fatty acid esters by the action of lipases results in the common flavor defect known as lipolytic or hydrolytic rancidity, and is distinct from oxidative rancidity (127,128). Lipolysis in dairy fats can be extremely detrimental, due to the number of highly volatile, short chain fatty acids present, especially butyric acid. Lipases are unique among enzymes, in that they are active at the lipid-serum interface. In milk, lipases are ineffective unless the fat globule membrane is damaged or weakened in some way. Lipolysis may be caused by the lipoprotein lipase (LPL) that is endogenous to milk, or by bacterial lipases. The properties of the fat globule membrane are most important to lipolysis. Mastitis, which alters milk composition, also increases sensitivity of the fat globule to lipolysis. Other factors that destabilize the fat globule membrane, especially agitation and/or foaming, also promote lipolysis. Lipolysis is accelerated by the replacement of the native membrane with surface active material (mainly casein micelles and whey proteins) from the plasma (128). This effect is at least partly due to redistribution of LPL from the plasma to the fat globule membrane, and accounts for greatly increased lipolysis after homogenization. In the milk from some animals, lipolysis may proceed without subsequent thermal or mechanical activation. This effect, frequently referred to as spontaneous lipolysis, is unlikely to occur in herd or pooled milks, because it is prevented by the mixing of affected milk with three to five times its volume of normal milk.

The major conditions that influence spontaneous lipolysis are late lactation, insufficient fresh forage, and low yielding cows. Cooked flavors in dairy products, including ice cream mix, are caused by using milk products that have been heated to too high a temperature, or by using excessively high temperatures in mix pasteurization. The flavor is typified by scalded milk, and is caused by sulfhydral groups from denaturation of disulfide bonds in whey proteins. If it is mild, it can dissipate with time as the sulfhydral groups oxidize, so it is most often noticeable directly after heat processing. A mild cooked flavor is not objectionable, but intense heating can cause the defect to linger and become increasingly objectionable. Ice cream can sometimes absorb off-flavors from its storage environment. Volatile compounds like smoke, ammonia, paint or diesel fumes have been known to be detectable in ice cream after inadvertent exposure to these odors. It is thus important to recognize that storage environments must be kept free of strongly volatile materials.

B. TEXTURE DEFECTS Considerable effort goes into processing ice cream so that the final product has the desired consumer appeal. From a structural standpoint, this involves controlling ice crystallization, air incorporation, and fat destabilization. During storage, however, significant changes can occur to the structural elements that lead to loss of quality. Textural defects common to ice cream include recrystallization of ice crystals, lactose crystallization (sandiness), and shrinkage. 1.

Recrystallization

In ice cream, numerous small crystals are desired for the smooth texture that they impart. Thermodynamically, however, this state is inherently unstable due to the very high surface area of ice crystals. In principle, this system would be in a lower energy state if the ice phase took the form of a single, very large crystal to minimize the surface area (or more correctly, the surface energy). Thus, there is a thermodynamic driving force for the small crystals in ice cream to disappear, leaving fewer and larger ice crystals. Recrystallization is seen as an increase in mean size and widening of the range of sizes (Figure 154.25), and is accompanied by a decrease in the number of crystals (96). The driving force for this rearrangement is based on the Kelvin equation, which states that the equilibrium temperature of a crystal surface is dependent on its radius of curvature. Thus, smaller ice crystals have a slightly lower equilibrium temperature than larger crystals. In a mixture of ice crystals as found in ice cream, the small crystals are less stable than the larger ice crystals. During storage, the smaller ice crystals melt away at the same time that the larger ice crystals grow larger, as shown


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Frequency (%)

Storage time

Crystal size

FIGURE 154.25 Typical changes in crystal size distribution during storage (96). The arrow represents a decrease in frequency of crystals found within a size range with increasing mean size.

schematically in Figure 154.26(A). This increase in size of larger ice crystals at the expense of smaller crystals is often called Ostwald ripening, or simply ripening. However, calculations of the difference in equilibrium temperature between small and large ice crystals in ice cream show that this difference is only significant for very small crystals (10,122). The difference in driving force, expressed as a difference in equilibrium temperature, between crystals of only 1 µm in radius is less than 0.05°C. For a crystal of 10 µm radius, the temperature difference is less than 0.005°C. Thus, the driving force for

Ostwald ripening of ice crystals in ice cream is very small. In fact, Donhowe and Hartel (72) did not observe true Ostwald ripening in extensive studies of mechanisms of ice recrystallization during storage of ice cream under accelerated recrystallization conditions on a microscope slide. It was found that other mechanisms were more important in ice cream. Nevertheless, it is this slight difference in equilibrium temperature between large and small crystals that, over long periods of time, can lead to significant changes in the state of ice crystals in ice cream (and other frozen foods). The main static (constant temperature) mechanisms for recrystallization of ice crystals during storage of ice cream include accretion and isomass rounding (10). When storage temperature is constant, these two mechanisms are responsible for recrystallization of ice crystals in ice cream (72). Isomass rounding is very similar to Ostwald ripening, but is based on regions of a single crystal with different radii of curvature. A spherical ice crystal would not undergo isomass rounding, since the radius of curvature is uniform at all points of the sphere. In other words, a sphere has the minimum surface area to volume ratio. Ice crystals in ice cream are not spherical in nature (see Figure 154.5), so have a higher surface area to volume ratio. Ice crystals in ice cream are somewhat irregularly shaped, based on the mechanisms of ice formation in the freezer barrel. Thus, there is a driving force for the sharper edges (protruberances) to melt away, and for the flatter sides to grow out until the ice crystal approaches a more spherical state (Figure 154.26(B)). This process has been observed for ice crystals in ice cream held at relatively warm temperatures (⫺5°C) (72). In this case, the ice crystal dispersion in ice cream progressed from the initial irregular-shaped crystals

(A)

(B)

(C) Colder

Warmer

Colder

Warmer

Colder

Warmer

(D)

FIGURE 154.26 Mechanisms of recrystallization: (A) Ostwald ripening, (B) isomass rounding, (C) accretion, (D) melt-refreeze.


Fre

ezin

Temperature

to essentially spherical crystals over time. Because the driving force for this transition is very small (the differences in size characteristics are very small), the process is relatively slow compared to other recrystallization mechanisms. Another important mechanism of recrystallization under constant temperature conditions is accretion. It has been estimated, based on the physical number and sizes of ice crystals and air cells, that ice crystals in freshly hardened ice cream are separated, on average, by a serum film that is less than 10 µm thick (6). This close proximity leads to an instability in the region between the two crystals that leads to bridge formation, and eventually to accretion (Figure 154.26(C)). Accretion has been found to be the main mechanism of recrystallization during the initial stages when ice crystals are closely packed together. Once the crystals have become larger and more separated, the importance of accretion diminishes (72,75). Although it is informative to understand these static mechanisms for recrystallization, ice cream is rarely (if ever) stored under conditions where temperature is constant. As documented in Section II.F, temperatures are continuously changing during storage and distribution of ice cream. Even when stored under “constant” temperatures, most refrigeration systems evoke some temperature fluctuation as compressors cycle on and off. Thus, the process of melting and refreezing is continually occurring, and this process can have a dramatic impact on the ice crystals. In fact, the melt-refreeze mechanism of recrystallization is probably the most important process leading to the change in ice crystals in ice cream during frozen storage (59,72). As temperature fluctuates in ice cream, the amount of ice (phase volume) changes accordingly. If the temperature fluctuations are relatively slow, the ice phase volume changes according to the equilibrium freezing point depression curve. This can be seen schematically in Figure 154.27 (96). When temperature increases, the amount of ice present decreases according to the freezing point depression curve. All ice crystals melt away to some extent, but the smallest crystals melt away a little faster (due to the lower equilibrium temperature) and may eventually disappear (melt away completely). Once a crystal has disappeared, it no longer returns and no new crystals nucleate (driving force is too low). The mass initially contained in that ice crystal must now be redistributed on the remaining crystals when the temperature is lowered and the ice phase volume increases. This process is seen schematically in Figure 154.26(D). The melt-refreeze mechanism is the primary mechanism for recrystallization in ice cream under conditions where temperature is changing (59,72). The rate of recrystallization in ice cream during storage and distribution is dependent on numerous factors, including the initial state of ice crystals in the ice cream, storage temperature and fluctuations, and formulation factors (10). Extended shelf-life requires that the ice crystals are maintained as small as possible for as long as possible.

g po

int d

epre

ssio

T1

n

T2

C1 (a)

C2

Solute concentration (%)

I2 Amount of ice frozen

154-42

I1

T2 (b)

T1

Temperature

FIGURE 154.27 Effects of fluctuations in temperature (from T1 to T2) on a) change in the concentration of the unfrozen phase (C1 to C2), and b) change in amount of ice frozen (I1 to I2) (96).

Of the parameters that influence recrystallization, storage conditions and formulation factors are two of the most important. The rate of recrystallization is a strong function of temperature, with the rate decreasing significantly as storage temperature decreases (59,72). Each of the mechanisms of recrystallization described above progresses more slowly as the temperature is decreased. The result is that the rate of recrystallization decreases as storage temperature decreases. In fact, if ice cream is stored below its glass transition temperature, molecular mobility will be sufficiently low, and the recrystallization rate effectively goes to zero. The glass transition temperature of ice cream is about –32°C (85,105). However, the rate of recrystallization typically is quite low if storage temperature is maintained below about –20°C (72). The extent of temperature fluctuations also influences the rate of recrystallization through the effect on the melt-refreeze mechanism. Based on Figure 154.26, the effect of temperature fluctuations depends on the storage


temperature since the change in ice phase volume with a given change in temperature decreases as temperature decreases (72). Thus, storage at –20.0 ⫾ 2.0°C has much less effect on recrystallization than storage at –8.0 ⫾ 1.0°C. A heat shock index can be used to quantify this effect (129). Since the temperature changes during the various stages of storage and distribution, the rate of recrystallization changes during storage according to the local temperature and fluctuations. Furthermore, different points within a single package experience different thermal conditions and undergo recrystallization at different rates. Donhowe and Hartel (73) showed that ice crystals at the center of a half-gallon container of ice cream remained the smallest, whereas ice crystals near the package surface experienced the greatest rate of recrystallization. The thermal insulating capacity of ice cream, in effect, protects the interior of ice cream from external temperature fluctuations. Ben-Yoseph and Hartel (125) used typical temperatures and times in different stages of distribution of ice cream, and the rates of heat transfer into a package, to predict the ice crystal size at any point in a container of ice cream based on the recrystallization kinetics of Donhowe and Hartel (72). The effects of storage temperatures on ice crystal size at different points in the distribution system were clearly demonstrated. Of the formulation factors that influence recrystallization, stabilizer and sweetener types are the two most important. In fact, stabilizers are added to ice cream primarily to control recrystallization during storage. However, it is still not clear exactly how stabilizers affect recrystallization (see Section I.B.4). Several potential mechanisms have been hypothesized for the effect of stabilizers on recrystallization (10). These include (1) an increase in viscosity of the unfrozen phase, (2) specific inhibition of ice crystal growth rates, (3) physical obstruction due to formation of a weak gel structure (58,71), (4) a change in thermal properties of ice cream due to addition of stabilizer (82), and (5) a decreased perception of iciness due to addition of stabilizers (81). It is possible that each of these potential mechanisms plays a role in the effect of stabilizers on recrystallization. However, further work is needed to verify exactly how stabilizers act to inhibit ice recrystallization during storage of ice cream. The type of sweetener used in the mix formulation has also been found to influence the rate of recrystallization during storage of ice cream (74,84). The effect of sweetener type, however, is primarily related to the amount of water frozen into ice at any temperature. Hagiwara and Hartel (74) correlated recrystallization rate during storage of ice cream with the calculated amount of water frozen into ice for ice creams made with different sweeteners. Recrystallization rate decreased proportionally as the amount of water frozen into ice increased. Since the amount of water frozen at any temperature is directly related to freezing point, recrystallization rate also was seen to decrease as the freezing point temperature increased. Since recrystallization is a diffusionlimited process (based on migration of water molecules),

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more ice at a given temperature (and less water) leads to slower recrystallization, due to the lower mobility of the water molecules. The lower mobility correlates with an increase in glass transition temperature of the ice cream (74). 2.

Lactose Crystallization

The problem of “sandiness” in some ice creams during storage has been related to crystallization of lactose from the milk solids in the formulation (1,130). It is not only that lactose crystals appear in ice cream during storage, but that these lactose crystals must grow to sufficient size that they can be detected by the palate and distinguished from ice crystals (131). Based on various sources, it has been estimated that the critical size for lactose crystals in ice cream is about 15 µm. Above this size, their presence can be detected as a sandy or grainy characteristic that is different from the coarse texture associated with large ice crystals. When present in ice cream, lactose crystals dissolve at a much slower rate than ice crystals melt. Thus, the lactose crystals remain in the mouth even after the ice cream has melted; hence, the sandy mouthfeel. Lactose in ice cream crystallizes when the concentration in the serum phase (unfrozen concentrate) exceeds the solubility concentration of lactose. Since the solubility of lactose is very low (and decreases as temperature goes down), lactose is supersaturated and prone to crystallize at almost any level in ice cream stored at common freezer temperatures. In fact, thermodynamically, lactose should crystallize in just about all ice cream, since it is in the supersaturated state at storage temperatures. The fact that lactose does not crystallize in all ice cream during storage may be attributed to the slow kinetics of lactose nuclei formation at these conditions. The viscosity of the unfrozen phase is sufficiently high that lactose nucleation is inhibited for extended periods of time (and may not occur within the shelf-life of an ice cream product). Thus, two competitive forces are at work that govern crystallization of lactose in ice cream. The first is the increase in concentration driving force as temperature is decreased, which tends to promote lactose crystallization at lower temperatures. Working against this, however, is the decrease in molecular mobility as the temperature is decreased. Thus, there is a storage temperature where lactose crystallization is at a maximum. For a wide range of commercial ice creams, this temperature occurs at about ⫺10 to ⫺12°C (130,132,133). Storage in this temperature range leads to the most rapid lactose crystallization in ice cream. Storage at both higher and lower temperatures requires longer times for onset of lactose nuclei formation (132). Of the formulation factors responsible for lactose crystallization, the initial milk solids level in the mix is probably the most important. An upper limit of 15.6 to 18.5% msnf has been suggested to prevent lactose crystallization, with the higher limit for products that move

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quickly through the distribution chain (1). The presence of sucrose and stabilizers may have an inhibitory effect on lactose crystallization, perhaps through their effect on viscosity of the unfrozen phase during storage. However, addition of powdered or particulate ingredients (e.g., nuts) after initial freezing tends to promote lactose crystallization through two potential mechanisms. Any particulate material added may act as nucleation sites for lactose and promote graining, and it is widely recognized that agitation of a supersaturated sugar solution enhances the likelihood of nucleation (134). 3. Shrinkage In some situations, ice cream that has been improperly handled exhibits shrinkage, where the ice cream pulls away from the walls of the container. Many parameters have been implicated in the mechanism of shrinkage, including formulation factors like improper use of proteins, emulsifiers and stabilizers, and external factors like atmospheric pressure (49). Shrinkage results from a loss of discrete air bubbles as they coalesce and begin to form continuous channels, eventually leading to collapse of the product itself into the channels (48). Shrinkage tends to occur most often after the ice cream experiences a decrease in pressure, as when ice cream is shipped across mountains or transported by plane, which first causes a volume expansion followed by collapse. The extent of air channeling, and hence a measure of ice cream susceptibility to collapse and shrinkage, can be measured by determining the response in volume of the ice cream to pressure changes, given that the volume of discrete bubbles will correlate directly to pressure changes, while the volume of air channels will not (135). According to the ideal gas law, the size (volume) of an air bubble is related to the external temperature and pressure, assuming the volume is free to change. As temperature is decreased, at constant pressure, the volume of an air bubble will decrease. As pressure is increased, at constant temperature, the air bubble should also contract. For example, when ice cream exits the draw of a continuous freezer, pressure is reduced (pressure within the freezer is higher than atmospheric pressure), and all of the air bubbles should expand slightly. At this point, though, the viscosity of the ice cream is sufficiently low that this expansion can easily be accommodated by the surrounding matrix, and the air bubbles approach an equilibrium at atmospheric pressure. Cartons of ice cream are filled to their final weight and volume at this point, and any changes in volume during later storage and distribution may lead to negative changes in the ice cream appearance. After hardening, when the surrounding matrix has stiffened considerably, subsequent changes in pressure (or temperature) can lead to changes in the forces between the air cells and the surrounding matrix. Expansion or shrinkage, depending on the conditions, may be the result.

Goff et al. (136) reported on the effects of vacuum storage on expansion and shrinkage of ice cream. Containers of ice cream at –16°C were exposed to reduced pressure (8 in Hg) for 3 hours and then stored for 6 days at –16°C. Volume changes were measured 3 hours after release of vacuum, and again at the end of 6 days of storage. Expansion of the ice cream was observed after the vacuum storage, in accordance with the ideal gas law. However, after 6 days of storage those same ice creams exhibited shrinkage. In all cases, ice creams made with higher overrun had the greatest expansion and subsequent contraction. At –16°C, the unfrozen matrix must still be sufficiently pliable that a change in atmospheric pressure can cause a change in volume of the ice cream. Interestingly, although the period of vacuum exposure caused expansion, the ultimate result when pressure was brought back to atmospheric was shrinkage of the ice cream volume. This suggests that the unfrozen matrix expanded with the increased air bubble size initially, and then relaxed to a smaller volume than originally found. Goff et al. (135) related this to the nature of the interface between the air bubble and the unfrozen serum. They suggested that components like proteins, stabilizers, and emulsifiers play an important role in determining the viscoelasticity of this interface and subsequent changes in ice cream volume during pressure or vacuum storage.

IV. CONCLUSIONS Ice cream is one of the most complex food products, since it contains multiple phases (ice crystal dispersion, foam, emulsion, viscous unfrozen matrix, and potentially, a weak gel system and a glass). Formation of the different phases is controlled during freezing, but the process of forming one phase generally influences the formation of the other phases. Thus, manufacturing of ice cream requires careful control of both ingredient formulation and processing conditions. Since ice cream and related products are some of the few food products consumed in the semi-frozen state, the freezing process is most important to ultimate smooth texture. As ice cream readily undergoes ice recrystallization, especially during periods of temperature fluctuation, precise control of frozen storage and distribution conditions is also critical for the preservation of optimal textural quality. For all these reasons, ice cream type products present processing, storage, and distribution characteristics that are unique amongst the frozen foods.

ACKNOWLEDGMENT The information in this chapter has been modified from “Ice cream and frozen desserts,” by H. D Goff and R. W. Hartel, in Handbook of Frozen Foods, Editors: Y. H. Hui et al., Marcel Dekker, New York, 2004.


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155

Edible Fats and Oils Processing and Applications

Richard D. O’Brien Fats and Oils Consultant

CONTENTS I. II.

Introduction ........................................................................................................................................................155-2 Fats and Oils Characterization ..........................................................................................................................155-2 A. Edible Fats and Oils, Nonglyceride Components......................................................................................155-2 III. Sources of Fats and Oils ....................................................................................................................................155-3 IV. Genetically Modified Vegetable Oils ................................................................................................................155-3 V. Processing Flow Sequence ................................................................................................................................155-4 A. Extraction ..................................................................................................................................................155-5 B. Rendering ..................................................................................................................................................155-5 C. Refining Systems ......................................................................................................................................155-5 D. Degumming................................................................................................................................................155-6 E. Caustic Neutralization................................................................................................................................155-6 F. Bleaching....................................................................................................................................................155-6 G. Animal Fat Purification Systems ..............................................................................................................155-6 H. Hydrogenation............................................................................................................................................155-7 I. Post Bleaching............................................................................................................................................155-7 J. Fractionation ..............................................................................................................................................155-7 K. Interesterification ......................................................................................................................................155-7 L. Blending ....................................................................................................................................................155-8 M. Deodorization ............................................................................................................................................155-8 N. Liquid Oil Filling and Packaging ..............................................................................................................155-8 O. Shortening Plasticization and Packaging ..................................................................................................155-8 P. Margarine Mixing, Chilling, and Packaging..............................................................................................155-9 Q. Flaking and Spray Chilling ........................................................................................................................155-9 R. Bulk Fats and Oils Shipments....................................................................................................................155-9 VI. U.S. Edible Fats and Oils Consumption ............................................................................................................155-9 VII. Edible Fats and Oils Utilization ......................................................................................................................155-10 A. Shortening Products ................................................................................................................................155-11 B. Margarine and Spread Products ..............................................................................................................155-11 1. Consumer Margarines and Spreads ..................................................................................................155-12 2. Industrial Margarines and Spreads....................................................................................................155-12 C. Liquid Oils ..............................................................................................................................................155-12 1. Consumer Liquid Oils ......................................................................................................................155-13 2. Industrial Cooking Oil Applications ................................................................................................155-13 3. Industrial Salad Oil Applications ......................................................................................................155-13 4. High Stability Oils ............................................................................................................................155-13 References ..................................................................................................................................................................155-14

155-1

155-2

I.


INTRODUCTION

Fats and oils have been recovered for thousands of years from oil bearing seeds, nuts, beans, fruits, and animal tissues. These raw materials serve a vital function in the United States and world economics for both food and nonfood applications. Edible fats and oils are the raw materials for oils, shortenings, margarines, and other specialty or tailored products that are functional ingredients in food products prepared by food processors, restaurants, and in the home. The major nonfood product uses for fats and oils are soaps, detergents, paints, varnish, animal feeds, resins, plastics, lubricants, fatty acids, and other inedible products. Interestingly, many of the raw materials for industrial purposes are by-products of fats and oils processing for food products; however, some oils are produced exclusively for technical uses due to their special compositions. Castor, linseed, tall, and tung oils are all of vegetable origin and are produced for industrial uses only. The USDA Economic Research Service statistics indicate that, of the 27.472 billion pounds of edible fats and oils used in the year 2000, 76.6% was for food products and 23.4% was for nonfood products [16]. Fats and oils occur naturally in a wide range of sources and each source provides a separate and distinctive material. There are hundreds of oil bearing seeds and fruits, all animals produce fat, and marine sources provide oils; however, only a few are of economic importance. All edible fats and oils are water insoluble substances which consist predominantly of glyceryl esters of fatty acids, or triglycerides, with some nonglyceridic materials in small or trace quantities. The terms “fats” and “oils” are used interchangeably and the choice of terms is usually based on the physical state of the material at ambient temperature and tradition. Generally, fats appear solid at ambient temperatures and oils appear liquid. In the final analysis, it is the chemical composition that defines the characteristics of the individual fat or oil, which in turn determines the suitability of this ingredient for various processes and applications.

II. FATS AND OILS CHARACTERIZATION Both the chemical and physical properties of fats and oils are largely determined by the fatty acids that they contain and their position within the triacylglycerol molecule. Chemically, all fats and oils are esters of glycerin and fatty acids. Nevertheless, the physical properties of natural fats and oils vary widely. This is because (i) the proportion of the fatty acids vary over wide ranges, and (ii) the triacylglycerol structures vary for each individual oil and fat. Fats and oils are commonly referred to as triglycerides because the glycerin molecule has three hydroxyl groups where a fatty acid can be attached. All triglycerides have the same glycerin unit, so it is the fatty acids which contribute the

different properties. The fatty acid components are distinguished in three ways: (i) chain length, (ii) the number and position of the double bonds, and (iii) the position of the fatty acids within the glyceride molecule. Variations in these characteristics are responsible for the chemical and physical differences experienced with edible fats and oils. The structure of a fatty acid is commonly denoted by a systematic name after the nomenclature of its parent hydrocarbon, by its common name, or by a convenient shorthand designation showing the number of carbon atoms and the number of double bonds. The fatty acids carbon chain lengths vary between 4 and 24 carbon atoms with up to three double bonds. The most prevalent saturated fatty acids are lauric (C-12:0), myristic (C-14:0), palmitic (C-16:0), stearic (C-18:0), arachidic (C-20:0), behenic (C-22:0), and lignoceric (C-24:0). The most important monounsaturated fatty acids are oleic (C-18:1) and erucic (C-22:1). The essential polyunsaturated fatty acids are linoleic (C-18:2) and linolenic (C-18:3). The triglyceride structure of an edible fat or oil is affected by the fatty acids present and the point of attachment of each fatty acid to the glycerin. Triglycerides with three identical fatty acids are called monoacid triglycerides. Triglycerides containing more than one type of fatty acid are called mixed triglycerides. A mixed triglyceride containing three different fatty acids has three regioisomeric forms and six stereo-isomeric forms, depending on which fatty acid is in the middle, sn-2, or beta position of the glycerol portion of the molecule and which fatty acids are in the alpha or outer positions (sn-1 and sn-3). The distribution of the fatty acids is considered to be nonrandom when the saturated fatty acids are positioned predominately in the sn-1 and/or sn-3 positions and the unsaturated fatty acids are positioned predominately in the sn-2 position [17]. The fatty acid compositions of natural fats and oils vary significantly depending not only on the plant or animal species but also within the same species. Among the factors that affect the vegetable oil fatty acid compositions are climate conditions, soil type, growing season, plant maturity, plant health, microbiological, seed location within the flower, and the genetic variation of the plant. Animal fats and oils composition vary according to the animal species, diet, health, fat location on the carcass and maturity [12].

A. EDIBLE FATS AND OILS, NONGLYCERIDE COMPONENTS The primary constituents of extracted fats and oils are triglycerides but they also contain varying amounts of nonglyceride materials. Some of the nonglyceride components are undesirable, and can be considered a food safety hazard, while others are very beneficial. Therefore, the objective in all of fats and oils processing is to remove the


objectionable materials with the least possible damage to the desirable constituents. In most cases, free fatty acids, phospholipids, moisture, color pigments, oxidation products, waxes, trace metals, proteins, pesticides, meal, dirt, and other gross impurities are the materials that need to be removed. Most vegetable oils contain tocopherols, which are natural antioxidants that protect the oils from oxidation and should be retained. For some products, neither the color pigments nor waxes are detrimental and need not be removed. The major product quality concerns are with free fatty acids, phospholipids, oxidation products, proteins, and trace metals; all materials that affect the odor, flavor, and flavor stability of edible fats and oils products. In the U.S., fats and oils color is usually a major concern from a cosmetic sense to include pigments adsorption as a major impurities concern especially for products marketed directly to consumers. The major food safety concerns are with residual pesticides, mold, bacteria, and impurities developed during processing or with mishandling.

III. SOURCES OF FATS AND OILS Humans have survived as hunters and gatherers for a majority of their known existence on earth. It was only during the last 10,000 years that they learned to domesticate plants and animals. During this period, the evolution of cultivated plants has been shaped to the needs of modern man. Today’s agricultural crops are mankind’s creation. Humans cannot survive without them, nor can the crops that have been developed survive without human care. The combined largest source of vegetable oils are the seeds of annual plants grown in relatively temperate climates. Most of these annual plants are cultivated not only as a source of oil, but are also utilized as protein-rich foods. A second source of vegetable oil are oil-bearing trees. Olive, coconut, and palm oils are extracted from the fruit pulp rather than the seed of the fruit. Palm also has seeds, which provide palm kernel oil. All of the oil-bearing tree fruits require a relatively warm climate; i.e., tropical for coconut and palm and a warm climate for olive trees. Most of the oil-bearing tree fruits and kernels provide the highest oil yields. Oilseeds are annual plants which must be replanted each year, whereas the fruit oils are harvested from trees with long life spans. Olive trees are the most hardy and can live several hundred years. Coconut trees start to bear fruit after 5 to 6 years; their life expectancy is as long as 60 years. Palm trees start to bear fruit after 4 to 5 years and continue for another 20 years. Edible meat fats are supplied almost entirely by three kinds of domesticated animals, i.e., lard from pigs, tallow from cattle and sheep, and milk fat or butter from cows. These animals are raised in the greatest quantities, where they thrive the best, in temperate climates. Animal husbandry has evolved to the stage that these domestic animals not only require a temperate climate but also

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intensive agriculture to provide a plentiful supply of foodstuffs to produce the desired quality and quantity.

IV. GENETICALLY MODIFIED VEGETABLE OILS Plant breeding to modify the genetics of crops has been practiced for centuries. Historically, plant breeders have used crossing and selection techniques to enhance yields, oil contents, climate adaptation, and to effect changes in oil quality, composition, and resistance to pests or pesticides. Introduction of high-oleic safflower in 1964 and low erucic acid rapeseed oil, which became known as canola oil in 1978, are examples of successful fatty acid composition modifications using this technology. Mutagenesis, another plant breeding technique, where the seed is treated with a chemical or gamma-radiation to alter its physiological functions, was utilized by the Russians to develop high-oleic sunflower oil. These traditional tools used by plant breeders have been combined with biotechnology to broaden their capabilities. The traditional breeding methods cause thousands of genes to be transferred at each cross, whereas molecular genetic engineering can now transfer or alter a single gene. Genetic engineering can also transfer a gene from one species to another, which is impossible with the traditional methods. The genetic modification of oilseeds by conventional breeding techniques, combined with molecular genetic transformations, provide a much broader array of possibilities to improve food products. One of the first modified oil compositions produced commercially with this process was high-laurate canola oil. High-laurate canola was engineered by inserting a single gene from the California bay laurel tree that provided a substantial quantity of lauric fatty acid (C-12:0) in the oil [5]. This genetic engineering feat proved that a gene from one plant could be transferred to another to produce an oil with specific fatty acid groups in selective positions for either performance or nutritional effects. Agronomically, high-laurate canola was a total success but it failed in the marketplace. Two reasons were suggested for its failure. First, potential customers for genetically modified products were reluctant to commit because of consumer opposition to genetically modified crops. Second, the specialty oil was marketed at a premium price, which was twice that of most other oils [1]. Genetic varieties have been developed to modify the oilseed’s fatty acid profile to create new value-added oils. Regardless of the oilseed variety, most of these efforts have followed the same directions: (i) low-saturates for dietary needs; (ii) low-linoleic for flavor stability; (iii) high-oleic for health and oxidative stability; and (iv) highsaturates to replace hydrogenation. Currently, most of these modified varieties have captured very little market share or have never been commercialized. The major

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reason these improved oils have not found acceptance is the high cost. Some of the key factors which drive up the costs for these modified oils are [9]: ●

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Lack of competitive field yields—most modified oilseeds provide only about 85 to 95% of the yield potential of the regular oilseed variety. Farmers require a high premium to grow these lower yielding varieties. Identity preservation systems—separate handling systems are required at every stage from seed handling, planting, growing, harvesting, transportation, storage, extraction, and final processing. Low trait stability—environmental effects have caused inconsistent oil compositions in modified oilseed products.

Processing plants bearing oils (fruits, nuts and seeds) Crude oils extraction Cleaning, washing Drying, dehulling, flaking, cooking Pressing, centrifuging Sterilizing, stripping Expeller, expander Solvent extraction

Oils refining Degumming, caustic refining Bleaching Dewaxing, fractionation, hydrogenation, interesterification Blending Deodorization or steam refining Processing animal fatty tissues

Currently, the commercially available genetically modified oilseed crops are primarily varieties with improved agronomic traits, such as herbicide tolerance and pest resistance. In the U.S. these bioengineered herbicide-tolerant or pest resistant soybeans, cotton, canola, corn, and sunflowers have the same oil and protein compositions as their traditional counterparts, and do not require post-harvest segregated handling. Global acreage devoted to growing these genetically modified (GM) crops continues to rise. The United States produces 68% of the GM crops worldwide; Argentina, 22.5%; Canada, 6.1%; and China, 2.9%. Estimates, by the USDA National Agricultural Statistics Service, indicate that 74% of the soybean crop, 32% of the corn acreage, and 71% of the cotton planted in the United States in 2002 were GM hybrids [2].

V. PROCESSING FLOW SEQUENCE The fats and oils, extracted from the oilseeds, nuts, beans, fruits and animal tissues, vary from pleasant smelling products that contain few impurities to very offensive smelling, highly impure materials. Fortunately, researchers have developed technologies for processing the fats and oils products to make them more suitable for foods and other applications. Developments in lipid processing technology have produced ingredients that have been instrumental in the development of many of the current food products available that provide the functional and nutritional requirements of discerning and better informed consumers. Processes have been developed to make them flavorless and odorless and lighter in color, modify the melting behavior, rearrange the molecular structure, remove potential disease causing impurities, capture possible harmful materials, and provide other changes to make them more desirable for the intended application.

Rendering Filtration, water wash, caustic refining Bleaching Dewaxing, fractionation, hydrogenation, interesterification Blending Deodorization or steam refining

Management of end products from oils and fats refining Liquid oil filling and packaging Margarine mixing, chilling, packaging Shortening plasticization and packaging Bulk fats and oils shipments Flaking and spray chilling Plasticization and packaging

FIGURE 155.1 General steps in processing oils and fats from plant and animal products.

Edible fats and oils processing involves a series of processes in which both physical and chemical changes are made to the raw material. Figure 155.1 illustrates most of the potential processing flow sequencing to produce the various fats and oils products. Processing of fats and oils is initiated by an extraction or rendering process to remove the fat or oil from the seed, bean, nut, fruit, or fatty tissues. Vegetable oil’s processing after extraction almost always includes neutralization or refining, bleaching, and deodorization with the major differences being the choice of equipment and techniques utilized. Rendered animal fats are normally clarified to remove impurities, bleached and deodorized, again with differences in equipment and techniques providing the major differences. Clairification, neutralization, bleaching and deodorization are all purification processes which affect the flavor, flavor stability and appearance of the fat or oil product while removing harmful impurities. A review of the major fats and oils processes follows.


A.

EXTRACTION

Cleaning is the first step in the processing of vegetable oils. Typically, oilseeds contain stems, pods, leaves, broken grain, dirt, sand, small stones, and other extraneous seeds. These foreign materials reduce the oil content, adversely effect oil quality and increase the wear and damage potential to the extraction equipment. Shaker screens are used to separate the particles on the basis of size, whereas aspiration separates on the basis of density and buoyancy in a stream of air. Tramp iron, extraneous metal acquired during harvesting, storage or transportation is removed to prevent damage to the equipment by the placement of magnets in chutes just ahead of vulnerable processing equipment. Extraction of oil from materials of plant origin is usually done by pressing with the use of a continuous screw press or by extraction with volatile solvents. Prior to 1940, mechanical pressing was the primary method used. Mechanical pressing had limits because the oil recovery is poorer than with solvent extraction and the high temperatures generated damaged both the oil and the meal. The solvent method allows a more complete oil extraction at lower temperatures. Solvent extraction plants can be either batch or continuous. The continuous extraction plants can be percolation, immersion or direct extraction plants. Generally, the oilseeds may be divided by oil content; above and below 20% oil content. In most cases, oilseeds with a low oil content are subjected to both continuous and batch solvent extraction. High oil content seeds are normally extracted in two stages; first pressing and then solvent extraction; however, many single step continuous direct solvent extraction systems are in current use. To be used legally in the United States, oilseed extraction solvents and food processing substances must have been subjected to an approval by the U.S. Food and Drug Administration (FDA) or the U.S. Department of Agriculture (USDA), be generally recognized as safe (GRAS) for this use, or be used in accordance with food additives regulations promulgated by the U.S. FDA. Commercial hexane has been in major use since the 1940s as an oilseed extraction solvent on the determination that it is GRAS, and it may also be subject to a prior sanction. Like many other food-processing substances, there is no U.S. FDA regulation specifically listing nhexane as GRAS or having prior sanction. However, it has been cleared as a solvent in a number of other food products, one of them a cocoa butter substitute with a 5 ppm maximum limit. Because edible fats and oils are subjected to deodorization and other purification processes as a part of the manufacturing process before being used as a food product, they should not contain any of the extraction solvent, if proper practices are followed [19].

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B.

RENDERING

The fatty tissue from meat animals which is not a part of the carcass or that trimmed from the carcass in preparation for sale is the raw material from which lard and tallow are obtained. Separation of fat from the fatty tissues of animals is called rendering. The rendering process consists of two basic steps. First, the meat by-product is heated to evaporate the moisture, melt the fat present and condition the animal fibrous tissue. Two alternative cooking temperatures are used: fat temperatures below 120°F and fat temperatures above 180°F. A more complete separation of the fat and protein is accomplished with the higher temperature processing but a better quality protein is obtained with the lower temperature processing. Normally, the value of the protein dictates that the lower temperature poorer separation technique be used which probably leaves trace quantities of protein in the rendered lard or tallow. After cooking, the fat is separated from the solid proteinaceous material. In batch rendering the cooked material is allowed to separate and the fat to drain followed by filtration to complete the separation. Continuous rendering, introduced to replace the batch systems, normally consists of a continuous cooker which requires less cooking time and is more energy efficient with better quality control [11].

C.

REFINING SYSTEMS

Processors have the option of approaching edible oil purification in two ways; either chemical or physical refining. The two systems utilize very similar processes with the major difference being the method used for free fatty acid removal. Chemical refining, the conventional method used for removal of the nonglyceride impurities from edible fats and oils, consists of optional degumming, caustic neutralization, bleaching and deodorization. The alkali refining process produces good quality oil and is flexible with the ability to treat different oils and different qualities of individual oils. However, caustic refining has three major drawbacks: (1) the soap produced promotes a tendency for emulsion formation which will occlude neutral oil to increase oil losses; (2) oil losses are particularly high when processing oils with free fatty acids over 3.0%; and, (3) disposal of the soapstock produced has become more difficult. The second process, which has become known as physical refining, consists of removing the fatty acids from the oil by steam distillation under vacuum after the phosphatides have been removed by a degumming process followed by a pretreatment process before bleaching. The major advantages for physical refining are the elimination of soapstock, lower capital costs and fewer processes to operate and maintain. The objective of the initial processing step in either refining method is the

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removal of phosphatides, color bodies, and trace metals. Removal of these non-triglyceride impurities is crucial to ensure good product quality. Herein lies the major drawback for the physical refining system; i.e., complete phosphatide removal with degumming and bleaching is very difficult. Some of the other problems with physical refining systems can be: (1) additional bleaching earth is usually required; (2) pesticides are co-distilled with the fatty acids during steam refining; (3) phosphoric acid treatment may darken the gums produced and incomplete removal can produce off-flavors in the oil after deodorization; (4) steam distillation or deodorizer units must be designed to handle higher concentrations of free fatty acids; (5) cottonseed oil cannot be physically refined because the gossypol pigment must be removed with alkali refining; and, (6) it may be necessary to steam refine before hydrogenation or other processing to adjust melting characteristics followed by a second deodorization step. Physical refining is favored for processing high free acidity oils with low phosphatide contents; it has been demonstrated to produce good quality product from coconut, palm kernel, palm, lard, tallow, and some of the seed oils [20].

Caustic neutralization is ordinarily accomplished by treating the fat or oil with diluted sodium hydroxide. This treatment forms soapstock with the free fatty acids, phosphatides, trace metals, pigments, and other nonglyceride impurities that can be separated by settling or centrifugal force from the neutralized oil. The neutral oil is usually water washed and again separated by settling or centrifuged to remove trace impurities and residual soaps from the neutralization and separation processes. After water washing, the oil is either dried with a vacuum dryer or immediately bleached to remove the trace quantities of remaining water.

F. BLEACHING

Degumming is the treatment of crude vegetable oils with water, salt solutions. or dilute acids such as phosphoric, citric, or maleic to remove phosphatides, waxes, and other impurities. Degumming converts the phosphatides to hydrated gums, which are insoluble in oil for separation as a sludge by settling, filtering, or centrifugal action. Phosphatide removal is the first process for the physical refining system, and can also be used in chemical refining. However, with chemical refining the processor has the option of removing the phosphatides for their by-product value as lecithin or treating them as impurities to be removed along with free fatty acids during caustic neutralization.

Edible fats and oils bleaching is popularly and correctly regarded as the partial or complete removal of color; however, bleaching is also an integral process in both the chemical and physical refining systems. Bleaching is relied upon to clean up the traces of soap and phosphatides remaining after caustic neutralization and water washing for the chemical refining system. For physical refining, the technical feasibility depends upon bleaching as a pretreatment to remove phosphatides, trace metals, waxes, and the color pigments. Another, very important function of bleaching in both refining systems, is the removal of peroxides and secondary oxidation products. The usual method of bleaching is by adsorption of the pigments and other nonglyceride impurities on bleaching earth. In a typical process, the bleaching materials are added to the oil in an agitated vessel, either at atmospheric pressure or under a vacuum. The oil is heated to bleaching temperature and held to allow contact time with the bleaching earth. After the adsorbent has captured the color pigments, soap, phosphatides, trace metals, and polar materials, it becomes an impurity which must be removed from the oil with a filtration system. Control point impurities analyses are used to monitor the removal of the potential food safety hazard.

E.

G.

D. DEGUMMING

CAUSTIC NEUTRALIZATION

The conventional caustic neutralization process is the most widely used and most well known purification system. The addition of an alkali solution to a crude oil brings about a number of chemical and physical reactions: (1) the alkali combines with the free fatty acid present to form soaps; (2) the phosphatides absorb alkali and are coagulated through hydration; (3) pigments are degraded, absorbed by the gums, or made water soluble by the alkali; and, (4) the insoluble matter is entrained with the other coagulable material. Efficient separation of the soapstock from the neutralized oil is a significant factor in caustic neutralization which is usually accomplished with centrifugal separators. The conventional caustic soda neutralization systems have the flexibility to efficiently refine all of the crude oils presently utilized for food products [8].

ANIMAL FAT PURIFICATION SYSTEMS

Traditionally, the method used to purify meat fats has been a form of physical refining. The two main impurities in meat fats are proteins carried over from the rendering process and free fatty acids. The pretreatment phase for meat fats is the removal of the proteinaceous materials. Typically, this is easily accomplished by adding small amounts of diatomaceous earth and/or bleaching earth followed by filtration. An alternative clairification or pretreatment method is to water wash the fat to remove the proteins. This method also requires bleaching or at least drying to remove the moisture remaining in the oil after water washing. A third method for meat fat clarification is caustic refining. Chemical refining is usually reserved for poor quality animal fats or for specialty products used undeodorized to preserve the characteristic meat fat


flavor. The caustic refining system consists of caustic neutralization, water washing and vacuum drying.

H.

HYDROGENATION

The hydrogenation process is an important tool for the edible fats and oils processor. With hydrogenation, liquid oils can be converted into plastic or hard fats more suitable for a particular food product. There are two reasons to hydrogenate a fat or oil; (1) to change the physical form for product functionality improvement, and (2) to improve oxidative stability. Hydrogenation involves the chemical addition of hydrogen to the double bonds in the unsaturated fatty acids. The reaction is carried out by mixing heated oil and hydrogen gas in the presence of a catalyst. After the hydrogenation end point has been achieved, the hardened oil is cooled and filtered to remove the nickel catalyst. Most hydrogenations are performed in batch reactors due to the variation in raw materials and the desired end products. Normally, batch hydrogenation is performed in an agitated tank reactor with heating and cooling capabilities designed to withstand pressures of 7 to 10 bar. First, the catalyst is suspended in the oil. Then, hydrogen gas, dispersed as bubbles, must be dissolved in the oil to reach the surface of the catalyst. The three reaction variables, pressure, temperature, and rate of agitation, are controlled to reduce batch-to-batch variation for preparation of the desired hydrogenated product or basestock. The typical analytical evaluations for endpoint control which measure consistency are refractive index, iodine value, and various melting points. A food safety control point would be the incomplete removal of the nickel catalyst after the reaction is completed; however, this is not a critical control point because the post bleaching process immediately following hydrogenation is designed to remove the remaining trace catalyst impurities.

I.

POST BLEACHING

A separate bleaching operation, immediately following the hydrogenation process, has three purposes: (1) insurance that all traces of the prooxidant hydrogenation catalyst that have escaped the filtration system after hydrogenation have been removed; (2) to remove undesirable colors generally of a greenish hue, accentuated during hydrogenation by heat bleaching of the red and yellow pigments; and, (3) removal of peroxide and secondary oxidation products. Post bleach systems are usually batch systems for the same reasons as for hydrogenation systems; production of a wide variety of hydrogenated basestocks.

J.

FRACTIONATION

Edible fats and oils are fractionated to provide new materials more useful than the natural product. Fractionation

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may be practiced to remove an undesirable component, which is the case with dewaxing and winterization, or to provide two or more functional products from the same original fat or oil, as is the case with cocoa butter equivalents or substitutes and high stability oils. The three fractionation process types practiced commercially to produce the value-added products are: (1) dry fractionation; (2) solvent fractionation; and, (3) aqueous detergent fractionation. Dry fractionation, which includes winterization, dewaxing, hydraulic pressing, and crystal fractionation processes, is probably the most widely practiced. Solvent or aqueous detergent fractionation processes provide better separation of specific fractions for the more sophisticated fats and oils products. All of these fractionation processes practice the three successive stages of fractionation: (1) cooling the oil to supersaturation to form the nuclei for crystallization; (2) progressive growth of the crystalline and liquid phases; and, (3) separation of the crystalline and liquid fractions. A food safety control point identified for the solvent fractionation system would naturally be removal of the solvent used. Complete solvent removal is assured with steam distillation in the deodorization process which is downstream.

K.

INTERESTERIFICATION

The interesterification process can alter the original order of distribution of the fatty acids in triglyceride-producing products with melting and crystallization characteristics different from the original oil or fat. Unlike hydrogenation, interesterification neither affects the degree of saturation nor causes isomerization of the fatty acid double bond. It does not change the fatty acid composition of the starting material but rearranges the fatty acids on the glycerol molecule. The process of interesterification can be considered as the removal of fatty acids from the glyceride molecules, shuffling them, and then replacement on the glyceride molecules at random. This change in the distribution of the fatty acids affects the structural properties and melting behavior of the fats and oils. Commercially, the interesterification process has been utilized for the production of confectionery fats, margarine oils, cooking oils, frying fats, shortenings, and other special application fats and oils products. Two types of chemical interesterification process are practiced: random or directed. Random rearrangement of fats and oils can be accomplished using either a batch or continuous process. Both random interesterification processes perform the three important rearrangement steps: (1) pretreatment of the oil; (2) reaction with the catalyst; and, (3) deactivation of the catalyst. In the directed rearrangement process, one or more of the triglyceride products of the interesterification reaction is selectively removed from the ongoing reaction. Continuous processes are normally used for directed rearrangements for better

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control. Trisaturated glycerides are crystallized and separated from the reaction which upsets the reaction equilibrium so that more trisaturated glycerides are produced.

L.

BLENDING

Different stocks are blended to produce the specified composition, consistency, and stability requirements for the various fats and oils products, such as shortenings, frying fats, margarine oils, specialty products, and even some salad or cooking oils. The basestocks may be composed of hydrogenated fats and oils, interesterified products, refined and bleached vegetable oils, purified animal fats, and/or fractions from winterization, dewaxing, or another form of fractionation. The products are blended to meet both the composition and analytical consistency controls identified by the product developers and quality assurance. The consistency controls frequently include analytical testing for solids fat index, iodine value, various melting points, fatty acid composition, and other evaluations designed to insure compliance with customer requirements. The blending process requires scale tanks and meters to proportion the basestocks accurately for each different product. The blend tanks should be equipped with agitators and heating to assure a uniform blend for consistency control [8].

M.

of various designs are utilized by edible fats and oils processors to produce deodorized oil. All of the systems utilize steam stripping with four interrelated operating variables: (1) vacuum, (2) temperature, (3) stripping steam rate, and (4) holding time.

N.

LIQUID OIL FILLING AND PACKAGING

Most salad and cooking oils are packaged shortly after deodorization in containers for home, restaurant, or large food processor use. The processing necessary for most oils are oxidative stability preservation measures, such as nitrogen protection, temperature control, light avoidance, and the addition of any additives required by the individual products. The oil is filtered for a final time in-line to the bottle filler. The effectiveness of this final filtration is monitored with laboratory filterable impurities testing of packaged product samples obtained utilizing a statistical sampling plan. Food safety concerns for retail liquid oils were lessened with the packaging change from glass to plastic containers. Glass breakage and contamination of other containers were major concerns when glass bottles were used. Exposure of the oil to the atmosphere is limited to a micro-second for most filling lines with a tamper-evident seal applied to the container before the cap is applied.

DEODORIZATION

With conventional edible oil processing, deodorization is the last in a series of process steps used to improve the taste, odor, stability, and food safety of the fats and oils by the removal of undesirable substances. In this process, the fats and oils products are steam-distilled under vacuum. The object is to remove the volatile impurities from the oil. The foremost concern from a quality aspect is the volatile impurities which have objectionable flavors and odors; however, deodorization is also very important from a food safety aspect. Steam distillation removes any trace pesticide and “heavy” metals contents obtained during the growing process. Deodorization is primarily a high-temperature, high-vacuum, steam distillation process to remove volatile, odoriferous materials present in edible fats and oils. It is the last major processing step through which the flavor and odor and many stability qualities of a fat or oil product can be changed. From this point forward, efforts must be directed toward retaining the quality that has been built into the fat and oil product with all of the preceding processes [8]. The odoriferous substances in fats and oils are generally considered to be free fatty acids, peroxides, aldehydes, ketones, alcohols, and other organic compounds. Experience has shown that the removal of flavor, odor, and other undesirables correlates well with the reduction of free fatty acids. Therefore, all commercial deodorization consists of steam stripping the oil for free fatty removal. Currently, batch, semicontinuous, and continuous systems

O. SHORTENING PLASTICIZATION AND PACKAGING Plasticized shortening products can be defined as fats with a consistency that can be readily spread, mixed, or worked. Considerably more is involved in the plasticization of shortening and margarine than merely lowering the temperature to cause solidification. Slow cooling of these products produces a grainy, pasty, non-uniform mushy product that lacks the appearance, texture, and functional characteristics associated with plasticized products. Development of these characteristics are a function of controlled crystallization or plasticization. The final consistency of a shortening is the culmination of all the factors influencing crystallization and plasticization: chilling, working, tempering, pressure, and gas incorporation. The plasticization process involves the rapid chilling and homogenization of a shortening mixture. Most shortenings are quick-chilled in closed thin-film scraped-wall heat exchangers with extrusion valves to deliver a smooth homogeneous product to the package at 17 to 27 atm pressure. Nitrogen is injected at 13 ± 1% into most shortenings to increase the product’s workability and provide a white, creamy appearance. After packaging, many processors temper shortenings at temperatures slightly above the packaging temperature to allow the crystal structure of the hard fraction to reach equilibrium and form a stable matrix. After tempering, shortenings are usually stored and shipped at controlled temperatures of


70 to 80°F (21.1 to 26.7°C) to avoid crystal change and loss of the plastic properties [7].

P. MARGARINE MIXING, CHILLING, AND PACKAGING Margarine was developed as and continues to be a butter substitute. It is a flavored food product containing 80% fat, made by blending selected fats and oils with other ingredients, such as milk, salt, color and fortified with vitamin A, to produce a table, cooking, or baking fat product that serves the purpose of dairy butter, but is different in composition and can be varied for different applications. Now, spreads have been developed as margarine substitutes. The major difference between spreads and margarine is that spreads are not required to contain a minimum of 80% fat. Processing for margarines and spreads begins with the preparation of an emulsion of the ingredients. Emulsions are prepared by adding the oil soluble ingredients to a heated margarine oil formulation in an agitated emulsion tank. Concurrently, a pasteurized aqueous phase is prepared by mixing all of the water soluble ingredients together in another vat. The water phase is then added to the oil phase to make the emulsion. The emulsion is rapidly chilled with scraped-wall heat exchangers similar to those used for shortening products. The plasticized products are then formed into prints, or filled into the various containers for consumer, restaurant, or food processor use. Most margarine and spread products are stored at refrigerator temperatures immediately after packaging, with the exception of some specialized baking products [8].

Q.

FLAKING AND SPRAY CHILLING

Fat flakes describe the higher melting fat and oil products solidified in a thin flake form for ease of handling, quick remelting, or for a specific function in a food product. Chill rolls and processed oil formulations have been adapted to produce several different flaked products that can provide distinctive performance characteristics in specialty formulated foods. The flaked products, produced almost exclusively for restaurant and food processor consumers, are hardfats or stearines, shortening chips, icing stabilizers, confectioners fats, hard emulsifiers, and other customer-specific products. The flake products are solidified on a chill roll, which has been described as an endless moving chilling surface held at a temperature below the crystallization point of the applied fat or oil product to form a congealed film on the outer surface. Specifically, chill rolls are usually 4 foot diameter hollow metal cylinders, in various lengths, with a machined and ground smooth surface, internally refrigerated, that revolve slowly on longitudinal and horizontal axes, with several options for feeding the melted oil onto the surface. After application, a thin film of liquid fat is

155-9

carried over the roll, and as the revolution of the roll continues, the fat is partially solidified. With all chill roll designs, the solidified fat is scraped from the roll by a doctor blade positioned ahead of the feeding mechanism. Flakes are packaged in kraft bags, corrugated cartons with vinyl liners, or other suitable containers for storage and shipment [7]. Spray chilled or powdered fats are specialized products developed for ease of incorporation, handling, melting efficiency, uniform delivery with addition systems, encapsulation, and other special purpose uses. The spray chilling process consists of atomizing a molten fat in a crystallization zone or tower, maintained under temperature conditions where a very fine mist of the melted fat is contacted with cooled air or gas to cause crystallization without marked supercooling [8].

R.

BULK FATS AND OILS SHIPMENTS

Food processors that use fats and oils in large quantities often have the facilities to handle this liquid ingredient in bulk. All of the products packaged for shipment and use can be provided to the customers in tank cars or tank trucks, except margarine and spread mixes, which contain milk and salt. The customers for these bulk products must have fats and oils bulk handling systems to receive, store, and handle the liquid products.

VI. U.S. EDIBLE FATS AND OILS CONSUMPTION Climate and availability certainly influenced the eating habits of our ancestors. Inhabitants of central and northern Europe obtained their edible fats from animals, while people in southern Europe, Asia, and Africa acquired their edible oils from vegetable sources. The food products developed in these different regions used the available fats and oils products. Consequently, the cuisine of the central and northern Europe countries developed around the use of solid fats like butter, lard, and tallow for breads, pastries, and many other baked products. Similarly, the diets of inhabitants from the warmer climates were developed around liquid oils for food products like sauces, dressings, etc. These trends appear to continue to be the preference of their descendants. Immigrants to the United States brought their food preferences with them and introduced them to others from different regions of the world. Fats and oils technology has further increased the varied and rich American diet by improvement of existing products and development of new food products. The resultant North American eating habits have made the U.S. a consumer of almost every available fat and oil. The American consumer is offered these fats and oils as a liquid oil, margarine, shortening, or as an ingredient in a prepared food product. The fourteen

155-10


TABLE 155.1 U.S. Edible Fats and Oils Usage and Per Capita Consumption Millions of Pounds Year Canola Coconut Corn Cottonseed Olive Palm Palm Kernel Peanut Safflower Soybean Sunflower Lard Tallow Butter Total Per Capita Consumption, pounds Vegetable Oils Animal Fats Total

1950 129 223 1445 79

1960

1970

1980

26 103

172 310 1225 51 1 53 62

1446

3011

788 445 891 67 182 94 193 100 6253

1032 673 523 58 299 NR 112

2050 156 1327 6984

1889 328 1113 8215

1645 518 1075 12251

9114 64 1023 995 1017 14910

24.0 21.9 45.9

26.7 18.5 45.3

39.0 14.1 53.0

45.0 12.2 57.2

1990

2000

577 897 1149 851 211 256 362 197 58 12164 200 825 955 1095 19797

1774 968 1711 674 455 375 243 244 102 16210 357 962 1498 1022 26565

52.8 9.4 62.2

63.1 11.5 74.6

NR ⫽ not reported.

major U.S. fats and oils sources are listed in Table 155.1, which reviews the annual usage of both animal and vegetable oils over the past 50 years [14–16]. Fats and oils consumption has been categorized into visible and invisible sources. Visible fats and oils are those isolated from animal tissues, oilseeds, or oil fruits and used for food preparation as shortening, margarine, or salad oil. Invisible fats and oils are consumed as part of meats, poultry, eggs, dairy products, fish, fruits, or vegetables, and account for approximately 60 percent of fat consumption. The pounds per person values reported in Table 155.1 are those from visible sources only. Visible fats and oils usage has more than tripled in the United States over the past 50 years, not only due to population increases, but also from increased consumption. Average per person consumption has increased by almost 50% during this period. The fats and oils usage data in Table 155.1 reflect some distinct trends: (a) a move away from animal fats to vegetable oils; (b) replacement of previously established fats and oils with different source oils; (c) introduction of new vegetable oils; (d) a rise and fall of some individual source oils; (e) source oil changes reflecting the results of medical studies; (f) introduction of new oil seed varieties; and more.

VII. EDIBLE FATS AND OILS UTILIZATION Fats and oils are the raw materials for margarine, shortening, liquid oil, and other specialty or tailored products that

Fats and oils products, per capita consumption data Butter Margarine Lard & tallow Shortening Liquid oils Other

1,950 8.6

1,960 8

1,970 5.3 11.0

1,980 3.6 9.1

1,990 3.5 8.7

2,000 3.6 6.6

4.9

9

12.6 11.0 8.6 0

8 13 9 2

4.7 17.3 15.5 2.4

3.4 18.3 21.3 1.5

2.2 22.3 24.2 1.2

5.9 23.1 33.7 1.6

FIGURE 155.2 Fats and oils products per capita consumption during 1950–2000.

become essential ingredients in food products prepared in the home, restaurants, and by food processors. Butter, lard, and tallow are fats that are used as raw materials for margarines or shortenings, as well as for direct use with little or no processing. The direct usage of animal fats has decreased considerably since 1950, as shown in Figure 155.2 [14,15]. Butter usage decreased 58% from 1950 to 2000. Lard and tallow direct use also had a substantial decrease (82.5%) through 1990, but rebounded for only a 53% overall decrease from 1960 to 2000. Margarine, developed as a butter substitute, has also experienced a decrease (40%) in popularity since 1980. Shortening usage more than doubled between 1950 and 1990, but slowed to only a 3.6% increase for the decade ending 2000. The usage rate for liquid oils almost tripled since 1950 to absorb all of the other fats and oils product losses and then some. Overall, the per capita fats and oils visible consumption rate


155-11

increased 63% for the last half of the twenty-first century, with a move toward liquid oils. The increased popularity of liquid oils is more than likely due to: 1. Diet modifications to reduce saturated fats, trans isomers, and cholesterol 2. Awareness of the high polyunsaturated or essential fatty acid content of liquid oils 3. More convenient handling of the liquid oils than that of solid fats 4. Improved product formulations and processes to accommodate liquid oils 5. Reduced dependence on the crystalline properties of solid fats for functionality through the use of emulsifiers

A.

●

SHORTENING PRODUCTS

Originally, shortening was the term used to describe the function performed by naturally occurring solid fats like lard and butter in baked products. These fats contributed a “short” or tenderizing quality to baked products by preventing the cohesion of the flour gluten during mixing and baking. Shortening later became the product identification used by all vegetable oils processors in the United States to abandon the lard substitute concept; hence, shortening was an American invention. As the shortening product category developed, the limited application also expanded to include all baked products. Today, in the U.S., shortening has become virtually synonymous with fat to include many other types of edible fats designed for applications other than baking. In most cases, products identified as shortening will be 100% fat; however, there are exceptions such as puff pastry and roll-in shortenings which may contain moisture. Generally, a fat product containing at least 80% fat and the required vitamin A content, is margarine. Products that do not meet this criteria have been identified as shortening since shortening does not have a U.S. Standard of Identity. Currently, a description for shortening would be: processed fats and oils products that affect the stability, flavor, storage quality, eating characteristics and the eye appeal of prepared foods by providing emulsification, lubricity, structure, aeration, a moisture barrier, a flavor medium, or heat transfer [8]. Most shortenings are identified and formulated according to usage. The packaged shortening forms that have emerged to satisfy the requirements of the consumers and the food industry are plasticized, pumpable liquid, flakes, powders, chips, and beads. Almost all of these shortening products can also be shipped to large customers in liquid bulk quantities. A brief description of each of the shortening forms follows [7]: ●

Plasticized Shortenings - General purpose plasticized shortenings are still identified as all-purpose, unemulsified, emulsified,

●

●

●

●

B.

animal-vegetable blends, all vegetable, or the like; while the trend is to formulate foodservice and food processor shortenings to perform a specific function for the intended food product. These shortenings are also identified by their intended usage, i.e., a baking application such as cakes, icings, puff paste roll-in, and others; frying applications, specific dairy analog products, household use, and so on. Liquid Shortenings - The pumpable liquid shortening designation covers all fluid suspensions that consist of a hard fat, usually beta tending, and/or a high melting emulsifier dispersed in a liquid oil. This shortening type was developed to pour or pump at room temperature for volumetric measurement or metering for either packaged or bulk-handled products. Flakes - Hardfat, hardbutter, hard emulsifier, and stabilizer flakes are high melting fats- and oils-based products solidified into thin flake form for ease in handling and quick melting, and are used to perform many different functions in food products. Chips - Shortening chips are made thicker and larger than flakes for incorporation into baked products to provide a flaky product similar to danish pastry without the labor intensive roll-in process. Powders - The higher melting fats and oils products can be spray chilled to produce powders. Most of the products flaked can also be powdered for ease in handling or encapsulation of a food product for protection and/or delayed release in a finished food product. Beads - Shortening beads have irregular granular shapes that can be metered at more uniform rates with vibratory or screw feeders and resist stratification or separation in mixtures with other granular materials.

MARGARINE AND SPREAD PRODUCTS

Margarine is a prepared food product developed because of a butter shortage in France. Its evolution to a highly accepted table spread and ingredient for cooking, baking, and prepared foods is a prime example of fats and oils technology. Margarine has evolved from an imitation of dairy butter to a nutritive food which provides a concentrated source of energy, a uniform supplement of vitamin A, a source of essential fatty acids, satiety, a universally accepted flavor, and a compliment to other foods. United States FDA and USDA regulations define margarine as a plastic or liquid emulsion food product containing not less than 80% fat and 15,000 international units per pound of vitamin A, and may contain optional ingredients with

155-12


specific functions. The usual optional ingredients are water, milk or milk products, emulsifiers, flavoring materials, salt and other preservatives, and colorants. Low fat spreads, originally introduced in the 1960s as diet margarines, are available with a multitude of fat levels between 20 and 70%. Low calorie and low fat marketing created a consumer interest in spreads after the diet margarines had been rejected by consumers for poor melting and eating characteristics. Functionally, the spread products are intended to be used as a tablespread or for cooking and most packages have a statement that the spread is not intended for baking or frying. 1.

Consumer Margarines and Spreads

Margarine prints generally in quarter pound and one pound solids were the basic margarine products available until soft margarines were introduced in 1962. The soft margarines, with higher unsaturated fatty acid levels, were packaged in plastic tubs in both regular and whipped versions. The whipped margarines were easier to spread and provided less calories per serving due to the 30% nitrogen content. The nutritional appeal of the soft margarines was carried even further with the introduction of spreads, which began to capture market share in the 1970s; the spreads market share increased from less than 5% in 1976 to more than 74% in 1995. The major uses for consumer margarines and spreads continue to be as a tablespread, cooking ingredient, seasoning agent. The consumer-directed functional aspects of the margarine and spread products are spreadibility, oiliness, and melting properties. Spreadibility continues to be one of the most highly regarded attributes of consumer margarine products, second only to flavor. Oil-off is the most serious for print products, as the inner wrappers become oil soaked and oil may even leak from the outer package. The melting properties of the margarine oil ingredient, the emulsion tightness, and the processing, tempering, and storage conditions which help determine crystal development and stability have a direct affect upon the mouth feel and release of the flavoring materials, as well as the consistency. Scratch baking in the home decreased considerably with the introduction of good quality prepared mixes and frozen ready-to-eat products. Nevertheless, for any home baking, the U.S. consumer will usually choose shortening or a print margarine. Measurement of soft margarine requires a different scale than stick products because of the creaming gas content: 5% in regular and 30 to 35% in whipped soft margarines. Also, spreads have exceptionally poor baking functionality due to the high moisture/low fat content. 2.

Industrial Margarines and Spreads

Foodservice and food processor margarine and spreads are usually considered industrial products. The most popular

foodservice margarine is the consumer stick margarine formulation packaged in 1-pound solids, which is used for cooking and seasoning. Individual serving or portion control spread products are also popular foodservice dining room products. Additionally, a bakers’ margarine formulated with an all-purpose shortening base is used by many foodservice kitchens for their baking requirements. Food processor margarine and spread products are formulated for more specific uses than either the foodservice or consumer products. The stick margarine formulations are packaged in 50 pound cube cases for use in prepared foods. Margarines are also formulated and plasticized with Danish pastry roll-in capabilities, like the shortening products discussed in section V.A, to take advantage of the flavor, color and moisture incorporated into the emulsion. Spread type products were used by food processors before the consumer had accepted them, but for different applications. One of the applications is for self-basting of meat and poultry products during baking. Another is a biscuit topping with special dairy flavor notes and buttermilk curd. Others employ different flavors, spices, or other special ingredients for specific applications, products, or processes.

C.

LIQUID OILS

A liquid oil is usually identified by its physical state at ambient temperature, irregardless of whether the source material is animal, vegetable, or marine. Some source oils appear to disagree with this designation until the mean temperature at the place of origin is considered. For example, oil products from palm and coconut trees are a solid at ambient temperatures in cool climates, but a liquid at the prevailing temperatures in the tropical climates where these plants grow. Therefore, the definition of a liquid oil would be: any oil that is a clear liquid without heating. Liquid oils are further classified by their functionality traits; cooking, salad, and high stability. The definition for each of these classifications is: ●

●

Cooking Oil - An edible oil that is liquid and clear at room temperature, or 75°F (23.9°C), that may be used for cooking. Cooking oils are typically used for pan frying, deep fat frying, sauces, gravies, marninates, and other nonrefrigerated food preparations where a clear liquid oil has application. Cooking oils usually congeal or solidify at refrigerator temperatures. Salad Oil - An edible oil that is suitable for the production of a mayonnaise or salad dressing emulsion and which is stable at low temperatures. This requirement has been refined to require that, in order to qualify as a salad oil, an oil sample must remain clear without clouding for at least 5½ hours while submerged in an ice bath.


●

1.

High Stability Oil - An edible oil that possesses an exceptional oxidative or flavor stability, and is a clear liquid at room temperature. The measure of oxidative stability used for high stability oils is the Active Oxygen Method (AOM) or AOCS Method Cd 12-57. High stability oils will withstand the AOM abuse for periods in excess of 75 hours, and some longer than 300 hours, as opposed to the 8 to 20 AOM hours for cooking and salad oils.

Consumer Liquid Oils

Cooking and salad oils available for home use are bottled and marketed through grocery stores and other retail outlets. The source oils available to the retail consumer are canola, corn, cottonseed, olive, peanut, safflower, soybean, sunflower, blends of these source oils, and some other specialty oils. Most of the oils are only refined, bleached, and deodorized, with the exception of those that require dewaxing or winterization to remain clear liquids on the grocery store shelves, like canola, corn, cottonseed, and sunflower. A steady growth in the consumption of cooking and salad oils is evident from the USDA Economic Research Service statistics in the Oil Crops Situation and Outlook Yearbook. In fact, salad and cooking oils were the sole fats and oils growth area for the year 1997. The trend away from solid fats to liquid oils indicates that the U.S. consumer is reacting to the cautions of the medical profession regarding the relationship of fats and oils to coronary disease. As a result, consumers have replaced solid shortenings and margarines with liquid oils. 2.

temperatures. Salad oils were developed for use in mayonnaise, and are a necessity for the preparation of other salad dressings, sauces, and other food products that are emulsions prepared at cool temperatures or must withstand clouding or congealing at refrigerator temperatures after preparation. Most of these products require high quantities of oil in the formulation, 30 to 80%, to provide the eating characteristics and consistency desired. 4.

High Stability Oils

The primary prerequisites of a high stability oil is liquidity at ambient temperatures and resistance to oxidation. Most oils which are liquid at room temperature contain high levels of unsaturated fatty acids. They are most susceptible to oxidation, which limits application to products where an extended shelf-life is not a requirement. Technology has identified two techniques to enhance the stability of liquid oils: (1) hydrogenation and fractionation to separate the hard fraction from the liquid oil fraction, which retains a high stability, and (2) the use of plant bleeding techniques to produce liquid oils with very high monounsaturated fatty acid levels. The applications established for the high stability oils are [8]: ●

●

Industrial Cooking Oil Applications

Cooking oils are utilized wherever liquidity is permissible or important and the application does not require a clear liquid oil at cool temperatures. Cooking oils may be used for pan frying, deep fat frying, gravies, and other applications. Cottonseed oil has a unique flavor property that makes it a desirable frying oil for snack foods. Corn oil is regarded as exceptional in flavor and quality, with a healthy image for incorporation into processed foods, and also for snack foods frying. Peanut oil maintains a respectable cooking oil market for snack frying and foodservice frying operations, especially for fish and chips. Some of the other applications for cooking oils are packing canned meats and fish products, pan-release products, bread, buns, and sweet doughs. 3.

155-13

Industrial Salad Oil Applications

Salad oils are required in most dressing products, sauces, and other food products prepared or stored at cool

●

●

●

●

Deep Fat Frying - The high stability oils have substantially increased frying stability by limiting the opportunities for oxidation due to the absence of polyunsaturates. Frying stability for the high stability oils is near the performance for heavy duty frying shortenings, with the convenience and fried food appearance of a liquid oil. Protective Barrier - Surface application to food products with the high stability oils provides protection from moisture and oxygen invasion, prevents clumping, and imparts a glossy appearance. Specific applications include raisins and other fruits, breakfast cereals, nut meats, snacks, croutons, bread crumbs, spices, and seasonings. Carrier - Colors, spices, flavors, and other additives may be blended in the high stability oils to preserve the flavor, color, and activity without development of off-oil flavors for long periods. Pan-release Agents - As a major ingredient in the preparation of oxidative stable spray or brushing lubricants for baking pans, confectionery products, and other materials. Food Grade Lubricants - The high stability oils are food grade alternatives to the mineral oil products for lubrication of equipment that contacts food products. Compatibility - Unlike solid fats, the high stability oils are compatible with all types of fats and oils since crystal type is not a concern.

155-14


REFERENCES 1. Anonymous, High-laurate canola oil sold as boiler fuel, INFORM 12:1019, 2001. 2. Anonymous, GM acres reach 120 million acres, INFORM 13:234, 2002. 3. Anonymous, U.S. biotech seed plantings to increase in 2002, INFORM 13:453, 2002. 4. Chaudry, M. M., Nelson, A. I., and Perkins, E. G., Distribution of Aldrin and Dieldrin in Soybeans, Oil, and By-Products During Processing, J. Am. Oil Chem. Soc. 53(11):695–697, 1976. 5. Del Vecchio, A. J., High-laurate canola, INFORM 7:230–243, 1996. 6. Mounts, T. L., Evans, C. D., Dutton, H. J., and Cowan, J. C., Some Radiochemical Experiments on Minor Constituents in Soybean Oil, J. Am. Oil Chem. Soc. 46(9):472–484, 1969. 7. O’Brien, R. D., Soybean Oil Crystallization and Fractionation, in Practical Handbook of Soybean Processing and Utilization, edited by D. R. Erickson, AOCS Press and United Soybean Board, Champaign, IL, 1995, pp. 260–264. 8. O’Brien, R. D., Fats and Oils Formulating and Processing for Applications, Technomic Publishing Co., Lancaster, PA., 1998, pp. 1–4, 47–54, 129–131, 168–175, 182–183, 251–253, 258–260, 264–266, 483, 525, 652–653. 9. O’Brien, R.D., Fats and Oils Formulating and Processing for Applications, Second Edition, CRC Press, Boca Raton, FL, in press. 10. Parker, W. A., and Melnick, D., Absence of Aflatoxin from Refined Vegetable Oils, J. Am. Oil Chem. Soc. 43(11):635–638, 1966.

11. Prokop, W. H., Rendering Systems for Processing Animal By–Product Materials, J. Am. Oil Chem. Soc., 62(4):805–811, 1985. 12. Sonntag, N.O.V., Structure and Compositions of Fats and Oils, in Bailey’s Industrial Oil and Fat Products, Vol. 1, 4th Edition, D.Swern, ed., John Wiley & Sons, Inc., New York, NY, 1979, pp. 69–72. 13. Smith, K. J., Polen, P. B., DeVries, D. M., and Coon, F. B., Removal of Chlorinated Pesticides From Crude Vegetable Oils by Simulated Commercial Processing Procedures, J. Am. Oil Chem. Soc. 45(9):866–869, 1968. 14. USDA, Fats and Oils Situation, Economic Research Service, July, 1977, p. 14–15. 15. USDA, Fats and Oils Situation, Economic Research Service, August, 1985, p. 8. 16. USDA, Oil Crops Situation and Outlook Yearbook, Economic Research Service, Oct, 2002, p. 61. 17. Wan, P.J., Properties of Fats and Oils, in Introduction to Fats and Oils Technology, Second Edition, O’Brien, R.D., Farr, W.E., and Wan, P.J. editors, AOCS Press, Champaign, IL, 2000, pp. 21–24. 18. Vail, R., Fundamentals of HACCP, Cereal Foods World, 39(5):393–395, 1994. 19. Wakelyn, P. J., Regulatory Considerations for Oilseed Processors and Oil Refiners, in Introduction to Fats and Oils Technology, Second Edition edited by P. Wan et al., AOCS Press, Champaign, IL, 2000, pp. 319–321. 20. Young, F. V. K., Physical Refining, in Edible Fats and Oils Processing: Basic Principals and Modern Practices: World Conference Proceedings, edited by D. R. Erickson, American Oil Chemists’ Society, Champaign, IL, 1990, pp. 124–135.

156

Fat Hydrogenation in Food Processing

Jan Sajbidor

Faculty of Chemical and Food Technology, Slovak University of Technology

CONTENTS I. Historical Background ........................................................................................................................................156-1 II. Basic Principles of Edible Oil Hydrogenation....................................................................................................156-2 III. Effects of Process Conditions ............................................................................................................................156-3 IV. Hydrogenated Fat in Human Nutrition ..............................................................................................................156-4 References ....................................................................................................................................................................156-5

Increasing the degree of saturation of fatty oils via hydrogenation is the most important process of the fatty oil industry, particularly in the production of edible fat products. This reaction makes possible the synthesis of food products such as shortening and margarine. Vegetable oils are too soft for margarines because of their liquid nature, while on the other hand saturated fats are too hard. Depending on the end use, most shortening fat systems require an intermediate hardness. Industries use this process to turn cheap oils into simulated butter products. Hydrogenation is an excellent process for consistency change and prolongs shelf stability. However, there are medical side effects due to the trans isomers produced that can cause many health problems if too much is consumed. Hydrogenation of edible oils and fats are chemical processes in which hydrogen is added to double bonds of unsaturated fatty acids in lipid. Lipid is usually in the form of triacylglycerol, but other structures containing ethylenic linkages can be also hydrogenated [7]. The general aim of hydrogenation processes is to adjust their melting properties and improve their stability [32]. Besides fat and hydrogen, this process requires a catalyst — usually nickel deposited on a silicate support. Though already a classical process, widely applied since the early 20th century, it is not yet possible to a predict the molecular composition of the hydrogenated oil as a function of feed stock composition, catalyst type and concentration, reaction pressure, temperature and time. This is partly due to the complexity of the process, with a large number of hydrogenation reactions occurring in parallel, and partly due to the simultaneous occurrence of isomerization and double bond conjugation reactions. The complexity of the reaction is further illustrated by noting

that the partial hydrogenation of soybean oil results in the production of a minimum of different linoleic, linolenic and oleic esters, the cis and trans forms of which could produce more than 4000 different triacylglycerols. Understanding hydrogenation is important because it is a major reaction, which leads to many everyday products. Besides margarine and shortenings, hydrogenated oils end up in such things as ice cream, candy, chocolate, potato chips and baked goods.

I.

HISTORICAL BACKGROUND

Although reactions involving catalytic hydrogenation of organic substances were known prior to 1897, the property of finely divided nickel to catalyze the fixation of hydrogen on hydrocarbon double bonds was discovered by the French chemist, Sabatier. Thus, unsaturated hydrocarbons in the vapor state could be easily converted into saturated ones when passing hydrogen gas over a catalytic metal. Soon after this report a liquid-phase hydrogenation of fatty oils was patented in 1903 in England by German chemist Normann [23] and this title was passed to the British firm Joseph Crossfield and Sons. In 1909, Procter and Gamble Company acquired the American rights to the Crossfield patents. Soon after that Procter and Gamble introduced its hydrogenated shortening, “Crisco,” on the market. The first hydrogenated products were a blend of totally hydrogenated cottonseed oil and refined liquid cottonseed oil. This created a product that had the consistency of lard but which was less likely to liquefy at warmer temperatures. The technique of partial hydrogenation was developed in the 1930s and it complemented the development of 156-1

156-2


a high-yield solvent extraction method to render fats from vegetables and seeds. The advent of hydrogenation led to possibilities for a new branch of science — oleochemistry. In the mid-1930s, a stainless steel stirred autoclave for hydrogenating tallow fatty acids was constructed. One of the earliest, and still exceedingly important oleochemical products, was fabric softener, whose principal ingredients were fatty amines, manufactured by hydrogenating fatty nitriles derived from fatty acids. Esterification of fatty acids, followed by hydrogenolysis, produces fatty alcohols, another of the major products in the manufacture of oleochemicals.

II. BASIC PRINCIPLES OF EDIBLE OIL HYDROGENATION The basic chemical equation for hydrogenation of an unsaturated carbon-carbon double bond is shown below. While it appears very simple, in reality it is extremely complicated. –CH ⫽ CH– ⫹ H2 → –CH2 ⫺ CH2– It includes the following steps: 1. Transfer and/or diffusion of individual reactants (unsaturated fatty acids and hydrogen); 2. Hydrogen adsorption on the surface of a catalyst; 3. Addition of hydrogen on double bonds accompanied with their cis/trans and positional isomerization; 4. Desorption of reactants from catalyst surface and transfer to the bulk of liquid oil. During a hydrogenation reaction three different modifications can occur: 1. A double bond can be changed to a single bond, e.g., changing a 2-polyunsaturated fatty acid into a monounsaturated fatty acid, or a monounsaturated fatty acid into a saturated fatty acid. 2. The location of the double bond can be moved up or down the fatty acid chain, and/or the configuration of the double bond can be changed to either cis or trans. 3. Highly polyunsaturated fatty acids are the most susceptible to the process of hydrogenation, as they contain more double bonds than other fatty acids. Depending on the conditions applied during the process, hydrogenation can be classified as either a selective or non-selective process. Reaction selectivity is defined as the conversion of a diene to a monoene, compared with the conversion of a monoene to a saturate. The simple

reaction model for triene hydrogenation was published by Bailey in 1949, and later mathematically defined by Albright [1]. This model considered each reaction to be first order and irreversible. It could be used for measuring the relative reaction rate constants (K) for each hydrogenation step during the batch hydrogenation of oils containing triene fatty acids. K1 Linolenic

K2 Linoleic

䉴䉴䉴

K3 Oleic

Stearic

Ll ⫽ Ll0e⫺K1t

冢

冣

K1 Ol ⫽ Ll0 ᎏᎏ K2 ⫺ K1

冢

e 冣冢Kᎏᎏ ⫺K 冣

冢

e 冣冢Kᎏᎏ ⫺K 冣

冢

冣

K1 冢e⫺K1t ⫺ e⫺K2t冣⫹ L0e⫺K2t L ⫽ Ll0 ᎏᎏ K2 ⫺ K1

K1 ⫺ Ll0 ᎏᎏ K2 ⫺ K1

K2

3

冢

K2

3

⫺K1t

⫺ e⫺K3t冣

1

冢

⫺K1t

⫺ e⫺K3t冣

2

K2 冢e⫺K2t ⫺ e⫺K3t冣⫹ Ol0e⫺K3t ⫹ L0 ᎏᎏ K3 ⫺ K2 Where: Ll0 ⫽ concentration of linolenic acid in time t ⫽ 0 L0 ⫽ concentration of linoleic acid in time t ⫽ 0 Ol0 ⫽ concentration of oleic acid in time t ⫽ 0 K1–3 ⫽ relative reaction rate constants (K) for each hydrogenation step t ⫽ time In a theoretical sense, an oil hardened with perfect preferential selectivity would, first of all, have its linolenic acids reduced to linoleic acids before any linoleic was reduced to oleic acid; then all linoleic acids would be reduced to oleic, before any oleic acids were saturated to stearic acid. Unfortunately, this does not happen in the actual practice. For practical application, if the relative reaction rate constant calculated for linoleic to oleic divided by the reaction rate constant for oleic to stearic is 31 or above, the hydrogenation is termed selective, if below 7.5, it is non-selective. Hydrogenation can be partial or complete. This reaction does not proceed at room temperature, and requires a catalyst to overcome activation energy. These catalysts can be heterogeneous, such as nickel, platinum, or palladium, or homogeneous, such as the Wilkinson’s catalyst. These reactions give off heat, which can determine the degree of saturation and predict stability as well. Transition metal complexes make good catalysts, since they bring the reactants together and break the hydrogen bond. These reactions are stereoselective, in that the hydrogens are added in such a way as to predict the outcome.


Hydrogenation can take place only when the liquid unsaturated oil, the solid catalyst and the gaseous hydrogen have been brought together in a heated stirred reactor. Interaction between chemisorbed hydrogen on the catalyst surface and the double bond of fatty acyl is the first step of heterogeneous hydrogenation. Another hydrogen atom may add to the adjacent position and the saturated molecule is desorbed; or if there is no hydrogen atom available, hydrogen may be removed from a chain carbon atom by the catalyst. Addition of one hydrogen atom results in the opportunity of free rotation to reorient the geometry of the molecule from cis to the thermodynamically more stable trans configuration. Whether cis or trans is formed depends on the geometric positioning of the carbon chains attached to the double bonded carbons. The double bond in the original position may also be converted to trans. The double bonds in the new position may also be shifted. As hydrogenation proceeds, the isomerized double bonds tend to be shifted farther and farther along the chain, and the trans isomer content increased until the monoenes are saturated. Hydrogenation of the polyunsaturated fatty acid chain is similar to that for the saturated fatty acid chain. During partial saturation of polyenes, besides hydrogenation of one double bond, the positional migration or trans formation of the other bonds can occur. Methylene-interrupted dienes on the catalyst surface can be conjugated or hydrogenated to saturated equivalent. If the mixture to be hydrogenated contains monoenes, dienes, and polyenes, there will be competition among the different unsaturated systems for the catalyst surface. By simple arithmetic probability, an ethylenic linkage from one of the more unsaturated esters will be preferentially adsorbed from the oil to the catalyst surface, isomerized and/or hydrogenated, and then desorbed to diffuse to the main body of the oil. Factors that affect the hydrogenation and consequently the resultant products, are the temperature of the oil mixture, hydrogen gas pressure, catalyst activity, catalyst concentration, agitation of the mixture, and time duration of the process [7]. The degree of selectivity in hydrogenation also affects the crystal stability of the resulting fat. Commonly, the selective reaction conditions cause more isomerization to trans isomers and less stearic acid development to effect high solids at the lower temperatures [24]. Since the trans form of an unsaturated fatty acid has a higher melting point in comparison to the cis form, the occurrence of the trans form in the product helps to create desirable solid levels. Although it has a beneficial effect on the quality of the product, it also increases the risk of coronary heart diseases [27]. A study carried out by Yap [36] showed that selectively hydrogenated canola oil formed a mixture of betaprime and beta crystals, whereas non-selective hydrogenation resulted in the beta form of crystals. Incorporation of trans fatty acids through selective hydrogenation favors beta-prime crystallization. Therefore, it

156-3

becomes difficult to obtain desirable acceptability in terms of melting profile, low trans-acids, and favorable polymorphic behavior (and indirectly rheological behaviour) by sticking to only one technique of hydrogenation. Thus, hydrogenation conditions are manipulated to choose the most desirable set of processing parameters.

III.

EFFECTS OF PROCESS CONDITIONS

In the hydrogenation process, the composition and properties of the final product depend on various operating factors. Temperature has a significant influence on the rate of hydrogenation, selectivity and trans-isomer production. It has been reported that selectivity is directly proportional to the temperature applied during hydrogenation [7]. This fact is the consequence of different activation energies for various hydrogenation reactions. Reduction of double bonds in the unsaturated fatty acid chain is an exothermal process. σ bonds formed between the carbon and hydrogen atoms are, together, stronger than the hydrogen-hydrogen σ bond and π bonds being broken. The heat of hydrogenation can be measured and is simply the amount of heat evolved when one mole of the unsaturated compound becomes hydrogenated. Increasing temperature accelerates the rate of saturation and influence on hydrogen solubility of oil. The common temperature interval for partial catalytic hydrogenation of edible oils is between 160–210°C. High temperature of the oil during hydrogenation favors greater selectivity and thus results in more trans fatty acid generation. Heat of hydrogenation is an important measure of stability. The trans isomer gives off the least amount of heat, while the hydrogenation of the terminal double bond gives off the most. The cis isomer is in the middle of the two. All three consume one mole of hydrogen and yield the same product. The trans isomer is usually more stable than the cis isomer because the substituents are farther apart in the trans isomer than in the cis, and it is sterically more favorable. Among important factors influencing the hydrogenation process is the type of catalyst. Currently, the most widely-used commercial catalyst for edible oil hydrogenation is active nickel supported on an inert substance [6], [35]. High catalyst concentration favors selectivity with large amounts of trans isomer formation. There is, however, some concern about the toxicity of traces of nickel remaining in the oil [31]. Other catalysts including copper on silica [15], nickel–silver [17] or copper chromite [29] have been investigated. Noble metal catalysts are not generally used because of their high cost. However, their high activity in small quantity and the possibility of reuse with fixed bed reactors may offset the cost disadvantage. Palladium on carbon has been used for some commercial hydrogenation because of its high linolenic and linoleic activity at low temperature [22]. Platinum on carbon had been found to be highly active but with low selectivity

156-4


producing the saturated fat [5]. A more recent report [16] found that a modified platinum on carbon catalyst by incorporating a small amount of ammonia in the hydrogen has high selectivity with low trans-isomerization. Rubin et al. [30] reported on the first mixed system containing both homogeneous and heterogeneous catalysts for edible oil hydrogenation. Using Ni and methyl benzoate chromium tricarbonyl [MeBeCr(CO)3], they showed it was possible to retain the advantages of both catalysts while using them in combination. MeBeCr(CO)3 is highly stereoselective toward cis-monoenes. However, because it hydrogenates via a cisoid mechanism in which methylene interrupted double bonds must be conjugated prior to hydrogenation, the reaction rate is limited by the slow conjugation reaction. When MeBeCr(CO)3 was paired with Ni, minimal cis–trans isomerization was maintained while higher hydrogenation rates, more characteristic of Ni, were observed. Polymer stabilized noble metal nanoclusters for selective hydrogenation of small molecule substrates such as unsaturated aldehydes and hydrocarbons have been studied [34]. These catalysts were also reported to possess high enantiomeric selectivity [38]. Polymer stabilized platinum has been found to have ~5 times higher activity than Pt/C with increased selectivity for partial hydrogenation of allyl alcohol at 25°C and atmospheric pressure. In general, metal nanoclusters have properties and activity that are quite different from the corresponding conventionally prepared supported and unsupported metal catalysts. Their potential as catalysts for selective hydrogenation for the oil and fat in the oleochemical industries is only scarcely being explored. Hydrogen solubility in oil is directly proportional to its pressure. Mattil [19] reported that high hydrogen gas pressure during hydrogenation increased the rate of hydrogenation and caused a decrease in the selectivity of the reaction. Such conditions favor less TFA (trans fatty acids) formation. Change of hydrogen pressure from 103 to 310 kPa for example can reduce hardening time by at least 60%. All edible oils contain trace amounts of poisons deactivating reaction sites of catalyst. The minimum amount of catalyst necessary for their neutralization is called the threshold concentration. Once this threshold level has been reached, additional catalyst increases reactivity in a mathematically predictable manner. Under conditions of high temperature and pressure, a more than doubling of catalyst concentration (from 0.005% nickel to 0.0125%) increased the reaction rate above 50%. This is a very important factor in keeping solid catalyst in oil bulk and facilitating solubilization of hydrogen in the oil. Increases in the degree of agitation favors non-selectivity hydrogenation and suppresses the formation of high melting trans-isomers [3]. Beal and Lancaster [4] studied the effect of agitation and batch size on the rate of hydrogenation, and on the stability of the fat. They observed that the rate of hydrogenation increased with an increase in the degree of agitation of an oil or mixture of oils. Furthermore, the stability of the fats increased

with an increase in the hydrogenation batch size. Achieving the optimum degree of mixing is not difficult in the laboratory; but it can be difficult in the plant. Researchers conducting optimization tests of the hydrogenation process, have investigated reactor configurations [12], alternative energy sources such as microwave, magnetic, ultrasonic, etc., in addition to heat [11] or alternative sources of hydrogen, such as metal hydrides or soluble hydrogen donors [22]. In 1992, Yusem and Pintauro [37] developed an edible oil hydrogenation electrolytic process using atomic hydrogen produced at the cathode. Since hydrogen was generated in situ directly over the catalytic surface, it eliminated the need for enhancement of hydrogen transfer rates. As a result, high temperatures and pressures were not required.

IV. HYDROGENATED FAT IN HUMAN NUTRITION The medical viewpoint of hydrogenation is strongly discussed, especially for the role of trans fatty acids (TFA). In 1993, a report published in Lancet by Willett et al. [35] an extensive study of more than 85,000 nurses concluded that women who ate four or more teaspoons of margarine a day had more heart attacks than women who rarely ate margarine. The main goal of this controversial study was the correlation of dietary vegetable oil-based trans fatty acids intake with coronary heart disease. The results started biochemical, toxicological and epidemiological research aiming to elucidate the real nutritional and health impact of trans fatty acids. On the other hand, it is known that cows’ milk or dairy products contain trans isomers of fatty acids because of intestinal bacterial activity [18]. Depending on the diet, milk fat has 2–9% total TFA isomers vaccenic acid [28]. There are many other side effects of trans fatty acids including allergic reaction, arteriosclerosis, increased risk of cancer, decrease in insulin response, lowered quality of breast milk and slight immune dysfunction. Because of the effects of TFAs on the metabolism of gamma-linolenic and arachidonic acid [13], ingestion of trans isomers can affect the metabolism of prostaglandins and other eicosanoids and may alter platelet aggregation and vascular function as well [2]. TFAs also show competitive interactions with essential fatty acid metabolism (EFA) by inhibiting its incorporation into membrane phospholipids and reducing the conversion of EFAs to eicosanoids. In 1991 Koletzko [14] supposed that isomeric trans fatty acids could actually reduce tumor growth and metastasis. Up to the present time, there is little evidence that TFAs are related to the risk of cancer at any of the major cancer sites [10]. On the contrary, association between some forms of cancer in humans and the intake of hydrogenated vegetable fats has also been reported [8]. The general conclusion is that increasing the intake of TFAs (at expense of cis fat) does not produce an adverse outcome with respect to cancer risk.


It has been demonstrated that TFA ingestion increases low-density lipoprotein (LDL) cholesterol to a degree similar to that of saturated fats [20]. The increase in LDL concentration has been attributed in part to the down-regulation of the LDL receptor. In contrast to other forms of fats, trans isomers decrease high-density lipoprotein (HDL) cholesterol [9]. This can be responsible for a markedly increased risk for coronary heart disease, a relationship that is different and must not be confused with serum low-density or high-density lipoprotein (LDL or HDL) cholesterol levels [21]. Trans fatty acids can make platelets stickier, which increases the chance of a clot in a blood vessel. This is the cause of strokes, heart attacks and circulatory occlusions in other organs like the lungs. Our body, fortunately, has natural ways to protect itself from massive TFA intake. There are certain enzymes which recognize the conformational difference and reject the trans fatty acids. Enzymes can refuse to use these molecules for processes for which they are not suited. Our brain is also protected from the trans fatty acids and, as well, an unborn child will have no exposure to the side effects imposed by them, as the placenta is impermeable to trans fatty acids [25]. Our body will also break them down as quickly as possible for energy use to ensure that the cis fatty acids are reserved for more important, vital roles. Strong hydrogenation of soybean oil allows for the formation of variable small amounts of conjugated linoleic acids (CLA). They have been claimed to affect immunomodulation and body composition alteration, and to prevent or cure atherosclerosis and stomach, colon, skin, and prostate cancer. CLA has also been linked directly to increased insulin sensitivity, normalized glucose tolerance, improved hyperinsulinemia and lowered levels of circulating free fatty acids. Recent findings suggest that not only does CLA affect many different pathways, but that individual isomers of CLA act differently. Several studies have demonstrated that the cis-9, trans-11 isomer is responsible for the anticarcinogenic effects of CLA [26], [33]. Obviously, we have to wait for human clinical studies to confirm all these claims.

REFERENCES 1. Albright, L.F.: J. Am. Oil Chem. Soc. 47 (1970), p. 490. 2. Asherio, A., Hennekens, C., Buring, J., Master, C., Stamper, M., Willett, W.: Circulation 89 (1994), p. 94. 3. Bailey, A.E., Feuge, R.O., Smith, B.A.: Oil and Soap 19 (1942), p. 169. 4. Beal, R.E., Lancaster, E.B.: J. Am. Oil Chem. Soc. 31 (1954), p. 619. 5. Berben, P.H., Borninkhof, F., Reesink, B., Kuijpers, E.: Inform 5 (1994), p. 668. 6. Chu, Y.H., Lin, L.H.: J. Am. Oil Chem. Soc. 68 (1991), p. 680. 7. Coenen, J.W.E.: J. Am. Oil Chem. Soc. 53 (1976), p. 382.

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8. Enig, M.G., Munn, R.J., Kenney, M.: Fed. Proc., 37 (1978), p. 2215. 9. Hayashi, K., Hirata, Y., Kurushima, H., Saeki, M., Amioka, H., Nomura, S., Kuga, Y., Ohkura, Y., Ohtami, H., Kajiyama, G.: Atherosclerosis, 99 (1993), p. 97. 10. Ip, C., Marshall, J.: Nut. Revs., 54 (1996), p.138. 11. Jart, A.: J. Am. Oil Chem. Soc. 74 (1997), p. 615. 12. King, J.W., Holliday, R.L., List, J.R., Snyder, J.M.: J. Am. Oil Chem. Soc. 78 (2001), p. 107. 13. Kinsella, J, Brucker, G, Mai, J., Schrime, J.: Am. J. Clin. Nutr., 34 (1981), p. 2307. 14. Koletzko, B.: Die Nahrung 35 (1991), p. 229. 15. Koritala, S.: J. Am. Oil Chem. Soc. 49 (1972), p. 83. 16. Kupel, J. (to Unilever Ltd.), Eur. Pat. Appl. 80200577.7 (1980). 17. Le Febvre, J. Baltes, J.: Fette, Seifen, Anstrichm. 77 (1975), p. 125. 18. Mackier, R., White, B., Bryant, M.: CRC Crit Rev Microbiol 17 (1991), p. 449. 19. Mattil, K. F.: in D. Swern (Ed.), Bailey’s industrial oil and fat products (pp. 794–823). Interscience Publishers, New York (1964). 20. Mensink, R.P., Katan, M.B.: N. Engl. J. Med., 323 (1990), p. 439. 21. Mensink, R.P., Zock, P.L., Katan, M.B., Hornstra, G.: J. Lipid Res., 33 (1992), p. 1493. 22. Naglic, M., Smidovnik, A., Koloini, T.: J. Am. Oil Chem. Soc. 75 (1998), p. 629. 23. Normann, W.: Brit. Pat. 1,515 (1903). 24. O’Brien, R.D.: Technomic Pub. Co. Inc, Pennsylvania (1998). 25. Ohlrooge, J.B., Emken, E.A., Gulley, R.M.: J. Lipid Res., 22 (1981), p. 955. 26. O’Quinn, P.R., Nelssen, J.L., Goodband, R.D. Tokach, M.D.: Anim. Health Res. Rev. 1 (2000), p. 35. 27. Ovesen, L., Leth, T., Hansen, K.: J. Am. Oil Chem. Soc. 75 (1998), p. 1079. 28. Parodi, P.W.: J. Dairy Sci. 59 (1976), p. 1870. 29. Rieke, R.D., Thakur, D.S., Roberts, B.D., White, G.T.: J. Am. Oil Chem. Soc. 74 (1997), p. 333. 30. Rubin, L.J., Koseoglu, S.S. Diosady, L.L., Graydon, W.F.: J. Am. Oil Chem. Soc. 63 (1986), p. 1551. 31. Savchenko, V.I., Makaryan, I.A.: Platinum Metal Rev. 43 (1999), p. 74. 32. Ucciani, E.: in: M. Guisnet, J. Barrault, C. Bouchoule, D. Duprez, C. Montassier, G. Pérot (Eds.), Heterogeneous Catalysis and Fine Chemicals, Elsevier, Amsterdam, 1988, p. 33. 33. Wahle, K.W.J., Heys, S. D.: Prost Leukotrien Essent Fatty Acids, 67 (2002), p.183. 34. Wang, Q., Liu, Q., Wang, H.: J. Colloid Interface Sci. 190 (1997), p. 380. 35. Willett, W.C, Stampfer, M.J, Manson, J.E.: Lancet 341 (1993), p.581. 36. Yap, P.H., deMan, J.M., deMan, L.: J. Am. Oil Chem. Soc. 66 (1989), p.1784. 37. Yusem, G., Pintauro, P.N.: J. Am. Oil Chem. Soc. 69 (1992), p. 399. 38. Zuo, X., Liu, H., Guo, D., Yang, X.: Tetrahedron 55 (1999), p. 7784.

157

Manufacture of Asian (Oriental) Noodles

Shin Lu

Department of Food Science, National Chung Hsing University

Wai-Kit Nip

Department of Molecular Biosciences and Bioengineering, Univeristy of Hawaii-Manoa


Introduction ......................................................................................................................................................157-1 Size Classification, Types of Wheat-Based Oriental Noodles and Some Chemical Properties ......................157-2 General Procedures in the Manufacture of Wheat-Based Asian (Oriental) Noodles ......................................157-2 Manufacture of Dry Asian (Oriental) Wheat- and Rice- Based Noodles ........................................................157-4 A. Wheat-based Noodles ..............................................................................................................................157-4 B. Rice-based Dry Asian (Oriental) Noodles................................................................................................157-8 V. Manufacture of Fresh Asian (Oriental) Wheat- and Rice-Based Noodles ......................................................157-8 VI. Manufacture of Pre-Cooked Asian (Oriental) Noodles..................................................................................157-11 VII. Manufacture of Selected Non- Wheat- or Rice-Based Asian (Oriental) Noodles ........................................157-11 VIII. Factors Affecting Product Quality of Asian (Oriental) Noodles ....................................................................157-13 References ..................................................................................................................................................................157-13

I.

INTRODUCTION

The term “Asian (oriental) noodles” is used very broadly to describe mostly noodle-like products produced mainly in Eastern, Southeastern or Pacific Asian countries using common wheat flour, rice (or rice flour) or other starch materials as the main structural ingredient. Even though the terms “noodles” and “pasta” are often used interchangeably, they are technically different. The common wheat-based “Asian or oriental noodles” differ from the Western style pasta that uses durum wheat flour as the main structural ingredient. The term “noodles” also differs from the US definition of noodles that contains egg solids as part of the Standard of Identity, and readers for this chapter should be aware of these differences. At this time, there is no Standard of Identity for Asian (oriental) noodles in the USA. Asian noodles vary considerably in size, appearance (color and shape), ingredients, chemical properties, and methods of manufacturing. These variables are introduced briefly in this chapter. There are a few excellent reviews on Asian (oriental) noodles available and readers should consult these references for further information (1–8). Factors affecting the production of these Asian (oriental) noodles are

also studied to some extent, and some references are provided at the end of this chapter for further information (9–37). It will not be surprising that considerable amounts of literature in Japanese, Korean, and Chinese are also available, but not easily accessible at the time of this writing. It is generally believed that noodles originated in China several thousand years ago, and the present day form of noodle was developed at least two thousand years ago. In the Chinese language, the term “mien (mian)” is used to describe noodle-type products (with a few exceptions in shape) made from common wheat flour (main structural ingredient). In fact, the Chinese character for noodles has “mia (wheat)” on its side as part of its character structure. Similar products made from rice, mung bean, and other ingredients are all grouped under the term “fen” (rice noodles), for example, “mi fen” (rice noodle), “tung fen” (mung bean threads), and “ho fen” (oily rice stripes). The Chinese character for “fen” is written with “mi (rice)” on its side as part of its character structure, indicating it originated from rice or starch material. It is also believed that “mien” and “fen” spread from China to its neighboring countries. This is supported by the terms with similar sounds used in these 157-1

157-2


countries: “men” (Japan); “mie” (Indonesia); “mee” (Thailand, Singapore, and Malaysia), “Pho” (Vietnam).

II. SIZE CLASSIFICATION, TYPES OF WHEAT-BASED ORIENTAL NOODLES AND SOME CHEMICAL PROPERTIES Table 157.1 shows the classification of “Asian noodles” in two ethnic groups with examples: Chinese and Japanese. The two classifications are similar. Table 157.2 shows the appearance of various types of wheat-based oriental noodles with typical examples, components, and quality of cooked products. It should be noted that these are only examples, and they may fall into different categories when some ingredients are substituted TABLE 157.1 Classification of Wheat-Based Noodles from Two Ethnic Groups Ethnic Group Japanese

Chinese

Class Very thin noodles Thin noodles Standard Flat noodles Very thin noodles

Thin noodles Flat noodles

Wide flat noodles

Thick noodles

Examples Somen Hiya-mugi Udon Kishi-men, Hira-men Longxu mian (China), Yinsi (silver threads) mien (Hong Kong) Xi mien (China, Hong Kong, Taiwan) Yangchun mien (China) Kuan (broad) mien (China, Taiwan) Dai mien (China) Cu mien (China, Hong Kong, Taiwan) Cu mien [Shanghai (China)], similar to Udon

or omitted. For example, Chinese cat’s ear noodles can be made with or without buckwheat. When they are made with buckwheat, they fall into the brownish buckwheat category, and when they are made with just common wheat flour, they fall into the white-salted category. Table 157.3 compares the basic chemical properties of some common wheat-based Asian noodles. The major properties include protein content in the wheat flour, amount of water used in making the dough, amount of salt used in the formulation, and presence or absence of alkaline agents (sodium and/or potassium carbonate). All these factors affect the eating quality of the final product. It is obvious, with the addition of alkaline agent(s), the pH of the final product will be shifted to the alkaline range. The addition of alkaline salts to the formulation not only alters the pH and color, but also improves the water absorption properties of the final product. In addition, addition of alkaline agents improves the texture of the cooked product, making it more chewy with less of a tendency to soften and paste after cooking. The flavor of the cooked product is also typical of an alkaline odor. The majority of consumers in Hong Kong, for example, prefer this type of Asian (oriental) noodles to the common whitesalted noodles. However, the reverse is true for most of the consumers in central and northern China. Oil is used to coat a few freshly-made oriental noodletype products (see below). For instant noodles, the range of oil content in the final product depends on whether the noodles are oil-fried after steaming.

III. GENERAL PROCEDURES IN THE MANUFACTURE OF WHEAT-BASED ASIAN (ORIENTAL) NOODLES In general, wheat-based Asian (oriental) noodles are made by sheeting and rolling procedures in small factories

TABLE 157.2 Comparison of Major Types of Dry Wheat-Based Noodles Types White-salted Yellow-alkaline

Typical Examples Japanese udon Regular plain noodles Cantonese-type noodles Taiwanese-type noodles

Brownish buckwheat

Japanese soba, Chinese cat’s ear noodle

Instant

Japanese ramen, Cantonese E-mien (deep-fried noodle) Cantonese shrimp egg noodle, Imitation

Savory

Components Common wheat flour, salt, water, egg (optional) Common wheat flour, water, alkaline salts (sodium and/or potassium carbonate), salt (optional), egg (optional), yellow coloring (optional) Buckwheat flour, water, limewash or alkaline salts (optional), salt (optional), yam flour (optional) Common wheat flour, water, salt, alkaline salts (optional), oil/fat (optional), Common wheat flour, salt, water, alkaline salts (optional), savory ingredients

Quality of Cooked Product Soft, elastic texture and smooth surface Firm, chewy, springy texture, and bright yellow appearance

Firm, chewy, tender texture

Elastic, chewy

Firm, chewy, springy texture, color dependent on savory ingredient used


157-3

TABLE 157.3 Chemical Properties of Various Types of Dry Wheat-Based Noodles Ingredients Types White-salted Yellow-alkaline Brownish buckwheat (60–70 parts common buckwheat to 30–40 parts wheat flour) Instant Savory

Protein in Flour

Water Added

Salt Added

Alkaline Agent(s)

8 to 10% 10 to 12% 12 to 14 %

30 to 35% 30 to 35% 45 to 48%

2 to 3% Variable Variable

No Yes Yes/No

6.5 to 7 9 to 11 6–5 to 7

None None None

8 to 12% 10 to 12%

30 to 36% 30 to 35%

Variable Variable

Yes/No Yes/No

6.5 to 7 9 to 11% (alkaline type) 6.5 to 7% (regular type)

15 to 21.5% (fried-type) 1.5 to 1.8% (dried type)

pH

Oil Added

that is different from the extrusion procedure used commonly in the production of pasta (see chapter on dried pasta in this handbook). Figures 157.1–157.7 show the activities in the production of the dough, sheeting, rolling,

and cutting of the dough sheets into noodle stripes. It should be noted that dough development is achieved to some degree in these sheeting and rolling processes (Figure 157.4).

FIGURE 157.1 Putting flour in the mixer.

FIGURE 157.3 Sheeting of dough into thin dough sheets.

FIGURE 157.2 Making of a soft dough from various ingredients.

FIGURE 157.4 Rolled dough sheets ready for further processing.

157-4


FIGURE 157.7 Cutting of dough sheets into raw noodle stripes.

FIGURE 157.5 Double rolling of dough sheets to form the gluten structure.

FIGURE 157.6 Connecting the upper rolls of dough sheets to the bottom roller.

IV. MANUFACTURE OF DRY ASIAN (ORIENTAL) WHEAT- AND RICE- BASED NOODLES Dry Asian (oriental) noodles are common products with the advantages of stability and being easy to transport. However, they take a longer time to cook than the fresh product. Examples of selected wheat-based and ricebased Asian (oriental) noodles are described below with comments.

A.

WHEAT-BASED NOODLES

The majority of Asian (oriental) noodles are sold in the dry form in plastic/cellophane wrappings or in cardboard boxes with cellophane wrapping. Chinese people are the major consumers of Asian (oriental) noodles, especially the regular white-salted and rice noodles.

Table 157.4 is a generalized scheme for production of dry noodles in China. It should be noted that savory-type noodles are also available in many varieties. The generalized scheme for production of dry noodles is a procedure that involves mixing, resting, sheeting, rolling, cutting, and drying. Similar products, such as Japanese somen, udon, and buckwheat sobo are produced in a similar manner, except that in some cases, extrusion procedures are used instead of sheeting and rolling. Table 157.5 describes a procedure used in Taiwan to produce dry “La Mien” (stretched noodles). Making traditional “La Mien” (hand-swung noodles) manually is a very skillful technique (see below), accomplished by swinging the dough into very thin noodles. The “La Mien” thus produced is usually consumed right away by cooking in boiling water, followed by addition of seasonings, soup and other ingredients. However, due to the popular demand of such products, a dry form has been developed by varying the manufacturing process from hand-swinging to manually stretching, and the La Mien thus produced is dried for easy transportation and long term storage. It also can be produced in much larger quantity than the traditional handswinging process. Figures 157.8 to 157.15 show the unique operations in the production of dry Taiwanese La Mien (hand-stretched noodles) in a small factory. Table 157.6 is a generalized production scheme for dry Taiwanese Yi-mien, an alkaline-type noodle with the addition of alkaline agent(s). This production scheme is also similar to that for Cantonese alkaline noodles except for the ingredients used (egg is used fairly commonly for Cantonese alkaline noodles) and the addition of a shaping process (also a common procedure in Cantonese alkaline noodles). Shaping is not common in other forms of oriental noodles except instant noodles. Figure 157.16 shows the sun drying procedure in the production of Taiwanese Yi-mien in a small factory. Deep-frying the freshly-made noodles not only removes the moisture, but also cooks and alters the structure of the final product. The frying process makes the structure very porous, allowing it to more easily absorb


157-5

TABLE 157.4 Generalized Scheme for Production of Dry WheatBased Noodles in China

TABLE 157.5 Generalized Scheme for Production of Dry Taiwanese La Mien (Stretched Noodles)

Weigh out the basic ingredients: wheat flour (100 parts) water (25–32 parts, dependent on gluten content of wheat flour, at 30°C) salt (2–3 parts) alkaline agent (optional, 0.1 to 0.2% of flour weight) Weigh out additional ingredients (optional): Egg – 10% of flour weight (fresh egg); 8% of flour weight (frozen shelled egg), or 12.5% of flour weight (egg powder) Milk – 14 to 25% (fresh milk), or 2 to 3% (milk powder) Dried meat floss – 5% by flour weight plus 3% salt Tomato sauce – 5% Soymilk – made from 15 kg soybean for each 50 kg of flour Fish stock – made from 2.5 kg of fish plus 1 kg salt for 50 kg of flour Mung bean milk – made from 15 kg mung bean for each 50 kg of flour Chili powder – 1.5% by weight of flour plus 3% salt Monosodium glutamate (MSG) – 0.5 to 1.0% Egg white Egg yolk Butter Beef powder Prawn meat L-lysine hydrochloride Chicken broth Spinach juice Calcium powder Mix up the flour, water, and other ingredients fully for 10–15 minutes to allow for hydration of the protein, starch, and other biological components to a uniform-colored, crumbly mixture without any pockets of dry flour. Linear velocity of dough mixer is adjusted to 2–3 m/sec. Rest the dough for 10–15 minutes by mixing at low speed of 0.6 m/sec (10 rpm) at room temperature. Sheet and roll the dough 6 to 7 times to reduce the dough to desirable sheet thickness (0.6, 0.8, 1.0, or 1.5 mm). Cut the dough sheets to desirable width (0.8 to 1.0, 1.5, 2.0, 3.0, or 6.0 mm). Dry the noodle stripes at a temperature below 50°C for about 2 hours, or at 38°C for about 7.5 hours (at controlled relative humidity between 70 to 80 %), or at 45°C for 3.5 hours (at 80% relative humidity) to 13.5 to 14% moisture content. Machine-cut the dried noodles. Weigh the noodles at 250 or 500 g each. Pack the weighed noodles into appropriate plastic bags, followed by sealing of the bags.

Weigh out appropriate amount of medium strong wheat flour (9–10% protein) Weigh out appropriate amount of salt (6%) Mixing of the wheat flour and salt with appropriate amount of water (32–36%) Sheet the dough and cut the sheets into threads Coil dough threads onto two sticks (like spinning cotton into yarn) 20 cm apart, followed by stretching to 40 cm and then 60 cm, respectively At 60 cm in distance, age the noodle stripes are aged (matured) for 2.5 hours Stretch the noodle stripes again to 120 cm in distance Sun dry or mechanically dry the noodle stripes to about 30% moisture Fold the noodle stripes 4 times Further reduce moisture content to about 13% Packaging of final product in plastic or laminated packages

FIGURE 157.8 Dough stripes in a bowl.

Adapted from Refs. 3, 4, 5.

boiling water. The fried product puffs up and expands considerably during the frying process. This makes the final product very easy to rehydrate in the cooking process, generally in a matter of seconds after putting it into boiling water. Cantonese E-mien (E-fu-mien) has been a delicacy among the Cantonese community for decades. It is now available in regular laminated packs or

FIGURE 157.9 Pulling out the dough stripes from the bowl of noodle stripes.

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FIGURE 157.10 Coiling the dough stripes on two sticks 20 cm apart.

FIGURE 157.13 Extending the noodle stripes to 120 cm apart for sun drying.

FIGURE 157.11 Extending the noodle stripes to 40 cm apart.

FIGURE 157.14 Partially sun-dried noodle stripes folded back to 60 cm apart.

FIGURE 157.12 Extending the noodle stripes to 60 cm apart.

as a vacuum-packed product in supermarkets in the US. It is also available as a freshly-made product directly from the factory. Table 157.7 shows a generalized production scheme for Cantonese E-mien (E-fu-mien). Nissin Foods Company in Japan first invented Instant Chicken Ramen (noodles) in 1958, and packaged in laminated/cellophane pouches. This product that can be rehydrated in a bowl with boiling water in 3 minutes. It is produced by first steaming the noodle portions, followed by


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brief frying in tropical oil. The seasoning was packed in a small packet. The fried noodle and the seasoning then were packed in a laminated/cellophane pouch. This form of package was later improved by using Styrofoam cups. However, both types of packaging are available today. Because of the concern in consuming tropical oils, a modified production process of drying the steamed noodles instead of frying was introduced. A separate packet of oil is included in the package for optional use by the consumer. Various flavors are available now. Table 157.8 is an example of a generalized production scheme for instant noodles in Taiwan, adapted from the Japanese procedure.

FIGURE 157.15 Partially sun-dried noodle stripes folded back to 30 and 15 cm (final), respectively.

TABLE 157.6 Generalized Scheme for Production of Dry Taiwanese Yi-Mien Weigh out appropriate amount of strong wheat flour (>11% protein) Mix the four with 0.4 to 0.6% alkaline agent (sodium and/or potassium carbonate) and water to form a weak dough Cover the dough and leave the dough for maturation for about 30 minutes Sheet and roll the dough to desirable thickness (about 2–3 mm) Fold the dough into plates of dough sheets Cut the multi-layered dough sheets to make the noodle stripes Noodle stripes are then shaped and sun-dried or mechanically dried to about 13% for long term storage (see also scheme for Taiwanese La Mien production)

FIGURE 157.16 Sun drying of Yi-mien on bamboo trays to reduce the moisture for long term storage.

TABLE 157.7 Generalized Scheme for Production of Cantonese E-Mien (Deep-Fried Noodles) Weighing of ingredients [wheat flour (medium strength), salt, water, egg (optional, potassium carbonate (optional)] Mixing of dry ingredients for a short time Adding water to adjust moisture content to about 36% Mixing of dough for 10–15 minutes Sheeting the dough through sheet rollers to reach 1–2 mm in thickness Cutting of sheeted dough to strips of 1 mm in width to form long strings of noodles Weighing of noodles to standard portions Frying of noodle portions in a continuous oil fryer Cooling of oil-fried noodles Packaging of fried noodles into cellophane bags, cardboard boxes, or Styrofoam trays then wrapping with cellophane

TABLE 157.8 Example of Generalized Process for Dry Instant Noodle Manufacturing in Taiwan Weighing of ingredients [wheat flour (medium strength), salt, water] Mixing of dry ingredients for a short time Adding of water to adjust moisture content to about 36% Mixing of dough for 10–15 minutes Sheeting the dough through sheet rollers to reach 2 mm in thickness Cutting of sheeted dough to strips of 1 mm in width to form long strings of noodles Weighing of noodles to 100 g portions Steaming of portioned noodles for 2–3 minutes in temperaturecontrolled steamer (95°C) Frying of steamed noodle blocks at 150°C for 1.5 minutes in a continuous oil fryer (fried-typed instant noodles) (net weight 80 g) or Drying of steamed noodle blocks in controlled temperature chamber to 80 g net weight Cooling of oil-fried or dried instant noodle Packaging of fried or dried noodles in plastic/cellophane bags, or Styrofoam cups Adding packages of seasonings (dry, wet, and/or oils) into container Sealing of container

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The Instant Ramen developed by Nissin Foods Company was well accepted by the Japanese. It expanded its operation into other countries in the late 1960s and early 1970s. It is one of the most popular snack or regular meal items in many countries. Currently, instant rice noodles (“mi fen”) are also available. In comparing the production schemes for Cantonese E-mien and instant noodles, the instant noodle process can be considered as an improvement of the Cantonese E-mien process, as the noodle volume of instant noodle remains essentially the same, as compared to the expanded volume in Cantonese E-mien (3–4 times). This has the advantage of much smaller product volume. Also, seasoning are included in the product package. In addition, dried and not fried instant noodles are available. This satisfies the health-concerned consumers. However, Cantonese E-mien has the advantage of much reduced cooking time and unique texture. It probably will remain as a unique cuisine-type product, as compared to the instant noodles which are part of the mainstream food chain.

B.

RICE-BASED DRY ASIAN (ORIENTAL) NOODLES

Rice noodles (“mi fen” or rice sticks) are a deviation of Asian (oriental) wheat-based noodles in that rice is the basic structural component. In addition, traditional procedures for the production of rice noodles involve the wet-milling of rice to remove the soluble constituents in the rice kernel, and the gelatinization of the rice starch. This process is tedious, and involves the problem of liquid waste disposal, even though it is not high in biological oxygen demand. A modified procedure is to use rice flour directly instead of the wet-milled rice flour, thus avoiding the liquid waste disposal problem. It should be noted that dry milled rice flour is not the same as the wet-milled rice flour, and the quality of the final product is not expected to be the same. It is generally believed that wet-milled rice flour has a smoother texture after gelatinization. Table 157.9 describes the general steps involved in the production of dry rice noodles. Consumers should be aware that in recent years, some manufacturers have begun to use corn starch or other starches partially or completely instead of rice flour to make similar products, and still call them “rice sticks.” They have similar properties, but are not as good as the original rice noodles (rice stick).

V. MANUFACTURE OF FRESH ASIAN (ORIENTAL) WHEAT- AND RICE-BASED NOODLES Considerable amounts of fresh Asian (oriental) noodles are produced for the retail market, restaurant trade, and at a household level. The makings of some of these products are described below. Won Ton Mien is one of the alkaline wheat-based noodles produced for the fresh retail markets common in

TABLE 157.9 Generalized Scheme for Production of Dry Rice Noodles in Taiwan Process A Clean polished high-amylose rice to remove foreign matters Soak rice kernels in water for 3 hours Grind the soaked rice into a slurry with a suitable amount of water, avoiding the production of excessive damaged starch granules Pour the rice slurry into a cloth bag and press the filled bag with a mechanical press to remove water Mix the de-watered rice solids in a heated mixer for 50 minutes and partially cook the rice solids to a soft mass (addition of corn starch and/or wheat is optional) Mix the soft rice mass a second time to further soften Transfer the soft rice mass to a presser to form thick sheets, followed by extruding the sheets into rice noodles, cooling and loosening the extruded rice noodles immediately to avoid sticking together. Steam-cook the extruded rice noodles for about 50 minutes Cut the cooked rice noodles with a knife when still warm Shape the cut rice noodles into bundles or blocks Load the shaped rice noodles onto trays in carts Mechanically dry the shaped rice noodles for 8 hours Cool thoroughly before packaging into specific containers for retail or storage Process B Instead of soaking, grinding, and de-watering the rice, rice flour is used directly in mixing and partial cooking The rest of the steps are essentially the same as Process A

Taiwan. Similar products are also produced elsewhere. The production steps are similar to production of dry white-salted noodles, with basic mixing, sheeting, rolling, and cutting steps. It should be noted that a differentlyshaped product called “pian er mien” (sheeted mien) is produced by similar procedures with or without alkaline salts; instead of cutting the dough sheets into thin stripes, the sheets are cut into about 1 inch squares. Also, the fresh Won Ton Mien can be dried into dry Won Ton Mien, like the other wheat-based “mien.” Table 157.10 describes the basic steps in the production of Taiwanese Won Ton Mien. Figures 157.17 and 157.18 show the typical operation procedures in its production. Fresh oily wheat-based “mien” are unique, in that oil is added to the cooked “mien” or “fen” to provide a special mouthfeel and al dente. They are common in southern China and southeastern Asian countries. The oily, alkaline wheat-based “mien” are sometimes called Hokkien-type noodles. The procedures for preparing the raw “mien” are essentially the same as other alkaline wheat-based “mien.” They are then boiled once or twice, or steamed until they are completely cooked before coating with oil and food coloring. Table 157.11 describes the procedures of manufacturing Taiwanese oily wheat-based noodles in a small


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TABLE 157.10 Generalized Scheme for Production of Fresh Taiwanese Won Ton Mien

TABLE 157.11 Generalized Production Scheme for Fresh Oily Noodle Production

Weigh out appropriate amount of strong wheat flour (>11% protein) Mix the four with 0.35% alkaline agent (sodium and/or potassium carbonate) and water to form a weak dough Cover the dough and leave for maturation for about 30 minutes Sheet and roll the dough to desirable thickness (about 1 mm) Fold the dough into plates of dough sheets Cut the multi-layered dough sheets to make the noodle stripes or noodle squares Noodle stripes and noodle squares are then sold fresh

Weigh out appropriate amount of medium strong wheat flour (9–10% protein) Weigh out appropriate amount of sodium carbonate (about 0.35 to 0.5%) Mix wheat flour and sodium carbonate with water (about 32–36%) to make a weak dough Leave the dough for maturation for about 30 minutes Sheet and roll the dough to desirable thickness (about 2 mm) Cut the dough sheets into noodle stripes (about 2–3 mm wide) Steam the noodle strips for about 1.5 minutes until the noodle stripes are completely cooked Cool the cooked noodles to room temperature Mix the cooked noodles with small amounts of yellow coloring and liquid oil Package the oily noodles in suitable containers for marketing

FIGURE 157.17 Sheeting of dough to 1.0 mm thick sheet.

FIGURE 157.19 Cutting of dough sheets into noodle shape.

FIGURE 157.18 Dough sheets folded back and forth before cutting.

factory. Figures 157.19 to 157.22 show the unique steps in the manufacture of such product. Another oily product is the Cantonese oily rice-based “ho fen” (rice stripes). It is produced first by preparing a rice

FIGURE 157.20 Cut noodles on conveyor belt.

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a delicacy, as they take skill to make. The hand-swung La Mien is considered a fresh “mien,” as it is cooked right after making. The addition of alkaline agent(s) is optional. Table 157.13 describes the procedures used in the making of hand-swung La Mien. It should be noted that the process can be modified to make the production less labor intensive, as described earlier. TABLE 157.12 Generalized Production Scheme for Fresh Cantonese Oily “ho fen” (Thick Rice Stripes)

FIGURE 157.21 Cooling of cooked noodles for a short time in a water bath.

Prepare a rice slurry with rice flour and water. Put a small amount of oil on stainless or bamboo trays to coat the trays evenly. Pour rice slurry on the trays to form a thin layer (about 1–2 mm thick). Place the trays on racks in the steamer. Steam the trays of thin layers of rice slurry to gelatinize the starch. Remove the trays from the steamer and cool. Roll up the gelatinized rice sheet from each tray with a spatula (about 10 cm in depth). Cut the layered rice sheets into 1 cm wide stripes (“ho fen”). Wrap the rice “ho fen” in paper.

TABLE 157.13 Generalized Scheme for Production of Hand-Swung La Mien in China

FIGURE 157.22 Mixing of cooked noodles with liquid oil in a rotating drum.

slurry from rice flour, followed by steaming a thin layer of the slurry on an oil-coated stainless tray or bamboo sheet. The gelatinized “fen” is then folded into layered slabs, followed by cutting of the slabs into stripes. These noodles are much thicker and broader than the Chinese “hand-cut mien” (see later) or Japanese “udon.” Oily rice-based “ho fen” is very soft and smooth in texture. The granular size of rice flour used has a definite effect on the quality of the final product, as the difference in granular size can be detected easily. The original “ho fen” was made with wet-milled rice flour with a very fine texture. However, it is much more costly to make, and has the liquid waste disposal problem similar to dry rice noodles (rice sticks). Table 157.12 describes the basic steps in the making of Cantonese “ho fen.” Original Chinese hand-swung La Mien (La Mian) was made by skillful masters or chefs. They are considered

Weigh out medium strong wheat flour (about 10% protein, 1 kg), salt (20 g), water (550 g), and sodium carbonate (10 g) Put wheat flour in a bowl Dissolve salt in 500 ml (500 g) water with temperature at about 25°C (summer) to about 35°C (winter) Add salted water gradually to the flour Mix wheat flour and salted water to a crumbly consistency and knead the dough until uniform Cover the dough with a clean cloth and rest for 30 minutes at 20–30°C Dissolve sodium carbonate in 50 ml (50 g) water Add the sodium carbonate solution to the rested dough and knead the dough until the sodium carbonate solution is uniformly distributed into the dough Roll the dough into a long rope Hold each end of the dough rope with each hand and swing the dough rope up and down The elongated dough rope is folded and twisted Repeat the swinging, folding and twisting 6 to 7 times to create a uniform dough piece Place the dough piece on a table and dust the dough rope evenly with wheat flour Hold the two ends of the dough rope with one hand and insert the four fingers of the other hand into the loop Stretch by gently shaking the rope until the rope is 10–16 cm long Repeat the dusting and stretching until 6 stretches with 64 strands of La Mien is achieved (for very thin La Mien, up to 12 stretches are used) The La Mien then can be put into boiling water for a few minutes before consumption by mixing with other ingredients Adapted from Refs. 1, 4, 5.


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Noodles of various shapes other than the traditional stripes are made commonly by the Chinese people, especially those in the rural areas in the northern provinces. Table 157.14 describes three common Chinese hand-cut “mien.” The addition of whole egg, egg white, or egg yolk is optional, depending on consumer preference in different locations. The hand-sheeted and cut noodle is the most common. “Cat’s ear mien” and “knife-cut mien” are less common, and are considered specialty products. It should be noted this “knife-cut mien” has a thicker center, providing a special al dente texture.

sodium benzoate used as preservatives, and sterilized for long term storage. Frozen precooked “saimen” (alkaline noodles) have also been available for some time. The “saimen” is precooked in water or steamed, cooked, cooled, packaged and sealed in plastic bags before freezing. The shelf-life of this product is good.

VI. MANUFACTURE OF PRE-COOKED ASIAN (ORIENTAL) NOODLES

For decades, there have been products on the market that are made from materials other than wheat or rice, but are included in the Asian (oriental) noodle category. Table 157.15 lists some of these examples with their ethic origin and main ingredients. These products are sometimes called “starch noodles” as they are made from starch of various origins. One of the most fascinating Asian (oriental) noodles made from materials other than wheat or rice are mung bean threads, sometimes called cellophane or transparent noodles by Westerners. This product has the unique property of being transparent like clear cellophane after it is cooked. The best mung bean threads can stay in their original shape and remain intact for about 2 days after being cooked and kept in the soup. This is because of their unique starch gelling properties, which also provide very good al dente properties. Table 157.16 describes the procedures used to make traditional mung bean threads. True (pure) mung bean threads are made from mung bean only. However, the products today on the market may contain mung bean and other starch materials like broad beans and other starches. The bottlenecks of making true mung bean threads include the intensive labor required and liquid waste disposal. The liquid waste is fairly rich in nutrients as it contains all the vitamins, minerals and proteins in the mung bean. In the past, this waste was used as animal feed. Attempts have also been made to recover the protein from this liquid waste. Because of these problems, it is understandable that materials other than mung beans, and improvements in technology are being considered. Korean sweet potato vermicelli (“dang myun”) is a product similar to mung bean threads with transparency after it is cooked. However, the vermicelli is colorless, but kind of light brownish green. It also has very good al dente properties. Japanese “harusame” is also a similar starch noodle product as it is made also made from starches from potato, sweet potato, rice or mung bean. Another unusual Asian (oriental) noodle is the translucent Japanese “Shirataki” noodle made from devil’s tongue yam (elephant yam or konjac/konjak yam). This product is marked as a form different from others. It stays in liquid in a sealed container, is pasteurized and has to be kept refrigerated. This product is considered a low-calorie

In recent years, precookd, ready-to-eat udon in sterilized pouches has been available. The cooked udon is sealed with water in pouch-type containers with lactic acid or

TABLE 157.14 Generalized Scheme of Hand-Cut Wheat-Based Noodles Hand Sheeted and Cut Noodles Weigh out special wheat flour (1000 g), cold water 400 g, and potato starch (50 g) Mix the flour with cold water to make a dough Knead the dough to a smooth and even condition Flatten the dough with a rolling pin on a smooth surface into a rectangular shape by rolling in different directions to extend the dough evenly Dust the dough sheet with potato starch Repeat the rolling and dusting several times to stretch the dough to about 3 mm thick The dough sheet is then folded accordian-like before cutting to 1.5 mm wide The noodles are shaken to remove the potato starch The noodles are then cooked in boiling water until done before consumption by mixing with other ingredients Cat’s Ear Noodle (Mao-er-dao) Procedure similar to above for making dough Cover the dough with a damp cloth and let stand for 20 minutes Sheet and roll the dough to 1 cm thick Cut the sheets into 5 cm parallelograms or squares Pinch the small slanted squares into the shape of a cat’s ear before cooking in boiling water Knife-Cut Noodles (Dao Xian Mian, Paring Noodles with a Knife) Weigh out appropriate amount of wheat flour (e.g., 600 g) Mix the flour with appropriate amounts of salt (5 ml) and water (10 ml) Knead the wheat flour, salt and water into a stiff dough cylinder Hold the dough cylinder in one hand and pare long noodle stripes out of the dough with a knife. (Note: The stripes are not of even thickness, thicker in the center and thinner on the sides.) Cook the noodle stripes in boiling water Adapted from Refs. 3, 4, 5.

VII. MANUFACTURE OF SELECTED NONWHEAT- OR RICE-BASED ASIAN (ORIENTAL) NOODLES

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TABLE 157.15 Oriental Noodles Made from Materials other than Wheat Flour and Rice Flour Name Bean threads cellophane noodles transparent noodles Chinese vermicelli fen si, fen sai, fun sai soo hoon, suhoon green bean thread noodles tung boon su un panit sotanghon won sen bun tau tanghoon Harusame Sweet potato vermicelli dang myun (tang myun) Korean buckwheat noodles naeng myun Shirataki sirataki ito konnyaku yam noodles devil’s tongue noodles Soba Buckwheat noodles Tapioca sticks tapioca starch noodles hu tieu bot loc Tientsin fen pi (sheets)

Ethnicity Chinese

Indonesian Tagalog (Philippines) Thai Vietnamese Malaysian Japanese

Main Ingredient(s) Mung bean starch

Korean

Potato, sweet potato, rice, or mung bean starch Sweet potato

Korean

Buckwheat, potato starch

Japanese

Devil’s tongue yams

Japanese

Buckwheat, common wheat

Vietnamese

Tapioca starch

Chinese

Mung bean

TABLE 157.16 Generalized Scheme for Traditional Production of Dry Mung Bean Threads (“fun see,” Cellophane, or Translucent Noodles) Clean mung beans to remove foreign matter Soak mung beans in water for 4 to 5 hours in summer and 10 hours in winter Finely grind the soaked mung beans with added water using a stone grinder Dilute the bean slurry with three times the amount of water and let sit for about 8–9 days to ferment (dissolving the nitrogenous and other undesirable matter) Remove the liquid when it gets foamy, leaving the sediment at the bottom Add clean water back to the sediment and mix Repeat the process for 7–8 days until the mung bean starch is pure Filter out the mung bean starch in cloth bag by gravity Divide the mung bean starch into two portions Add small amount of cold water to the first half of mung bean starch to make a slurry Add boiling water to make a thin paste Mix in the second half of mung bean starch with stirring to form a sticky and elastic paste Transfer the sticky and elastic paste to a perforated funnel Press the paste in the funnel so that the paste is extruded out through the small openings Drop the extruded mung bean threads into boiling water immediately and gelatinize the starch into transparent threads Scoop out the transparent mung bean threads and spread them on bamboo trays, keeping the threads in an orderly arrangement Sun dry the mung bean threads to dryness Package the dried mung bean threads into suitable containers for retail or storage


food as it utilizes the gums (hydrocolloids) in the devil’s tongue yam as the main structural material. Fresh elephant yam contains glucomannan (a soluble dietary fiber) and starch at a ratio of about 2:1, and has excellent water holding capacity. It is popular in Japan and Taiwan, and also available in the oriental markets in the US. Elephant yam is the original material used to make Konjac/kojac gum, a GRAS food ingredient.

VIII. FACTORS AFFECTING PRODUCT QUALITY OF ASIAN (ORIENTAL) NOODLES In the manufacture of Asian (oriental) noodles, one or more of the following common procedures are applied, depending on the kinds of product to be produced: ● ●

● ● ● ● ●

● ● ● ● ●

Selection of raw materials Mixing of ingredients to form a dough, slurry or paste Resting of dough Sheeting and rolling Extruding Shaping Pressing of slurry or paste through perforated funnel Steaming Boiling of cut noodle stripes Frying of noodles Cooling Drying of final product

In addition to the above-mentioned variables in a manufacturing process, the producer has to consider production cost, environmental issues, consumer preference, and market competition, as well as proprietary formulations and practices. This makes it very complicated to compare quality of similar types of product. Scientific measurements are helpful, and these have been applied. However, a food technologist or scientist is more interested in the composition of ingredients, kinds of oil used and amount absorbed, application of food additives to prolong shelf-life, rheology of dough, consistency of slurry, work needed to knead the dough, and other factors. Table 157.17 is a summary of the major factors that could affect the quality of Asian (oriental) noodles based on the literature published (9–37). The list of references at the end of this chapter does not answer all the questions a food technologist or scientist wants to ask, but it will provide some indications of what is known and what needs to be studied in the future. Asian (oriental) noodles, with beginnings in a primitive, cottage-type industry, is now a big industry, especially for instant noodles. These noodles have captured the attention of consumers worldwide. With the improvement in technology, it is expected that this industry will grow

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TABLE 157.17 Factors Affecting Product Quality of Oriental Noodles Group I. Ingredients Wheat flour used (especially protein content) Buckwheat flour used Rice flour used (especially amylose to amylopectin ratio) Mung bean used Water quality Amount of salt used Kinds of frying oil used Amount of coating oil used Amount and kinds of alkaline salts used Kinds and amount of additional ingredients used (starchy materials, savory ingredients) Group II. Dough Quality Rheology of dough Viscosity of slurry Group III. Processing Conditions Mixing of ingredients Kneading of dough Drying temperature, duration and condition Dough resting condition and duration Cutting actions for dough or noodle stripes Stretching actions on dough Relative humidity in drying chamber and environment Steaming temperature and duration Frying temperature and duration Sheeting and rolling actions Starch extraction conditions Extruding condition Cooking conditions

further and gain further acceptence by more consumers in various regions.

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6. SK Kim. Instant noodle technology. Cereal Foods World 41(4):213–218, 1996. 7. S. Nagao. Processing technology of noodle products in Japan. In: JE Kruger, RB Matsuo, JW Dick. eds. Pasta and Noodle Technology. St. Paul (MN): Amer Assoc Cereal Chemists. 1996, pp. 169–194. 8. J. Udesky. The Book of Soba. Tokyo (Japan): Kodansha International Ltd., 1988. 9. Anon. The use of stabilizers in instant wheat noodles. Asian Pacific Food Industry (Nov): 80, 82, 84, 1995. 10. BK Baik, Z Czuchajowska, Y Pomeranz. Role and contribution of starch and protein contents and quality to texture profile analysis of Oriental noodles. Cereal Chem 71:315–320, 1994. 11. BK Baik, Z Czuchajowska, Y Pomeranz. Discoloration of dough for oriental noodles. Cereal Chem 7292: 198–205, 1995. 12. FP Bejosano, H Corke. Effect of Amaranthus and buckwheat proteins on wheat dough properties and noodle quality. Cereal Chem 75:171–176, 1998. 13. Bhattcharya M, Corke H. Selection of desirable starch pasty properties in wheat for use in white and yellow alkaline noodles. Cereal Chem 73:721–728, 1996. 14. Crobie G. The relationship between starch swelling properties, paste viscosity, and boiled noodle quality in wheat flours. Jour Cereal Science 13:145–150, 1990. 15. JE Dexter, RR Matsuo, BL Dronzek. A scanning electron microscopy study of Japanese noodles. Cereal Chem 56:202–208, 1979. 16. NM Edwards, MG Scanlon, JE Kruger, JE Dexter. Oriental noodle dough rheology: relationship to water absorption, formulation, and work input during dough sheeting. Cereal Chem 73(6):708–711, 1996. 17. FCF Galvez, AVA Resurreccion, GO Ware. Process variables, gelatinized starch and moisture effects on physical properties of mungbean noodles. Cereal Chem 59(2):378–381, 386, 1994. 18. DW Hatcher, JE Kruger, MJ Anderson. Influence of water absorption on the processing and quality of oriental noodles. Cereal Chem 76(4):566–572 1999. 19. PS Jin. Food Industry (In Chinese). Taipei (Taiwan): Jing Zhong Book Store. 1956, pp. 35–38. 20. WS Kim, PA Seib. Apparent restriction of starch swelling in cooked noodles by lipids in some commercial wheat flours. Cereal Chem 70:367–372, 1993. 21. CM Konik, LM Mikkelsen, R Moss, PJ Gore. Relationship between physical starch properties and yellow alkaline noodle quality. Starch/Starke 46:292–299, 1994. 22. CM Konik, DM Miskelly, PW Gras. Starch gelling power, grain hardness and protein: Relationship in

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36.

37.

sensory properties of Japanese noodles. Starch/Starke 4:139–144, 1993. S Lu S, FK Kuo. Effects of emulsifier and starch on the quality of oriental noodle. (In Chinese) Journal of Food Science 18:313–323, 1991. S Lu, FK Kuo FK, FJ Kao FJ. Effects of acid, alkali and salt on the quality of oriental noodles. (In Chinese) Journal of Food Science (Taipei) 18:104–115, 1991. S. Lu. The physical and chemical properties of dough sheets from two kinds of flour. (In Chinese) Chinese Agric Chem Society Journal (Taipei) 28:219–236, 1990. DM Miskelly, HJ Moss. Flour quality requirements for Chinese noodle manufacture. Journal Cereal Science 3: 379–387, 1985. R Moss, PJ Gore, IC Murray. The influence of ingredients and processing variables on the quality and microstructure of Hokkien, Cantonese and instant noodles. Food Microstructure 6(1):63–74, 1987. HJ Moss, DM Miskelly, R Moss. The effect of alkaline conditions on the properties of wheat flour dough and Cantonese-style noodles. Journal Cereal Science 4: 261–268, 1986. M Oda, Y Yasuda, S Okazaki, Y Yamauchi, Y Yokoyama. A method of flour quality assessment for Japanese noodles. Cereal Chem 57:253–254, 1980. NH Oh, PA Sieb, AB Ward, CW Deyoe. Noodles. IV. Influence of flour protein, extraction rate, particle size, and starch damage on the quality characteristics of dry noodles. Cereal Chem 62:441–446, 1985. NH Oh, PA Seib, KF Finney, Y Pomeranz. Noodles. V. Determination of optimum water absorption of flour to prepare oriental noodles. Cereal Chem 63(2):93–96, 1986. NH Oh, PA Seib, CW Deyoe, AB Ward. Noodles. I. Measuring the textural characteristics of cooked noodles. Cereal Chem 60:433–438, 1983. K Rho, PA Seib, OK Chung, DS Chung. Retardation of rancidity in deep-fried instant noodles. Jour Amer Oil Chemists’s Society 63(2):251–256, 1986. K Shelke, JW Dick, YF Holm, KS Loo. Chinese wet noodle formulation: A response surface methodology study. Cereal Chem 67(4):338–342, 1990. H Toyokawa, GL Rubenthaler, JR Powers, EG Schanus. Japanese noodle qualities. I. Starch components. Cereal Chem 66:387–391, 1989. H Toyokawa, GL Rubenthaler, JR Powers, EG Schanus. Japanese noodle qualities. I. Flour components. Cereal Chem 66:382–386, 1989. KR Vadlamani, PA Seib. Reduced browning in raw oriental noodles by heat and moisture treatment of wheat. Cereal Chem 73(1):88–95, 1996.

158

Extruding and Drying of Pasta

Frank A. Manthey

Department of Plant Sciences, North Dakota State University

Wesley Twombly

Nuvex Ingredients, Inc.

CONTENTS I. II.

Introduction ........................................................................................................................................................158-1 Ingredients ..........................................................................................................................................................158-2 A. Semolina ......................................................................................................................................................158-2 1. Durum Milling......................................................................................................................................158-2 2. Semolina Quality ..................................................................................................................................158-2 B. Other Ingredients ........................................................................................................................................158-4 C. Water Quality ..............................................................................................................................................158-4 III. Pasta Processing ..................................................................................................................................................158-4 A. Mixing..........................................................................................................................................................158-4 1. Hydration ..............................................................................................................................................158-4 2. Mixing Equipment................................................................................................................................158-5 B. Extruding ....................................................................................................................................................158-5 1. Screw Design........................................................................................................................................158-6 2. Screw Function ....................................................................................................................................158-6 3. Dough Flow ..........................................................................................................................................158-6 4. Frictional Heating ................................................................................................................................158-7 5. Dough Viscosity....................................................................................................................................158-7 6. Die Assembly........................................................................................................................................158-8 7. Extruder Output ....................................................................................................................................158-9 C. Pasta Drying ................................................................................................................................................158-9 1. Drying Stages ......................................................................................................................................158-9 2. Moisture Migration during Pasta Drying ..........................................................................................158-11 3. Maillard Reactions during Pasta Drying ............................................................................................158-11 4. Checking in Pasta ..............................................................................................................................158-11 5. Pasta Defects/Troubleshooting ..........................................................................................................158-12 IV. Good Quality Pasta............................................................................................................................................158-12 References ..................................................................................................................................................................158-13

I.

INTRODUCTION

Before the 1800s, pasta was made by hand. The first mechanical devices for pasta manufacturing were invented in the 1800s (1). Around 1850, the first handoperated pasta press was built. By the early 1900s, mixers, kneaders, hydraulic piston-type extrusion presses, and drying cabinets were available for batch manufacturing of pasta. In 1933, the first continuous single-screw pasta press was invented. Before 1974, pasta was dried using low temperature drying profiles that mimicked open-air

drying conditions typical of the region around Naples, Italy. It required 18 to 20 hours to dry pasta when using a low temperature drying profile. High temperature drying (60 to 80°C) of pasta was introduced in 1974 and ultrahigh temperature (80 to 100°C) drying was introduced in the late 1980s. Drying at high or ultrahigh temperatures has reduced drying time of long goods (e.g., spaghetti) to about 10 and 6 hours, respectively. Today, pasta manufacturing is totally automated with pasta presses capable of producing spaghetti at 3,500 kg/h and macaroni at 8,000 kg/h. 158-1

158-2


II. INGREDIENTS A. 1.

SEMOLINA Durum Milling

Semolina is the primary product of durum milling. A durum mill consists of a break-roll system, a sifter system, and a purifier system. Each break-roll consists of a pair of corrugated rolls that rotate at different speeds. The speed differential between the two rolls, typically 2.5:1, provides a shearing action that removes the bran and germ from the endosperm (2,3). Granulation of semolina is dependent on the number of corrugations per unit length. The sifter system sizes the granulated material through a series of sieves and returns large particles to a break-roll for further reduction. The fine material is collected as flour. The intermediate material goes to the purifier system, which uses air and sieves to further size and clean the semolina. Aspiration separates bran particles and other impurities from the semolina. Bran has a greater surface area to unit weight than semolina. Sieves associated with the purifier system size the semolina into specific particle size ranges (millstreams). The final granulation depends on the durum mill and the millstreams collected. The coarse granulation of semolina results in better flow properties than does the very fine particle size of flour (ⱕ212 µm). Federal Code of Regulation (4) defines semolina as the food prepared by grinding and bolting cleaned durum wheat to such fineness that, when tested by a prescribed method, it passes through a No. 20 sieve (840 µm), but not more than 3% passes through a No. 100 sieve (149 µm). It is freed from bran coat, or bran coat and germ, to such an extent that the percent of ash therein, calculated to a moisture-free basis, is not more than 0.92%, and its moisture content is not more than 15%. Semolina used for pasta processing is much smaller than the 840 µm maximum size and typically ranges in particle size from 450 to 150 µm. The particle size distribution of semolina from five commercial mills shown in Table 158.1 indicates that 60 to 70% of the semolina granules are between 425 and 250 µm. The short mixing times

used by new pasta presses require a fine granulation because small granules hydrate quicker than large granules. However, the hard grinding required for fine granulation can result in starch damage, which can increase cooking losses and decrease cooked firmness (5). Uniform granulation is important for even hydration. Hydration of semolina having a wide range of particle sizes will tend to result in the small particles absorbing too much water and the large particles absorbing too little. Both over-hydration and under-hydration will adversely affect dough development and will result in poor pasta quality. 2.

Semolina Quality

Semolina quality is determined by speck count, color, grit content, ash content, moisture content, protein content and quality, and microbial load. Defects in semolina are directly transferred to the pasta product (6,7).

a. Physical quality Speck count is a quality control measure for semolina commonly determined at the mill. Specks in semolina are generally brown or black. Bran is a common source of brown specks. Black specks originate from bran of diseased kernels, weed seeds, dirt, and insect parts. Black specks are more noticeable than brown specks in semolina and in pasta. Speck counts of five commercial semolinas ranged from 17 to 30 specks/10 in2 (Table 158.1). Speck counts greater than 40 specks/10 in2 are considered undesirable (8). High speck counts indicate a possible problem with grain cleanliness and grain quality and/or with the mill. Speck count is typically determined by a visual count of specks in a given area. The visual count of specks in semolina is generally determined under a constant light source for a fixed area of semolina that has been packed flat. Visual speck counts can vary greatly depending on the individual (9). Some mills have begun using digital image analysis to determine speck count (9,10). Ash content reflects the purity of the semolina. Semolina with high ash content generally will have a high speck count. Bran, weed seeds, and soil are high in ash.

TABLE 158.1 Quality and Particle Size Distribution (%) of Semolina from Different Mills1,2 Commercial Sample 600

425

Mesh size, µm1 250 180

149

⬍149

Protein (%)

Ash (%)

Specks no/10 in2

A B C D E

12.6 18.0 13.5 12.2 29.3

61.9 53.7 69.8 69.8 50.6

4.0 3.8 1.6 1.6 1.3

2.0 3.0 2.5 2.6 2.8

12.7 11.9 13.8 12.7 13.7

0.78 0.74 0.79 0.74 0.77

24 21 17 23 30

0.2 0.3 0.0 0.0 0.8

17.0 17.8 13.0 13.8 14.5

Corresponding mesh size and sieve number: 600 µm mesh ⫽ No. 30 sieve; 425 µm mesh ⫽ No. 40 sieve; 250 µm mesh ⫽ No. 60 sieve; 180 µm mesh ⫽ No. 80 sieve; 149 µm mesh ⫽ No. 100 sieve.

1

2

Protein and Ash based on 14% moisture.


For this reason, ash is commonly used in semolina specifications to ensure low contamination. In general, ash contents up to 0.80% are acceptable (11). Grit is metal, stone, or glass particles found in semolina. Grit can clog screens, block the die orifice, and/or damage the Teflon coating of the die. Damage to the die will result in defects in pasta such as grooves or tears. Grit that contaminates pasta poses a health threat as it can damage consumers’ teeth.

b. Compositional quality Semolina contains up to 80% starch and 2 to 3% nonstarch polysaccharides (12,13). Durum starch is composed of 70 to 75% amylopectin and 25 to 30% amylose (14,15). The impact of variations in amylopectin-amylose ratio on pasta extrusion is probably minimal, since dough temperature during extrusion ranges from 45 to 50°C, which is below gelatinization temperatures for durum starch. Starch is important in determining cooking quality of pasta (16), as variations in starch properties impact water uptake, gel consistency, and gluten matrix integrity during cooking. Starch damage in the semolina is a result of milling and of α-amylase catalyzed breakdown of starch during preharvest sprouting. Starch damage after milling is generally ⱕ5% (2,17). Some starch damage can occur during extrusion (18,19). Damaged starch is associated with increased water absorption and increased cooking losses. Nonstarch polysaccharides are primarily composed of arabinoxylan. Nonstarch polysaccharides have high water binding capacity. For example, nonstarch polysaccharides isolated from hard red spring wheat absorbed 6.3 to 6.7 times their weight of water (20). Semolina contains 1 to 2% lipid (21). Semolina lipids affect cooking loss and are important for color. Fatty acids can complex with amylose, which reduces water solubility of amylose and lowers cooking loss. Carotenoid pigments, particularly xanthophylls and lutein, are found in semolina and give pasta its characteristic yellow color (22,23). Semolina color can be evaluated by quantifying pigment concentration or by using a reflectance colorimeter. Johnston et al. (24) reported that reflectance values corresponded well with spectrophotometer values for pigment content. The apparent color of semolina varies with particle size. The yellow appearance decreases with a decrease in particle size, even though the yellow pigment content remains the same. The decrease in yellow color is due to greater light scattering with small than with large particles. Pasta quality is greatly affected by semolina protein content and quality. Semolina typically contains 12 to 16% protein (25,26). For good pasta quality, protein content of at least 12% on a 14% mb is preferred. The target minimum used by the durum wheat breeding program at North Dakota State University for advancing durum lines is 12.5% protein on a 14% mb.

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Semolina protein is composed of about 20% metabolic proteins (enzymes) and about 80% storage proteins. Storage proteins are composed of two classes of proteins, gliadins and glutenins, which, when hydrated and mixed, form gluten. Gluten gives dough its unique viscoelastic structure. Gliadin proteins are monomeric and range in size from 30,000 to 80,000 kDa (27). Gliadin proteins provide cohesion to the gluten matrix and are responsible for extensibility and viscosity of gluten (28,29,30). Glutenins are polymeric proteins consisting of subunits ranging in size from 12,000 to more than 130,000 kDa and can form polymeric protein complexes with molecular weights ranging from a few hundred thousand to several million (31). Unlike gliadins, subunits of glutenin proteins are capable of aggregation due to the formation of intermolecular disulfide bonds between subunits (32). Glutenins are responsible for the strength and elastic properties of gluten (33).

c. Gluten/dough quality Gluten quality is assessed by the wet gluten/gluten index (approved Method 38-12) and sodium dodecylsulfate (SDS) microsedimentation tests (34,35). The wet gluten/gluten index test involves washing semolina with a 2% salt solution. During washing, the semolina is kneaded to develop the gluten and to help remove the starch. After washing, the remaining wet gluten is centrifuged in a special centrifuge tube that contains a perforated plate. Gluten index is the ratio of the weight of gluten remaining on the top of the perforated plate divided by the weight of the wet gluten. The stronger the gluten, the less likely the gluten will be forced through the perforations during centrifugation. Gluten index has been used to identify weak, strong, and very strong gluten lines (36). In general, gluten index values ⬍5 indicate weak gluten and gluten index values ⱖ85 indicate very strong gluten. Durum grown in North Dakota and Montana typically has gluten index values of 45 to 55. The SDS microsedimentation test involves hydrating ground durum wheat in a SDS/lactic acid solution. Proteins of high molecular weight and those having strong interactions with starch are insoluble in the SDS/lactic acid solution and form a sediment. The height of the sediment is measured. A high SDS microsedimentation value is associated with good protein quality. A SDS microsedimentation value ⬍30 indicates weak gluten while a value ⱖ35 indicates strong gluten. Dough properties of semolina generally are assessed by mixograph in North America and by farinograph or alveograph in other parts of the world. Mixograph records the torque transmitted through the dough, which is recorded by a pen on chart paper (37). Mixograms from semolina of four cultivars grown in the Northern Plains are presented in Figure 158.1. Mixogram peak represents maximum dough

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FIGURE 158.1 Mixograms for semolina from durum wheat cultivars: Rugby (a), Munich (b), Lebsock (c), and Belzer (d). Durum wheat cultivars were grown in North Dakota.

development. Time to peak gives an indication of time required to fully develop the dough. The drop in the curve after the peak gives an indication of dough stability. Both peak height and width of the curve at peak reflect dough strength. Rugby (Figure 158.1a) is known to be a weak gluten cultivar. Even though Rugby has excellent protein content, it has weak dough properties (low peak height and narrow band width) compared to Belzer (Figure 158.1d). Most cultivars grown in North Dakota, Montana, and Canada are considered to have strong gluten, and produce mixograms similar to Munich and Lebsock (Figure 158.1b,c).

B.

OTHER INGREDIENTS

Other ingredients sometimes are added to semolina to improve its nutritional quality or to make specialty pastas. In the United States, pasta is enriched with vitamins (thiamin at 8.8 to 11 mg/kg, riboflavin at 3.75 to 4.85 mg/kg, niacin or niacinamide at 59.4 to 74.8 mg/kg, and folic acid at 1.98 to 2.64 mg/kg) and minerals (iron at 28.5 to 36.3 mg/kg and calcium (optional) at 1100 to 1375 mg/kg). Wheat bran, oat flour, and barley flour are examples of materials added to pasta to increase dietary fiber content (38,39,40). Flours of edible legumes, buckwheat, amaranth, and lupin have been added to improve the content and nutritional quality of protein in pasta (41,42,43). Vital wheat gluten, disodium phosphate, surfactants, and lipids have been added to improve cooking or textural quality of pasta, particularly pasta that is refrigerated, frozen, or canned (44,45). A variety of nonwheat and non-cereal products have been added to pasta to improve its nutritional quality (46). Code of Federal

Regulations (21 CFR Part 139) provides a list of ingredients that can be added to pasta products (4).

C.

WATER QUALITY

Water used in pasta processing should be pure, without chemical (including heavy metals) or bacterial contamination, have no off-flavors, and be slightly acidic, pH 6.6 to 6.9 (44). Water can have a maximum mineral content of 400 to 500 mg/L. Presence of iron salts should be avoided. Mineral impurities should not exceed 180 to 200 mg/L calcium and magnesium carbonates, 70 to 90 mg/L sulfates, 25 to 30 mg/L silicates, 5 to 10 mg/L chlorides, and 10 to 40 mg/L of organic matter (44,47,48).

III.

PASTA PROCESSING

Modern pasta presses are capable of producing 3,500 kg/h of long goods (spaghetti, vermicelli, and linguine) and up to 8,000 kg/h of short goods (macaroni, rigatoni, and ziti). Pasta processing can be divided into four stages: mixing, kneading, shaping, and drying.

A.

MIXING

1.

Hydration

The goal of the mixing stage is to uniformly blend and properly hydrate ingredients. Semolina is typically hydrated to 30 to 32% moisture content. The hydration level often has to be adjusted higher or lower to obtain proper consistency of dough containing nontraditional


ingredients during extrusion (42,49). When nontraditional ingredients are present, the amount of water added will depend on the overall moisture content and the water binding properties of the various ingredients. For example, nonstarch polysaccharides have a high water binding capacity, which can affect water distribution in dough systems during pasta processing and drying. Due to high levels of nonstarch polysaccharides in bran, whole wheat and bransemolina mixtures can require greater hydration to achieve proper dough development during extrusion than does semolina. Thus, it might take more water and time to properly hydrate whole wheat, compared to semolina, during the hydration/mixing stage of pasta processing (49,50,51). Rate of hydration is promoted by warm water, warm semolina, and small semolina particle size. Semolina temperature is generally only a concern in winter, as semolina stored in unheated bins can be very cold. Cold temperature will slow the rate of hydration. Semolina containing fine granulation will hydrate quicker than semolina with coarse granulation, because of the increased surface area and increased starch damage associated with small granulation. Uniform granulation is important for proper hydration. When semolina contains both large and small particles, the small particles tend to over-hydrate and large particles tend to under-hydrate. Over-hydration results in a soft, sticky dough. Pasta extruded from over-hydrated semolina requires more energy to dry and can stretch and stick together on the drying rods. Under-hydration results in a stiff dough which requires more energy for extrusion, generates more heat during processing, and can result in breakage problems for long goods hung on drying rods, as well as cutting problems for short goods. Complete hydration of the semolina particles is very important for proper dough development. Protein must be hydrated before gluten can form. As the storage proteins (gliadin and glutenin) hydrate, they change their conformation and begin to unfold and interact by forming intra-molecular and inter-molecular bonds. Without adequate hydration, regions in the dough and pasta will exist where no gluten is formed, which results in discontinuity of the gluten matrix. These regions of discontinuity are areas of structural weakness and will appear as white starchy areas in extruded pasta. 2.

158-5

evenly coat the semolina particles with water before entering the main mixer. The principal function of the main mixer is to provide time for thorough hydration. Most mixers contain two counter-rotating parallel shafts with paddles set so that they move the hydrated material forward (Figure 158.2). The retention time in the mixing chamber depends on the speed of the paddles and the length of the mixing chamber. The time must be sufficient to allow for proper absorption of water by the semolina or semolina-ingredients. Conventional mixers have a retention time of 10 to 20 min. New systems are being developed that reduce the retention time to 2 to 3 min or less (52). Pasta lines using short retention time mixers require fine granulation of semolina to achieve rapid hydration. Most dry pasta manufacturers apply a vacuum (⫺63 to ⫺80 kPa) either at the mixer or just before the extrusion barrel (53). Vacuum promotes hydration by eliminating surface tension associated with air and reduces pigment oxidation by lipoxygenase enzymes (23). Vacuum also prevents air from being trapped inside the developing dough. Air trapped inside extruded pasta will appear as a series of very fine bubbles resulting in a hazy appearance. If dried at high or ultrahigh temperatures, these bubbles will act as focal points for stress and ultimately result in checking of the finished pasta. Fresh or frozen pasta manufacturers generally do not use a vacuum system in the process. Their product is opaque. The air bubbles in the product do not seem to have any significant impact on the end product appearance or cooking quality. The added equipment to produce a vacuum, combined with the maintenance required, do not seem to justify the cost.

B.

EXTRUDING

Hydrated semolina passes through several zones in the extruder: conveying, compacting, kneading, relaxing, and

Mixing Equipment

The mixer is divided into two sections: premixer and main mixer. The flow of dry ingredients into the premixer is regulated by volume or weight using a volumetric or gravimetric feeder, respectively. Warm water (35 to 40°C) is sprayed onto the semolina in the premixer. It is important that the water not be sprayed onto the metal parts of the premixer, as semolina will accumulate on wet metal. Ingredient buildup in the mixer represents a potential source of microbial activity. High speed premixers are commonly used to rapidly and

FIGURE 158.2 Mixing chamber. (Photograph courtesy of Northern Crops Institute, Fargo, ND.)

158-6


extruding. Conveying, compacting, and kneading are associated with different regions of the extrusion screw. At the end of the screw, an extension tube often is used to allow the dough to relax before being extruded through the die. 1.

Screw Design

The extrusion screw is made of stainless steel or chromeplated steel. Traditionally, extrusion screws used in pasta processing were deep flighted with constant root diameter and uniform pitch the entire length of screw (Figure 158.3). Deep flights provided high screw conveying capacity and allowed high back pressure flow in the screw. Harper (53) reported a flight angle of 12 degrees for pasta extrusion. A sharper flight angle would increase mixing and decrease the conveying efficiency. Extrusion screws are designed with length-to-diameter ratios between 6:1 and 9:1 (53). The long length-to-diameter ratio results in low mechanical energy/unit throughput. Screws used to extrude pasta typically have diameters ranging between 12 to 20 cm. Extrusion screws have become more sophisticated. Some newer pasta presses have screws with variable pitch and variable root diameter. These screws often begin with a large dough cavity and gradually get smaller in pitch and larger in root diameter toward the end of the screw (52). As pitch decreases, the number of flights on the screw increases, which increases the screw surface-to-volume ratio and increases the conversion of mechanical energy to heat through friction. Similarly, increasing root diameter decreases flight depth, which increases the amount of energy and pressure applied to the dough. Thus, the new screws increase the amount of work applied to the dough, which has allowed a reduction in screw speeds from 20 to 40 rpm with traditional presses, to typically 18 rpm, and still maintain output (52).

FIGURE 158.3 Extrusion screw (a) and kneading plate (b). (Photograph courtesy of Northern Crops Institute, Fargo, ND.)

2.

Screw Function

The beginning of the screw is involved with conveyance. This part of the screw is not fully encased by the extruder barrel, but is exposed to allow the hydrated semolina from the mixer to be deposited onto the screw. The screw is choke-fed. The hydrated semolina is conveyed forward into the barrel as the screw turns. Compaction occurs once the hydrated semolina is conveyed inside the barrel. Pressure rapidly increases from 0 to 2 MPa after about two turns of the screw (54). Hydrated semolina exposed to 2 MPa begins to transition from a granular material into a fully compacted dough. Temperature changes during compaction are due to heat dissipation by friction against the barrel, which is then transmitted to the hydrated semolina. Le Roux et al. (54) reported that temperature rise was localized near the barrel wall and that the average temperature increase was about 5°C for a channel length of 10 cm under typical experimental conditions. After compaction, the remaining length of the screw is involved in kneading and conveyance of the dough toward the die. Pressure continues to slowly increase as the dough progresses toward the end of the screw. Deep flights provide high conveying capacity and high back pressure (up to 12.7 MPa) in the screw (48). At the end of the screw, pressure declines slightly as the dough moves into the extension tube, where it is allowed to relax before being extruded through the die. For a commercial press, the pressure at the die is generally 10 MPa.

3.

Dough Flow

Dough is conveyed in the channel of the screw. Dough moves toward the leading edge of the flight next to the cylinder wall and flows away from the flight near the root of the screw, which causes the dough to spiral down the channel. Forward flow and back pressure act together to knead the dough. During kneading, the gluten molecules are stretched and aligned according to rotational movement of the screw (55). The flow of dough is not uniform in the channel. Dough near the root of the screw moves much more slowly than the material near the cylinder wall (55,56). The material against the metal wall is worked and heated more than the material at the center of the screw channel. Le Roux et al. (54) did not observe any recirculating regions within the channel, but noted that a large zone existed at the channel bottom where the product moved very slowly, less than 10 mm/s when the barrel velocity was 50 mm/s. The uneven flow rate results in irregular dough development in the extrusion screw. At the end of the screw, the protein matrix is irregular, but is interconnected with some alignment of starch granules along the direction of flow (55). To reduce the heterogeneity of the dough, some


158-7

FIGURE 158.4 Extension tube (a), extrusion head (b), and kneading plate (c). (Photograph courtesy of Northern Crops Institute, Fargo, ND.)

FIGURE 158.5 Longitudinal grooves machined along the inner surface of the extrusion barrel. (Photograph courtesy of Northern Crops Institute, Fargo, ND.)

screw designs include a cut-flight or a kneading plate at the end of the screw (Figure 158.3). Kneading plates are stainless steel with small holes. Kneading plates split the dough into streams that recombine on the other side of the plate. An extension tube is sometimes placed after the kneading plate when extruding long goods (Figure 158.4). The dough flowing through the kneading plate enters the extension tube, which allows the dough a brief rest before entering the extrusion head where the dough flow is diverted downward 90° and forced through a die. At the beginning of the extension tube, after going through the kneading plates, the protein matrix is quite continuous and starch granules are clearly aligned. By the end of the extension tube, the dough is translucent and cohesive and is considered fully developed (55). Friction between the dough and the extrusion barrel and between the dough and the screw is necessary for compression of the dough and for conveyance of the dough through the extrusion barrel. Without friction, the screw would turn and the dough would remain stationary relative to the screw. To have proper conveyance of the dough, the friction associated with the barrel must be greater than the friction associated with the screw. Longitudinal grooves are machined along the inner surface of the extrusion barrel (Figure 158.5) to enhance the friction between the dough and the barrel. To reduce friction between the dough and the screw, the screw surface is made of polished stainless or chrome-plated steel. Experimental values of the coefficient of friction corresponding to the screw smooth surface have been estimated to be between 0.2 and 0.4. Le Roux et al. (54) reported that a screw with a deep flight and a uniform pitch and root diameter needed a barrel coefficient of friction greater than 0.5.

temperature during extrusion is 45 to 50°C (48,57). Protein begins to denature at dough temperatures ⬎50°C. Denatured storage proteins (gliadins and glutenins) are unable to form gluten. High dough temperature during extrusion will cause a soft sticky product when cooked. Excess heat generated by friction during extrusion is removed by use of a water jacket which surrounds the extrusion barrel. A high volume of warm water is used to maintain both the barrel and dough temperature near 45°C. Circulating cold water in the water jacket would result in overcooling the dough at the barrel surface, which would adversely affect dough viscosity. Frictional heating of dough can be calculated by knowing the moisture content of hydrated semolina and the specific mechanical energy (SME, kJ/kg) for pasta extrusion. Heat capacity of wheat dough can be estimated using the equation presented by Baird and Reed (58),

4.

5.

Frictional Heating

Heat is generated by friction between the dough and metal surfaces of the barrel and the screw. The target dough

cp (kJ/kg°C) ⫽ 1.44 ⫹ 2.74Xw where cp is heat capacity and Xw is moisture content. If the moisture content of hydrated semolina is 31%, then the heat capacity of the hydrated dough would be 2.29 kJ/kg°C. If the SME is assumed to be 70 kJ/kg (59,60), then the heat generated during extrusion would be 70 kJ/kg divided by the heat capacity of the hydrated dough (2.29 kJ/kg°C) to give a temperature rise of 31°C. If the hydrated semolina entered the extrusion screw at 35°C, it would exit the extruder at 66°C. With a target dough temperature of 45°C, two thirds of the heat would need to be removed by a circulating water jacket. These results are similar to those reported by Harper (53) and Hoskins (56). Dough Viscosity

Dough systems have non-Newtonian flow properties. Dough is a viscoelastic system. Viscoelastic properties of

158-8


dough are dependent on gluten strength of the semolina used, temperature, hydration, and amount of work applied to the dough. Dough viscosity decreases with increased temperature and hydration. Dough viscoelastic properties are decreased when overworked. This can be seen in the mixograms in Figure 158.1, where dough strength declines with time after reaching peak height. Apparent viscosity of a dough system can be described by:

The die is attached to the end of the extrusion head (Figure 158.6). A die is composed of a support and multiple inserts (Figure 158.7). Die support is made from bronze or stainless steel. The support must be capable of withstanding a tremendous amount of pressure over time without yielding. Stainless steel can tolerate higher pressures, but tends to retain more heat than bronze (62). Most industrial die supports are made from stainless steel.

Inserts are miniature dies that are housed inside holes that have been bored into the supports (Figure 158.7). Inserts are generally made from bronze, due to their low heat retention. The advantage of using inserts is that worn inserts can be replaced easily without having to return the entire die to the manufacturer. The number of inserts in the die will determine production output and must be balanced with the pasta press. Too many inserts can weaken the die, which could cause the die to bend under pressure, could reduce the density of the product, and could cause strands to overlap during extrusion. Too few inserts can cause excessive back pressure that can damage the die and/or the extruder and can reduce production output. Die supports are typically 100 mm thick, and their inserts are typically 20 mm thick (63). Thus, dough flows into the hole in the die support, then into and through the orifice in the insert. Pasta comes in hundreds of shapes, which are determined by the flow of dough through the die orifice during extrusion. For example, inserts for spaghetti contain a round orifice, whereas inserts for shells have a horizontal orifice that is slightly larger at the center. The dough will flow faster at the center than at the ends of the orifice in the shell die, which causes curvature (62,63). The orifice in the insert is often coated with a fluorocarbon polymer such as Teflon. A Teflon-coated orifice will have a low coefficient of friction, which will reduce back pressure needed for extrusion and increase the rate of extrusion (53,56). Dies with Teflon inserts are used when a smooth translucent surface is desired. Surface texture affects cooking and culinary properties of the product. Pasta with a rough surface has a greater exposed surface area, which tends to absorb water quicker and retain more sauce when compared to pasta with a smooth surface.

FIGURE 158.6 Extrusion head (a) and die (b). (Photograph courtesy of Northern Crops Institute, Fargo, ND.)

FIGURE 158.7 Die support (a) and inserts (b). (Photograph courtesy of Northern Crops Institute, Fargo, ND.)

µ a ⫽ mv n⫺1 where µ a ⫽ apparent viscosity, m ⫽ consistency, v ⫽ shear rate, and n ⫽ flow index. Using a dough made from semolina, Le Roux et al. (54) demonstrated that the power law remained valid over a wide range of shear rates, typically 0.1 to 1000 s⫺1, which encompasses the range expected in pasta extrusion. A typical shear rate for a commercial pasta press has been estimated to be 5 s⫺1 (59). Power law (or flow) index, n, increased with hydration, but remained within 0.4 to 0.5 (54). Food extrudates exhibit flow indices between 0.25 and 0.5 (61). Within normal extrusion temperatures, 45 to 55°C, consistency, m, varies with hydration and temperature according to exponential laws. 6.

Die Assembly


The shape of the die support depends on the product produced. Short goods generally have a round die support, while long goods generally have a rectangular support. Extruded long goods are collected by a spreader, which spreads long goods on sticks and cuts the long goods from the die. Since product discharge is not uniform across the die, the strands are cut to a uniform length and the trim is reintroduced into the mixer via the trim return system. Pasta hanging on the sticks is conveyed to the dryer. Dried pasta is cut to length and the trim is reground and reintroduced into the semolina at amounts up to 15% (64,65). Extruded short goods are cut by a special rotary cutter with one or more blades. The cut pasta falls onto trays or a conveyer where it moves into the dryer. Short good length is determined by extrusion rate and speed of the rotary cutter. An uneven rate of discharge across the die will result in variable lengths of the short goods. 7.

Extruder Output

In-depth mathematical descriptions and discussion of extrusion can be found in several references (53,66,67). Extruder output is described by the following equation: Extruder output ⫽ drag flow ⫺ pressure flow ⫺ leakage flow. Drag flow is the forward movement of the dough due to the relative motion between the screw and the barrel (68). Drag flow increases with increased screw speed and flight depth to screw diameter ratio. Pressure flow is the backward flow of dough in the screw channel due to a pressure gradient. Leakage flow is the backward flow between the flights and the extruder barrel due to a pressure gradient. Pressure flow is proportional to the pressure gradient across the screw length (69). An increase in flight depth to screw diameter ratio increases drag flow more than pressure flow. Flight depth to screw diameter ratio for a commercial pasta press is typically 0.25 to 0.33 (59). Both drag flow and pressure flow increase with increased pitch angle (70). Leakage flow is related to pressure flow. Leakage flow occurs within the clearance between screw flights and the barrel and within the longitudinal grooves in the barrel wall. A typical clearance between the screw and barrel is 0.5 mm (66). The clearance will increase over time due to wear. An increase in clearance will be manifested by a decrease in output and an increase in energy transferred to the product, with associated increase in dough temperature. Hoskins (56) estimated that the ratio between pressure and drag flow in a pasta press screw is about 0.6. If pressure flow and leakage flow are combined, then drag flow and pressure flow can be estimated if press output is known.

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For example, assume that a pasta press extrudes 3,500 kg/hr of long goods. Then: Drag Flow ⫺ Pressure Flow ⫽ 3,500 kg/hr and Pressure Flow/Drag Flow ⫽ 0.6. Drag Flow ⫻ 0.6 ⫽ Pressure Flow Drag Flow ⫺ Drag Flow ⫻ 0.6 ⫽ 3,500 kg/hr Drag Flow (1 ⫺ 0.6) ⫽ 3,500 kg/hr Drag Flow ⫽ 8,750 kg/hr Pressure Flow ⫽ 5,250 kg/hr The amount of energy transferred to the product during extrusion is designated as SME (kJ/kg). SME transferred to the pasta is calculated as the mechanical energy (kJ/s) to extrude pasta divided by the amount of pasta processed (kg/s). Mechanical energy required to operate the empty press is subtracted from the mechanical energy required to operate the press under load. Dough temperature and hydration level greatly affect SME required for extrusion. Abecassis et al. (60) reported that an increase in dough temperature or hydration decreased dough viscosity and subsequently decreased SME. A SME of 70 kJ/kg is typical for pasta extrusion, under normal operating conditions (54,59,60).

C.

PASTA DRYING

Like many other foods, pasta is dried to give a longer storage time. Shelf-life of pasta products is commonly listed as two years. Vitamins degrade over time. When the vitamin claim on the nutritional label is no longer valid, then the processors must declare the pasta expired. Therefore, fortification level and the levels claimed on the label determine the shelf-life. Typically, industry will dry pasta to 12% moisture, although the Federal Code of Regulations (4) allows the moisture content to be as high as 13%. Pasta is a difficult product to dry. Pasta’s low moisture, coupled with dimensional changes during drying, can result in checking (stress cracks in the product). Checking occurs when the stresses in the product exceed the strength of the pasta. As drying temperatures increase, the properties of the pasta change, due primarily to inactivation of enzymes and protein denaturation. A list of changes can be found in Pasta Technology Today (47) and in Pasta and Noodle Technology (71). The pasta drying process is generally divided into three main stages: predrying, final drying, and cooling/stabilizing stages. 1.

Drying Stages

a. Predrying The predrying stage begins when the product exits the die. Typically, ambient air (possibly heated) flows at the face of the die, which provides some surface drying of the

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product. This is referred to as the initial predrying. Initial predrying serves several purposes. The primary purpose is to dry the surface of the product sufficiently to prevent pieces from sticking together. Initial predrying increases the rigidity of the pasta surface, which minimizes collapsing or deforming of hollow products (such as elbows, shells, and ziti) when they drop onto a solid surface or have the weight of other pieces resting on them. The amount of moisture removed during the initial predrying step is small (⬍1%), but is a necessary step for maximum pasta quality. After initial predrying at the die, the product is conveyed to the predrying section of the dryer. In the case of long goods, the product is collected onto rods, which are mechanically conveyed directly into the dryer. In the case of short goods, the product can either be dropped directly into the dryer (if the extruder is positioned above the dryer) or conveyed to the dryer, usually by a mesh belt conveyor or other conveying system able to keep airflow on the pasta. The moisture content of the pasta entering the predryer section of the dryer is ~29 to 31%. The predrying stage is ~10% of the total drying time and removes about one-third of the total water in the pasta. The high drying rate is possible because the pasta is still in a plastic state, which prevents a buildup of stress in the pasta. Depending on the temperature of the dryer, the pasta will remain plastic as moisture content is decreased to 18% (for ultrahigh temperature, ⱖ80°C, drying) or 21% (for low temperature, ⬍60°C, drying). Water is one plasticizing agent, which allows the product to deform without creating residual stresses. Temperature is another factor that helps plasticize the product. The moisture content at which the pasta transitions from plastic to elastic state decreases as temperature increases. The predrying stage (and the final drying stage) will generally have a “resting” stage (sweating stage) scheduled in the drying profile or built into the dryer design. During the “resting” stage, the driving force for the drying (temperature and humidity of the air) is low enough that the product is not losing water to the surrounding air. The moisture inside the product redistributes during this time. The core, which has higher moisture content than the surface of the product, will lose water to the surface of the pasta product. Redistribution of the moisture helps even out the dimensional change and minimizes stress due to moisture loss in the product. If enough moisture reaches the surface, the surface becomes plastic and stresses are relieved.

b. Final dryer The product will have ~18 to 21% moisture content upon entering the final dryer and will exit the dryer at ~12%. The rate of moisture removal in the final dryer is critical, because the product is in an elastic state. If drying is too fast, the stresses near the surface of the product will

exceed the strength of the pasta and checking will occur. While stress accumulated during the final drying can result in checking, the root cause of stress in the product may be due to events that occur earlier in pasta processing. For example, product thickness will increase as the insert wears during extrusion. A thick product is more difficult to dry; and if no changes are made in the drying profile, checking will occur.

c. Cooling/stabilization stage The product is brought to near ambient temperature and exposed to ~50% relative humidity during the cooling/stabilization stage. The product is equilibrated with the ambient environment to minimize the possibility of checking due to environmental stresses. Moisture is more evenly distributed as the product moves through the cooling stage. Some of the water near the core of the product will migrate toward the surface, which will relieve some of the stresses that accumulated during the drying process. One difficulty in humid environments is that the surface of dried pasta will begin to absorb moisture from the atmosphere. Stress occurs as the absorbed moisture causes the surface of the product to expand. This stress, coupled with the residual stresses in the product from drying, may result in checking of the product. Even with little or no residual stress, checking will occur in pasta if the relative humidity is ⱖ75% (72). To prevent checking in humid environments, dried pasta may need to be packaged within 3 hours of exiting the dryer. Advances in drying technology have resulted in three drying intensity categories that describe processing temperature and relative humidity ranges: conventional drying, high temperature drying, and ultrahigh temperature drying. Conventional drying (low temperature drying) imitates the conditions that occur in the Mediterranean region of Italy, the origin of pasta. The drying of pasta originally took place in the open air or in a ventilated room. High temperature drying is a drying cycle where the maximum temperature applied is between 60 and 80°C. Whereas, ultrahigh temperature (very high temperature or tres haute temperature) is a drying cycle where the temperature applied reaches ⱖ80°C. As drying temperature increases, the relative humidity of the drying cycle increases and drying time decreases. Spaghetti (a thick product) may take about 24 hours to dry with low temperature drying, 12 hours with high temperature drying, and 5 hours or less with ultrahigh temperature drying. The exact drying cycle used is dependant on the product being dried and the equipment manufacturer, so exact drying profiles are not provided here. The drying profiles used by the Durum Wheat Quality and Pasta Processing Laboratory in the Department of Cereal and Food Sciences at North Dakota State University can be found in Yue et al. (14).


2.

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Moisture Migration during Pasta Drying

Factors that have a strong influence on drying rate include pasta thickness (doubling the thickness of the pasta will reduce the drying rate 75%) and air temperature, while factors that have a small influence on drying rate include relative humidity and airspeed (73). Pasta is a dense, continuous product with very few capillaries. Like similar products (soap, gels, pastes), pasta drying is unsteady-state and can be modeled using Fick’s second law:

∂X ∂ 2X ᎏ ⫽ Dᎏ ∂t ∂ x2 ∂ 2X 1 ∂X ∂X ᎏ ⫽D ᎏ ⫹ ᎏ ᎏ r ∂r ∂t ∂ r2

冨

(for a slab) or

冨

(for a cylinder)

where X ⫽ moisture content, t ⫽ time, x ⫽ ½ the thickness of the product, and r ⫽ radius of the product. Fick’s second law describes drying in materials where liquid water is diffusing toward the surface of the material. Research indicates that Fick’s second law is not a perfect model for the pasta drying process, because the moisture gradient in the pasta is steeper than the Fickian model predicts (74,75). However, Fick’s second law may be a reasonable starting point. A wide range exists for diffusivity values (at least two orders of magnitude between the highest and lowest reported values), but the majority of results include diffusivity values in the range of 25 ⫻ 10⫺12 m2/s to 50 ⫻ 10⫺12 m2/s (73,74,76). Diffusivity is unaffected by total pressure in the drying environment, indicating that moisture migrates in pasta as liquid or adsorbed water and not as water vapor (76). 3.

applying high temperature in the dryer until the pasta reaches a water activity of 0.7 (81). The Maillard reaction is and will continue to be a concern in pasta production. The industry has accepted high temperature and ultrahigh temperature drying as optimal drying cycles, producing the highest quality pasta. This higher quality comes with the risk of its own defect: “browning” during drying. Most pasta produced under high temperature and ultrahigh temperature drying cycles will not have the browning defect, but there are factors that can increase the likelihood of Maillard reactions occurring. Factors directly caused by the semolina or the drying cycle have been researched to some degree (77,79,80). However, there are factors not easily anticipated in a research environment, such as “holding” of product in the dryer due to other equipment problems, effect of equipment wear on the amount of starch damage in the product, and poorly calibrated sensors. While the Maillard reaction may not be a concern for most pasta processors today, it would be unusual not to see some reddish-colored pasta on the shelf in a local supermarket. 4.

Checking in Pasta

There are two types of checking in pasta: predryer checking and final dryer checking. Predryer checking, as the name implies, occurs in the predryer section. This defect will appear as spots in the finished product. The spots will generally be round and be near the surface of the product. These spots may appear similar to spots due to insufficient vacuum during extrusion. Final dryer checking is easily identifiable. In the spaghetti, final dryer checking will appear as an oval on the surface of the spaghetti, and can be described as a “crescent,” “canoe,” or “half-moon” (Figure 158.8). In other products, final dryer checking

Maillard Reactions during Pasta Drying

Maillard reactions result in the development of a red or orange color, which is significantly different from the golden color expected in pasta. Maillard reactions can also result in the development of off-flavors in pasta. The Maillard reaction requires a reducing sugar and a free amino group. The reducing sugars for the Maillard reaction are provided by damaged starch. The damaged starch can be the result of growing conditions, milling, mixing with water, or extrusion (77,78,79). The free amino group is generally from a lysine residue in protein. Maillard reactions can occur during high temperature and ultrahigh temperature drying. Water activity of 0.75 to 0.80 (80) and/or a moisture content of ~15% (77) are optimum moisture conditions for the Maillard reaction to occur in pasta. Pasta in equilibrium with a high relative humidity environment (97% rh, water activity ⫽ 0.97) is unlikely to develop an off-color during drying (79). The development of off-colors also may be limited by not

FIGURE 158.8 Final dryer checking in rotelle (a), elbow macaroni (b) and spaghetti (c). Note partial and full oval shaped checking in spaghetti and checking on the outside and on the face of vanes of rotelle.

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will be visible as cracks in the pasta. The stresses causing final dryer checking are created during final drying, but the actual checking of the product may not occur for up to 10 days after drying. Both predryer and final dryer checking are caused by removing moisture too quickly from the pasta. Predryer checking can be corrected by increasing the relative humidity and or the residence time in the drying chamber. These changes may ensure that the moisture content going into the final dryer is appropriate for that dryer. Because each dryer is different, knowing the operating conditions and properties of the product when the production line is running correctly cannot be over-emphasized. While general values of moisture content at various stages in the drying can be found for “typical” dryers, knowing the exact conditions in the dryer that you are trying to troubleshoot is extremely valuable. Final dryer checking can be corrected by increasing the relative humidity in the final dryer section. Increased relative humidity in the final dryer section may slow drying enough to require increased drying time. Final dryer checking can be due to improper predryer conditions. Final drier checking may also be due to pasta leaving the predryer at too high of a moisture content.

Frequently, only minor changes in the dryer will correct a checking problem. For example, a pasta manufacturer was producing a penne product that was checking badly. The plant producing this product eliminated the checking by increasing the relative humidity in the final dryer section by 3%. The root cause of this plant’s problem appeared to have been worn die inserts. The thickness of the product had increased enough to cause these drying problems. 5.

Pasta Defects/Troubleshooting

Defects in pasta production do occur, and being able to locate the source of the problem is important in solving the defect problem. Having accurate data on the optimal drying parameters as measured and controlled in the process being investigated cannot be over-emphasized. Having a “baseline” for comparison will help to solve many problems quickly. Table 158.2 lists some of the more common defects found in dried pasta along with the likely cause(s).

IV. GOOD QUALITY PASTA The goal of pasta production is to make a dried pasta that is translucent (although some portions of the market prefer pasta extruded through a bronze die, which will result

TABLE 158.2 Troubleshooting Pasta Defects Defect

Possible Cause

Specks in the pasta

Raw ingredients

White spots and streaks on the pasta (see Figure 158.9)

Wide semolina particle size distribution Very dry semolina Insufficient hydration time Loss of vacuum on the extruder

Circular bubbles in the pasta

Predryer stress Deformed pasta Cracks (checking) in pasta

“Red” or “orange” color in the pasta

Varying dough moisture at die Poor die pressure distribution Drying too fast in the final dryer section “canoe” or “crescent” shape in spaghetti (see Figure 158.8c). Cracks in other products (see Figures 158.8a,b) Mechanical damage (sections of pasta chipped out of the ends of tube products (see Figure 158.10) Post-dryer checking Rough mechanical handling

Maillard reaction occurring in the pasta

Possible Solution Check for grit and black or brown specks. Ensure it meets the plant’s specifications. Narrow the size distribution of semolina Use semolina with a higher moisture content Increase residence time in the mixing chamber Make sure vacuum system is clean and operating properly Ensure relative humidity the product is exposed to from the die and through the predryer is correct Ensure semolina and water flow rates are consistent Check for possible obstructions to flow at the die Check conditions in the final dryer, possibly increase relative humidity to slow the drying process Ensure product does not drop far or onto too solid a surface when conveyed Post-dryer checking can occur if ambient relative humidity is too low (⬍10%) or too high (⬎75%) and the product is exposed to the conditions for 3 hours or more Pasta may be dropped from too great a height, fracturing the product. In tubular products, this will result in squares or triangles being knocked out of the ends, instead of cracks on the entire length of product Check RH and temperature in the final dryer section Check semolina for excessive sugars or ash (indicating a high semolina extraction)


FIGURE 158.9 Rigatoni showing mechanical damage. Cracks in the pasta are only at the ends of the pasta and broken areas are roughly rectangular.

FIGURE 158.10 Hydration problem. Rotelle viewed from the end. Note hydration streaks about 1/3 the distance from the center of the product.

in a rough surface) and free of visual defects such as checking, hydration spots, and specks. This pasta should cook to a non-sticky, firm product with little cooking loss, and resist overcooking.

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56. CM Hoskins. Macaroni production. In: SA Matz. ed. Cereal Technology. Westport, CT: AVI Publishing, 1970, pp 246–299. 57. A Debbouz, C Doetkott. Effect of process variables on spaghetti quality. Cereal Chem 73:672–676, 1996. 58. DG Baird, CM Reed. Transport properties of food doughs. In: C Mercier, P Linko, JM Harper.eds. Extrusion Cooking. St. Paul, MN: Am Assoc Cereal Chemists, 1989, pp 205–234. 59. JM Harper. Food extruders and their applications. In: C Mercier, P Linko, JM Harper. eds. Extrusion Cooking. St. Paul, MN: Am Assoc Cereal Chemists, 1989, pp 1–15. 60. J Abecassis, R Abbou, M Chaurand, M-H Morel, P Vernoux. Influence of extrusion conditions on extrusion speed, temperature, and pressure in the extruder and on pasta quality. Cereal Chem 71:247–253, 1994. 61. L. Levine. Extruder screw performance, Part V. Cereal Foods World 46:169, 2001. 62. D Maldari, C Maldari. Design and performance of pasta dies. Cereal Foods World 38:807–809, 1993. 63. A. Barozzi. Pasta dies: Design techniques – production systems. Italian Food Beverage Technol 8 (October):22–31, 1996. 64. BJ Donnelly. Pasta regrinds: Effect on spaghetti quality. J Agric Food Chem 28: 806–809, 1980. 65. K Fang, K Khan. Pasta containing regrinds: Effect of high temperature drying on product quality. Cereal Chem 73:317–322, 1996. 66. JM Harper. Extrusion of Foods, Vol. I. Boca Raton, FL: CRC Press, 1981, pp 7–92. 67. L Levine. Extrusion Processes. In: DR Heldman, DB Lund. eds. Handbook of Food Engineering. New York: Marcel Dekker, 1992, pp 621–666. 68. GJ Rokey. Single-Screw Extruders. In: MN Riaz. ed. Extruders in Food Applications. Lancaster, PA: Technomic, 2000, pp 25–50. 69. L Levine. Filling extruder screws and developing pressure in screws. Cereal Foods World 43:665–666, 1998.

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159

Seafood Products – Science and Technology

Barbara Rasco

College of Agricultural, Human and Natural Resource Sciences, Washington State University

Gleyn Bledsoe

Institute of International Agriculture, Michigan State University

CONTENTS I. II. III. IV. V. VI. VII.

General Information ........................................................................................................................................159-1 Processing Technologies ..................................................................................................................................159-4 Cold Storage ....................................................................................................................................................159-4 Live Handling ..................................................................................................................................................159-4 Handling and Shipping Live Fish ....................................................................................................................159-5 Harvesting ........................................................................................................................................................159-5 Refrigeration ....................................................................................................................................................159-6 A. Vacuum Packaging ..................................................................................................................................159-6 VIII. Freezing ............................................................................................................................................................159-7 A. Types of Freezing ....................................................................................................................................159-8 B. Packaging ................................................................................................................................................159-9 IX. Cured and Salted Products ..............................................................................................................................159-9 X. Smoking..........................................................................................................................................................159-10 XI. Dehydration ....................................................................................................................................................159-11 XII. Fermentation ..................................................................................................................................................159-12 XIII. Thermal Processing ........................................................................................................................................159-12 XIV. Summary ........................................................................................................................................................159-13 Acknowledgments ......................................................................................................................................................159-13 References ..................................................................................................................................................................159-13

I. GENERAL INFORMATION Dr. George Pigott coined the phrase “aquatic food products” because it was difficult to find one word to adequately define edible animals and plants from the aquatic environment. “Seafood” denotes only food from the sea or marine environment, omitting freshwater plants and animals. Similarly “fish” is not an all encompassing term for all of the different animals (both vertebrate and invertebrate) and plant products humans take from the water and eat. The FDA recently defined fish to mean: “fresh or saltwater finfish, crustaceans, other forms of aquatic life (including, but not limited to, alligator, frog, aquatic turtle, jellyfish, sea cucumber, and sea urchin and the roe of such animals) other than birds or mammals, and

all mollusks, where such animal life is intended for human consumption.” (Title 21 of the Code of Federal Regulations. Part 123.3(f)).

Over 350 species of mollusca (e.g. clams, oysters, snail, octopus), arthropoda (e.g. lobsters, crabs, shrimp and crayfish), reptilia (e.g. turtles, alligators), amphibia (frogs), gastrapoda (whelks), holothurians (sea cucumbers), and chordata (finfish) are used as food (1,2). Furthermore, aquatic plants and marine mammals are important components in the diets of people from many cultures. Interesting, unconventional, unique and somewhat bizarre traditional foods are made from aquatic products. Numerous tissues are consumed besides the muscle from aquatic animals. Some examples of the wide variety of aquatic food products are presented in Table 159.1 using 159-1

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TABLE 159.1 Examples of Aquatic Foods Item Whole animal

Plants Liver/hepatopancreas

Tongue Heart/kidney Stomach/throat Brain/head Spinal column Eyes Gills Gonadal tissue, roe

Gonadal tissue, milt Testicles Penis Skin Connective tissue Adipose tissue Oil (head or adipose)

Bodily fluids Fin Shell Exoskeleton Bone

Source

Examples

Mollusc Crustacea Fish Insects Marine plants, various Marine plants Finfish, various Marine mammals Crustacea Finfish, various Marine mammal Finfish, various Marine mammal Finfish, various Finfish, various Marine mammal Finfish, various Finfish, various Finfish, various Mollusk Fin fish Crustacea Reptile Mollusk Finfish Marine mammal Marine mammal Finfish, various Finfish, various Marine mammal Finfish, various Marine mammal Mollusks Finfish Finfish, various Mollusk, various Crustacea, various Finfish, various

species common to Westerners. This is nothing close to an exhaustive list of the different types of products made from aquatic plants and animals, but it provides some insight into how diverse aquatic food products can be. For many species the muscle tissue plus certain organs are eaten together. For example, lobster, crab or scallop meat is eaten with the hepatopancreas and roe (if present) by individuals who like it. The entire organism is commonly consumed (e.g. oysters, limpets, some clams, small fish). For some species only certain organs are consumed, such as sea urchin roe. Roe is eaten in a salted or seasoned form as caviar from a variety of different fish and invertebrates. The most common vertebrate species for the production of roe products

Clams, oysters, octopus, limpet Small crab Sardines, smelt Sea vegetables, nori Food additives (agar, carrageenan) Cod, pollock, salmon Seal, walrus, whale Lobster, crab Cod, halibut Seal, whale Salmon, tuna Seal, whale Cod Salmon, cod halibut (cheeks) Whale Sturgeon Salmon Salmon Sea urchin Sturgeon, herring, salmon Lobster, crab, shrimp Turtle eggs Oyster Salmon Seal Seal (East Asian medicinal) Salmon, rockfish Shark (cartilage) Seal, whale Herring, salmon, shark, cod Whale (blubber), seal (blubber and extracted oil) Squid (ink) Blood Shark Oyster (as nutritional supplement) Shrimp Smelt, sardine

are flying fish roe (for tobiko used on sushi), sturgeon and paddlefish for black caviar, salmon for salmon caviar or ikura, herring roe for kosunoko, cod roe for tarako, and pollock roe for mentaiko (also spelled mentiko). Salted fish roe from cod or capelin are used as a component of pastes or spreads. Lower grade sturgeon can also be pressed and sliced or mixed with butter or soft cheese and used as a spread. Roes can be partially dehydrated; for example, sujiko, which made by salting and then pressing skeins of sockeye salmon roe. A dried roe product is made from mullet roe and is called karasumi. High levels of wax esters give mullet roe a unique chewy texture. Flavored or seasoned roes are becoming increasingly popular. For example, chili flavored mentaiko is a widely used


condiment in Korea, and salmon roe treated with soy and other seasonings is popular in Japan and throughout Asia. Sauces and salad dressings often contain fish roe. Fish roe and milt are served in soup or lightly sautéed. Milt can be smoked and made into spreads. Uni, is a colored, flavored alum treated gonadal tissue from the sea urchin used primarily for sushi. Steamed fish, soups and stews are made from the whole fish, with tissue from the head (such as the cheeks and tongue) and the eyes reserved for special guests. Cheek tissue, particularly from larger fish such as halibut are commonly served separately. Halibut cheeks are the muscle from underneath the eye of the fish. Since halibut can reach up to 250 pounds, the cheeks can be several ounces each. In the recent past, halibut cheeks were sometimes mislabeled as scallops or sea scallops, but with the current high demand, halibut cheeks are a popular food in their own right. Certain tissues of a fish may be fried (skin, intestines) and served as such. Skin and intestine are often incorporated into stews, soups, or ground meat preparations. These tissues are dried for use in a seasoning or base. In addition, fish skin can be cured into leather for belts, wallets and shoes. The most common types of leather in Western markets are from shark and skate, sturgeon, eel and salmon. Specialty dishes use fish connective tissue as a featured ingredient. Fish maw soup (from finfish stomach and throat tissue) and shark fin soup both take advantage of the thickening properties of these connective tissues for the preparation of various dishes. Gelatin can be recovered from fish skin and connective tissue for use in traditional foods and, if appropriately manufactured, as kosher gelatin. Shark cartilage is both a food component, and when dried, a nutritional supplement. A wide variety of condiments, sauces and seasoning are made from aquatic animal tissues. Most common are fish oils forming the base for margarine, dressings, sauces and condiments. The omega-3 fatty acids in high quality stabilized fish oil are a nutritional supplement commonly sold in capsule form and more recently as a stabilized ingredient in functional foods. The importance of fish oil in the diet is a continuation of a longstanding trend. Prior to the widespread availability of vitamin supplements and fortified milk, cod liver oil was a primary source of vitamin D. Oil from marine mammal tissue (e.g. seal oil or ooksook, and whale blubber) remains popular in the traditional diets of people above the 47th parallel in reflecting the dietary patterns of the samoyed peoples who migrated throughout Alaska, the Pacific Northwest, Scandinavia, British Isles, Russia and Japan. Marine mammal oils can be rendered at room temperature or rendered with heat. These oils may be consumed fresh or aged. These oils serve as a condiment or dipping sauce and are used in a similar fashion as olive oil in Mediterranean cuisines.

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Aquatic animal foods are valuable, and in some cases, incredibly expensive. Because of the value, widespread poaching of shark and sturgeon have led to severe restrictions and closures in these fisheries. Fortunately, sturgeon can be cultivated, hopefully saving important Caspian Sea species from extinction. Reasonably good Beluga sturgeon caviar at retail routinely runs $200 per ounce or more. The most expensive aquatic food product however is gold “Almas” caviar from Beluga sturgeon (Huso huso) purported to be over 100 years old, is packaged in 24 karat gold tins and sells for US$ 23,000⫹ per kilogram (3). There have been few efforts to cultivate shark. Sharks are slow growing and breed after several years of age. Some sharks bear live young, meaning that the number of offspring from each female is small. Fishing pressure on large sharks is threatening the viability of sub-populations, particularly in the waters surrounding Southern Asia. The value of dried shark fin varies greatly by size and appearance, but for good specimens, the value exceed hundreds of dollars per pound. A variety of nutritional supplements and traditional medicines are made from aquatic organisms. Besides fish oil, calcium supplements are made from oyster shell. Oyster milt and other reproductive tissues are purported to have medicinal properties. This includes the use of the seal penis in Asian medicine, a suitable, albeit a misbranded substitute for tiger penis in traditional Chinese medicine. Numerous marine plants and their extracts are common sources of vitamins, minerals, and phytoactive components with alleged beneficial properties. Aquatic products have been consumed from ancient times and were probably the first animal sources of protein in the human diet. Humans settled near water and have captured fish and harvested mollusks since prehistoric times. Commercial fishing was part of the ancient culture of Egypt where ancient fishing vessels have been unearthed that vary little from designs of vessels used in artesian fisheries today. Asian and European civilizations have consumed fish for thousands of years before recorded history. Native Americans can trace their use of fishery products back 3,500–10,000 years. Numerous archeological sites containing bone, shell, fishing hooks from bone and stone net weights have been recovered in the United States (4) with important sites at Lake Ozette in Washington state verifying the use of cedar bark nets among coastal tribes going back several hundreds of years. In addition to wild harvest, culturing fish and ranching or impounding fish in ponds dates back a thousand years or more. Among the Makah, live fish were harvested off the northern most tip of the continental United States and then placed into ponds and held until consumed. This technique provided the people with a source of food when the weather was too rough to fish. It also provided a means to have fish species that would otherwise be out of season. Similarly aquaculture and impounding of fish have been practiced for centuries on the islands of Hawaii and in

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Polynesia. Commercial aquaculture and fishing were key industries in ancient Asian socities and in the West during the Roman Empire with archeological sites indicating the widespread use of aquaculture in Italy and North Africa. Today aquatic food products are the major source of high quality animal protein to a quarter of the world’s population. The consumption of aquatic food products is increasing internationally, with aquaculture being the source of any new product to meet market demand. By 2020, a deficit of 10% or more in the world-wide supply of aquatic foods is predicted, even with the growth of cultured product increasing rapidly. International trade in aquatic food products is critical to the balance of trade of many countries. For example, the US imports 3.9 billion pounds of aquatic food products from over 160 different countries with half of this from China, Ecuador, Chile, Canada and Thailand. Imported seafood constitutes more than half of the seafood consumed in the United States (5). Rights to fish and the control of critical fisheries (e.g. cod in the North Atlantic) have provided the basis for numerous protracted trade disputes, and sometimes, all out war.

II. PROCESSING TECHNOLOGIES The primary advantages of preservation of seafood products are to extend product shelf life, ensure product safety and nutritional value, and maintain product quality. Specific market advantages obtained by preserving aquatic food products include: the ability to distribute food over long distances far from the point of harvest; to hold product when it is no longer in season, or to hold it in a form suitable for later processing or consumption.

III. COLD STORAGE To make these products widely available, aquatic food products are often refrigerated or frozen. The widespread use of refrigeration, freezing, and cold storage has meant that aquatic food products, normally available only seasonally and within a small region, can now be sent around the world any time of the year. Because of reliable integrated transport, live and high value products can be air shipped from relatively remote locations to major metropolitan areas within 48 hours of harvest. Shipping costs often exceed the value of the product. Until quite recently, aquatic foods were primarily harvested and consumed locally. These foods were generally available only during a limited season. These limited seasons or “fish runs” were critical to the human survival in many parts of the world. Entire cultures developed around the annual salmon runs in Asia, Alaska and the Pacific Northwest. Also important to the survival of many cultures have been the seasonal migrations of huge schools of fish such as herring, smelt, sardines or anchovetta and the

larger pelagic fish and the mammals that feed upon them. Harvests were primarily restricted to coastal areas as there were few methods available for preserving fish on-board high seas fishing vessels. An exception to this was the North Atlantic salt cod fishery that started in the 14th century and continues today. Animal food products deteriorate rapidly at ambient temperatures, and aquatic food products are generally even more susceptible to deterioration. Refrigeration works by slowing metabolic processes. Reducing temperature slows the growth of pathogenic and spoilage microorganisms and reduces the rate of deteriorative biochemical and chemical reactions in the muscle and other edible tissues (6). However, many animals and plant foods from the aquatic environment, particularly marine fish, are poikilothermic and are adapted to living at low temperatures (⫺1–10°C). Tropical fish are also poikilothermic since water temperatures for these species rarely exceeds 20°C. The endogeneous enzymes in these cold adapted “naturally” work at refrigeration temperatures, so spoilage reactions occur at a relatively rapid rate since refrigeration does little to impede them. In addition, spoilage bacteria associated with poikilothermic organisms continue to grow. This is why products from aquatic animals and plants deteriorate more quickly than foods from terrestrial sources and must be processed quickly and held under proper conditions to maintain highest quality.

IV. LIVE HANDLING With the exception of aquaculture where the fish can be harvested with limited stress, finfish are most commonly “stressed” when captured. In the capture fisheries, finfish are literally still “hunted” and capture techniques still include the use of nets (for example, seine, trawl, and gill nets to catch salmon, herring, or pollock); hooks and lines (for example, to catch halibut or swordfish); traps (for crab and shrimp), even harpoons (for bluefish tuna and marlin). This harvest induced stress leads to a reduced level of glycogen in the flesh when the fish are brought on board. New methods of reduced temperature and moderate amounts of carbon dioxide as an anesthesia prior to slaughter can reduce the stress to fish and improve muscle quality. As the fish pass through rigor, the ultimate pH of the fish tissue is higher than for meat, generally pH 6.4–6.6. Little glycogen is left in the muscle tissue for conversion to lactic acid during the glycolytic process that accompanies rigor. In contrast, land animals are generally rested prior to slaughter and have higher levels of glycogen, and a lower ultimate pH, around 5.5 for mammalian muscle and 5.9 for chicken. The higher ultimate pH in fish is one reason why fishery products are relatively more susceptible to microbial spoilage than other muscle foods stored under the same conditions. The endogeneous enzymes in


the fish muscle and viscera of most commercially important species are highly active at refrigeration temperatures. Also, the microbes that grow on the external surfaces, gills, and in the viscera are adapted to growing at relatively low temperatures and can cause rapid spoilage. Other factors specific to the biology of aquatic animals causes the muscle tissue to be in less than prime condition when harvested. These biological factors make proper refrigeration and freezing critical for maintaining product quality. Salmon, for example, are commonly captured as they return from the ocean to spawn in a fresh water stream, often many hundreds of miles inland. With salmon, the fish have stopped eating, and have also had to physiologically “readapt” to swimming in fresh water. The fish must mobilize their energy reserves (adipose fat, muscle fat, and muscle protein) for migration as well for producing roe (eggs) or milt (sperm). At a certain point during the spawning process, the salmon flesh becomes pale, soft, and flavorless. This severity of this problem is species, gender, and run dependent. Any aquatic food product should be refrigerated or cooled on ice as soon as possible after harvest. Live mollusks should be placed in refrigerated seawater, held in cold storage at 10°C or lower, or be placed in salt water ice. Live marine mollusks can be placed ON THE SURFACE of fresh water ice; however, placing live marine mollusks in fresh water ice will kill them. Mollusks can remain alive under these conditions for five days or more.

V. HANDLING AND SHIPPING LIVE FISH Handling and shipping live invertebrate fish is a common practice. Molluscan shellfish (clams, scallops, oysters, mussels, snails), crustaceans (crabs and lobsters) and other invertebrates (limpets, sea cucumber) are shipped by keeping the animals moist by wrapping them in seaweed, moist paper or liners, and reducing temperature slightly. Often these creatures are placed into circulating water tanks at the point of sale. The large marine Alaskan and Tasmanian king crab are available for retail sale as live animals and sell for over $100 per animal. Live transport of finfish is becoming more common. Here, fish are shipped in temperature controlled tanks with air or oxygen circulation and then transferred and held in “live tanks” at retail facilities or in restaurants. More exotic foods animals including live frogs and turtles are transported in a similar fashion. Most of the recent advances in oxygen permeable packaging for life fish transport have come from the pet trade with these techniques becoming more important for food fish.

VI. HARVESTING “White fish” such as cod, pollock, or whiting, are commonly harvested on the high seas by trawl and held on

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board in refrigerated seawater until they have gone through rigor. Certain species of larger and high value fish are harvested by hook and line including swordfish and halibut. Pacific cod are harvested using a long line technique, headed and gutted and frozen shipboard within 2.5 hr at ⫺20°F in a prerigor state. Product frozen prerigor is preferred in the Japanese market. If handled properly, fish caught by hook and line can be of higher quality than those harvested by net. Warm water aquacultured fish such as catfish or tilapia are collected from ponds and stunned by dropping the water temperature. Another way of stunning the fish is to place the fish into carbon dioxide saturated water (600 ppm or greater). After this, the fish are bled by cutting the gill rakers or by cutting the artery anterior of heart. This allows the heart to remain functioning and pump the blood out of the body. The fish are then placed circulating ice water for 5–20 min. so the blood can be completely removed, further processed for the fresh market or frozen. Bleeding a fish greatly improves muscle color, storage stability and flavor. Clearly the post harvest stress in cultured fish is less, since struggle can be reduced when the fish are harvested. To improve quality, cultured fish can be fasted for up to a couple of days before harvest, reducing the metabolic activity of the digestive enzymes. Fish generally pass through rigor “whole” and still retain visceral enzymes, and if these are present at high levels they can cause deterioration of the meat during storage. Besides stress, and the affect of harvest method used, seasonal variations play an important role. Wild caught fish can vary greatly in product quality depending upon when in the breeding cycle harvesting occurs. For Alaska pollock (Theragra chalcogramma), the fish harvested during the breeding season for mentaiko (or mentiko) have poorer quality flesh than fish harvested at other times during the year. Similarly, large variations in muscle quality are seen in salmonids. For example, the flesh texture of pink salmon (Oncorhynchus gorbuscha) tends to remain firmer for male compared to female fish in the same run, as a result of the mobilization of fat and energy reserves by the female fish for egg production. Also, salmon within the same run harvested at a mouth of a river contain more fat and have a much higher quality than fish harvested closer to the spawning grounds. Fish harvested at the mouth of a river from short runs also tend to be of lower quality than fish from longer runs. This is because fish with longer migration patterns require larger energy stores for migration and tend to have a higher fat content and a richer flavor. Normally, fish are allowed to pass through rigor before they are processed and are commonly held at 4–10°C until rigor has resolved. For the best quality product, fish should be processed as soon after resolution of rigor as possible. Because these animals and their accompanying microflora are adapted to cooler temperatures, deleterious biochemical reactions can occur quickly in fish. Care must be

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taken to ensure that butchering operations are as clean and sanitary as possible to avoid cross contamination between viscera and meat. Eviscerating must be conducted under cool conditions. Often fish processing facilities are kept at 45–50°F to maintain product quality.

VII. REFRIGERATION For this chapter, refrigerated is used to describe product temperatures above 0°C and frozen, product temperatures below 0°C. Holding and shipping live fish is technically a refrigeration process since products are held at temperatures between 3–10°C, depending upon the species. Rigor begins (onset of rigor) in fish within one to two hours depending upon species and ambient temperature. Onset of rigor is temperature dependent and occurs sooner at higher temperatures. Extremely large fish such as bluefin tuna weighing several hundred pounds go through rigor slowly like other large animals. As a comparison, the onset of rigor in beef muscle is within 10–24 hr postmortem at room temperature, in chicken in 2–4 hr, and in whale muscle, 50 hr (7). Fish pass through rigor within hours and are generally processed post-rigor. Fish should pass through rigor (resolution of rigor) before fillets are frozen to avoid toughening, shrinkage and to reduce drip loss when the product is thawed out and used (thaw rigor). One exception to processing pre-rigor for certain at-sea longline processors that process high value fish prerigor, is freezing fish within two or three hours of harvest. Another exception is in aquaculture, where fish are often processed prerigor. Fish must be carefully handled post rigor, since rough handling can tear the muscle tissue causing the myotomes to separate. This phenomenon is called gaping. Gaping is an important quality consideration in finfish harvested from cold waters. Gaping is most prevalent in fish allowed to pass through rigor at elevated temperatures (⬎17°C). Other manifestations of rough handling include discoloration and softening as a result of bruising, caused by rupturing blood vessels within the muscle tissue. Also, fractures of the vertebrae introduce blood spots into the muscle tissue. Controlling the temperature of muscle foods is important for maintaining quality during storage. Muscle fibers contract postmortem at physiological temperatures. However, the amount of contraction decreases and is lowest around 10–20°C. At temperatures lower than 10°C, muscle contraction increases again. Contraction of muscle fibers at low temperatures causes the quality defect of cold shortening making muscle tissue tough. Cold shortening occurs in prerigor muscle (below 10°C) because the sarcoplasmic reticulum cannot efficiently store calcium ions at these lower temperatures. Fish muscle, with the exception of that from large pelagic species, is not highly susceptible to cold shortening. However, a related problem is thaw shortening, which occurs when muscle is

frozen prerigor and then thawed rapidly. Because ATP is not depleted in the muscle cells when tissue is frozen prerigor, the muscle fibers contract rapidly during thawing releasing large amounts of tissue fluids (drip loss) with accompanying muscle toughening. There are a number of methods for storing fish at reduced temperatures (15°C or less) including: crushed ice, slush ice (water ice dispersed in water alone or in water containing additives (e.g. salt, organic acids, antimicrobials, sugar)), champagne ice (slush ice with gaseous carbon dioxide) and mechanical refrigeration. Even with refrigeration, aquatic food products have a limited shelf life (Table 159.2). Eviscerated (“dressed” or gutted) whitefish such as cod or halibut, and salmon have a shelf life of a week or less at 4°C, but fatty fish in the “round” containing visceral contents such as mackerel or herring should be stored no longer than a couple of days. The shelf life can be extended significantly using tightly controlled storage conditions at lower temperatures by a process called superchilling. This technique involves holding the product at 0 to ⫺1°C with variations of holding temperature less than ⫾ 0.5°C. Most fish muscle does not freeze above ⫺2°C.

A. VACUUM PACKAGING Vacuum packaging also increases shelf life of certain products. Storage of chilled vacuum packaged meats including smoked fish up to 10 weeks is possible at 4°C. The primary, but somewhat unwarranted concern with vacuum packaged seafood products, is the growth of Clostridium botulinum type E. This organism can grow at refrigeration temperatures 38°F (3°C) and relatively high concentrations of water phase salt (4.5–6%)(6). However, most products will have signs of decomposition before the risk of botulism becomes significant. Recent concern among regulatory agencies about the safety of vacuum packaged fresh fish and smoked fish products from Listeria monocytogenes is also misplaced.

TABLE 159.2 Refrigerated Shelf Life of Fresh and Cured Aquatic Food Products Approximate Days Remaining in Good Condition Products

32°F

60°F

Cod, fresh Salmon, fresh Halibut, fresh Finnan haddie Kippers Herring, salted Cod, dried salted

14 12 14 28 28 1 yr 1 yr

1 1 1 2 2 3–4 mo 4–6 mo

Adapted from Pigott and Tucker, 1990 (4).


Packing these foods under vacuum or with nitrogen flushing maintains product quality longer and also provides a packaging that is tamper evident. Concerns with thermal abuse can be addressed by labeling: “keep refrigerated at 38°F or less” and “use or freeze by” dating. Modern time temperature indicators or recorders can be used to ascertain whether a product has been thermally abused possibly jeopardizing its safety, and these monitoring techniques are becoming more common.

VIII. FREEZING One of the earliest food patents was issued in 1842 for refrigerated fish. However, mechanical refrigeration/freezing did not become a significant method for preserving aquatic food products until the early 1950’s. The development of shipboard refrigeration and freezing systems made high seas fisheries possible by permitting vessels to harvest finfish and crustaceans from distant areas and bring these aquatic food products to shore-based processing facilities and distribution centers. In a similar manner, the development of practical freezing technologies and refrigerated/frozen transportation systems allowed shore plants to be constructed near fishing grounds while providing service to worldwide markets (8). The recent rapid international expansion of aquaculture now provides fresher and less expensive aquatic foods to consumers throughout the year. Important cultured species including salmonids (Atlantic and Pacific salmons, rainbow trout), catfish, tilapia, sea bream, halibut, eels, sole/flounder, striped bass, molluscan shellfish, shrimp, and sea vegetables (e.g. nori, the common covering for sushi rolls) are commonly available around the world at any time. This would not be possible if it had not been for the development of practical refrigerated/frozen processing, sophisticated supply chain management and reliable air transportation. Unfortunately high quality frozen or refrigerated (fresh) aquatic foods are too often unavailable because of poor handling, poor processing, or inadequate temperature control (9). This is a problem that still plagues the industry and has since its inception. Refrigeration and freezing also made it possible to introduce new and extremely valuable products into commerce, for example caviar and fish roe products. Caviar products are cured with salt, but with few exceptions, refrigeration or freezing is required to maintain product safety and quality. Other extremely valuable aquatic food products which would not otherwise be available without freezing include king crab with the shell on, giant prawns, magaro (sashimi tuna or tuna to be consumed raw) and lox (lightly salt cured, cold smoked, effectively raw, salmon). In general, deterioration can be reduced if the temperature is 4°C or less and by ensuring holding temperatures are well controlled. To halt deterioration, the mobility of water

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within the food must be reduced. This is particularly significant for aquatic food products because the water content is high. Finfish contains 60–80% water on a weight basis and some aquatic products contain over 90% water. Individuals within the industry often remark that they sell some of the most expensive water in the world. Water retains the ability within any food product to serve as a solvent or reactant until a temperature of ⫺40°C is achieved and maintained. Even below ⫺40°C, product quality will still be affected by surface dehydration unless protected by packaging or physical barriers such as an ice glaze. Freezing muscle foods permits storage for one year or longer at ⫺20°C, assuming that temperature fluctuations in the storage freezer can be controlled [Table 159.3]. Rapid freezing is required for aquatic food products, even more so than for muscle tissue from terrestrial animals. Muscle proteins in fish are less tolerant to changes in the ionic strength of intracellular fluids that occur during freezing than other types of muscle food. Small intracellular ice crystals will form in rapidly frozen samples with less visible tissue damage. For slowly frozen samples, large intracellular ice crystals form which rupture cell membranes, increase drip loss and damage texture. Fish muscle myotomes are more susceptible to mechanical damage during freezing than the muscle tissue of terrestrial animals. This is due in part to the orientation of the myotomes and to the relative weak connective structures that hold them together. Rapid freezing is also critical for maintaining the quality of animals frozen whole such as shrimp, lobster and molluscan shellfish. These animals are often frozen without eviscerating, so it is critical to freeze tissue rapidly with as little tissue damage as possible to limit digestive enzymes from being released into the flesh. Usually, after the fish has been frozen, it is protected with a water glaze to limit surface dehydration. Packaging materials that permit moisture retention and exclude light are preferred. Freezing and on-board refrigeration has made it possible to expand commercial fisheries to new species that were not widely utilized until the late 1970’s. The

TABLE 159.3 Practical Storage Life for Aquatic Foods (Months) (4,9) Temperature

Fatty fish, glazed Lean fish Lean fish fillets Lobster, crab, shrimp in shell Shrimp, cooked peeled Clams, oysters

⫺12°C/ 10°F

⫺18°C/ 0°F

⫺24°C/ ⫺12°F

3 4 — 4 2 4

5 9 6 6 5 6

⬎9 ⬎ 12 9 ⬎ 12 ⬎9 ⬎9

Adapted from Institut International du Froid, 1986 (9).

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development of a factory trawler fleet and growth of whitefish fisheries around the world for surimi, fillets, and fish blocks production would not have been possible without the ability to harvest tons of fish at a time and keep them in refrigerated seawater storage until the fish could be processed on board. Similarly, without recent developments in freezing technology it would not be possible to hold the millions of pounds of frozen processed product on board ship until it can be delivered hundreds of miles to shore, and from there to consumers.

A. TYPES

OF

FREEZING

Different freezing methods are employed in seafood production. Some of these are outlined in Table 159.4, with common temperature and air velocity parameters provided for freezing different food products. Most aquatic food products are blast frozen, or frozen under conditions in which product is packaged and placed upon shelves inside a chamber. Very cold air at high velocity is blown around the chamber by powerful fans near the ceiling. After the product is frozen, it is removed from the blast freezer and placed in a storage freezer. Sometimes large fish, such as salmon, are frozen in a blast freezer without being packaged first. Glazing is also used to extend the shelf life of frozen whole, dressed fish, fillets, whole shrimp or molluscs. Glazing involves dipping or spraying water or an aqueous solution on the product after the surface has been frozen. Sometimes a cryoprotectant such as fructose, sucrose or sorbitol, an antioxidant such as ascorbic acid, or a thickening agent (e.g. alginate) are added to the glaze. Levels of glaze on whole fish can be as high as 9% by weight. The glaze sublimes during frozen storage, protecting the product from surface dehydration or freezer burn. The glaze also keeps oxygen from migrating into the food limiting lipid oxidation. The presence of a good glaze on seafood is positive factor, however to prevent economic fraud, seafood products are sold by weight after the glaze has been removed (or weight net of glaze).

TABLE 159.4 Freezing Methods for Different Muscle Food Products (9) Product fish, bulk fish fish

Freezer Type air blast/batch air blast/continuous tunnel plate cryogenic nitrogen carbon dioxide

T ⫺30–⫺40 ⫺40 ⫺30 ⫺40–50 ⫺196 ⫺78.5

Adapted from Institut International du Froid, 1986 (9).

Air Velocity (m/s) 17

Contact plate freezers are commonly use for freezing products which can be marketed as uniform slabs such as blocks of fish fillets, fish mince, fish roe, and surimi. Plate freezing is used upon factory processors because it is compact, efficient, and has relatively low operating costs. In a contact plate freezer, the product is placed in a rigid pan between two large metal plates that contain circulating refrigerant. These plates are pressed down upon the product as it freezes. Plate freezing is required for products that must have uniform dimensions including fillet block, mince/block, or mince used for sandwich portions, fish sticks, or nuggets. Very uniform dimensions are required by the secondary manufacturer who cuts the blocks into portions of uniform size and weight. Plate frozen products are frozen in aluminum pans of very specific dimensions. These pans are lined with coated paperboard block liners folded to fit inside the freezer pan. The fish product is arranged inside the liner, and the lid of the liner folded over and closed. The product is packed by weight. These pans are placed into a contact plate freezer. Commercial freezers on ships can be 10–12 plates and contain dozen of blocks per layer. It takes approximately 2–2.5 hours to freeze a 7.7 kg block of fish in a commercial plate freezer (⫺28°F). Cryogenic freezing or immersion freezing in liquid nitrogen or a carbon dioxide “snow” are popular methods for freezing high value items such as shrimp and molluscan shellfish. The freezing rate is extremely rapid, and for some products, this can cause the food to crack or split. The carbon dioxide forms a snow on the food, and then sublimes. Carbon dioxide is often preferred, since there is less thermal shock than with liquid nitrogen and less physical damage to the product. For seafood, the product is placed on a conveyor and passed through a carbon dioxide snow. For nitrogen freezing systems, the product is cooled with gaseous nitrogen before the liquid nitrogen is sprayed on it. After the product is frozen, it is packaged in plastic and allowed to equilibrate to the frozen storage temperature before it is transferred to a storage freezer. These products are generally glazed. Often vacuum packaging is used. Individually quick frozen shrimp are commonly frozen in carbon dioxide snow in South American plants, and in spiral blast freezers in Asian facilities. Each type of freezing can produce an excellent product and freezing rate is rapid. Shrimp are glazed with a spray of water after freezing. After the glaze sets, the shrimp are packaged in plastic barrier film of various types, packaged in a cardboard master case and held in a storage freezer, preferably at –20°C although this not always possible. Rapid freezing is critical for fish fillets or steaks to limit the formation of large intracellular ice crystals. Contact plate freezers would be used for frozen block, but tunnel freezer (blast freezer) operated as a batch or continuous system could also be used successfully. Chemical changes, specifically lipid oxidation, occur in fish tissue


during frozen storage. Even though the lipid content of “white fish” is less than 1%, the membrane lipids are highly susceptible to oxidation. This oxidation can lead to stale and rancid off-flavors. Gadoid fish including Atlantic and Pacific cod, hakes and haddock, contain high levels of trimethylamine oxide (TMAO). This compound is broken down by enzymes active during frozen storage that cause proteins in the muscle to cross-link and cause toughening. These enzymes are more active when the tissue has been damaged, which is another reason careful freezing is important. “Fishy” off flavors are a result of microbial decomposition occurring BEFORE the fish were frozen. Frozen storage temperature must be carefully controlled to limit ice crystal growth and water migration in the fish. Wide fluctuations in storage temperature enhance the rate of deleterious chemical and biochemical reactions in the fish that lead to off flavors. For other products, poor frozen storage conditions would result in the liberation of proteolytic and lipolytic enzymes from the viscera which would cause loss of quality during storage and after the product is thawed.

B. PACKAGING A wide variety of packaging materials are used for frozen aquatic food products. For frozen fish fillets, headed and gutted Pacific salmon, and frozen glazed crab, the product is loosely wrapped in plastic and placed inside a cardboard carton for shipment to distribution centers. Shrimp, individually quick frozen fillets, and breaded products, are commonly marketed in heat sealed plastic bags. Some large products, such as whole tuna, are not packaged at all. Certain traditional foods, including uni [sea urchin roe brined and treated with alum] and sujiko [brined, colored, whole skeins of salmon roe] are still marketed in small wooden boxes. Frozen Dungeness crab meat (muscle removed from cooked crab) and razor clams are packaged in cans with double seamed metal ends although there is a trend to package these products in plastic because of food safety concerns. People mistakenly believe that products in metal cans are shelf stable, which raises food safety concerns.

IX. CURED AND SALTED PRODUCTS The addition of salt is the initial step in the production of cured, smoked, fermented and many dehydrated products. The purpose of curing is to reduce water activity through the addition of the salt itself and by the dehydrating effect the salt has by removing water from tissue being treated. After salt is added, products are placed on inclined or perforated surfaces to drain. This step removes water, and although is not enough to make a product shelf stable, will increase shelf life or, if drying is to follow, allow this step to be more efficiently conducted.

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Salted aquatic foods are ancient products and still widely consumed today as specialty foods. In Japan, the most common salted fish are sardine, mackerel, salmon and roe, herring and roe, pollock and roe and squid. In China small pelagic fish, cuttlefish, shrimp, squid, jellyfish and molluscs are salted and dried. Marine fish of all types, most commonly cod, haddock, salmon, sardine, anchovies and herring are important products in Europe and North America and are cured and salted Cured products are ready-to-eat raw foods. The most common are from salmon. Gravlax is made by adding spices, sugar, salt and herbs to salmon fillets and allowing these to cure for several days under refrigeration. In Japan, teijin is made by dry salting or brining salmon for no more than a few hours. Lox is made by dry salting or brining salmon for several minutes to a couple of hours, and then smoking the product for a short period of time at temperatures less than 90°F. These foods are not normally cooked prior to consumption. Because of the potential for parasite contamination in wild-harvested salmon, it is recommended that only frozen fish be used. Fish roe products are another type of cured ready to eat foods (3). For a detailed description of roe products see Chapter 161 in this series. Production of roe products is still an art to some degree. Sturgeon roe is made by simply blending singled eggs with 4–5% finely ground salt by weight for a number of minutes at or somewhat below room temperature. The salted eggs are drained on a fine screen, during which time the eggs are carefully inspected and any defective eggs or connective tissue removed. After this, the product is packaged into cans that have slots in the side to allow fluid to drain from the container. The product is generally aged for at least 30 days prior to sale to develop oxidized flavor notes and a darker color, although there is a growing market for the highly desirable freshly salted eggs. Salted salmon roe or ikura were traditionally made using a dry cure method. But more common now is a process which carefully suspends singled out eggs in a saturated salt solution for two to less than ten minutes depending upon the species of eggs, degree of maturity, and desired final salt concentration. For sujiko, whole skeins are used. The skeins are soaked in saturated brine containing flavors, sodium nitrite (100 ppm) and hydrolyzed vegetable protein for about 20 minutes. After brining, the skeins are layered with fine salt in a plastic (formerly wood) container. Weights are placed upon these containers to compress the sujiko and to remove moisture. Barako is a similar product made by recovering broken salmon skeins from sujiko processing. Barako is sold as singled eggs and is not compressed. Herring roe or kazunoko [Clupea pallasii (Pacific herring), Clupea harengus (Atlantic or Baltic herring)] are also a product in high demand in Asia, particularly in Japan. Kazunoko or “yellow diamond” roe is made by curing whole herring egg skeins and is commonly

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prepared as sushi or as a garnish for rice dishes. The herring roe is not immediately removed from the fish. Instead, the fish are frozen, preferably by brine freezing, or a combination of brine freezing and blast or plate freezing, the objective being to preserve the natural shape and form of the roe sacs within the fish. The frozen herring is then shipped to processing plants where the kazunoko is produced. The freezing and frozen storage of the herring is part of the process of “conditioning” the herring making roe removal easier. At the processing plant, the herring are thawed, tempered, or “slacked out” by placing the fish in fresh water that is exchanged several times during the 24-h thawing process. This helps to remove blood and other undesirable constituents from the fish. The skeins are then removed (or “popped”) from the herring. While skein removal is commonly done by hand, automated systems are now used at many facilities both to sort the fish by gender and then to remove the egg skeins from the female fish. The skeins are sorted, brined, cured, and then packed in an approximately 5-gal plastic pails in 100% brine solution, which is topped off with a scoop (500 to 750 g) of loose salt. The product is then shipped and held under refrigerated temperatures of ⫺4°C or lower. The brining process traditionally involves many steps in which the skeins are held in totes of brine of increasing strengths, finishing with a saturated brine solution. All in all, the brining process normally takes 5 to 7 days with daily changes of brine. A primary purpose of this process, in addition to curing, is to remove any discoloration in the skeins due to blood, enzymatic activity, or contaminants. In some instances, hydrogen peroxide is used by secondary processors to bleach discolorations. Following brining, skeins are sorted by quality and size. Most kazunoko is shipped to Japan where it is drained, inspected once again, and packaged for retail sale. Gift packs consisting of individual matched pairs of skeins sell for $10⫹ per pair (approx. 5 oz). Individual brined herring eggs (capelin, cod, or tobiko also have the same application) are added to sea vegetable salads and to seafood salads containing, among other things, marine plants (sea vegetables), clams, limpets, or marinated octopus. Another product from herring roe is tarama, a mayonnaise-like condiment manufactured from emulsified fish eggs. Acceptable tarama can be produced from damaged skeins and from overly mature roe. A most interesting herring roe product is kazunoko kombu or herring roe on kelp and is a garnish for a variety of dishes, most commonly soups, salads or side dishes. It can be very expensive, often over $100 per pound. For the highest quality kazunoko kombu, a uniform, dense layer of herring eggs of similar size and color covers both sides of a piece of kelp. Traditionally, kazunoko kombu is harvested when herring spawn. Schools of herring release their eggs simultaneously, and

the eggs adhere to kelp until the fish larvae hatch. Kazunoko kombu is still harvested in the wild; however, most is now produced by harvesting live herring just prior to spawning and placing them into pens (called “pounds”) in which cut kelp has been suspended. When the fish spawn, the eggs adhere to this kelp to a thickness of up to one-half inch per side (or an inch in total thickness). The fish are then released back to the wild and the egg-coated kelp is washed, trimmed, cut to market size, and packed in brine. Due to a shortage of natural kazunoko kombu, there have been several attempts at developing acceptable substitutes. One of the somewhat successful attempts at such uses a surimi-based paste as an adhesive in the highly labor-intensive operation of attaching a layer of herring eggs to pre-cut pieces of kelp. The coated kelp is then placed in a form under slight pressure and heat to set it and then packed in light brine. Other aquatic plants are salted and used as food. Brown kelp (Laminaria japonica) is commonly washed, boiled, salted, dried, salted and repackaged. The largest market is China. Seasoned kelp is a popular snack in Taiwan and Japan prepared by cooking dried kelp in soy sauce, sugar, salt and spices. Wakame (Undaria pinnatifida and U. peterseniana) is a salted dehydrated brown seaweed reconstituted and added to miso soup (10). Salted jelly fish is made with a multistep salting process by treating brine and alum in increasing concentrations with a final step in dry salt. Uni, from sea urchin gonadal tissues, is also made with salt and alum.

X. SMOKING Smoking is a form of dehydration and is an ancient form of food preservation. Smoke imparts a flavor and color to the food but limited preservative effect. Fish with a higher fat content such as salmon, black cod or sablefish are the most popular smoked products in addition to specialty products such as cold smoked oysters. Fortunately, Listeria monocytogenes is somewhat sensitive to phenolic components in smoke. The shelf life of smoked product is similar to that of fresh product. Therefore, unless the smoked product has also been thermally processed (commercially sterile, canned or retort pouch), or dehydrated to a water activity low enough to inhibit pathogen growth (Awⱕ0.85) it must be refrigerated (6). Cold smoked products are ready-to-eat products that have not received a ‘cook step’ or pasteurization treatment. To cold smoke fish, cured fish is exposed to smoke at ⱕ 90°F (6). Fish can also be cold smoked without brining. Product prepared in this fashion is held refrigerated and then broiled or grilled. It is a preparation that works especially well for halibut, black cod and salmon and is popular for restaurants. A common smoking process is to treat fish fillets, steak or pieces of larger fish, or butterflied smaller fish


with dry salt or in a salt brine until a salt content of 1–3% is reached. Then the fish are drained and permitted to cure for several minutes or several hours depending upon the product. Curing allows intracellular fluids to drain from the fish, and permits further equilibration of salt within the muscle tissue. During curing, a pellicle or slightly hardened surface layer on the fish is formed from the migration of soluble proteins to the surface coupled with surface dehydration. After curing, the fish is exposed to smoke. The type of product will dictate the type of wood used, the heat, time and humidity of the smoking operation. With few exceptions, hard woods are used for smoking (e.g. oak, hickory, alder, maple, cherry, apple, mesquite). The key with cold smoking, is for the temperature remain low enough so that microbial growth is kept to a minimum. Smoking can take a couple of hours for lox to several days for traditional Indian smoked products, some of which are still made over fires in traditional drying sheds. Cold smoked products are sometimes thermally processed in jars. Thin strips of cold smoked salmon, eel or lamprey are packed in jars with oil and then subjected to a commercial sterilization process. Hot smoked products are cured and smoked as described above, then, in addition, receive a pasteurizing treatment. A heating cycle follows at the end of the smoking cycle. Depending upon the product, smoke may or may not be applied during this heating step. Hot smoked products are commonly vacuum packaged. Either hot or cold smoked product may also be canned or processed in retort pouches producing a shelf stable product.

XI. DEHYDRATION Dried fish has historically been a critical item of commerce. Even today, as much as 25% of the world’s fish harvest is dried. Access to cod fishing grounds was the cause of major international disputes throughout the 19th century. This is because dried salt cod (bacalao) was the major protein source for workers at sea, during the period of slave trading in the New World, and for the military in 16th–19th century Europe. Dried fish is light and easy to transport and did not decompose without refrigeration. Markets are still strong for dried cod in southern Europe, West Africa, Brazil and the Caribbean. Per capita consumption of dried fish is high in island states, Subsaharan Africa, and in nations with a high per capita fish consumption such as Portugal, where there has also been a historically large cod fishery. Dehydrated product includes dried and flaked fish and are popular products in Asia. Flaked fish is usually reconstituted prior to use, primarily in soups. Dried bonito flakes form the base for popular condiments in Thailand and Japan. Dried fish (including shrimp, squid and cuttlefish) are often consumed as a snack in Asian communities, among Native Americans, and to a more limited degree in

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Africa and South America in areas where fish consumption is high. Salted and dried fish are traditionally made by layering fish with dry salt at levels of 1:4 by weight of salt to fish, allowing the fish to cure and drain from anywhere from a couple of days to a few weeks. Then the fish are removed from the brine, the surface liquid removed, and placed on drying racks, often outside in the sun, until enough water is removed to make the product shelf stable. Often, fish are strung through the tail or impaled on small wooden sticks to dry, commonly without being salted in advance. In earlier times, fish were split and placed upon rocks to dry in the sun and wind. Sun drying is still used today with major commercial operations for drying cod in the cold North Atlantic winds off the Lofoten Islands in Norway. Drying racks are up to sixty feet high. Similar operations for air drying fish are found in Newfoundland and Labrador. In Hong Kong and in other parts of Asia, small fish to large squid are commonly sun or air dried. In fact, the vast majority of dried fish products are still ‘naturally’ sun or air dried. Even though this drying technology is simple it is not without sophistication. Multistage drying processes are conducted in facilities of simple construction. Among the Native American tribes along the Columbia River, salmon fillets are sun or air dried until a certain desired consistency is reached, then the fish are cut into strips, sometimes salted, and placed on dowels in drying racks. Drying, usually in combination with smoking, occurs over a number of days inside a drying chamber or shed. Ambient temperature and wind velocity control drying time. Often times, dehydration is combined with other food preservation processes such as curing or salting. Jerkies are thin hard or somewhat pliable strips of fish made by a cold smoking fish treated with salt or salt-sugar mixtures. Aquatic plants are often dehydrated. Nori or laver (Enteromorpha spp; Ulva spp., Monostroma nitidum) are green seaweeds sold in dried sheets that form the outer surface of sushi rolls. Small slices coat the surface of rice crackers and are included in dried seasonings for rice, soups and sauces. Nori is either mechanically or sun dried and is sometimes toasted. Dried or toasted nori is a component in seasoning pastes containing soy sauce and sugar. Drying is a less important preservation method for fish in developed nations than in the past. Reconstitution and use of salt-preserved foods, such as salt cod or dried cuttlefish, in food preparation is involved and labor intensive. Many dried, salted foods have fallen out of favor in part because current meal preparation times are short – in the West averaging less than 15 minutes per meal! However, the increasing popularity of authentic Asian, Spanish and Caribbean cuisine are exposing more individuals to traditional salted and dried fish products, reintroducing these foods to many people.

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All known mechanical drying methods have been used for fish products. The highest value are for freeze dried products such as small shrimp used in instant soups. Microwave drying technologies may replace freeze drying as a cost effective alternative to these methods are further developed. Fluidized bed drying makes a highly acceptable product as well. Flaked fish is prepared on drum dryers and by convective drying methods.

XII. FERMENTATION Aquatic animal tissues are commonly fermented (9) at both low and higher salt contents. Lactic acid fermentation are utilized to some degree in the production of Asian fish sauces, fish balls and sausages. Fish sauce (from whole fish) or fish pastes (shrimp paste) are salty and highly flavored, and clearly an acquired taste for the Western palate – like single malt scotch and cigars. Fish sauce with specific flavor profiles, clarity and color are national and regional specialties in Thailand (nam-pla), Vietnam and Kampuchea (nuoc-mam), Indonesia (petis), Malaysia (budu), Philippines (patis), China (yu-lu) and Japan (shotturn). Traditionally, fish sauce is made by layering whole small fish with salt (20–40% by weight) in a ceramic crock with a perforated bottom. A weight is placed on top of the fish to remove air and to force the liquid produced during fermentation to drain off. The crock is held for several days at high ambient temperatures. To control fermentation temperature in certain regions, fermentation vessels may be buried in the ground, in this case, preparation of fish sauce may take a number of months. Fish sauce may be filtered or a certain amount of insoluble residue may remain in it. It may or may not be pasteurized. Common types of fish are sardines, anchovies, ambassids and shrimp. In Japan, fish sauce preparations may also contain soy sauce or wheat koji, small clams, or oysters to modify the flavor and aroma. Japanese fish sauces may also contain added sources of proteolytic enzymes to assist with the fermentation process. Fish pastes can be made from the residue remaining after fish sauce production or by fermentation of fish and wheat bran by Aspergillus oryzae. Fish sauce residue is mixed with red rice and fermented producing the pink condiment, bagoong, popular in the Philippines. Similar products are made in southeast Asia by blending fish sauce residue with glutinous rice, or roasted rice and molasses (mam-cho). Proteolytic fermentations using endogeneous microflora conducted at cold temperatures include fermented whole fish and fish viscera in Asia, and the stink fish and stink eggs of the Northwest US coast up through Alaska and into Siberia. Fermented organs (e.g. sea urchin gonad) or muscle (fermented squid) are popular foods in Japan (9). Fermented salmon belly flaps and viscera (the

traditional lomi lomi in Hawaii) or fermented pyloric caeca are specialty items in Japanese and Filipino communities. These fermentations are generally conducted with little or no added salt. Among the Native people of the Pacific Northwest, Alaska and into Siberia, seal flippers, marine mammal muscle, and fish heads were traditionally packed into seal skins and buried in the ground for several months. Fermented seal flippers prepared by salting the flippers and packing them into barrels remains a popular food in the Pribilof Islands. Problems arose in these Native communities with the advent of plastic bags and central heating, when people began making these traditional foods indoors within a shorter period and at a higher temperature creating a risk for Clostridium botulinum intoxication. After this problem was discovered, practices were modified again to make the traditional foods safely. Stink eggs are another traditional food of the indigenous peoples of the Northwest. In one type of preparation, salmon eggs are fermented by placing them into a small cloth sack coated with flour to exclude air. After several days, the eggs become liquified producing a condiment. Marine plants are incorporated as ingredients alcoholic beverage production in Japan.

XIII. THERMAL PROCESSING By the mid 19th century, fish was being thermally processed in metal, lead soldered, and sealed cans. Thermal processing for aquatic foods includes retort pouches and more recently microwave sterilization. Canned tuna and salmon are still common, and new convenient forms of these products are making inroads into lunch markets. Canned tuna remains the most popular fish product by volume, consumed in the United States. Improvements in the quality, availability and price of fresh and frozen fish products, along with cheaper poultry products, have negatively impacted the canned seafood market, particularly salmon, which is still widely consumed around the world and remains an important product for the Alaskan fishing industry. Producers of canned salmon are seeking new markets in the food service sector with retort pouch products, and with new market forms of canned salmon including seasoned products, canned smoked products, and skinless/boneless products and microwave processed shelf stable portions. The market for canned salmon is not expected to grow, with the possible exception of high-end pet foods. Likewise, the markets for other types of canned seafood products (clams, shrimp, tuna, anchovies, sardines, etc.) are expected to remain stable or to decline. Recent innovations in commercial sterilization should create new markets for shelf stable aquatic foods. Salmon processed in a microwave retort with overpressure has properties similar to fresh steamed salmon. The dramatic improvement in quality is a result of the dramatically


reduced come-up time for the thermal process that reduces overheating at the surface of the container and resulting loss of texture and flavor. Similarly, dielectric processing using microwave (915 MHz) or radio frequency (27 MHz) energy shows similar promise for pasteurizing heat labile aquatic foods such as caviars and smoked fish that begin to thermally denature around 70°C.

XIV. SUMMARY Aquatic food products are among the most varied and interesting food products we consume. Improvements in live haul and refrigerated transport have made year round availability of fresh aquatic food products a reality in world markets. Traditional dried, fermented and cured products are becoming popular in more markets as people become exposed to foods from other areas and regions. The aquatic environment, particularly aquaculture, will be the major source for increased sustainable production of protein foods as world population grows, making technical advances applicable in this area critical to food self sufficiency.

ACKNOWLEDGMENTS We appreciate the support of the International Marketing Program for Agricultural Commodities and Trade (IMPACT) at Washington State University, National Fisheries Institute, and Aquaculture Idaho/Washington for this work and for development of aquatic food products technology.

REFERENCES 1. NAS. 2003. Scientific Criteria to Ensure Safe Food. National Academy of Sciences. Washington, DC.

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2. FAO, 2004. Fishery Statistics, Catches and Landings. Food and Agriculture Organization, Rome, Italy: National Marine Fisheries Service, Fisheries Statistics and Economics Division. Department of Commerce, Silver Springs, MD. http://www.st.nmfs.gov 3. Bledsoe, G.E., Bledsoe, C.D. and Rasco, B.A. 2003. Caviars and fish roe products. Critical Reviews in Food Science and Nutrition. 43(3):317–356. 4. Pigott, G.M. and Tucker, B. 1990. Seafood – Effects of Technology on Nutrition. Marcel Dekker, Inc. New York, NY. 5. Anon. 2001. Food Safety. Federal Oversight of Seafood Does Not Sufficiently Protect Consumers. United States General Accounting Office. Report to the Committee on Agriculture, Nutrition and Forestry US Senate. Washington, DC. GAO-01–204. 6. FDA, 2001. Fish and Fishery Products Hazards and Control Guide. Third Edition. Office of Seafood, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Public Health Service, Department of Health and Human Services, Washington, DC. 7. Foegeding, EA, Lanier, TY and Hultin, HO. 1996. Characteristics of edible muscle tissues. In: Food Chemistry. Third Edition. Ed. OR Fennema. Marcel Dekker. New York, p. 910. 8. Burgess, G.H.O., Cutting, C.L., Lovern, J.A. and Waterman, J.J. 1967. Fish Handling and Processing. Chemical Publishing Co. Inc., New York, NY. 9. Anon. 1986. Recommandations Pour la Preparation et la Distribution des Aliments Congeles. 3rd ed. [Recommendations for the processing and handling of frozen foods]. Institut International du Froid 177, boulevard Malesherbes, F-75017, Paris, France. 10. Huang, Y-W. and Huang, C-Y. 1999. Traditional oriental seafood products. Ch 9. In: Asian Foods. Science and Technology. Eds. C.Y.W. Ang, K.S. Liu, Y-W. Huang. pp. 251–274.

160

Surimi and Surimi Analog Products

Barbara Rasco

College of Agricultural, Human and Natural Resource Sciences, Washington State University

Gleyn Bledsoe


CONTENTS I. Background..........................................................................................................................................................160-1 II. Surimi Manufacture ............................................................................................................................................160-2 III. Analog Manufacture ............................................................................................................................................160-5 References ....................................................................................................................................................................160-7

I.

BACKGROUND

Surimi is composed of the myofibrillar proteins recovered by washing minced fish and was developed in the early 1960’s as a source of fish muscle protein for neriseihin or kneaded seafood a popular food in Japan (1,2,3). Japan remains the primary market for surimi where various types of boiled, grilled or fried fish patties, cakes or balls constitute up to one-third of all seafood consumed. Pigott (3) describes the most common product forms as follows: Kamaboko–washed fish flesh mixed with flavorants, possibly color (usually pink) and gelling agents; shaped, and steamed. It is commonly sliced and added to udon soup. Chikuwa (broiled kamaboko) – is an open cylinder of kamaboko placed onto a skewer and broiled. Satum-age (fried kamaboko) – kamaboko is shaped into different forms and sizes and fried. May or may not have other ingredients added. In Western markets, surimi is most commonly recognized as the base material for production of imitation crab or kanibo. But because surimi is a protein gel, it can be used in all sorts of food products. The most common are breaded, broiled or fried surimi-containing hors d’oeuvres or entrée items. Surimi forms the base for flavored meat, seafood, or vegetable mousses, spread and dips. It can also be extruded into different shapes mimicking shrimp, scallops or lobster and vegetables such as mushrooms. Surimi has been used in low fat imitation cheeses and dairy desserts, fish sausages and fish-based hams. Ham and sausage kamaboko, the first analog products replacing pork with fish and adapting products which are part of Asian cuisines to Western tastes. Other objectives have been to reduce the fat or caloric content of the beef or pork products they imitate. Emulsions

using surimi or washed or formed salmon mince or chunks, can also resemble ham or hard sausages. Reduced fat pepperoni and hard sausages have been made from fish, but are formulated most successfully when tallow or pork fat is added, defeating one of the major purpose for using fish as a base, since the level of saturated fat remains high. Surimi can replace dairy emulsifiers and binding agents in foods where allergens are a concern. A novel application for surimi is the production of a kosher artificial black (sturgeon, lumpfish or paddlefish) caviar. A few of these foods have had limited commercial success, and hopefully more will follow. Prior to the development of surimi, kamaboko was made from fresh fish in coastal communities in Japan including the conger eel, lizardfish and croaker with records for its production to the 12th century AD (2). Guild artisans produced surimi using a traditional labor intensive process involving a careful filleting of fish, removal of bone, skin, viscera, and the dark stomach lining. The fish was minced, washed several times with water to remove soluble proteins, salts, and lipids. Washing improves gel strength and elasticity, essential properties for high quality analog products (1). In the traditional process, the washed fish mince is drained and forced through a fine sieve. This last step removed any remaining bone and skin fragments. The resulting fish cake was ground with salt in a stone mortar to solubilize the myofibrillar proteins. Flavorings and starch are also blended into the fish muscle matrix forming a pliable dough, which is shaped into half cylinders upon a flat base consisting of a small piece of a native Sawara cypress making kamaboko. The wood contributes an aromatic flavor, and provides a cutting surface for the kamaboko after it is cooked. The kamaboko sets into a firm gel by cooking it in steam. 160-1

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Recovery of the Japanese economy after World War II significantly increased kamaboko consumption, with production climbing to 268,000 tons in 1954, and to 408,000 tons by 1960 (2) and by 1965, there were over 40 shore-based kamaboko manufacturing plants in northern Japan (3). Accompanying this growth in demand was less availability of fish from in-shore grounds. Kamaboko requires relatively fresh fish and fish frozen at sea produces a less satisfactory product. Further, it was not economically feasible to produce any of the myriad of formulated consumer products, such as kamaboko or chikuwa, on board fishing vessels. A combination of these factors led to the development of the intermediate product, surimi. In modern commercial operations, surimi is made by recovering the myofibrillar proteins by washing minced fish muscle, stabilizing the washed mince with a combination of cryoprotectants and then freezing it. Cryoprotectants are added to retain the functional properties of the muscle proteins during frozen storage. A typical cryoprotectant combination includes: 4–5% sucrose (granular cane sugar), 4–5% sorbitol (in powdered form), and a 0.3% blend of phosphate salts. These cryoprotectant blends are usually proprietary. Other additives may include calcium lactate, sodium bicarbonate, ascorbic acid, or antioxidants. With cryoprotectants, surimi can be stored frozen for a year or more at ⫺20°F. Other factors led to an increase in the demand for surimi analogs in the 1980’s, most importantly a shortage of king crab which led to the development and commercial production of kanibo or imitation crab, which remains one of the most popular analog products on the world market in the form of “crab legs” and “crab meat” salad pieces or chunks is a popular product in the United States. “Crab legs” are made from small ropes or logs of texturized, colored, crab flavored surimi. Other “imitation” seafood products are much less common in US markets including imitation scallops, shrimp, lagnostino, and lobster. Unfortunately, surimi production is one of the most inefficient uses of fish and one that comes with a high capital and operating cost. Yields have improved and can exceed 20% recovery on well run lines. The market is currently moving away from pollock surimi production, due to low market prices for surimi, although other species are becoming more common as a source of raw material. The demand for fillets and fish portions compete with surimi for raw material. Fish that are not used for surimi production are used for individually quick frozen or shatter pack fish fillets (specifically deep-skinned fillets), fillet blocks, or minced fish blocks. Most at sea processing vessels have the capacity to make both surimi and fillet products. Many vessels also produce fishmeal from the meat that cannot be efficiently recovered as well as the skin, bone, viscera. The

ratio of surimi to fillets manufactured will depend upon the market for the respective products. Currently, the market for fillets is stable and the price for surimi is low causing a bias toward fillet production.

II.

SURIMI MANUFACTURE

Concurrent with development of offshore surimi production was the emergence of the Alaska pollock (Theragra chalcogramma) fishery. Alaska pollock is still the most common source of fish for surimi although other fish are commonly used. Alaska pollock could be harvested in large quantities with limited amounts of by-catch and could be purchased for a low price compared to other “white fish” such as Pacific cod (Gadus macrocephalus), and during part of the year provided a lucrative roe (mentaiko or mentiko) market. Alaska pollock is currently the most plentiful commercially harvested species in the world with an exploitable biomass estimated at over 6,800,000 metric tons and a target catch weight in most years exceeding one million metric tons. Surimi production is stable at roughly 200,000 metric tons per year. Surimi manufactured at sea on board a factory trawler is generally superior to that produced in shore-based facilities because the fish is fresher. The yield of surimi from at-sea processors also tends to be higher than for shore based plants because of the higher quality of fish. Shore based processors were established in the 1990’s for political reasons tied to fishery allocations and not necessarily with the best interest of the fishers or the resource in mind. To hopefully obtain better control over management of the fishery, the United States foreclosed foreign fishers from the North Pacific fishing grounds off the coast of Alaska in the late 1970’s. The Fishery Conservation and Management Act (1976) and the International Fishery Conservation Act, extended the sovereignty of coastal nations over fish from 3 miles to 200 miles leading to the creation of a factory trawler fleet, that in the mid 1990’s had over 60 vessels. In response, Japanese companies formed joint ventures with American companies to produce surimi and exert market control over the pollock resource. As part of this effort, U.S. subsidiaries were established to operate shore-based surimi processing plants in Alaska. The shore-based plants could be completely foreign controlled, whereas factory trawlers were required by law to have at least 75% US ownership. To make matters worse, The Fishery Conservation and Management Act (1976) was amended in 1998, shifted a larger portion of the harvest to these foreign owned shore based processors. This significantly reduced the number of fish available to US owned at-sea processing vessels. This amendment was passed under the guise that a reallocation of fish would create more jobs in the local Alaskan economy; however, it has instead resulted in


the loss of much of the US fleet along with the high wage jobs they created. Currently less than half of the pollock surimi production occurs on at-sea processors, the reverse of the situation in 1997. Pollock is currently harvested in the US in three to four seasons. In the first or “A” season, roe bearing pollock are harvested. The pollock roe (mentaiko or mentiko) is a very valuable product and is in high demand in Japan and Korea. During the “A” season in January–April, the muscle tissue is in relatively poor condition because the fish are spawning; therefore the surimi is also of a poorer quality. The pollock harvested in the “B” season during late summer, are not spawning and produce a higher quality surimi. The “C” season runs during the fall, and late in this harvest, some fish are beginning to spawn. A “D” season for early summer is also proposed during some seasons. In a typical operation, Alaska pollock is trawl-caught and held on-board in refrigerated seawater (RSW) until they have progressed through rigor (Figure 160.1). The fish are then processed as soon as possible post-rigor but never longer than twenty-four hours after harvest. When surimi is to be manufactured at a shore based facility, the fish are held on-board in refrigerated seawater (RSW) at approx. 2°C, and delivered to the plant within 48 hours. Product held more than this will not make high quality surimi. The fish are off-loaded by pumps, and held at the plant in RSW until the fish pass through rigor, which takes around 5 hr. Other fish species besides Alaska pollock are playing a more significant role in surimi production (see Table 160.1). The international demand for Alaska pollock has increased, both as a source for surimi and also as a relatively inexpensive source of fish fillets that can substitute for cod. One species that has received particular attention in the United States is the Pacific whiting (Merluccius productus). This fish is commonly harvested in Washington, Oregon and British Columbia coastal waters. A relatively

Harvest ↓ Holding in RSW ↓ Filleting, eviscerating and skinning ↓ Mincing ↓ Washing ↓ Refiner ↓ Cryoprotectant addition ↓ Freezing

FIGURE 160.1 Outline of a Surimi Manufacturing Process.

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large biomass and low price, lack of competing uses and developed markets, and the proximity of the principal harvest, processing areas and markets, led to the utilization of Pacific whiting for surimi production. Recent Pacific whiting landings range between 310,000–350,000 metric tons. Management of the whiting fishery provides for separate in-shore and off-shore harvest allocations that, in turn, encouraged joint ventures by both U.S. fishing companies and Japanese firms with local shore processors in Oregon and Washington. In the Canadian whiting fishery, Canadian fishers harvest fish which are then processed on board foreign processing vessels or at on-shore facilities within 24 hr of harvest. For political reasons, US vessels are currently excluded from this fishery. The quality of Pacific whiting is affected by seasonal variations in muscle quality and in variations in pH as a result of spawning. A unique quality problem with Pacific whiting is an endogeneous protease produced in reaction to a muscle parasite (Myxosporidian proteinas). This parasite causes problems with proteolytic muscle disintegration reducing the gel-forming ability of myofibrillar proteins in whiting surimi. This protease is a cysteine cathepsin and weakens Pacific whiting surimi gel structure by hydrolyzing myosin, it is most active around 55°C (3). Fortunately, protease inhibitors (ca. 1% by weight) can be added to the minced whiting muscle in conjunction with the usual cryoprotectants (sucrose, sorbitol and phosphate salts) to maintain gel forming ability. Calcium lactate may also enhance the effectiveness of the inhibitors. Calcium salts, such as calcium lactate, enhance gel formation by accelerating myosin heavy chain crosslinking via cross-links between negatively charged groups on protein molecules. The most effective protease inhibitors include bovine blood plasma proteins, egg white, potatobased inhibitors (4,5) or whey added to a cryoprotectants blended at up to 1.5%. Recent concerns with “mad cow” disease (BSE) have resulted in the collapse of the blood plasma additive market. Allergen labeling requirements affect the attractiveness of egg white as an inhibitor. Together, these factors may create a demand for a new generation of surimi additives. The production of surimi is highly mechanized and closely resembles the first few traditional steps in kamaboko manufacture. The yield of surimi can vary significantly from 14 to 30% of the original weight of the fish. A common process for surimi manufacture is outlined in Figure 160.1. In the initial steps for surimi production, bones from fish fillets are removed mechanically in a “deboner.” This is a perforated drum that minces the fish and removes any bones by forcing the tissue through 3–5 mm perforations. The muscle tissue passes through to the inside of the drum, and the bone and any remaining skin remains on the outside. This material may be recovered for pet food or fish meal production. Other types of meat-bone separators

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TABLE 160.1 Some Fish Species Evaluated for Surimi Production Atlantic croaker (Micropogonias undulatus) New Zealand Hoki (Macruronus novaezelandiae) Southern blue whiting (Micromesistius australis) Pacific whiting (Merluccius productus) Atlantic cod (Gadus morhua L.) White hake (Urophycis tenuis) Arrowtooth flounder (Atherestes stomias) Pacific herring (Clupea harengus pallasi) Atlantic menhaden (Brevoortia tyrannus) Atlantic mackerel (Scomber scombrus) Sardine (Sardina pilchardus) Horse mackerel (Trachurus japonicus) Yellow striped trevally (Selaroides leptolepis)

White croaker (Argyrosomus argentatus) Lizard fish (Synodus sp.) Antarctic whiting (Merluccius australis) Hake (Merluccius merlussius) Pacific cod (Gadus macrocephalus) Red hake (Urophycis chuss) Yellowfin sole (Limada aspera) Capelin (Mallotus villosus)

Pink salmon (Onchorhynchus gorbuscha) Northern squawfish (Ptychocheilus oregonensis) Marlin (Kakaira sp.)

Chum salmon (Onchorhynchus keta)

Thresher shark (Alopias pelagicus) Silvertip shark (Carcharhinus albimarginatus) Hammerhead shark (Sphyma lewini) Milkfish (Chanos chanos) Sea bass (Diplectrum fresum)

Spotted shark (Galeocerdo curier) Silvertip shark (Carcharhinus brachyurus) Dogfish shark (Squalus ancanthias) Threadfin bream (Nemipterus tolu) Bacoco (Pomadasys branicki)

Weakfish or sea trout (Cyanoscion nothus) Ronco (Micropropagon undulatus)

Lisa (Mugil cephalus)

have also been used to a lesser degree including sieve type separators. Minced fish is composed of approximately 2/3 myofibrillar proteins and these proteins can form strong gels under the right conditions. In protein gels, the myofibrillar proteins are solubilized by blending, chopping or stirring salt into the minced muscle tissue. Soluble muscle proteins (sarcoplasmic proteins) including enzymes and heme proteins, blood, and lipid are removed during the washing steps (4) and these must be removed during surimi production if a high quality gel is to be formed. Sarcoplasmic proteins and residual lipids impede gel formation and can accelerate protein denaturation during frozen storage. The washing step is conducted two to four times under agitation at 5–10°C. Typically, in shipboard operations, the fish mince is washed with 1.8 to 3.6 volumes of (5–10°C) water in a countercurrent two-step washing system. Water is removed from the washed minced fish by passing the mixture through rotating screens or through a decanting centrifuge. Having excess washing hydrates the meat and makes water removal difficult during dewatering steps and impedes gel formation as well. Salt (0.01–0.3%) may be added to the final wash to make water removal easier (3). The recovered minced tissue contains approximately 25% solids. The salt content must be lower than that required to solubilize actomyosin prematurely forming a protein gel. Sodium bicarbonate (NaHCO3) may also be added to the rinse water to increase net recovery and to assist in the removal of unwanted constituents (6).

Jack mackerel (Trachurus murphyi) Sardine (Sardinops melanosticus) Bluefish (Pomatomus saltatrix) Unicorn leatherjacket (Alutera monoceros)

Refining is the final impurity removing stage of processing. A screw drive is used to force the washed mince through a cylindrical screen that has fine perforations. In a refiner, the washed fish mince is passed through a screw press achieving a final moisture content of 72–75%. Yield is low in surimi processing with as much as onethird of the fish flesh lost during the washing steps. Typically, less than 25% of the “round” (whole, uneviscerated) fish is recovered as surimi. Although much has been made of the potential to manufacture surimi from pollock frames and trim, this has yet to be successfully accomplished commercially. Almost all surimi is produced from the fillets, and the remainder of the fish muscle tissue is converted to fishmeal or discharged as waste (see Table 160.2). Recovery of additional solids by decanting wastewater streams and though secondary refining can increase overall recovery, but these steps must be conducted carefully if product quality is to be maintained. Cryoprotectants are blended into the surimi to maintain the gel forming properties of the myofibrillar proteins during frozen storage. The cryoprotectants commonly added are: approximately 5% sorbitol, 4–5% sucrose, and 0–0.3% phosphate (generally in the form of a blend of tetrasodium pyrophosphate and sodium tripolyphosphate). High quality pollock surimi contains approximately 72–75% water, 18% protein, 4% sucrose, 4% sorbitol and 0.3% polyphosphates.


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TABLE 160.2 Material Recovery in Surimi Production from Pollock (Data from (3)) Step

Yield

Whole fish Deheading and eviscerating Mechanical deboning Washing Refining Dewatering/screw press Mixing Extruding

100% 60% 47% 45% 22% 20% 24% 24%

Sorbitol and sucrose act as cryoprotectants and stabilize the protein gel network in surimi during freezing. Sucrose inhibits ice crystal formation and water migration from proteins during frozen storage. Sorbitol and sucrose promote preferential hydration of protein molecules and effectively increase the surface of area of the protein. Other monosaccharides used include glucose, lactose and fructose. Initially 10% sucrose was used as a cryoprotectant, however this produced surimi that was too sweet for Western palates and resulted in the formation of brown off colors during frozen storage from Maillard reactions. Sorbitol, which has a bland flavor, was substituted for half of the sucrose, and found to be effective.The level of sucrose and sorbitol vary depending upon the type and condition of fish and the desired flavor characteristics of the finished product. Gelling properties of surimi are improved by adding phosphate salts. Phosphates partially decouple actin myosin complexes formed during rigor. By adding phosphate, the gel forming ability and functional properties of a gel approach that of pre-rigor tissue. Phosphate addition can counteract loss of gel strength resulting from starch addition during the surimi analog process. Phosphates also increase moisture retention and increases the ability of a protein to reabsorb liquid when the surimi is thawed or tempered. Phosphate will increase the pH slightly, which will also lead to improved gel forming ability, gel strength and cohesiveness due to an increase in water holding capacity at a higher pH. Phosphate will also sequester magnesium, iron and zinc ions that interfere with gel formation. It can also sequester calcium, and this may or may not impact gelforming ability. Polyphosphates added at 0.5% provide the greatest gel strength, but 0.3% is optimal for gel strength and flavor (3,6) with sodium tripolyphosophate (STP) and trisodium pyrophosphate (TSPP) used in combination (4). Sodium bicarbonate (NaHCO3) is also used in the leaching steps of darker fleshed fish to aid in the removal of solubles and lipids in herring surimi and as cryoprotectant in pollock and whiting surimi. Antioxidants have been added to maintain protein functionality during frozen storage, the most common being ascorbic acid which may promote disulfide bond formation.

Normally, surimi is extruded into plastic bags (often 17 b) and frozen in contact plate freezers for 2.5 to 3.0 hours, with a target temperature of ⫺20°C. After freezing, the surimi may be packed two bags to a case and transferred to a storage freezer.

III.

ANALOG MANUFACTURE

A common formulation for a gelled fish product will have approximately 60% surimi. However, by adding certain starches imitation crab analogs containing as little as 33% surimi can be commercially produced. A protein content of 11% will provide a strong gel suitable for most analog products. To make an imitation seafood product from surimi, the surimi is tempered to slightly less than 0°C, blended with salt and other additives, including flavors and colorants; formed, extruded, or texturized by multiple folding or the use of spinnerets; and heat set, and packaged, generally under vacuum. These products are ready to eat. As a safety precaution, almost all analog products produced for sale in the United States, are pasteurized and are held either refrigerated or frozen. A common process is outlined in Figure 160.2. Surimi is tempered, or taken from a frozen state to a condition where it is pliable, under controlled conditions until it can be easily mixed in a silent cutter. This is a large bowl shaped device with moving blades which chop the protein gel into pieces and incorporate salt and other additives before the surimi mix is extruded to form myofibrillar protein gels, similar to what occurs in sausage manufacture. The texture and flavor of the surimi-based product is affected by the quantity and type of salts added, and the pH of the surimi. Therefore, tempered surimi is combined with salt to solubilize actomyosin. Starch is an extender

Frozen storage of surimi ↓ Tempering ↓ Mixing ↓ Salt addition ↓ Addition of other additives ↓ Extrusion ↓ Cooking ↓ Pasteurization ↓ Refrigerate or freeze

FIGURE 160.2 Surimi analog production.

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for surimi and reduces formulation cost. More importantly however, starch can strengthens the gel, and participates in gel formation as a dispersed phase. Egg white also increases gel strength and gel elasticity. Egg white can improve color and appearance of the surimi-based product after extrusion. Flavors, flavor potentiators, colorants and additional phosphate, sweeteners, mirin (Japanese rice vinegar) and antioxidants such as ascorbic acid (Vitamin C) may be added depending upon the desired product traits. After addition of various components, the surimi gel is transformed into a thick paste or protein gel that is subsequently heat set. When heat set, this results in the formation of a three-dimensional, continuous structural matrix. Salt (NaCl) solubilizes actomyosin. Complete solubilization is required to make a resilient surimi gel. Salt binding to myofibrillar proteins creates electrostatic repulsive forces between protein molecules, loosens the structure of a protein network and permits greater water binding. The optimal salt content varies with the fish species used. Generally, 2.5–3% salt provides optimal gel strength, however 2–2.5% produces products with a more acceptable flavor. The preference for salt content is product and market dependent. Higher concentrations of salt reduce the thermal stability of fish proteins allowing them to gel at lower temperatures. Very high levels of salt decrease gel strength due to protein precipitation, a phenomenon known as “salting out.” Freeze denaturation of the proteins in surimi during frozen storage will reduce protein solubility and increase the amount of salt required to solubilize actomyosin during analog manufacture. The minimum concentration of salt for extracting actomyosin from fish muscle is pH dependent. At pH 7.0, approximately 2% salt is needed. Salt solubilizes myofibrillar protein forming a thick sol or paste. The presence of the chloride ion may shift the isoelectric pH to a lower value increasing protein solubility at the existing pH. In surimi starch modify texture and improve gel strength of low quality surimi. Adding starch can reduce formulation cost by increasing water retention in the surimi product. It can also bind water, improve adhesion and stabilize the surimi gel. Adding excess starch makes surimi based gels brittle. Up to 20% starch can be added without adverse affects on gel strength. The mechanism for interaction between starch and protein in surimi gels is not well understood. Swollen starch granules are dispersed within the protein gel matrix. Starch granules swell but complete gelatinization does not normally occur. The surimi gels set at temperatures below the starch gelatinization temperature. Potato and wheat starches are commonly used. Potato starch has a low gelatinization temperature and produces high strength surimi gels. Pre-gelatinized tapioca starch and thin boiling starch produce surimi with reduced gel strength (3).

Some surimi analog products contain egg white. Egg white modifies the rubbery texture of surimi caused by the addition of starch. It also provides the surimi with a whiter and glossier appearance. Egg white makes the partially heat-set analog more elastic and stretchable, this is an advantage for “ropelike” products like imitation crab legs. The amount of egg white added depends upon the fish species used and the quality of the fish used. Egg white added at 10% produces a gel with a high yield stress, gels containing up to 20% are softer, and greater than 20% there is a decrease in gel strength and gels become brittle. Egg white contributes to the structure of surimi analog gels by filling interstitial spaces in the fish protein network. Because egg white is an allergen, it must appear on the label of surimi analog products. Class II food recalls of analog products have been initiated in the United States because of failure of companies to list egg white on the ingredient statement. Carotenoproteins in crab turn orange-red when crab is cooked. This is the reason a colorant is used on the surface of kanibo [imitation crab]. A natural or artificial color is added to surimi paste and applied to the outside surface of ropes of crab leg analog following extrusion. Flavors are added as liquid concentrates, pastes, or free flowing powders to the surimi mix during blending. Natural and artificial flavors are used. Crab meat extract and extracts from shell and processing by-products have been used. Crab-like flavors derived from non-shellfish sources have also been used to produce a kosher imitation crab. The level of salt, level and type of phosphate, and level of sugar and sorbitol have an impact on the flavor of surimi analogs. Hydrolyzed protein, dipeptides, or amino acids added to provide meaty, sweet or slightly bitter flavor to analog products. Monosodium glutamate (MSG) may be added as a flavor enhancer. Nucleotides are also added as flavor modifiers, commonly guanosine (0.0035%) and inosine monophoshate (0.01%). Nucleotides enhance flavor potentiating properties of MSG. Heat setting occurs in 3 stages represented by distinct textural changes. At 40°C, the setting is attributed to hydrophilic interaction of protein molecules. At 60°C, the gel weakens somewhat due to action of endogeneous proteases. Intermolecular and intramolecular protein bonding occurs around 80°C increasing gel strength. For the production of crab meat analogs, the surimi paste containing the desired additives (with the exception of red colorants) is sheeted in a thin layer and then heat set. After this first heat set, the sheet is scored with a device that looks like a large comb. The sheet is not cut completely through. This scoring forms long thin strips that resemble crab muscle fibers. Several of these strips are rolled together to form “muscle fiber bundles.” These are set and then a portion of the outside surface is colored red with a blend of surimi and food coloring. The ropes


are then cut into logs (approx. 4 inches in length), or into small cylinders or diagonal cut product for salad chunks. A second extrusion process involves extruding spaghetti thin strips of a surimi containing mixture into an acid bath. This sets the surimi and when this material is cut into small pieces and reformed, has the texture and mouthfeel of muscle fibers. This product is commonly mixed with salad chunks for use in seafood salads. Kamaboko is formed into half-rolls, surface dyed, and heat set. Other products, such as chikuwa (which look like a huge fish ziti or rigatoni) are extruded and cooked. Surimi texture is affected by how the product is heated and by the heating rate. Cooked surimi based analogs are more stable in the frozen state than surimi. Surimi based products pass through three stages of gel formation during conventional heat processing. The conventional isothermal method of producing analogs is divided into three stages of gel formation. These three stages are treated as separate unit operations called: forming, heat-setting and cooking. The first stages of gel formation occur as the surimi is initially heated. The gel increases in strength up to 13°C. In the range from 13°C to approximately 30°C, few changes in gel properties are observed. A second heat set occurs between 30–40°C and a final cook between 50–90°C during which time the maximum gel strength is realized. Imitation crab products are commonly vacuum packaged in plastic or nylon packaging. The products are pasteurized inside the package in a hot water bath. This is a food safety precaution because surimi analogs are ready to eat foods. In addition to killing vegetative cells of bacterial pathogens, pasteurization reduces the number of spoilage flora leading to an extended refrigerated shelf life. Surimi-based products are distributed as refrigerated or frozen foods. For frozen product, individual packages are frozen in a blast freezer, packaged into cases and held

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in frozen storage, preferably at ⫺20°C. Surimi-based products have a shelf life of approximately 1 year under these conditions. Quality changes, which occur to the product during frozen storage, include flavor changes resulting from oxidation of lipids and lipid soluble constituents, toughening, and loss of product integrity and product texture. The gel-forming ability of fish is affected by the frozen storage treatment as well as the fish species, freshness, and biological conditions of the fish prior to harvest. Poor frozen storage can encourage enzymatic lipid oxidation, cause protein denaturation and negatively impact gel formation.

REFERENCES 1. Lee, C. 1986. Surimi Manufacture and Fabrication of Surimi-Based Products. Food Technol. 40(3): 115–124. 2. Okada, M. 1992. History of Surimi Technology in Japan. In Surimi Technology, (Ed). Lanier, T., and Lee, C. Marcel Dekker, Inc. New York. 3. Pigott, G. 1986. Surimi: The ‘High Tech’ Raw Materials from Minced Fish Flesh. Food Reviews Int. 2(2): 213–246. 4. Wasson, D. 1992. Fish muscle proteases and heat induced myofibrillar degradation. A review. J. Aquatic Food Prod. Tech. 1(1):23–41. 5. Weerasinghe, V.C., Morrissey, M.T. and An, H. 1996. Characterization of active components in food grade proteinase inhibitors for surimi manufacture. J. Agric. Food Chem. 44:2584–2590. 6. Bledsoe, G.E., Rasco, B.A., and Pigott, G.M. 2000. The effect of bicarbonate salt addition on the gel forming properties of Alaska pollock (Theragra chalcogramma) and Pacific whiting (Merluccius productus) surimi produced under commercial conditions. J. Aquatic Food Prod. Tech. 9(1):31–45.

161

Caviar and Fish Roe

Gleyn Bledsoe


Barbara Rasco

College of Agriculture, Human and Natural Resource Sciences, Washington State University


Introduction ......................................................................................................................................................161-2 Processing Roe into Caviar ..............................................................................................................................161-2 Recovery and Yield of Roe ..............................................................................................................................161-2 Sturgeon Caviar ................................................................................................................................................161-2 A. Processing Sturgeon Roe ..........................................................................................................................161-5 B. Paddlefish Caviar ......................................................................................................................................161-6 V. Other Fish Roe Products ..................................................................................................................................161-7 A. Catfish Roe ..............................................................................................................................................161-7 B. Salmon Roe ..............................................................................................................................................161-7 1. Processing Salmon Roe-Sujiko ........................................................................................................161-8 2. Processing Salmon Roe-Ikura ..........................................................................................................161-8 3. Processing Salmon Roe, Marinated Roes and Other Products ........................................................161-8 C. Lumpfish Roe ..........................................................................................................................................161-9 1. Processing of Lumpfish Caviar ........................................................................................................161-9 D. Tobiko or Flying Fish Roe........................................................................................................................161-9 E. Whitefish Roe and Similar Products ......................................................................................................161-10 F. Cod Roe ..................................................................................................................................................161-10 G. Shad Roe ................................................................................................................................................161-10 H. Mullet Roe ..............................................................................................................................................161-10 I. Orange Roughy Roe ..............................................................................................................................161-10 J. Herring Roe or Kazunoko ......................................................................................................................161-11 K. Pollock Roe or Mentaiko........................................................................................................................161-12 L. Hake Roe ................................................................................................................................................161-12 M. Rock Sole Roe ........................................................................................................................................161-12 N. Sea Urchin Roe (Uni) ............................................................................................................................161-12 O. Sea Cucumber Roe ................................................................................................................................161-13 P. Roe from Crustaceans ............................................................................................................................161-13 VI. Chemical Composition of Caviar Products ....................................................................................................161-13 A. Proximate Composition of Fish Roe ......................................................................................................161-13 B. Lipid Composition of Fish Roe and Fish Roe Products ........................................................................161-13 C. Cholesterol Content of Fish Roes ..........................................................................................................161-16 VII. Grading and Quality Attributes of Roe Products ..........................................................................................161-16 VIII. Packaging Roe Products ................................................................................................................................161-17 IX. Food Safety Issues Associated with Roe Products ........................................................................................161-17 X. Conclusion ......................................................................................................................................................161-19 Acknowledgments ......................................................................................................................................................161-19 References ..................................................................................................................................................................161-19

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161-2

I.


INTRODUCTION

Fish roe products are popular traditional foods, often of very high value, with growing domestic and international markets. Caviars are the salt-cured and preserved eggs of finfish or aquatic invertebrates. Fish eggs are commonly called “roe,” particularly when they are present in skeins. Roe products are usually refrigerated or frozen although some are shelf stable, as a result of thermal processing, pickling, salting or dehydration. Roe are sometimes consumed along with other tissue in a single dish for example whole scallops in the shell, shrimp or lobster with coral (or roe), rock sole with roe, and she crab soup. Caviars are generally made after the eggs have been singled out by screening or otherwise separated from any supporting connective tissue. The eggs are then brined and cured, and sometimes flavored and/or colored. The most widely recognized and valued caviar is made from sturgeon harvested from the Caspian Sea region. Only sturgeon caviar can be labeled in the U.S. simply as “caviar.” Caviar from other fish or aquatic animal species must be identified with a qualifying term including the common name of the fish used. For example, caviar from salmon (or ikura) must be labeled “salmon caviar.”

salmonid roes develop tough, rubbery outer shells, while the sturgeons become soft when overly mature. Also, the flavor and consistency of the lipid moieties and proteins change with maturity making the mouth feel of caviar prepared from overly mature roe less desirable.

III.

RECOVERY AND YIELD OF ROE

Recovery of roe from whole fish can vary a great deal and is dependent upon the species, method of reproduction, stage of maturity, availability of appropriate feed, the level of stress and various environmental elements. For a female pink salmon, the yield of eggs is approximately 15%, and for a gravid sturgeon as high as 25%. Normally salmon roe is measured as the percent recovered from the entire harvest including both males and females or bucks and hens as they are referred to in trade. For midseason (mid- spawning) pink salmon runs recoveries are as low as 3% (range 3 to 10%, average 6%) (6). Mature roe herring are purchased by the ton on a basis of 10% roe recovery. A herring harvest testing out higher than 10% would receive a premium and one of less than 10%, a reduction in the final price. A more detailed comparison of egg yields for a number of species may be derived from the values presented in Table 161.1.

II. PROCESSING ROE INTO CAVIAR

IV. STURGEON CAVIAR

Roe products are to be made from wholesome, undamaged eggs, have a proper color and glossiness, texture, a desirable mouth feel, and their characteristic flavor with limited fishy, bitter, or oxidized flavor notes. The preferred mouth feel varies with species. In the case of ikura or salmon roe, a distinct fracture or “pop” when the egg is broken with the teeth or palate, a smooth, honey-like mouth feel is desired, while with sturgeon caviar a buttery texture that tends to melt in the mouth is desirable. Often hundreds or thousands of individual eggs are enveloped within ovarian membranes and these skeins or whole ovaries can be processed into products such as sujiko. More commonly, the individual eggs are separately recovered, brined and cured. Pastes or spread are common products made by blending eggs with butter, mayonnaise or salad dressing bases, lemon juice or soy or miso based sauces. Roe can be dehydrated. Dried mullet roe is one such food possessing an odd rubbery texture. Technically, caviar should only be used to describe fish eggs that are separated from the connective tissue of the ovaries and then salted and cured. Roe, regardless of the type, must be an optimal level of maturity to produce caviar. Immature roe tend to be bitter (e.g., herring, salmon) and may not take up salt uniformly. If salt uptake is not uniform, the product can readily spoil during storage since psychotropic spoilage micro flora, specifically certain types of lactic acid bacteria can grow within the lower salt pockets of the stored product. Overly mature roe may be soft, lose its elasticity, and may not form a plump, full egg after brining. Overly mature

More than 20 species of sturgeon are harvested for caviar. All are important sources of high quality and expensive sturgeon caviar. The most famous are the caviars produced from the Caspian white or beluga (Huso huso), Osetra (or Osietr or Ossietre) (Acipenser sturi or A. guldenstadti (1), Sevruga (A. stellatus or sevru), and Chinese or Kaluga sturgeons (Huso dauricus, A. dauricus or A. mantschuricus. Other important sources of caviar are the Russian sturgeon (A. gueldenstaedti), Amur River sturgeon (A. schrenki), ship sturgeon (A. nudiventris), and Siberian sturgeon (A. baerii dauricus, or A. mantschuricus) (2). Depending upon the species and environmental conditions, it can take 15 to 20 years for a female fish in the wild to become sexually mature and suitable for caviar production. A single one-ton Beluga sturgeon can produce 350 pounds of caviar worth hundreds of thousands of dollars on the wholesale market (10). Prices for premium Caspian Sea beluga caviar have always been high, and through 2003 prices of US$700/100 g with prices of US$150–400/100 g for osetra and sevruga caviars depending upon quality common in high end retail markets around the world. Prices for the world’s most expensive and extremely rare white or gold colored “Almas” or diamond Beluga caviar from fish, possibly 100 years old or more, was US$2,330/100 g in 2002. Almas caviar is traditionally sold in 24 karat gold tins. It is unclear whether Almas caviar is still available in the marketplace. Current prices of all forms of sturgeon caviar have skyrocketed following new CITES restrictions placed upon trade in beluga caviar in 2005. Current prices

Caviar and Fish Roe

161-3

TABLE 161.1 Egg Yield and Size for Fish Roe Species White Fish Whitefish (Coregonus sp.) Cod (Gadus morhua) Alaska or walleye pollock (Theragra chalcogramma) Herring (Clupea harengus) Haddock (Melanogrammus aeglefinus) Whiting (Merlangus merlangus) Saithe (Pollachius virens) Capelin (Mallotus vilosus) Carp (Cyprinus sp.) Sand eel (Ammodytes lancea) Pike (Esox sp.) Flounder (Psuedopluronectes sp.) Lumpfish (Cyclopterus lumpus) Tobiko (Cheilopogon furcatus) Salmon Chum (Onchorynchus keta) Pink (Onchorynchus gorbuscha) Coho (Onchorynchus kisutch) Sockeye (Onchorynchus nerka) Chinook (Onchorynchus tshawytscha) Sturgeon Cultured beluga (Huso huso) White sturgeon (Acipenser transmontanus)

Yield (Ave % wt)

Diameter (mm)

14

0.9–1.4 1.3–1.4

14 18

1.3–1.5 0.9–1.5 1.2–1.4 1.0–1.1 0.9–1.1 1.0–1.2 0.8–1.6 ~0.3 2.5–2.8 0.8–1.2 2–5 ⬍2

20

23

8–13 7–11 7–12 6–8 10

4–5 3.5–5 3.5–4 4–4.5 6–7

25 (max) 20

2.5 mm

Data from: (6)(15).

(March 2005) for sturgeon caviar at Caviar House, Heathrow Airport, London, UK were as follows: Osetra: US$220–635/100 g Sevruga: US$250–400/100 g Beluga: US$612–665/100 g Despite a strong demand for sturgeon caviar, supplies of wild harvested product have been decreasing. Over the last few decades, sturgeon (Huso spp.) harvest from the Caspian Sea has dwindled, and currently production cannot keep up with consumer demand. Naturally occurring populations of Acipenser sp. in Europe and Central and Eastern Asia have also decreased in recent years, resulting in part from political instability in Iran and new states formed from the republics of the former Soviet Union. Because of this, cultured fish species particularly Acispenser have received increased attention as sources of black caviar. Sturgeon culture is currently employed to restore natural runs, enhance natural run through hatcheries, or by production of adult animals in culture facilities for meat and roe. These efforts will hopefully ensure that sturgeon fisheries remain viable or become viable

again. Currently there are culture operations for Huso and Acispenser sp. in the nations surrounding the Caspian Sea, Eastern Europe, China, North and South America. However, in addition to CITES, the US Department of Fish and Wildlife has taken the short-sighted position of barring sale of beluga caviar in the US, effectively stopping culture efforts of Huso sp. in the United States, because the agency is concerned that it will not be able to differentiate whether or not caviar is from these culture operations or from wild harvested product. Regulation of caviar production is nothing new, with the fishery highly regulated for centuries. Russia and Iran are the largest producers of black caviar in what have been historically tightly controlled politicized fisheries. The Russian government has regulated production of black caviar since 1675 when Tsar Alexi prohibited Cossacks from direct marketing their caviar to foreigners (4). Many individuals associated with caviar production were executed who objected to Soviet control of the industry in the early 1920s. Fortunately, the Soviet Union was able to maintained reasonable control of caviar harvest and production, maintaining product quality and supporting close monitoring of fishing effort to protect fish stocks during this era. But after the break up of the Soviet Union, management of the fishery fell apart. Iran is the other major producer of sturgeon caviar. Their territorial waters do not appear to have suffered to the same extent from poaching and boot leg production as areas under the control of the former USSR republics including Russia. Sturgeon caviar is a subject of Persian lore back to biblical times, but the fishery was not highly regulated until 1893. In 1893, the Lianozov brothers managed to secure a fishing concession from the Persian government to harvest sturgeon and process the roe into caviar under a quality control system similar to that in place in Russia. The Soviets seized the Lianozov caviar operations in 1925, ignoring the fact that the operations were in Iran, not the USSR, creating a series of conflicts with Reza Shah Pahlavi who took umbrage at this incursion. By 1927, the Persians signed an agreement with the USSR government and caviar operations recommenced (4). The ouster of Mohamed Reza Shah Pahlavi in 1979 precipitated over 20 years of political chaos in Iran including an eight year war with Iraq that following directly upon the heels of the Islamic Revolution. Governmental control of caviar production in Iran deteriorated after the Revolution along with programs to protect the fishery. Fortunately, the situation has improved in recent years. Iran continues to be a major producer of caviar with well established markets in Europe and recently in the U.S. markets following a two decade trade embargo. Unlike Iran, the current situation in the former Soviet Union is grim. The construction of dams on the Volga River and the devastation to the fishery caused by virtually uncontrolled poaching after the breakup of the USSR has brought the Caspian sturgeon fishery to the point of collapse with extinction of the beluga, osetra and sevruga

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sturgeon becoming a very real threat. Projections are that 90% of the beluga sturgeon population has been lost in the past 20 years. In late 2001 only 28 beluga sturgeon were found over an entire Caspian Sea collection area with over 85% of the fish harvested being immature suggesting a highly depleted population (5). Although there are several well intended efforts to restore the depleted runs, the generally poor economic conditions in the effected countries has resulted in a reduction of funding to maintain sturgeon hatcheries in Russia and the former Soviet republics, and this has made matters worse. Demands for the surrounding range states to cooperate in setting realistic quotas and for conducting comprehensive surveys of sturgeon stocks have fallen on deaf ears. Recent harvest quotas were largely arbitrary and do not reflect fishing pressure on the Caspian Sea stocks (5). The situation with poached sturgeon has become so desperate that Russia has considered establishing a governmental monopoly on sturgeon harvest as a means to control poaching to some extent and maintain a viable fishery. Predictions are that illegal fishing generates $2 to $4 billion dollars a year in Russia and the former Soviet republics (Anon, 2001). Interpol has been involved in assessments of illegal trade in sturgeon although currently available data on illegal harvesting, trade and enforcement is very limited, citing the “Paris Agreement” CITES SC45 Doc 12.2 (5). Control of the fishery is difficult. For example, the Dagestani Coast Guard confiscated 64 tons of fish and 184 kg of caviar from poachers in 2000 and 10 tons of sturgeon through April 2001. In response, a crowd of at least 300 poachers stormed a coast guard station in Izberbash, Dagestan, to forcibly retrieve their confiscated boats and fishing nets in a well-organized attack that local officials described as part of the ongoing war with the local “caviar mafia” (6). Poachers used their wives and children as human shields in this attack. The seizure was a result of a newly instituted Russian restriction on sturgeon fishing and the caviar trade. More problems are sure to follow in light of new fishing restrictions. Mature and immature sturgeon are commonly available in bazaars and for sale on the street, with most of this harvest unregulated. As a result of the threat to the fishery, international organizations have called for measures from severe restrictions to the complete prohibition of import/export sales of caviar from Caspian Sea species. The UN’s Convention on International Trade in Endangered Species (CITES) and Russia, Kazakhstan, Turkmenistan and Azerbaijan reached agreement in January 2002 to severely restrict production of caviar from their waters (7). Regardless, much bootlegged product remains on the market. The US imports roughly 80% of all beluga caviar (5) and there have been numerous incidents of caviar being smuggled into the U.S. in recent years. In 2000, the U.S. Fish and Wildlife Service seized one ton of illegal

imported product through its enforcement powers under the Endangered Species Act, destroyed it, and fined the importers over 10 million dollars. Misbranded and adulterated products are also common in the US market. Decomposed, adulterated, and misbranded osetra, kaluga, sevruga, and beluga caviars have made their way to U.S. markets, and, when detected, have been seized. In 2002, the US Fish and Wildlife Service proposed a ban on beluga caviar imports into the United States stating that: “Despite the CITES listing, beluga sturgeon populations have continued to decline, and the population structure is increasingly skewed towards sub-adult fish, with a critical lack of spawning-age adult female fish.” The US Fish and Wildlife Service proposed to list the species as endangered1 due to “...loss of habitat throughout historic spawning areas due to dam construction and river-modification projects, over-harvest, widespread poaching and illegal trade, and pollution, [which] imperil the continued existence of this species.” The net effect of the turmoil in the Caspian Sea fishery has been an increase in aquaculture or farmed produced sturgeon caviar. The product is accepted as a responsible way to continue the production of this delicacy and to protect the remaining wild stocks. Although captive breeding programs for sturgeon in Russia began in the 1930s, development was much later in other countries. There are currently commercial aquaculture operations for sturgeon in Germany, Hungary, Romania, Italy, France, Spain, Portugal, Israel, Chile, Argentina, Russia, China, Iran, the Czech Republic, Uruguay and the United States. France and the Czech Republic each produce roughly 10 tons of caviar annually, with French production expected to double in the next 2 years (6). Most of these operations rely upon primary species such as beluga while commercial hybrids of the beluga include the bester, a cross between a female beluga sturgeon and a male sterlet (Acipenser ruthenus, also listed as Acerpensiformes ruthenus) are also cultured. There are also emerging commercial aquaculture operations of the single strain sterlet (Acipenser ruthenus) in Hungary, Poland, other European countries, and Florida directed toward meeting the international demand for gold caviar.

1 An endangered species is any species which is in danger of extinction throughout all or a significant portion of its range (16 U.S.C. 1532(6)). Factors which support listing the beluga sturgeon as endangered include: (1) loss of 85–90% of its historic spawning habitat through dams, river channelization etc., (2) over fishing, (3) disease causing tumors, reproductive abnormalities and large fish kills throughout their range, (4) inadequacy of existing regulations. For example, arbitrary catch limits, no maximum size limits which would protect spawning fish, poor control of by-catch mortality, insufficient penalties for poaching and illegal trading, and (5) an additional problem is lack of genetic diversity in hatchery stock in which fewer than 10 adult females may provide the base for regional hatchery operations for any given year class. 67 Fed Reg. 49657-49660.ecting sturgeon li.

Caviar and Fish Roe

Commercial harvesting of sturgeon in North America appears to have started about 1750 in New Jersey, however, the production of caviar did not enjoy much attention until the mid-1800’s. Initially starting on the Atlantic coast using Atlantic sturgeon (Acipenser oxyrhynchus), the production expanded to the Pacific coast from California to British Columbia where the white sturgeon (Acipenser transmontanus) was most common. The industry was virtually eliminated in just 50 years by overharvesting. US caviar production is currently seeing a significant revival primarily due to the farming of white sturgeon primarily in California and Idaho. The most common source of black caviar in North America is the white sturgeon (Acipenser transmontanus). This is also the most common sturgeon found on the North American continent. However, the production of caviar from the native wild stock is not normally permitted due to the near extinction of certain sturgeon species and subspecies in some North American watersheds. Therefore, almost all commercial production of sturgeon for either caviar or meat is from cultured sturgeon. Captive breeding programs for white sturgeon along with federal, state, and tribal management projects for wild stocks in the Snake, Columbia, and Missouri River systems should reverse a decline in wild fisheries stocks. In the Pacific Northwest, Native American tribes, along with US Fish and Wildlife, are the primary leaders in sturgeon restoration efforts. The Nez Perce, Kalisbell, and Yakama Nations have wild white sturgeon broodstock in captivity and are developing fishery enhancement programs for the sturgeon. Some of the tribal organizations are expanding their restoration efforts to include production and growout facilities for fish suitable for caviar production. By coupling restoration efforts with production, there is a greater likelihood that programs will remain viable because they would become financially self-sustaining. U.S. culture of sturgeon began in earnest in the late 1970s. In 1979, the U.S. began an intensive aquaculture program for white sturgeon as part of the Aquaculture and Fisheries Program at the University of California, Davis (8). For this program, wild female brood stocks were harvested and their eggs surgically removed. The eggs were fertilized and the first hatchlings produced in 1980. After this first success, commercial aquaculture firms began raising sturgeon for caviar from A. transmontanus possible between 6 to 10 years of age (8); other sources indicate 8 to 9 years (9). Dozens of sturgeon farms have been started in California (10) and Idaho. Southern states, including South Carolina, Florida and Louisiana, began evaluating programs for culturing Atlantic and Gulf sturgeons during the 1980s with current production and pilot operations in place. Cultured sturgeon are harvested at 1.5 to 6 years for meat production and 7 to 10 years for caviar. The Gulf of Mexico sturgeon (Acipenseroxyrinchus desotoi) a subspecies of the Atlantic sturgeon distributed from the

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Mississippi River to Tampa Bay, was commercially harvested for caviar in small quantities in inland Florida (12). As with other North American sturgeons, culture programs have been established for the Gulf sturgeon. Unfortunately, determining the sex of even mature sturgeon can be relatively difficult. Currently, the most practical method of sex determination requires a small incision in the upper part of the fish’s abdomen and a visual examination of the gonadal tissue. Male fish are generally diverted to harvest for meat and the female fish are returned to growout facilities where they remain until they have reached sexual maturity. At maturity, female fish may weigh from 40 to 80 kg. Premium caviar from farm-raised sturgeon is valued at a price equal to that of imported osetra or sevruga with the lowest prices around $36.00 per ounce (3) and production in California farms projected at 30,000 pounds annually.

A.

PROCESSING STURGEON ROE

Preferably, black caviar processing begins with the removal of the roe immediately after the fish has been killed. Wild sturgeon are generally harvested by seining and are transported alive to the processing facility. In some instances, the roe is removed from the sturgeon on board the harvest vessel. It is not necessary to kill the fish however to take the roe. Russian technologists long ago discovered that as much as two thirds of the roe can be removed by Cesarean section and after a short recovery period the fish can be returned to the wild or to the aquaculture tanks (6), thus roe can be harvested again from these fish during later spawning cycles. There are many instances in which roe has been successfully harvested over ten times from the same Beluga female and Sternin reported that eggs were taken from a single bester seven times over a period of 15 years (11). Processing caviar from cultured sturgeon begins when the fish are removed from the growout tank and transported immediately to the processing room where they are stunned and the roe is immediately removed. Bleeding the fish prior to roe recovery improves quality. The roe is then normally controlled in lots which identify the individual fish from which the roe was harvested. The roe is kept on ice and then processed as rapidly as possible under sanitary conditions in a cool environment. The total processing time from extraction to primary packing is normally less than 2½ hours. Following extraction, the first step in caviar production is the separation of the individual eggs from the connective tissue. This is normally accomplished by rubbing the skein on single stainless steel screen over a stainless steel bowl. The opening dimensions of the screen are approximately equal to the size of the eggs being separated. Traditionalists in Russia still prefer linen thread screens. Nylon has also been used with success. In contrast to singling out of salmon eggs where two or even

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three layers of screens are used, only a single screen is commonly used for sturgeon eggs. The singled eggs are then inspected, picked over, and briefly washed in a chilled, mild saline solution (ca. 3%) to remove extraneous debris. This rinsing step should not be more than 15–20 seconds or the quality and the shelf life of the end product may be reduced. The eggs should be briefly drained following the rinsing. The eggs are then placed in a stainless steel bowl, weighed and 3–5% by weight of fine, non-iodized salt, is added and blended by hand. They are then immediately spread out in a 2–5 cm thick layer on a stainless steel, fine mesh, and screened rack for curing. The curing step is very brief and need not be more that 5–15 minutes. From curing the caviar is placed in two-part, specially coated, tins for aging and sale. The tins are slightly overfilled and the top placed on and pressed down to gently compress the caviar so as to drive out any trapped air. A broad rubber ban is placed about the connecting segment of the can and cans are placed in refrigerated storage, initially on their sides, for continued draining and curing. This step takes approximately 28 days for the caviar to develop a mild and distinctly oxidized flavor favored by caviar aficionados. It should be noted, however, that many Americans prefer the flavor of the caviar present immediately after the initial 15 minute curing which leads us to another subject. Just what is good caviar? Salinity (total salt) of 3 to 3.5% is achieved in the final product after the excess fluids are drained off (11). The final salt concentration will vary depending upon egg maturity, freshness, brining temperature, and brining time. Salting affects the physical characteristics of the egg and increases the hardness of the egg sheath. Egg sheath assays show that the hardness doubles after brining and curing. Sturgeon caviar can also be pressed (pausnaya). Small or damaged eggs may also be lightly salted and compacted into a product that resembles a thick marmalade and is used as a spread. Damaged eggs are also added to butter or soft cheese and can be incorporated into sauces or pâtés. Fine caviar has much akin with fine wine and individual preference can be very different. Superb caviar to one person may not be so to another. In general, however, the best caviar should not taste salty, fishy or musty. It should have a mild flavor described by some as being slightly “nutty.” The texture should be similar to that of butter and should almost melt in one’s mouth. High quality caviar should have no detectable membrane or residual shell material present and a pleasant aftertaste. There should be no metallic or other off-flavors. Caviar also tends to readily absorb off-flavors, particularly from contact with certain metals. Traditionally, therefore, it is consumed with an ivory, mother of pearl or horn spoon and served in glass or porcelain bowls. Caviar should be stored in the refrigerator, tightly closed in the jar or specially coated metal tin in which it was purchased.

Some individuals like to place the container inside of a sealed plastic bag as well. A few additional notes on processing; for maximum quality and storage stability, sturgeon roe should be handled using exemplary sanitation practices and processed at as low a temperature as possible. The processing, curing and storage areas should be as clean as an operating theater and the temperature of the area kept well below 50°F while processing. The eggs should not be allowed to come in contact with metals other than stainless steel. The water used in the processing should be as aseptic as possible and not contain chemicals that will impart any off-flavors. Only non-iodized salt should be used and salt that has not been coated so as to reduce caking. Kosher-style salt seems to work quite well. The flesh of harvested sturgeon is first reduced to what is referred in the seafood industry as a “bullet.” A bullet is formed by removing the fins, scutes (the armored plates on the sides of the fish), gills, blood, intestines and the spinal cord from the fish. The bullet is commonly processed further into fillets, steaks, roasts, and hot or cold smoked meat products. The skin can be tanned into rather durable leather.

B.

PADDLEFISH CAVIAR

Caviar from the sturgeon’s poor cousin, the paddlefish, is showing significant growth in the Southeastern and Western US as a source of black caviar. In North America, paddlefish (Polyodon spathula), the shovel-nose catfish (Hemisorubim platyrhynchos) in addition to fresh water sturgeons (Acipenser sp.) are harvested from wild stocks for black caviar production. Paddlefish are found mainly in the Mississippi–Missouri River systems, reaching as far north as Minneapolis and St. Paul, Minnesota; as far east as Pittsburgh, Pennsylvania; and as far south as New Orleans, Louisiana. Their roe is processed in a similar manner as sturgeon roe and the quality of these products can be very high, although the products are generally less expensive than sturgeon caviar, ranging between $7 to $15 per ounce (3). However, wholesale prices of U.S. domestic paddlefish caviar (2002) reached these levels with retail sales prices being in the range of $20–35/oz. Restriction on sturgeon caviar should cause prices for these products to increase. Domestic production of North America paddlefish roe is roughly 60,000 pounds. The commercial culture of paddlefish is viable. The fish are raised both for their meat and roe. The meat is commonly hot smoked. Paddlefish feed on plankton, and this has led to a unique method of polyculture for these fish in the U.S. Paddlefish can be placed at a relatively low density in catfish ponds where they feed on the natural algal blooms and on blooms resulting from the breakdown of uneaten feed and fecal material. This type of polyculture has the potential to reduce the problem with benthic off-flavors in catfish

Caviar and Fish Roe

resulting from consumption of geosmin containing algae. The paddlefish must large enough before introduction into ponds to discourage predation by the catfish. The roe from paddlefish harvested from recreational fisheries in Montana are sold by non-profit civic organizations and the government to commercial processors and the derived funds are used to support local fisheries and wildlife programs.

V. OTHER FISH ROE PRODUCTS The increased popularity of sushi, coupled with a heightened interest in haute, international, and fusion cuisines, has spurred the development of expanded markets and new products from fish and fish roe. Important quality parameters for roe are a small sized egg (generally); a mild flavor and an appropriate mouth feel including a good “pop.” The most marketable roe products can withstand distribution and handling procedures, frozen storage, and have a reasonable shelf life under refrigeration. Fish from many species can be colored or flavored to match popular products such as black caviar or tobiko, which are in short supply. Data for size and yield of various fish roe are presented in Table 161.1.

A.

CATFISH ROE

Channel catfish (Ictalurus punctatus) has been evaluated as a black caviar substitute (13). Catfish roe from Ictalurus sp. and Clarius sp. resembles that of paddlefish or sterle and ranges in color from dark charcoal to gray and rarely to a light gold. The eggs have a greater variation in average diameter than do most other species and many are of a smaller size. The eggs also tend to be much more difficult to separate at earlier stages of maturity, however, can be made in to an excellent spread that has a number of gourmet applications.

B.

SALMON ROE

“Red” or salmon caviars, called “ikura” in Russia and Japan are popular around the world. Ikura is the style of salmon caviar where individual eggs are separated from connective tissue and cured. Other forms of salmon roe preparations include marinated roe, smoked and flavored roe products, barako, and sujiko. Sujiko are salted and flavored whole skeins. The majority of salmon caviar is produced from Pacific salmon, with chum salmon (Oncorhynchus keta) and pink salmon (O. gorbuscha) being the most popular. Salmon caviar is also produced from the other Pacific salmon: coho (O. kisutch), sockeye or red (O. nerka), and king or Chinook (O. tshawytscha). Both the largest volume and value is from chum salmon with ikura production at 2000 to 3000 MT per year. Salmon eggs are a major source of income for Alaskan harvesters and processors; the price paid for chum and pink salmon is

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often so low as not to warrant harvest or processing, however, the roe always has value. It is against the law in North America to simply harvest the roe and not to utilize the remainder of the carcass, a process known as “roe stripping;” however, some jurisdictions have permitted the practice in times of severe economic depression. The practice is not as harmful to the salmon runs as one might think at first glance, as these salmon are at the termination of their life cycle and spawn but once and then die. The harvest size is calculated to not adversely impact future runs. Still the practice of discarding the carcasses should be avoided and some use should be found for the fish. Atlantic salmon (Salmon salar) and Arctic char (Salvelinus alpinus) are used to a lesser degree for caviar production (6). The Pacific masu or cherry salmon (O. masou masou) is a minor source of ikura, restricted to small regions of Japan and Korea (11). Returns of salmon are cyclic with a peak in the late 1990’s followed by a collapse in 2000, putting the valuable roe in incredibly short supply. Chum salmon returns for the 2001 harvest season were similarly grim. Harvests increased in 2002 and 2003, but prices were somewhat depressed due to a poor economy and near deflationary conditions in important East Asian markets. The market potential for 2004 was brighter, due to a stronger economy, and to the resurgence in interest in wild harvested salmon resulting from a number of unjustified food scares tied to cultured salmon in 2003 and 2004. High protein low carbohydrate fat diets spurred a trend for greater salmon consumption and bolstered prices for wild salmon. This trend is expected to continue through 2005. Most of the salmon roe harvested in Alaska is exported, primarily to Japan, Korea, China and Western Europe although the US domestic market is growing. Some is processed into ikura in Alaskan facilities, but a large quantity of the “green” or unprocessed roe is simply packed in bulk, frozen, and then exported. Farmed Atlantic salmon (Salmo salar), coho salmon (O. kisutch), rainbow trout (O. mykiss formerly Salmo gairdneri), and its ocean run variant steelhead trout (also O. mykiss and referred to as salmon trout) are available in large quantities, exceeding 1 billion pounds per year. Due to the current glut of salmon on the world market, Atlantic salmon culturists in Norway and Finland are holding fish to sexual maturity, harvesting the roe, and producing a very high quality salmon caviar. The market for the product is growing, particularly in Europe and in South America. Salmon trout is already a popular source of red caviar in Europe where much of the product is from cultured fish. There have been some complaints in Japanese ikura markets that roe from cultured fish have an “aquaculture” smell, but that the appearance, color and sheen are excellent (6). Any legitimate issues surrounding the flavor of the aquacultured product can be addressed by altering fish diet. If there continues to be an oversupply of

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salmon on the world market this will lead to even a greater proportions of the harvest being directed toward the production of roe products. 1.

Processing Salmon Roe-Sujiko

Eggs from salmonid fish are much larger than sturgeon roe, for chum, the eggs range from 4 to 5 mm diameter to as large as 7 mm for Chinook salmon (Table 161.1) and are generally processed with less added salt. Salmon roe have a less “fishy” flavor and oxidized flavor than many sturgeon caviars (6) because salmon roe products are not aged. Salmon roe are most commonly processed into cured, individual eggs (ikura) or as whole egg skeins (sujiko). Sujiko is prepared by brining whole roe skeins for approximately 20 min in a solution of salt, nitrites, polyphosphates, and other additives and seasonings. After brining, the skeins are sorted by quality and size, and then alternately layered with fine salt in plastic or wooden containers (most commonly containing 5 kg of the finished product). The curing process involves compressing the skeins under a weight for 3 to 5 days at temperatures below 50°F (11°C). The freshly brined product contains approximately 20 ppm nitrite and after curing, the finished product should have no more than 5 ppm, which is the maximum concentration allowed for import into Japan. At this time, this is not an approved use of sodium nitrite in the United States, therefore, all product thus processed in the U.S. must be exported. Sockeye or red salmon is the primary species used for sujiko production, although chum and pink salmon are also used in large quantities. The early summer run of sockeye salmon in Bristol Bay constitutes the largest single supply of sujiko, and every effort is made to speedily process and ship it to Japan each summer for the Japanese summer festival of Obon at which sujiko is traditionally consumed. A byproduct of sujiko production is “barako,” or singled-out eggs from broken or rejected skeins of sujiko. To make barako, the broken skeins are removed following the sujiko brining process, and the eggs are simply singled by mechanically separating them from the skein membrane using a screen. 2.

Processing Salmon Roe-Ikura

a. Separation of eggs from skein material The process for separating the eggs from each other and from skein material is called screening. Screening is normally a manual, laborious, and time-consuming process. Using the conventional method, the roe is rinsed with 3% salt brine. Then the individual eggs are removed from the skein by mechanically forcing the eggs gently through specially designed three tiers of nylon or stainless steel screens. Enzyme-based processes for removing the connective tissue surrounding the eggs for ikura production decreases hand labor and may increase recovery.

b. Enzyme processes for egg recovery Enzyme preparations for roe recovery include high concentrations of collagenase. The enzymatic process mimics the release of collagenases and other activities by the female fish to dissolve the connective tissue surrounding mature roe when the eggs are released into the water column for fertilization. Recovery of a high yield of good quality eggs varies greatly with enzyme treatments. Proteolytic enzyme mixtures for removing the membranous egg skein material from the individual eggs have been developed for salmon roe. c. Brining and curing processes for ikura Brining and curing fish eggs is still an art and requires a great deal of skill. Salmon caviar usually contains 3 to 4% total salt for “malasol” or lower salt caviar. Higher salt products in the range of 4 to 6% are also common and are becoming a regulatory requirement in some markets due to concerns about Clostridium botulinum growth. Products with a salt content (2.8 to 3.5% salt) are becoming more popular as consumer preferences change, but these products tend to have a shorter storage life and must be prepared with this consideration in mind. For ikura, the separated eggs are agitated in brine (for saturated brine the egg/brine ratio is usually 1:3 (v/v) or less). The eggs are brined, generally 2 to 6 minutes between 98

55%; Fat in Dry Matter, 4–51%, Minimum) Acid-Coagulated Smear Coat or Surface Mold Normal Lactic Starter Surface Mold Cottage cheese Brie Camembert Quarg (USA) Quesco-Blanco Bel Paese Neufchatel Petit Suisse

Unripened Fresh Cottage (UK) York

Source: References 15, 32, 48.

TABLE 178.9 Approximate Weight of Cheese Varieties

TABLE 178.10 Requirements of Cheese Packaging Materials

Cheese Variety

Low permeability to oxygen, carbon dioxide, and water vapor Strength and thickness of film Stability under cold or warm conditions Stability to fats and lactic acid Resistance to light, especially ultraviolet Ease of application, stiffness, elasticity Ability to seal and accept adhesives Laminated films to retain laminated Low shrinkage or aging unless shrinkage is a requisite Ability to take printed matter Should not impart odors to the cheese Suitability for mechanization of packaging Hygienic considerations in storage and use Cost effectiveness as a protective wrapping

Approximate Weight (kg)

Hard to Semi-Hard or Semisoft Wensleydale Caerphilly White Stilton Single Gloucester Leichester Derby Sage Derby Cheddar Cheshire Dunlap Double Gloucester Lancashire

3–5 3–6 4–8 10–12 13–18 14–16 14–16 18–28 20–22 20–27 22–28 22

Internally Mold-Ripened (Blue-Veined) Cheese Blue Wensleydale Blue Vinney Blue Stilton Blue Cheshire

3–5 5–7 6–8 10–20

Soft Cheese Colwich Cambridge Melbury

0.25–0.50 0.25–1.0 2.5

Source: References 15, 17, 34, 42, 48, 52, 54, 55, 62.

different manufacturing steps a wide variety of cheeses with various characteristics. Table 178.11 summarizes the basic steps in the cheese manufacturing process (12, 15, 31, 32, 34, 42, 48, 52, 54, 55, 62). Table 178.12 summarizes the ripening conditions for various cheeses. Selected examples are introduced below to provide an overview of the complexity of cheese manufacturing (12, 15, 31, 32, 34, 42, 48, 49, 50, 52, 54, 55, 62).

Source: References 15, 32, 34, 35, 42, 48, 52, 54.

for curdling the milk. After the casein is recovered, it is salted and subjected to fermentation, with or without inoculation with other microorganisms to produce the desirable characteristics of the various cheeses. Variations in the

1. Cottage Cheese Manufacturing Cottage cheese is a product with very mild fermentation treatment. It is produced by incubating (fermenting) the standardized and pasteurized skim milk with the starter

Manufacture of Fermented Product

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TABLE 178.11 Basic Cheese Making Steps

TABLE 178.13 Basic Steps in Making Cottage Cheese

Standardize cheese milks Homogenize cheese milks Heat-treat or pasteurize cheese milks Add starter Add color and additives Coagulation/Curdling: Cut coagulum/curd Stir and scald Wash curd cheese Salt cheese Press cheese Coat, bandage, and wrap cheese Let cheese ripen Package for retail Store

Standardize skim milk Pasteurize milk with standard procedure and cool to 32°C Inoculate with Active Lactic Starter, Add Rennet, and Set Curd: Rennet addition—at 2 ml single strength (prediluted, 1:40) per 1000 kg milk within 30 minutes of starter addition Specifications Short Set Medium Set Long Set Starter concentration 5% 3% 0.5% Temperature of milk set 32°C 27°C 22°C Time from setting to cutting 5 hr 8 hr 14–16 hr Final pH and whey titratable acidity—4.6 and 0.52%, respectively Cut curd with 1.3, 1.6, or 1.9 cm wire cheese knife Cook Curd: Let curd cubes stand for 15–30 minutes and cook to 51–54°C at 1.7°C per 10 minutes Roll the curds gently every 10 minutes after initial 15–30 minute wait Test curd firmness and hold 10–30 minutes longer to obtain proper firmness Wash Curd: First wash with 29°C water temperature Second wash with 16°C water temperature Third wash with 4°C water temperature Drain washed curd (by gravity) for about 2.5 hours Salt and cream at 152 kg creaming mixture per 454 kg with final 0.5–0.75% salt content and 4% fat content (varies with products and optional) Package in containers Storage at refrigerated temperature

Source: References 12, 15, 31, 32, 34, 42, 48, 49, 52, 54, 55, 62.

TABLE 178.12 Cheese Ripening Conditions Storage

Relative

Types of

Period

Temperature

Humidity

Cheese

(days)

(°C)

(%)

Soft Mold ripened Cooked, e.g., Emmental Cold room Warm room Hard, e.g., Cheddar

12–30 15–60

10–14 4–12

90–95 85–95

7–25 25–60 45–360

10–15 18–25 5–12

80–85 80–85 87–95

Sources: References 12, 15, 31, 32, 34, 42, 48–50, 52, 54, 55, 62.

lactic acid bacteria to produce enough acid and appropriate pH for the curdling of milk. The curd is then recovered and washed, followed by optional salting and creaming. The product is then packed and ready for marketing. No further ripening is required for this product. This is different from most fermented cheeses that require a ripening process. Table 178.13 lists the various steps involved in the production of cottage cheese (15, 34, 42, 48, 52, 55, 62). 2. Cheddar Cheese Manufacturing Cheddar cheese is a common hard cheese without eyes used in the fast-food industry and in the household. Its production process is characterized by a requirement for milling and cheddaring of the curd. This cheese can be ripened with a wax rind or rindless (sealed under vacuum in plastic bags.) It is also categorized into regular, mild, or sharp based on the aging period (45–360 days). The longer the aging period, the sharper the flavor. It is packaged as a large block or in slices. Table 178.14 lists the basic steps in the manufacturing of cheddar cheese (15, 34, 42, 48, 52, 55, 62).

Source: References 15, 34, 42, 48, 52, 55, 62.

3. Swiss Cheese Manufacturing Swiss cheese is also a common cheese used in the fastfood industry and in the household. It is characterized by having irregular eyes inside the cheese. These eyes are produced by Propionicbacterium freudenreichii subsp. shermanii, which produces gases trapped inside the block of cheese during fermentation and ripening. A cheese with eyes like Swiss cheese has become the icon for cheese in graphics. Swiss cheese is also characterized by its propionic acid odor. The salting process for Swiss cheese utilizes both the dry- and brine-salting processes. Like cheddar cheese, it can be categorized into regular, mild, and sharp, depending on the length of the curing process. Table 178.15 lists the basic steps in the manufacture of Swiss cheese (15, 34, 42, 48, 52, 55, 62). 4. Blue Cheese Blue cheese is characterized by its strong flavor and by blue mold filaments from Penicillium roqueforti inside the cheese. It is commonly consumed as cheese or made into a salad dressing. In the manufacturing of blue cheese, as in that of Swiss cheese, salting is accomplished by the application of dry-salting and brining processes. It is characterized by a cream bleaching step to show off the blue

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TABLE 178.14 Basic Steps in Making Cheddar Cheese

TABLE 178.15 Basic Steps in Making Swiss Cheese

Standardize cheese milk. Homogenize milk. Pasteurization and additional heating of milk. Cool milk to 31°C. Inoculate milk with lactic starter (0.5–2% active mesophilic lactic starter). Add rennet or other protease(s)—198 ml single strength (1:15,000) rennet per 1000 kg milk. Dilute the measured rennet 1:40 before use. Agitate at medium speed. Set the milk to proper acidity—25 minutes. Cut the curd using 0.64 cm or wider wire knife. Stir for 5 minutes at slow speed. Cook the curd at 38°C for 30 minutes with 1°C for every 5 minute increment. Maintain temperature for another 4–5 minutes and agitate periodically at medium speed. Drain the curd at 38°C. Cheddar the curd at pH 5.2–5.3. Mill the curd slabs. Salt the curd at 2.3–3.5 kg salt per 100 kg curd in three portions in 30 minutes. Waxed Cheddar Cheese: Hoop and press at 172 kPa for 30–60 seconds then 172–344 kPa overnight. Dry the cheese at 13°C at 70% RH for 2–3 days. Paraffin the whole cheese at 118°C for 6 seconds. Rindless Cheddar Cheese: Press at 276 kPa for 6–18 hours. Prepress for 1 minute, followed by 45 minutes under 686 mm vacuum. Remove and press at 345 kPa for 60 minutes. Remove and vacuum seal in bags with hot water shrinkage at 93°C for 2 seconds. Ripen at 85% RH at 4°C for 60 days or longer, up to 9–12 months, or at 3°C for 2 months then 10°C for 4–7 months, up to 6–9 months.

Standardize cheese milk to 3% milk fat—treatment with H2O2-catalase optional. Pasteurize of the milk. Inoculate with Starters: Streptococcus thermophilus, 330 ml per 1000 kg milk Lactobacillus delbruechii subsp. bulgaricus, 330 ml per 1000 kg milk Propionibacterium freudenreichii subsp. shermanii, 55 ml per 1000 kg milk Add rennet, 10–20 minutes after inoculation—154 ml single-strength (1:15,000) rennet extract per 1000 kg milk, prediluted 1:40 with tap water before addition. Stir for 3 minutes. Let milk set (coagulate) for 25–30 minutes. Cut the curd with 0.64 wire knife; let stand undisturbed for 5 minutes; stir at medium speed for 40 minutes. Cook the curd slowly to 50–53°C for about 30 minutes and stir at medium speed, then turn off steam and continue stirring for 30–60 minutes with pH reaching 6.3–6.4. Allow the curd to drip for 30 minutes. Press the curd—with preliminary pressing, then at 69 kPa overnight. Salt the Curd: First salting—in 23% salt brine for 2–3 days at 10°C Second salting—at 10–16°C, 90% RH. Wipe the cheese surface from the brine soaking, then sprinkle salt over cheese surface daily for 10–14 days Third salting—at 20–24°C, 80–85% RH. Wash cheese surface with salt water and sprinkle with dry salt 2–3 times weekly for 2–3 weeks Rinded Block Swiss Cheese: Cure—at 7°C or lower (USA) or 10–25°C (Europe) for 4–12 months. Package in container and store at cool temperature. Rindless Block Swiss Cheese: Wrap or vacuum pack the blocks. Cure stacked cheese at 3–4°C for 3–6 weeks. Store at cool temperature.

Source: References 15, 34, 42, 48, 52, 55, 62.

mold filament with a lighter background and by needling the block of curd to spread the blue mold filaments. It also has a soft and crummy texture due to the needling process and to the gravity draining procedure used to drain the curd. The curing period of two to four months is shorter than for hard cheeses. Its shelf life of two months is also shorter than that of its harder counterparts. Table 178.16 lists the basic steps in the manufacture of blue cheese (15, 34, 42, 48, 52, 55, 62). 5. American Style Camembert Cheese American style camembert cheese is categorized as a soft cheese. It is characterized by a shell of mold filament on the surface produced by Penicillium camembertii. Brie cheese is a similar product. Addition of annatto color is optional. Like blue cheese, it is gravity drained. Therefore it has a soft, smooth texture. This cheese is surface salted and has a total curing period of three weeks before distribution. It is usually cut into wedges and wrapped individually for direct

Sources: References 15, 34, 42, 48, 52, 55, 62.

consumption. Table 178.17 lists the basic steps in the manufacture of American style camembert cheese (15, 34, 42, 48, 52, 55, 62). 6. Feta Cheese Manufacturing Feta cheese is a common cheese in the Mediterranean countries. It is a soft cheese characterized by its brine curing (maturation) process, which is not common in cheese making. Instead, it has a similarity to the manufacture of sufu (Chinese fermented tofu, see below in this chapter). Like other soft cheese, the curing period is only two to three months. Table 178.18 lists the basic steps in the manufacture of Feta cheese (50).

C. YOGURT Yogurt can be considered as a curdled milk product. Plain yogurt is yogurt without added flavor, stabilizer, or


TABLE 178.16 Basic Steps in Making Blue Cheese Milk Preparation: Separate cream and skim milk. Pasteurize skim milk by HTST, cool to 30°C. Bleach cream with benzoyl peroxide (optional) and heat to 63°C for 30 seconds. Homogenize hot cream at 6–9 mPa and then 3.5 mPa, cool, and mix with pasteurized skim milk. Inoculate milk at 30°C with 0.5% active lactic starter. Let stand for 1 hour. Add rennet—158 ml single strength (prediluted 1:40) per 1000 kg milk. Mix well. Let Coagulate or set, 30 minutes. Cut curd with 1.6 cm standard wire knife. Cook curd at 30°C, let stand 5 minutes, and then agitate every 5 minutes for 1 hour. Whey should have 0.11 to 0.14 titratable acidity. Drain whey by gravity for 15 minutes. Inoculate with Penicillium roqueforti spores—2 kg coarse salt and 28 g P. requeforti spore powder per 100 kg curd followed by thorough mixing. Add food grade lipase (optional). Salting: First salting—dip the curd in 23% brine for 15 minutes, then press or mold at 22°C, turning every 15 minutes for 2 hours and every 90 minutes for rest of day. Second salting—salt cheese surface everyday for 5 days at 16°C, 85% RH. Final dry salting or brine salting in 23% brine for 24–48 hours. Final salt concentration about 4%. Incubate for 6 days at 16°C, RH Wax and needle air holes or vacuum pack and need air holes. Mold filament development in air holes at 16°C for 6–8 days. Cure at 11°C and 95% RH for 60–120 days. Cleaning and Storing: Strip off the wax or vacuum packaging bag. Clean cheese, dry, and repack in aluminum foil or vacuum packaging bags. Store at 2°C. Product shelf life—2 months. Source: References 15, 34, 42, 48, 52, 55, 62.

coagulant. Its acceptance is limited to those who really enjoy eating it. With the development of technology, other forms of yogurt, such as flavored and sweetened yogurt, stirred yogurt, yogurt drinks, and frozen yogurt, are now available. Its popularity varies from location to location. It is considered as a health food when active or live cultures are added to the final product. Table 178.19 lists the basic steps involved in the manufacture of yogurt. Table 178.3, presented earlier, should also be consulted for reference to other ingredients (9, 58). Most commercially produced yogurt and its products contain sweeteners, stabilizers, or gums (Table 178.20); fruit pieces; natural and synthetic flavors (Table 178.21); and coloring compound (Table 178.22) (9, 58). Different countries also have different standards on the percent fat and percent solids-not-fat (SNF) contents in their yogurt products (Table 178.23) (9, 58).

178-9

TABLE 178.17 Basic Steps in Making American Style Camembert Cheese Standardize milk. Homogenize milk. Pasteurize milk at 72°C for 6 seconds. Cool milk to 32°C. Inoculate with 2% active lactic starter followed by 15–30 minutes acid ripening to 0.22% titratable acidity. Add annatto color at 15.4 ml per 1000 kg milk (optional). Add rennet —220 ml single-strength (prediluted 1:40) rennet per 1000 ml, then mix for 3 minutes and let stand for 45 minutes. Cut curd with 1.6 cm standard wire knife. Cook curd at 32°C for 15 minutes with medium speed stirring. Drain curd at 22°C for 6 hours with occasional turning. Inoculate with Penicillium camemberti spores by spray gun on both sides of cheese once. Press and mold curd by pressing for 5–6 hours at 22°C without any weight on surface. Surface salt cheese; let cheese stand for about 9 hours. Cure—at 10°C, 95% RH for 5 days undisturbed, then turn once and continue curing for 14 days. Packaging, Storage, and Distribution: Wrap cheese and store at 10°C, 95–98% RH for another 7 days. Move to cold room at 4°C and cut into wedges, if required, and rewrap. Distribute immediately. Source: References 15, 34, 42, 48, 52, 55, 62.

TABLE 178.18 Basic Steps in Making Feta Cheese Standardize milk with 5% fat, enzyme treated and decolorized. Homogenize milk. Pasteurize by standard procedure and cool to 32°C. Inoculate with 2% active lactic starter as cheddar cheese and allow to ripen for 1 hour. Add rennet at 198 ml single-strength rennet (prediluted, 1:40) per 1000 kg milk and let set for 30–40 minutes. Cut the curd with 1.6 cm standard wire knife and let stand 15–20 minutes. Allow curd to drip for 18–20 hour at 12–18 kg on 2000 cm2, with pH and titratable acidity developed to 4.6 and 0.55%, respectively. Prepare cheese blocks of 13 ⫻ 13 ⫻ 10 cm each. Salt in 23% salt brine for 1 day at 10°C. Can and box cheese blocks in 14% salt brine (sealed container). Cure for 2–3 months at 10°C. Soak cured cheese in skim milk for 1–2 days before consumption to reduce salt. Yield—15 kg/100 kg of 5% fat milk. Source: Reference 50.

The different variables described above make the situation complicated. The term “yogurt” in one country may not have the same meaning in another country. This creates difficulties for international trade. Consensus or agreement among countries, and proper labeling are needed to identify the products properly.

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TABLE 178.19 Basic Steps in the Production of Yogurt Standardize liquid milk. Homogenize liquid milk. Heat-treat or pasteurize liquid milk at 90°C for 5 minutes or equivalent. Cool pasteurized milk to 1–2°C above inoculation temperature. Add starter (inoculation), 1–3% operational culture. Add flavor, sweetener, gums, and/or color (optional). Incubate at 40–45°C for 2.5–3.0 hours for standard cultures. Break curd (optional). Cool to 15–20°C in 1–1.5 hours. Add live culture (optional). Package. Store at ⱕ10°C.

TABLE 178.20 Some Common Gums that Could Be Used in Yogurt Manufacturing Kind

Name of Gum

Natural

Agar Alginates Carrageenan Carob gum Corn starch Casein Furcelleran Gelatin Gum arabic Guar gum Karaya gum Pectins Soy protein Tragacanth gum Wheat starch Cellulose derivatives Dextran Low-methoxy pectin Modified starches Pregelatinized starches Propylene glycole alginate Xanthin Polyethylene derivatives Polyvinyl derivatives

Source: References 9 and 58.

D. FERMENTED LIQUID MILKS In milk-producing countries, it is common to have fermented milk products. These products were first discovered or developed by accident. Later, the process was modified for commercial production. Fermented liquid milks are similar to plain yogurt drinks. It is basically milk that has gone through an acid and or alcoholic fermentation. The final product is maintained in the liquid form rather than in the usual soft-gel form of yogurt. There are different fermented liquid milks available, but only sour milk, kefir, and acidophilus milk are discussed below. Readers should refer to the references listed below and other available literature on related products. 1. Sour Milk Manufacturing Table 178.24 presents the basic steps in the manufacturing of the most basic fermented liquid milk, sour milk. The milk is standardized, pasteurized, inoculated, incubated, homogenized, and packaged. It is a very straightforward

Modified gums

Synthetic gums

Sources: References 9 and 58.

procedure compared to those for the other two products, kefir and acidophilus milk (12, 15, 31, 32, 34, 49, 55, 62). 2.

Kefir Manufacturing

Kefir is a fermented liquid milk product characterized by the small amount of alcohol it contains and its inoculant, the kefir grains. It is a common product in the Eastern

TABLE 178.21 Some Common Flavors for Yogurt Retail Flavor Apricot Banana Bilberry Black currant Grape, Concord Lemon Peach Pineapple Raspberry Strawberry Source: References 9 and 58.

Natural Characteristic—Impact Compound NA 3-Methylbutyl acetate NA NA Methyl antranilate Citral g-Decalactone NA 1-p-Hydroxyphenyl-3-butanone NA

Synthetic Flavoring Compound Available g-Undecalactone NA NA trans- and cis- p-Methane-8-thiol-3-one NA 15 compounds g-Undecalactone Allyl hexanoate NA Ethyl-3-methyl-3-phenylglycidate


178-11

TABLE 178.22 Permitted Yogurt Colorings Name of Color

TABLE 178.25 Basic Steps in Kefir Processing Maximum Level (mg /kg)

Intigotine Brilliant black PN Sunset yellow FCF Tartrazine Cochineal Carminic acid Erythrosine Red 2G Ponceau Caramel

6 12 12 18 20 20 27 30 48 150


TABLE 178.23 Existing or Proposed Standards for Commercial Yogurt Composition [% Fat and % Solid-Not-Fat (SNF)] in Selected Countries % Fat Country Australia France Italy Netherlands New Zealand UK USA West Germany FAO/WHO Range

Low

Medium

Normal

% SNF

NA 0.5 1 1 0.3 0.3 0.5–1.0 0.5 0.5 0.3–1.0

0.5–1.5 NA NA NA NA 1.0–2.0 2 1.5–1.8 0.5–3.0 0.5–3.0

3 3 3 3 3.2 3.5 3.25 3.5 3 3–3.5

NA NA NA NA NA 8.5 8.5 8.25–8.5 8.2 8.2–8.5


TABLE 178.24 Basic Steps in Sour Milk Processing Standardize milk. Heat milk to 85–95°C, then homogenize. Cool milk to 19–25°C and transfer to fermentation tank. Add 1–2% start culture (inoculation). Allow shock-free fermentation to pH 4.65–4.55. Homogenize gel. Cool to 4–6°C. Fill bottles, jars, or one-way packs or wholesale packs.

Preparation of Mother “Kefir” Standardize milk for preparation of mother “kefir.” Pasteurize milk at 90–95°C for 15 minutes and cool to 18–22°C. Spread kefir grains at the bottom of a container (5–10 cm thick) and add pasteurized milk (20–30 times the amount of kefir grains). Ferment for 18–24 hours, mixing 2–3 times. Kefirs grains float to the surface. Filter out the kefir grains with a fine sieve, wash the grains with water, and save for the next fermentation. Save the fermented milk for the next-step inoculation. Preparation of Drinkable Kefir Blend fermented milk from above with 8–10 times fresh, pasteurized, untreated milk. Pour into bottles, then close the bottles and ferment mixture for 1–3 days at 18–22°C. [Another option is to mix the fermented milk with fresh milk at 1–5% and ferment at 20–25°C for 12–15 hours (until pH 4.4–4.5 is reached), then ripen in storage tanks 1–3 days at 10°C. Product is not as traditional but is acceptable.] Cool to refrigerated temperature. Store and distribute. Source: References 12, 15, 18, 31, 32, 34, 48, 49, 55, 62.

products of bacterial metabolism, together with curds of milk protein. Production of kefir is a two-step process: (1) the production of mother kefir and (2) the production of the kefir drink. Table 178.25 lists the basic steps in kefir manufacturing (12, 15, 18, 31, 32, 34, 48, 49, 55, 62). 3.

Acidophilus Milk

Acidophilus milk is considered to have probiotic benefits. Like yogurt, it is advertised as having live cultures of Lactobacillus acidophilus and Bifidobacterium bifidum (optional). These live cultures are claimed to provide the benefit of maintaining a healthy intestinal microflora. Traditional acidophilus milk has a considerable amount of lactic acid and is considered to be too sour for the regular consumers in some locations. Therefore, a small amount of sugar is added to the final product to make it more palatable. This later product is called sweet acidophilus milk. Table 178.26 lists the basic steps in the manufacture of acidophilus milk (12, 15, 31, 32, 34, 48, 49, 55, 62).

Source: References 12, 15, 31, 32, 34, 49, 55, 62.

III. MEAT PRODUCTS European countries and is considered to have health benefits. Among all the fermented dairy products, only this and similar products contain small amounts of alcohol. Also, in all the other fermented dairy products, pure cultures of bacteria, yeasts, and/or molds are used, but in kefir, the kefir grains are used and recycled. Kefir grains are masses of bacteria, yeasts, polysaccharides, and other

A. INGREDIENTS AND TYPES Fermented meat products such as ham and sausages have been available to different cultures for centuries. It is interesting to learn that the ways these products are produced are basically very similar in different cultures. Besides the meat, nitrite and salt, and sugar (optional), pure cultures are

178-12


TABLE 178.26 Basic Steps for Sweet Acidophilus Milk Processing Procedure 1: Standardize milk. Heat milk to 95°C for 60 minutes, cool to 37°C, and hold for 3–4 hours; reheat to 95°C for 10–15 minutes, then cool to 37°C. Inoculate with 2–5% bulk starter. Incubate for up to 24 hours or to 1% lactic acid. Cool to 5°C. Pack and distribute. Procedure 2: Standardize milk. Homogenize milk at 14.5 MPa. Heat to 95°C for 60 minutes. Cool to 37°C. Inoculate with direct vat inoculation (DVI) starter. Incubate for 12–16 hours or to about 0.65% lactic acid. Heat at ultra high temperature (UHT), 140–145°C for 2–3 seconds to eliminate undesirable contaminants. Cool to 10°C or lower. Package and distribute. Source: References 12, 15, 31, 32, 34, 48, 49, 55, 62.

sometimes used, especially in fermented sausages. Microorganisms do not merely provide the characteristic flavor for the products; the lactic acid bacteria also produce lactic and other acids that can lower the pH of the products. Pure cultures are sometimes used in hams to lower the pH and thus inhibit the growth of Clostridium botulinum. The raw meat for ham manufacturing is basically a large chunk of meat, and it is difficult for microorganisms to penetrate TABLE 178.27 Raw Ingredients for Fermented Meat Products Ingredient Meat Pork Beef Casing Salt Sugar Starter microorganisms Lactobacillus sakei, L. curvatus, L. plantarum, L. pentosus, L. pentoaceus Pediococcus pentosaceus, P. acidilactic Staplyococcus xylosus, S. carnosus Kocuria varians Debaryomyces hansenit Candida famata Penicillium nagiovense, P. chrysogenum Spices Other flavoring compounds Moisture retention salts Preservatives

Ham Yes No No Yes Optional Optional

Optional Optional Optional No

Source: References 6, 24, 28, 29, 51, 53, 60, 61, 64.

Sausage Yes Optional Yes Yes Optional Optional

Optional Optional Optional No

TABLE 178.28 Basic Steps in Dry Cured Ham Processing Prepare pork for dry curing. Mix the proper ratio of ingredients [salt, sugar, nitrite, and inocula (optional)]. Rub the curing mixture into the meat. Stack the green ham for initial dry curing at 36–40°C. Rerub the green ham and stack for additional curing at 36–40°C. [The ham should be left in the cure for the equivalent of 3days per pound of meat.] Soak the cured ham for 2–3 hours, then thoroughly scrub. Place green ham in tight-fitting stockinette and hang in smokehouse to dry overnight. Smoke at about 60 or 80°C with 60% RH for 12–36 hours. Cool. Vacuum pack and place in cool storage. Source: References 6 and 61.

into the center, unless they are injected into the interior. Microbial growth is mainly on the surface, and the microbial enzymes are gradually diffused into the center. By contrast, in sausages the cultures, if used, are mixed with the ingredients at the beginning, and the fermentation is carried out without difficulty. Besides, sausages are much smaller than hams. Table 178.27 lists some of the ingredients used in the manufacture of hams and sausages (6, 24, 28, 29, 51, 53, 60, 61, 64).

B. HAMS Hams, as indicated earlier, are made from large chunks of meat. Western cultures manufacture ham using either a dry cure and or a brine cure process, sometimes followed by a smoking process. Tables 178.28 and 178.29 list the basic steps involved with the dry cure and brine cure of hams, respectively. These two processes are similar except for the salting step (6, 61). Chinese hams are basically manufactured using a dry curing process. Procedures differ slightly, depending on TABLE 178.29 Basic Steps in Brine Cured Ham Processing Prepare pork for brine curing. Mix the proper ratio of ingredients (salt, sugar, and nitrite with inocula optional): 5 gallons of brine for 100 pounds meat. Soak the meat in the prepared brine, or stitch pump the brine into the meat (10% of the original weight of the meat) followed by soaking in the brine for 3–7 days vacuum tumbling or massaging (optional). Remove the meat from the cover brine and wash. Place green ham in tight-fitting stockinette and hang in smokehouse to dry overnight. Smoke at about 60 or 80°C and 60% RH for 12–36 hours. Cool. Vacuum pack and place in cool storage. Source: References 6 and 61.


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TABLE 178.30 Basic Steps in Chinese Jinghua Ham Processing

TABLE 178.31 Basic Steps in Dry (Fermented) Sausage Processing

Select pork hind leg, 5–7.5 kg. Trim. Salt, 7–8 kg salt per 10 kg ham. Stack and overhaul at 0–10°C for 33–40 days. Wash with cold water and brush. Dry in the sun for 5–6 days. Ferment (cure) for 2–3 months at 0–10°C (harmless green mold will develop on surface). Brush off the mold and trim. Age for 3–4 months, maximum 9 months; alternate aging process in temperature-programmable room with 60% RH for 1–2 months. Grade. Package and distribute. (Yield: about 55–60%.)

Select meat for processing. Chop and mix chopped meat with spices, seasonings, and inocula at temperature of about 10°C. Stuff the mixture in suitable casings. Make links. Cure or dry for 1–3 months in rooms with temperature, relative humidity, and air circulation regulated according to the type of sausage being produced. Package and place in cool storage.


the regions where the hams are made. The most famous Chinese ham is the Jinghua ham made in central China. Yunan ham, from southern China, also has a good reputation. In the old days, without refrigeration facilities during processing, transportation, and storage, it is believed that the ham completed its aging process during the transportation and storage stages. Today, with controlled temperature and relative humidity rooms, the hams are produced under controlled conditions. Table 178.30 lists the current process used in China for Jinghua ham (28, 64).

C. SAUSAGES Many European-type sausages are manufactured using a fermentation process. These sausages have their own characteristic flavors due to the formulations and curing processes used. It is not the intent of this chapter to list the various formulations. Readers should consult the references in this chapter and other references available elsewhere. Commercial inocula are available. Bacteria and some yeasts grow inside the sausage during the ripening period, producing the characteristic flavor. Molds can grow on the surface during storage if sausages are not properly packaged and stored in the refrigerator. Because these sausages are not sterilized, fermentation is an ongoing process, and the aged sausages carry a stronger flavor. Table 178.31 lists the basic steps in the manufacture of dry fermented sausages (24, 29, 51, 60).

IV. FERMENTED CEREAL PRODUCTS (BREADS AND RELATED PRODUCTS) A. KINDS

OF

PRODUCTS AND INGREDIENTS

In wheat-producing countries or areas, baked yeast bread is a major staple in people’s diets. This is common in the

Source: References 24, 29, 51, 60.

major developed countries. In other countries, other forms of bread may be the major staple. Baked bread may come in different forms such as regular yeast breads, flat breads, and specialty breads. Today, even retarded (chilled or frozen) doughs are available to meet consumers’ preference for a semblance of home-cooked food. For countries or areas with less available energy, other forms of bread such as steamed bread and boiled breads are available. Fried breads are consumed mainly as breakfast or snack items. Table 178.32 lists some examples of different types of breads (8, 22, 27, 45–47). Today, as a result of centuries of breeding selection, there are different types of wheat available to suit production environments in various regions with diverse climatic conditions. Wheat used for making bread is hard wheat, soft wheat, or a combination of both to meet product specifications. Wheat kernels are milled with removal of the bran and germ and further processed into wheat flour. Traditionally, this flour is the major ingredient for baking bread. For some health conscious consumers, whole wheat flour is the flour of choice for making bread nowadays. Wheat bran is also added to increase the fiber content of the product. Table 178.33 lists the proximate

TABLE 178.32 Types of Bread and Related Products Type Baked Breads Regular yeast breads Flat (layered) breads Specailty breads

Chilled or frozen doughs Steamed breads Fried breads Boiled breads

Examples Bread (white, whole wheat or muti-grain) Pocket bread, croissants Sourdough bread, rye bread, hamburger bun, part-baked bread, Danish pastry, stuffed bun Ready-to-bake doughs, retarded pizza doughs, frozen proved dough Chinese steamed bread (mantou), steamed stuffed buns Doughnuts Pretzels

Source: References 8, 22, 27, 45, 46, 47.

178-14


TABLE 178.33 Composition of Wheat, Flour, and Germ Material Wheat Hard red spring Hard red winter Soft red winter White Durum Flour, Straight Hard wheat Soft wheat Flour, Patent Bread Germ

TABLE 178.34 Bread Making—Functional Ingredients

Mositure Protein Fat Total Fiber % % % CHO % %

Ash %

13 12.5 14 11.5 13

14 12.3 10.2 9.4 12.7

2.2 1.8 2 2 2.5

69.1 71.7 72.1 75.4 70.1

2.3 2.3 2.3 1.9 1.8

1.7 1.7 1.7 1.7 1.7

12 12

11.8 9.7

1.2 1

74.5 76.9

0.4 0.4

0.46 0.42

12 11

11.8 25.2

1.1 10

74.7 49.5

0.3 2.5

0.44 4.3

Kind Basic Ingredient Wheat flour Yeast

Optional Ingredients

Additives Emulsifier

Flour treatment agents Preservatives

B. REGULAR BREAD Processing Aids

Table 178.35 lists the basic steps in bread manufacturing (8, 22, 45). There are three basic processes in commercial bread making: straight dough process, sponge-and-dough process, and continuous-baking process. The process to be used is determined by the manufacturer and the equipment available in the baking plant. Table 178.36 lists the basic steps in the different processes. The major difference is in the way the dough is prepared and handled (8, 22, 45). Because the dough may be prepared in various ways, the amounts of ingredients used differ accordingly. Table 178.37 lists two formulations, comparing the differences in ingredients that arise from differences in the dough preparation processes (8, 22, 45).

C. RETARDED DOUGH As indicated earlier, retarded dough is also available to some consumers. This type of dough is more accessible where refrigerators and freezers are more common. Dough is prepared so that the fermentation is carefully controlled, and the dough is packed inside the container. Storage of

Bread flour, whole wheat flour Compressed yeast, granular yeast, cream yeast, dried yeast, instant yeast, encapsulated yeast, frozen yeast, pizza yeast, deactivated yeast Saccharomyces cervisiae, S. carlsburgenis, S. exisguus

Salt Water


composition of wheat and some of their common wheat products (8, 22, 45). In the manufacture of various wheat-based breads and related products, the major ingredients are wheat flour, yeast, sourdough bacteria (optional), salt, and water. Other ingredients vary considerably with the types of products produced. These may be grossly classified as optional ingredients, additives, or processing aids. Each country has its own regulations and requirements. Table 178.34 lists basic ingredients, optional ingredients, additives, and processing aids used in the manufacturing of bread and related products (8, 22, 45).

Examples

Whole wheat flour, gluten, soya flour, wheat bran, other cereals or seeds, milk powder, fat, malt flour, egg, dried fruit, vitamins Sourdough bacteria: Lactobacillus plantarum, L. brevis, L. fermentum, L. sanfrancisco Other yeasts Diacetylated tartaric acid esters of mono- and diglycerides of fatty acids (DATA esters), Sodium stearyl-2-lactylate (SSL), distlled monoglyceride, lecithin Ascorbic acid, L-cysteine, potassium bromate, potassium iodate, azodicarbonamide Acetic acid, potassium acetate, sodium diacetate, sorbic acid, potassium sorbate, calcium sorbate, propionic acid, sodium propionate, calcium propionate, potassium propionate Alpha-amylase, hemicellulose, proteinase, novel enzyme systems (lipases, oxidases, peroxidases)


TABLE 178.35 Basic Steps in Regular or Common Bread Making Prepare basic and optional ingredients. Prepare yeast or sourdough for inoculation. Mix proper ingredients to make dough. Allow to ferment. Remix dough (optional). Sheet. Mold and pan. Proof in a temperature and relative humidity controlled chamber. Decoratively cut dough surface (optional). Bake, steam, fry, or boil. Cool. Package. Store. Source: References 8, 22, 45.


178-15

TABLE 178.36 Various Bread Making Processes

TABLE 178.37 Sample Bread Recipes

Straight Dough Baking Process: Weigh out all ingredients. Add all ingredients to mixing bowl. Mix to optimum development. Allow first fermentation, 100 minutes, room temperature, or at 27°C for 1.5 hours. Punch. Allow second fermentation, 55 minutes, room temperature, or at 27°C for 1.5 hours. Divide. Allow intermediate proofing, 25 minutes, 30–35°C, 85% RH Mold and pan. Allow final proofing, 55 minutes at 30–35°C, 85% RH Bake at 191–232°C for 18–35 minutes to approximately 100°C internal temperature.

White Pan Bread (Bulk Fermentation or Straight Dough Process):

Sponge-and-Dough Baking Process: Weigh out all ingredients. Mix part of flour, part of water, yeast, and yeast food to a loose dough (not developed). Ferment 3–5 hours at room temperature, or at 21°C for 12–16 hours. Add other ingredients and mix to optimum development. Allow fermentation (floor time), 40 minutes. Divide. Allow intermediate proofing, 20 minutes, 30–35°C, 85% RH, or 27°C for 30 minutes. Mold and pan. Allow final proofing, 55 minutes, 30–35°C, 85% RH Bake at 191–232°C for 18–35 minutes to approximately 100°C internal temperature. Continuous-Baking Process: Weigh out all ingredients. Mix yeast, water, and maybe part of flour to form liquid sponge. Add remaining flour and other dry ingredients. Mix in dough incorporator. Allow fermentation, 2–4 hours, 27°C. Pump dough to development chamber. Allow dough development under pressure at 80 psi. Extrude within 1 minute at 14.5°C and pan. Proof for 90 minutes. Bake at 191–232°C for 18–35 minutes to approximately 100°C internal temperature. Source: References 8, 22, 45.

this package is also carefully controlled. When the package is open, consumers can just follow the instructions on the package to bake their own bread. The technology is proprietary to the manufacturers, but there are some guidelines available (Table 178.38) (8, 22, 45).

D. FLAT (LAYERED) BREAD Flat bread is a general term for bread products that do not rise to the same extent as regular bread. Flat breads are common commodities in Middle Eastern countries and in

Ingredients Percent of Flour Weight Flour 100.0 Yeast 1.0 Salt 2.0 Water 57.0 Optional Dough Improving Ingredients Fat 0.7 Soya flour 0.7 Malt flour 0.2 White Pan Bread (Sponge and Dough Process): Sponge Ingredient Flour Yeast Salt Water

Percent of Total Flour 25.0 0.7 0.5 14.0

Dough Ingredients Flour Yeast Salt Water Optional Improving Ingredients Fat Soya flour Malt flour

Percent of Total Flour 75.0 2.0 1.5 44.0 0.7 0.7 0.2


TABLE 178.38 General Guidelines for Retarded Dough Production Reduce yeast levels as storage times increase. Keep yeast levels constant when using separate retarders and provers. Reduce yeast levels as the dough radius increases. Reduce yeast levels with higher storage temperatures. The lower the yeast level used, the longer the proof time will be to a given dough piece volume. Yeast levels should not normally be less than 50% of the level used in scratch production. For dough stored below ⫺5°C, the yeast level may need to be increased. Reduce the storage temperature to reduce expansion and weight loss from all dough pieces. Lower the yeast levels to reduce expansion and weight losses at all storage temperatures. Dough pieces of large radius are more susceptible to the effects of storage temperatures. The lower freezing rate achieved in most retarder-provers, combined with the poor thermal conductivity of dough, can cause quality losses. Proof dough pieces of large radius at a lower temperature than those of small radius. Lower the yeast level in the dough to lengthen the final proof time and to help minimize temperature differentials. Maintain a high relative humidity in proofing to prevent skinning. Source: References 8, 22, 45.

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the northern provinces of China. However, stuffed steamed breads are consumed as specialty items in various parts of China. Manufacture of steamed bread differs from that of regular bread mainly in the dough solidification process. Regular bread uses a baking process, whereas in steamed bread, steaming is used instead of baking. Consequently, in steamed bread, there is no brown crust on the bread surface because the temperature used is not high enough to cause the browning reaction. Steamed bread is always consumed hot or held in a steamer because the bread is soft at this temperature. Sometimes the bread is deep-fried before consumption. Steamed bread hardens when it cools down, making it less palatable. Various procedures are available for the production of steamed bread. Table 178.41 lists the basic steps in steamed bread processing in China (27).

TABLE 178.39 General Production Scheme for Flat Bread Ingredient preparation. Mixing of ingredients (dough formation). Fermentation. Dough cutting and rounding. Extrusion and sheeting (optional). First proofing. Flattening and layering. Second proofing. Second pressing (optional). Baking or steaming. Cooling. Packaging and distribution. Source: References 46 and 47.

countries or areas with less accessible energy. In developed countries, flat breads are considered specialty breads. The making of the dough is similar to that of regular bread. But, the dough is flattened and sometimes layered before it is baked directly inside the hearth or in an oven. Table 178.39 lists the general production scheme for flat breads (46, 47).

E. CROISSANTS AND DANISH PASTRIES Croissants and Danish pastries can be considered as products that result from modifications of the basic bread making process. The dough preparation steps are similar, but the ingredients are different. Table 178.40 compares the ingredients used in making croissants and Danish pastries. From this table, it is clear that even within each group, the ingredient formulation can vary considerably, producing a wide variety of products available in the market (8, 22, 45).

V. FERMENTED SOY PRODUCTS A. KINDS

OF


Soybeans have been available to the Chinese for centuries, and various fermented soy products were developed and spread to neighboring countries. These countries further developed their own fermented soy products. Soy sauce originating in China probably is the most famous and widely accepted fermented soy product. The credit for this wide acceptance also goes to the Kikkoman Company from Japan, which has helped spread soy sauce worldwide through their marketing strategy. Fermented whole soybeans such as ordinary natto, salted soybeans (e.g., Japanese Hama-natto and Chinese dou-chi), and tempe (Indonesia); fermented soy pastes (e.g., Japanese miso and Chinese dou-pan-chiang); and fermented tofus (e.g., sufu and stinky tofu or chao-tofu of Chinese origin) are

F. STEAMED BREAD (MANTOU) Steamed bread is common in the Chinese community. Plain steamed bread is consumed as the major staple in

TABLE 178.40 Formulations for Croissant and Danish Pastries Ingredients

Croissant

Danish Pastries

Flour Salt Water Yeast (compressed) Shortening Sugar Egg Skimmed milk powder Laminating margarine/butter

100 1.8–2.0 52–55.4 4–5.5 2–9.7 2–10 0–24 3–6.5 32–57

100 1.1–1.56 43.6–52 6–7.6 6.3–12.5 9.2–25 5–25 4–6.25 50–64


TABLE 178.41 Basic Steps in Steamed Bread Processing Selecting flour and ingredients such as milk powder and sugar (optional). Mixing dough. Fermentation: Full fermentation—1–3 hours Partial fermentation—0.5–1.5 hours No-time fermentation—0 hours Remixed fermentation dough—remixing of fully fermented dough with up to 40% of flour by weight. Neutralizating with 40% sodium bicarbonate and remixing. Molding. Proofing at 40°C for 30–40 minutes (no-time dough). Steaming for about 20 minutes. Steamed bread is maintained at least warm to preserve quality. Source: References 27.


178-17

TABLE 178.42 Raw Ingredients for Fermented Soy Products Ingredient Major Ingredients: Soy Soybean Soybean flour Salt Wheat Rice flour Major Microorganism(s): Mold Aspergillus oryzae Aspergillus sojae Mucor hiemalis, M. silivaticus M. piaini Actinomucor elegans A. repens, A. taiwanensis Rhizopus oligosporus R. chinesis var. chungyuen Bacteria Bacillus natto Klebsiella pneumoniae Bacillus sp. Streptococcus sp. Enterococcus sp. Lactobacillus sp. Halophlic yeasts Saccharomyces rouxii Torulopsis versatlis Halophilic lactic bacteria Pediococcus halophilus Bacillus subtilus Other Ingredients: Additional flavor added Preservative added

Soy Sauce

Natto

Soy Nuggets

Soy Paste

Tempe

Soy Cheese

Stinky Tofu

Yes Optional Yes Optional No

Yes No Yes No No

Yes No Yes No No

Optional Yes Yes No Optional

Yes No No No No

Yes Optional Yes No No

Yes Optional No No No

Yes No No No No No No No

No No No No No No No No

Yes No No No No No No No

Yes Optional No No No No No No

No No No No No No Yes No

Optional No Yes Yes Yes Yes No Yes

No No No No No No No No

No No No No No No

Yes No No No No No

No No No No No No

No No No No No No

No Yes No No No No

No No No No No No

No No Yes Yes Yes Yes

Yes Yes

No No

Yes Yes

Yes Yes

No No

No No

No No

Yes Yes

No No

Yes Yes

Yes Yes

No No

No No

No No

Optional Optional

No No

No No

No No

No No

Optional No

No No

Source: References 16, 37–39, 56, 57, 59, 63, 65.

more acceptable to ethnic groups. Consumers worldwide are gradually accepting these products through cultural exchange activities. The manufacturing of these products varies widely. Table 178.42 summarizes the ingredients needed for the manufacture of common fermented soy products (16, 37–39, 56, 57, 59, 63, 65).

sauce is then extracted from the fermented soybeans for standardization and packaging. Table 178.43 lists a generalized scheme for the manufacture of soy sauce. More detailed information is presented in references listed in this chapter and available literature elsewhere (16, 37–39, 57, 65).

B. SOY SAUCE

C. FERMENTED WHOLE SOYBEANS

There are many types of soy sauce, depending on the ratio of ingredients (wheat and soybeans), the fermentation and extraction procedures, and the flavoring ingredients (caramel and others) used. However, the procedures for manufacturing are similar. Basically, soy sauce is made by fermenting cooked soybeans in salt or brine under controlled conditions to hydrolyze the soy proteins and starches into smaller flavoring components. The soy

1.

Ordinary (Itohiki) Natto

Ordinary natto is a typical Japanese fermented whole soybean product. The sticky mucilaginous substance on the surface of soybeans is its characteristic. It is produced by a brief fermentation of cooked soybeans with Bacillus natto, and it has a short shelf life. Table 178.44 lists the basic steps in the manufacture of ordinary natto. For

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TABLE 178.43 Production Scheme for Soy Sauce Select and soak beans. Cook clean or defatted soybean under pressurized steam at 1.8 kg/cm2 for 5 minutes. Cool cooked bean to 40°C. Roast and crush wheat. Mix prepared soybeans and wheat. Inoculate with Aspergillus oryzae or sojae. Incubate mixture to make starter koji at 28–40°C. Add brine (23% saltwater) to make moromi (mash). Inoculate with halophilic yeasts and lactic acid bacteria (optional). Brine fermentation at 15–28°C. Add saccharified rice koji (optional). Age moromi (optional). Separate raw soy sauce by pressing or natural gravity. Refine soy sauce. Add preservative and caramel (option). Package and store. Source: References 16, 37–39, 57, 65.

TABLE 178.44 Production Scheme for Itohiki (Ordinary) Natto Start with clean, whole soybeans. Wash and soak at 21–25°C for 10–30 hours. Cook soybean under pressurized steam at 1–1.5 kg/cm2 for 20–30 minutes. Drain and cool soybean at 80°C. Inoculate with Bacillus natto. Mix and package in small packages. Incubation: 40–43°C for 12–20 hours, or 38°C for 20 hours plus 5°C for 24 hours. Final product. Refrigerate to prolong shelf life. Source: References 38 and 39.

detailed information on ordinary natto, please refer to the references in this chapter (38, 39, 65). 2.

Hama-natto and Dou-chi

Hama-natto is fermented whole soybeans produced in the Hama-matsu area of Japan. Similar products are produced in Japan, prefixed with different names taken from the production location. A very similar product in the Chinese culture is “tou-chi” or “dou-chi.” It is produced by fermenting the cooked soybeans in salt, brine, or soy sauce, and then drying them as individual beans. Hama-natto includes ginger in its flavoring, whereas the inclusion of ginger flavoring is optional in dou-chi. Table 178.45 lists the basic steps in the production of Hama-natto and douchi. For further information, readers should refer to the references in this chapter and other available literature (37–39, 65).

TABLE 178.45 Production Scheme for Soy Nuggets (Hama-natto and Dou-chi) Start with clean, whole soybeans. Wash and soak for 3–4 hours at 20°C. Steam cook soybean at ambient pressure for 5–6 hours or at 0.81.0 kg/cm2 for 30–40 minutes. Drain and cool soybean to 40°C. Add alum (optional for douchi). Mix with wheat flour (optional for Hama-natto). Inoculate with Aspergillus oryzae. Procedure 1 (Hama-natto): Incubate for 50 hours at 30–33°C. Soak inoculated soybean in flavoring solution for 8 months. Incubate under slight pressure in closed containers. Procedure 2 (dou-chi): Incubate at 35–40°C for 5 days. Wash. Incubate for 5–6 days at 35°C. Remove beans from liquid for drying. Mix with ginger soaked in soy sauce (Hama-natto only). Package final product (soy nuggets). Refrigerate to prolong shelf-life (optional). Source: References 37–39, 65.

D. FERMENTED SOY PASTES Both the Chinese and Japanese have fermented soy pastes available in their cultures, and they are made in similar manner. However, the usage of these two products is quite different. The Japanese use their fermented soy paste, miso, in making miso soup, and to a lesser extent, for example, in marinating/flavoring of fish. Miso soup is common in traditional Japanese meals. The Chinese use their fermented soy paste, dou-pan-chiang, mainly as condiment in food preparation. Dou-pan-chiang can also be made from wing beans, and this is beyond the scope of this chapter. Table 178.46 lists the basic steps in the manufacture of miso. For detail information on miso and dou-pan-chiang, readers should consult the references for this chapter and other literature available elsewhere (16, 37–39, 56, 57, 65).

E. FERMENTED TOFU 1.

Fermented Soy Cheese

Sufu, or fermented soy cheese, is made by fermenting tofu that is made by coagulating the soy protein in soy milk with calcium and/or magnesium sulfate. It is similar to feta cheese in its fermentation process. Both products are matured in brine in sealed containers. Some packed sufu contains flavoring ingredients. Table 178.47 lists the basic steps in the manufacture of sufu. For detail information, readers should refer to the list of references in this chapter and the other available literature (37–39, 59).


TABLE 178.46 Production Scheme of Fermented Soybean Pastes (Miso) Start with whole, clean soybeans. Wash and soak at 15°C for 8 hours. Cook at 121°C for 45–50 minutes or equivalent. Cool and mash the soybeans. Prepare soaked, cooked, and cooled rice (optional). Prepare parched barley (optional). Inoculate rice or barley with Aspergillus oryzae (tane-koji, optional). Mix koji and rice or barley mixture. Add salt to koji and rice or barley mixture and mix. Inoculate halophilic yeasts and lactic acid bacteria (optional). Pack mixture (mashed soybean and koji) into fermenting vat with 20–21% salt brine. Ferment at 25–30°C for 50–70 days. Blend and crush ripened miso. Add preservative and colorant (optional). Pasteurize (optional). Package and store. Source: References 16, 37–39, 56, 57, 65.

2.

Stinky Tofu

Stinky tofu is a traditional Chinese food made by fermenting tofu briefly in “stinky brine.” The tofu is hydrolyzed slightly during this brief fermentation and develops its characteristic flavoring compounds. When this raw stinky tofu is deep-fried, these compounds

TABLE 178.47 Production Scheme for Sufu (Chinese Soy Cheese) Clean whole soybeans. Soak. Grind with water. Strain through cheesecloth to recover soymilk. Heat to boiling and then cool. Coagulate soymilk with calcium and/or magnesium sulfate. Cool to 50°C. Press to remove water (formation of tofu). Sterilize at 100°C for 10 minutes in hot-air oven. Inoculate with Mucor, Actinomucor, and/or Rhizopus sp. Procedure 1: Incubate in dry form for 2–7 days, depending on inocula. Incubate (ferment in 25–30% salt brine) for 1 month or longer. Brine and age in small containers with or without addition of alcohol or other flavoring ingredients.

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TABLE 178.48 Production Scheme for Stinky Tofu Clean whole soybeans. Soak. Grind with water. Strain through cheesecloth to recover soymilk. Heat to boiling and then cool. Coagulate soymilk with calcium and/or magnesium sulfate. Cool to 50°C. Press to remove water (formation of tofu). Press to remove additional water. Soak in fermentation liquid for 4–20 hours at 5–30°C. Fresh stinky tofu, ready for frying or steaming. Refrigerate to prolong shelf life. Source: References 37–39, 59.

volatilize and produce the characteristic stinky odor, thus the name “stinky tofu.” It is usually consumed with chili and soy sauces. Stinky tofu is also steamed with condiments for consumption. Table 178.48 lists the basic steps in the manufacture of stinky tofu. Readers should consult the references in this chapter for further reading (37–39, 59).

F. TEMPE (TEMPEH) Tempe is a traditional Indonesian food consumed commonly by its people. It is made by fermenting cooked soybeans wrapped in wilted banana leaves or plastic wraps. The mold Rhizopus oligosporus produces its mycelia, and these mycelia penetrate into the block of soybeans. The mold mycelia also surround the block. This kind of fermentation is similar to molded cheese fermentation. Tempe is gradually being accepted by vegetarians in the West as a nutritious and healthy food. It is generally consumed as a deep-fried product. Table 178.49 lists the basic steps in the production of tempe (38, 39, 63, 65). TABLE 178.49 Production Scheme for Tempe

Procedure 2: Incubate at 35°C for 7 days until covered with yellow mold. Pack in closed container with 8% brine and 3% alcohol. Ferment at room temperature for 6–12 months. Final product (sufu or Chinese soy cheese).

Start with whole, clean soybeans. Rehydrate in hot water at 93°C for 10 minutes. Dehull. Soak with or without lactic acid overnight. Boil for 68 minutes. Drain and cool to 38°C. Inoculate with Rhizopus oligosporus w/o Klebsiella pneumonia. Incubate on trays at 35–38°C, 75–78% RH for 18 hours. Dehydrate. Wrap.

Source: References 37–39, 59.

Source: References 38, 39, 63, 65.

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TABLE 178.50 Raw Ingredients for Fermented Vegetables Ingredient Vegetable Head cabbage Chinese cabbage Mustard green Turnip Jalapeño Pepper Chili pepper Pickle/cucumber Salt Starter culture (lactic acid bacteria) Added vinegar Added spices Other added flavors Preservative(s)

Sauerkraut

Western Pickles

~o Jalapen Peppers

Kimchi

Oriental Vegetables

Yes No No No No No No Yes Optional

No No No No No No Yes Yes Optional

No No No No Yes No No Yes Optional

Optional Major Optional Optional Optional Yes Optional Yes No

Optional Optional Optional Optional Optional Optional Optional Yes No

No No No No

Yes Optional Yes Optional

Yes Optional No Optional

No Optional Optional Optional

Optional Optional Optional Optional

Source: References 1, 3, 5, 10, 13, 14, 19, 25, 36, 43.

VI. FERMENTED VEGETABLES A. KINDS

OF


Fermented vegetables were produced in different cultures in the old days to preserve the harvested vegetables when they are not available or due to climatic limitations. Some of these products started as traditional cultural foods but became widely accepted in other cultures. It is interesting that most of these processes are similar. Salt is used in the production of the product or the salt stock. Natural lactic acid fermentation, to produce enough lactic acid to lower product pH, is the major microbial activity in these processes. With the amount of salt added and lactic acid produced, these two ingredients create an environment that can inhibit the growth of spoilage microorganisms. Available leafy vegetables, fruits (commonly used as vegetables), and roots are used as the raw materials. Starter cultures are used occasionally. Vinegar is used in some products. Chili pepper and other spices are used in many products. Preservatives may also be used to extend shelf life after the package is opened. Table 178.50 compares the ingredients used in different fermented vegetable products (1, 3, 5, 10, 13, 14, 19, 25, 36, 43).

B. SAUERKRAUT The term sauerkraut literally means sour (sauer) cabbage (kraut). It is a traditional German fermented vegetable product that has spread to other cultures; it is used on its own or in food preparations. Its sequential growth of lactic acid bacteria has long been recognized. Each lactic acid bacterium dominates the fermentation until its end product becomes inhibitory for its own development and creates

TABLE 178.51 Basic Steps in Sauerkraut Processing Select and trim white head cabbage. Core and shred head cabbage to 1/8 inch thick. Salt with 2.25–2.50% salt by weight with thorough mixing. Store salted cabbage in vats with plastic cover, weighed with water to exclude air in the cabbage. Ferment at 7–23°C for 2–3 months or longer to achieve an acidity of 2.0% (lactic). Heat kraut to 73.9°C before filling the cans or jars, then exhaust, seal, and cool. Store and distribute. Source: References 5, 13, 19, 25.

another environment suitable for another lactic acid bacterium to take over. The fermentation continues until most of the available fermentable sugars are exhausted. The production of sauerkraut is not risk-free and sanitary: precautions must be taken to avoid spoilage. Table 178.51 presents the basic steps in sauerkraut processing (1, 13, 19, 25).

C. PICKLES Western-style pickles are produced by salting the pickling cucumbers in vats in salt stocks for long-term storage, followed by desalting, and bottling in sugar and vinegar, with or without spices. The fermentation is still lactic acid fermentation. However, it is more susceptible to spoilage because air may be trapped inside the slightly wax-coated cucumbers. In the salt curing of cucumbers, spoilage can occur, and precautions should be taken to avoid its occurrence. Because of their high acidity and low pH as well as their high salt content, the products are generally mildly


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TABLE 178.52 Basic Steps in Fermented Pickles Processing

TABLE 178.54 Basic Steps in Fermented Chinese Vegetables

Size and clean cucumbers. Prepare 5 (low salt) or 10% brine (salt stock). Cure (ferment) cucumbers in brine for 1–6 weeks to 0.7–1.0% acidity (lactic) and pH of 3.4–3.6, dependent on temperature, with salinity maintained at a desirable level (15% for salt stock). Addition of sugar, starter culture, and spices is optional. Recover pickles from brine, then rinse or desalt (salt stock). Grade. Pack pickles into jars filled with vinegar, sugar, spices, and alum, depending on formulation. Pasteurize at 74°C for 15 minutes, followed by refrigerated storage; exhaust to 74°C at cold point, then seal and cool; or vacuum pack and heat at 74°C (cold point) for 15 minutes, then cool. Store and distribute.

Select and clean vegetables. Cut vegetables (optional).

Source: References 1, 3, 5, 13, 14, 19.

heat-treated to sterilize or pasteurize them. Table 178.52 lists the basic steps in the production of Western-style pickles (1, 3, 5, 13, 14, 19).

D. KIMCHI Kimchi is a traditional Korean fermented vegetable. Most kimchi is characterized by its hot taste because of the fairly high amount of chili pepper used in the product and its visibility. However, some kimchis are made without chili pepper, but with garlic and ginger as well as other vegetables and ingredients. Vegetables used in making kimchi vary with its formulation: Chinese cabbage, cucumber, and large turnip are more common. Either chili pepper, or garlic and ginger can be used to provide a hot sensation. Other ingredients may also be added to provide a typical flavor. The fermentation is still lactic acid fermentation. Traditionally, kimchi was made in every household in rural areas in Korea to provide vegetables for the winter, when other fresh vegetables were not readily available. Today, it is a big industry in Korea, and kimchi is available year-round. Even TABLE 178.53 Basic Steps in Kimchi Processing Select vegetables (Chinese cabbage, radish, cucumber, or others). Wash vegetables. Cut vegetables, if necessary. Prepare 8–15% brine. Immerse vegetables in brine for 2–7 hours to achieve 2–4% salt in vegetable. Rinse and drain briefly. Add seasoning. Ferment at 0°C to room temperature for about 3 days. Package (can also be done before fermentation). Store at 3–4°C. Source: References 36 and 43.

Procedure 1: Wilt vegetables for 1–2 days to remove moisture. Dry salt vegetables in layers with weights on top (5–7.5% salt). Ferment for 3–10 days. Wash. Dry or press fermented vegetables (optional). Add spices and flavoring compounds. Package. Sterilize (optional). Procedure 2: Wilt cut vegetables. Rinse fermentation container in hot water. Fill the container with cut vegetables. Add 2–3% brine and other flavoring compounds (optional). Ferment at 20–25°C for 2–3 days. Ready for direct consumption or packaging and cool storage. Source: References 10 and 36.

small kimchi refrigerators are now available to meet the demands of consumers living in cities. In other parts of the world where Koreans are residents, kimchi is available either as a household item or as a commercial product. Kimchi is usually not heat sterilized after packaging in jars. Pasteurization is optional. Kimchi is considered perishable and is stored refrigerated. Table 178.53 lists the basic steps in the manufacture of kimchi (36, 43).

E. CHINESE PICKLED VEGETABLES The Chinese also manufacture a wide range of pickled vegetables. Various kinds of vegetables are used as raw materials. The fermentation can be either a dry-salting or a brining process, depending on the product to be manufactured. However, the fermentation is still lactic acid fermentation. The major difference between Chinese-style pickled vegetable products and Western-style pickles is that desalting is usually not practiced in the manufacture of Chinese-style pickled vegetables. The desalting process is left to the consumers, if needed. Also, some Chinese-style vegetables are made into intermediate moisture products that are not produced in their Western-style counterparts. Table 178.54 lists some of the basic steps in the manufacturing of selected Chinese pickled vegetables (10, 36).

VII. APPLICATION OF BIOTECHNOLOGY IN THE MANUFACTURING OF FERMENTED FOODS With the advances in biotechnology, microorganisms with special characteristics for the manufacturing of fermented

178-22


foods have become available. The most significant example is the approval by the FDA of Chy-Max (chymosin produced by genetic manipulation) used in the production of cheese. Its availability greatly reduces the reliance on chymosin from young calves and produces economic savings. Other products with similar or other properties are also available in the market. Genetically modified lactic acid bacteria and yeasts used in fermented food production are also available nowadays to reduce production costs. Gradual acceptance by consumers is the key to the further development and success of biotechnology (2, 15, 20, 26, 31, 34, 52, 55, 62). Readers should refer to the references in this chapter and other references available for further information.

VIII. PROCESS MECHANIZATION IN THE MANUFACTURE OF FERMENTED FOODS Fermented foods produced by traditional methods are labor intensive and rely a great deal on the experience of the manufacturers. The main drawback is product inconsistency. In most developed countries, products such as many cheeses, yogurts, breads, sausages, and soy sauce are now made by highly mechanized processes to standardize the products (4, 7, 11, 21, 23, 30, 33, 40, 41, 44). This not only provides product consistency, but also reduces production costs. Consumers benefit from these developments. However, some consumers, even in developed countries, still prefer the traditional products, even at an increased cost, because of their unique product characteristics. There are also fermented products that are still made by traditional or semimechanized processes because mechanization processes have not been developed for them.

ACKNOWLEDGMENT The information in this chapter has been derived from Food Manufacture Manual, copyrighted and published by Science Technology System, West Sacramento, California, ©2004. Used with permission.

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179

Sour Cream and Crème Fraîche

Lisbeth Meunier Goddik Oregon State University

CONTENTS I. II.

Introduction ......................................................................................................................................................179-1 Sour Cream ......................................................................................................................................................179-1 A. Definition ..................................................................................................................................................179-1 B. Sensory Characteristics ............................................................................................................................179-2 C. Utilization ................................................................................................................................................179-2 III. Fermentation ....................................................................................................................................................179-2 IV. Gel Formation ..................................................................................................................................................179-2 V. Stabilizers ........................................................................................................................................................179-3 VI. Processing ........................................................................................................................................................179-3 A. “Short Cuts”..............................................................................................................................................179-4 B. Low Quantity ............................................................................................................................................179-4 C. Chymosin Addition ..................................................................................................................................179-4 D. Set Sour Cream ........................................................................................................................................179-4 E. Direct Acidification ..................................................................................................................................179-5 F. Low Fat and Non-fat Sour Cream ............................................................................................................179-5 VII. Shelf-Life..........................................................................................................................................................179-5 VIII. Sensory Defects in Sour Cream ......................................................................................................................179-5 A. Flavor ........................................................................................................................................................179-5 B. Body and Texture......................................................................................................................................179-6 IX. Crème Fraîche ..................................................................................................................................................179-6 Acknowledgment..........................................................................................................................................................179-7 References ....................................................................................................................................................................179-7

I.

INTRODUCTION

Sour cream is a relatively heavy, viscous product with a glossy sheen. It has a delicate, lactic acid taste with a balanced, pleasant, buttery-like (diacetyl) aroma (1). Various types of sour cream are found in many regions of the world. The products vary in regard to fat content and by the presence or absence of non-dairy ingredients. Furthermore, both cultured and direct acidification is utilized to lower pH. This chapter will cover sour cream as it is produced in the US and its French counterpart — crème fraîche.

II. SOUR CREAM A.

DEFINITION

The US Food and Drug Administration (21CFR 131.160) defines sour cream as follows (2): “Sour cream results from

the souring, by lactic acid producing bacteria, of pasteurized cream. Sour cream contains not less than 18 percent milkfat; ……. Sour cream has a titratable acidity of not less than 0.5 percent, calculated as lactic acid.” If stabilizers are used, the fat content of the dairy fraction must be at least 18 percent fat and above 14.4 percent of the entire product. Consumers’ desire for decreasing dietary fat content has created a market for low fat sour creams. Among these products, the reduced fat (at least 50% fat reduction), and non-fat are common, in part due to FDA’s labeling requirements for low fat products (21CFR101). Sales data over the past 25 years for the US market (3) are illustrated in Figure 179.1. The trend clearly shows increased sales. In 2000, nearly 400 million kg of sour cream were sold. Per capita sales of sour cream and dips were 1.4 kg. In comparison, per capita sales for yogurt, heavy cream, and half and half were 2.1 kg, 0.9 kg, and 1.7 kg, respectively (3).

179-1

Sour cream & dips sales (million kg)

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450 400 350 300 250 200 150 100 1975

1980

1985

1990

1995

2000

Year

FIGURE 179.1 Sale, in million kg, of sour cream and dips in the US between 1975 and 2000. Source: USDA, Agricultural Marketing Service.

B.

SENSORY CHARACTERISTICS

Traditionally, the flavor of sour cream was well characterized by “sour.” However, the trend for cultured dairy products is toward a milder flavor (4), which permits the sensation of aromatic compounds produced by lactic acid cultures. Lindsay et al. (5) found that important flavor compounds in sour cream include diacetyl, acetic acid, acetaldehyde, and dimethyl sulfide. Sour cream is highly viscous and should be smooth and free of particulate matter. As for appearance, a homogenous, glossy surface is preferred and no whey separation should be visible in the container (6).

C.

UTILIZATION

Sour cream is predominantly utilized as an accompaniment with warm entrees such as baked potatoes, burritos etc. This usage imposes certain demands on the sensory characteristics of the product, especially in regard to texture when in contact with warm surfaces. Sour cream must remain viscous without whey separation when placed on warm food. Some have even requested that baked potatoes can be reheated in the microwave with sour cream already added, and the sour cream should remain unaltered by this treatment. In addition, flavor characteristics become less significant when mixed with high intensity savory flavor notes such as those encountered in the Mexican cuisine. In fact, for some usages the absence of off-flavors may be considered as the primary flavor attribute. This general shift in emphasis away from flavor toward texture has led to a renewed interest in a “back to basics” sour cream such as crème fraîche, which is described later in this chapter.

III.

FERMENTATION

As with all fermented dairy products, the choice of starter culture is crucial for the production of high quality sour cream (7). Mixed strains of mesophilic lactic acid bacteria are used for sour cream. In general, both acid and aroma producers are utilized. Acid producers include Lactococcus (Lc) lactis ssp. lactic and Lc lactis ssp. cremoris. Lc lactis

ssp. lactis biovar diacetylactis (or Cit⫹ Lactococci) and Leuconostoc mesenteroides ssp. cremoris are commonly used aroma producers. The acid producers convert lactose into L-lactate through a homofermentative pathway. They can produce up to 0.8% lactic acid in milk (8) and are responsible for lowering pH in the fermented product. In contrast, aroma producers are heterofermentative and can convert lactose into D-lactate, ethanol, acetate and CO2. In addition, these strains convert citrate into diacetyl which is one of the major flavor compounds responsible for typical sour cream flavor. Diacetyl is subsequently partially converted into acetoin, which is a flavorless compound (9). Extensive research at starter culture companies have led to the development of Leuconostoc strains that show less of a tendency to convert diacetyl into acetoin, thus retaining high levels of diacetyl (D. Winters, personal communication. 2002). Use of such strains can extend the shelf-life of sour cream, as it takes longer for the product to turn stale. Leuconostocs also reduce acetaldehyde to ethanol (10,11). In fact, acetaldehyde has been shown to promote the growth of Leuconostoc mesenteroides ssp. cremoris (12,13). Acetaldehyde is typically associated with yogurt flavor (green apple), but is considered an off-flavor in sour cream. The choice of starter cultures will affect product texture as well. Strains of acid producers have been developed which increase viscosity through the production of exopolysaccharides (14). These polysaccharide chains contain galactose, glucose, fructose, mannose and other sugars. Quantity and type depend on the bacteria strain and growth conditions (15,16). The exopolysaccharides interact with the protein matrix creating a firmer network and increasing water binding capacity. The importance of this behavior was confirmed by Adapa and Schmidt (17) who found that low fat sour cream, fermented by exopolysaccharide producing lactic acid bacteria, was less susceptible to syneresis and had a higher viscosity. Production of high quality sour cream requires a fine balance of acid, viscosity, and flavor producing bacteria. While this balance varies among commercially available strains, a typical combination would be 60% acid producers, 25% acid and viscosity producers, and 15% flavor producers (D. Winters, personal communication, 2002).

IV. GEL FORMATION Fermentation leads to a significant increase in viscosity. Two physicochemical changes cause this behavior (18,19). The casein submicelles disaggregate because of solubilization of colloidal calcium phosphate. In addition, the negative surface charge on the casein micelles decrease as pH approaches the isoelectric point. This creates the opportunity for casein micelles to enter into a more ordered system. Besides the protein network, cream gains viscosity from the formation of homogenization


179-3

clusters (20). Following single stage homogenization at room temperature, milk fat globules will cluster and these clusters may contain up to about 105 globules (21). Casein molecules adsorb onto newly formed fat globule membranes and, in the case of high fat content, form bridges between fat globules. Clustering increases viscosity because 1) serum is entrapped between the globules and 2) formation of irregular shaped clusters.

V. STABILIZERS The gel structure may not be sufficiently firm to withstand abuse during transportation, handling, and storage. This could result in a weak bodied sour cream and whey syneresis in the container. These defects are especially noticeable for low fat products. To ensure consistent firm texture dairy processors often choose to add non-dairy stabilizers (22). Stabilizers commonly found in sour cream include polysaccharides and gelatin. Stabilizers must be food grade and approved. The type and quantity used vary widely dependant on fat content, starter culture, and required sensory characteristics of the final product. Types and quantities of potential stabilizer mixtures used in sour cream are outlined in Table 179.1. Especially the non-fat formulation contains other ingredients such as emulsifiers, color, and protein. Polysaccharides bind water and increase viscosity. Commonly used plant polysaccharides include carrageenans, guar gums and cellulose derivatives. Modified starches are frequently utilized as well. It is necessary to fully hydrate these polysaccharides to optimize their functionality. Depending on the ingredient, this may require efficient blending systems for incorporation of the ingredient into the cream, though care should be taken to avoid churning the cream. Complete hydration can sometimes only be accomplished following heating and cooling steps, which conveniently are done by the pasteurization process. Time may also be a factor for hydration to occur. Besides TABLE 179.1 Example of Stabilizer and other Ingredients Used in Sour Cream Product Sour Cream

Low fat sour cream Non-fat sour cream

Ingredients Modified food starch, grade A whey, sodium phosphate, guar gum, sodium citrate, calcium sulfate, carrageenan, locust bean gum Same as above Modified food starch, microcrys talline cellulose, propylene glycol monoester, gum Arabic, artificial color, cellulose gum

Source: Adapted from 27.

Usage Level 1.5–1.8%

1.75–2.0% 6.2–6.6%

binding with water molecules, polysaccharides may also interact with milk proteins and form a network, which limits the movement of water and increases viscosity. A short description of the stabilizers is provided below: a) Carrageenans: Extract of seaweed. Three types of carrageenans are commercially available, lambda, iota, and kappa, which differ based on the amount of sulfate. They have low viscosity at high temperature but viscosity increases during cooling. Lambda has the highest sulfate content, is soluble in cold milk, and forms weak gels. Iota is soluble in hot milk (55°C) and prevents syneresis. Kappa only dissolves in hot milk (⬍70°C) and forms brittle gels (23). b) Guar gum: Endosperm of seed from Cyanopsis tetragonolobus plant. Different types of guar gum are available to fit processing conditions. Maximum viscosity develops over time. All are soluble in cold milk. The main component is mannose with attached galactose units. c) Methylcellulose: A cellulose which improves freeze-thaw stability and prevents melt upon heating (22). d) Gelatin: In contrast to the polysaccharides described above, gelatin consists primarily of protein (84–86%) and is derived from animal sources such as skin and bones (24). Gelatin is an excellent gelling agent but some off-flavors are perceived when used at excessive concentrations.

VI.

PROCESSING

Throughout the processing of sour cream, extra care should be taken to protect the cream. Prior to pasteurization, rough cream treatment could lead to rancid off flavors due to lipolysis. Following fermentation, it is important to treat the coagulum gently to retain body and texture. This includes use of positive displacement pumps instead of centrifugal pumps, round pipe elbows instead of 90° angles, and use of gravity feed wherever possible. In addition, special cream pasteurizers may be used (Figure 179.2). Ingredients can be incorporated directly into standardized cream by mixing equipment such as a triblender. Another option is to incorporate the dry ingredients into the milk portion before standardizing the cream. The mix is preheated and homogenized (~ 65°C, 10–25 MPa) (25,26) immediately prior to pasteurization. Dairy homogenizers are normally double stage to prevent homogenization clusters. However, in sour cream production single stage homogenization is preferred to build up the body of the product. Additional viscosity is obtained if the cream is homogenized downstream from the pasteurizer though such a process increases the potential for postpasteurization contamination. Pasteurization is done at relatively

179-4


Cream (4°C)

Stabilizers

Mixing

Preheating (55°C)

Homogenization (10−25 MPa)

Pasteurization (85°C 45 sec)

Cooling (22°C)

Incorporation of starter culture

Fermentation in tank (14−18 hrs)

Breaking of coagulum (pH 4.6)

Cooling (12°C)

desired acidity (~pH 4.5 or titratable acidity around 0.7% – 0.8%) is achieved. Typically this takes 14–18 hours. The coagulum is broken by gentle stirring and the product is cooled either by pumping cooling water into the double jacketed area of the tank or by pumping the cream through a special plate cooler. The cream should be cooled to around 8–12°C, which slows starter culture activity before packaging. Prior to packaging, it can also be passed through a homogenizer screen (smoothing plug) or similar type of flow restrictor to smooth and improve texture (27). The final cooling to around 4°C must occur slowly in the package in the cooler in order to allow the cream to obtain the appropriate viscosity. It is essential that the cream not be moved during this cooling step. The above process assumes large-scale production. However, numerous process variations exist.

A.

Throughout the process described above, special attention is focused on gentle treatment of the product to assure proper body and texture. In reality, the stabilizers used today permit more flexibility in the process. A certain amount of product abuse can be tolerated without lowering the product quality because the stabilizers, when properly used, create a firm texture and prevent whey separation.

B. Homogenization (5−10 MPa)

Packaging

Storage (4°C)

FIGURE 179.2 Process flow chart of typical sour cream process.

high temperatures (85–90°C for 10–45 sec), well above what is required for destruction of pathogens. The more severe heat treatment lowers the potential for oxidative and rancid off-flavors during storage as well as it may help improve product viscosity. The cream is cooled to 22–25°C, pumped into the fermentation tank and starter culture is added. Gentle mixing should continue until culture and cream are properly mixed (maximum 30 min). At this point mixing is stopped until fermentation is complete. The fermentation tank may be double-jacketed to allow for better temperature control. However, in reality this is not essential if the temperature of the processing room remains relatively constant around 22°C. Fermentation temperature may vary slightly from plant to plant. Higher temperatures lead to faster fermentation and potentially a more acidic product while lower fermentation temperatures may give a more flavorful product. The fermentation is slowed down/stopped by cooling when the

“SHORT CUTS”

LOW QUANTITY

It is possible to significantly simplify the process when producing small quantities of product. Sour cream can be made with a double-jacketed pasteurization tank, a pump, and a fermentation tank with gravity feed to the filler. The absence of a final in-line cooling step would require an efficient cooling procedure for the packaged product.

C.

CHYMOSIN ADDITION

Low quantities of chymosin may be added at the same time as the starter culture. This creates a more “spoonable” sour cream. Lee and White (28) found that chymosin addition (e.g. 0.066 ml/L) to low fat sour cream resulted in increased viscosity and whey separation. Sensory scores were lower for the chymosin containing sour cream in regard to flavor, body/texture, and appearance. This indicates that it may be preferable to modify the stabilizer mixture rather than to add chymosin when trying to increase product viscosity.

D. SET SOUR CREAM The standardized, pasteurized cream can be mixed with starter culture and immediately filled into the package. The cream is then fermented within the final package which leaves the coagulum undisturbed. When the appropriate acidity is obtained, the products are cooled either by passing through a blast cooler or by placement in a cooler.


The advantage of this method is the possibility to lower or eliminate stabilizers and yet obtain excellent body and texture. The disadvantages are the large space requirement for fermenting the packaged product and the relatively slow cooling.

E.

DIRECT ACIDIFICATION

A product somewhat similar to sour cream can be obtained by direct acidification by organic acids such as lactic acid instead of fermentation. However, Kwan et al. (29) and Hempenius et al. (30) found that sensory panelists preferred cultured sour cream instead of chemically acidified cream. Product temperature at the time of acidification is critical and should be around 20–25°C. Higher temperatures increase the likelihood that graininess occurs and lower temperatures increase the time required for gel formation (27).

F. LOW FAT AND NON-FAT SOUR CREAM Vitamin A fortification is required in these products. The processes are often similar to traditional sour cream though non-fat sour cream mix should be homogenized at much lower pressure. The main difference is observed in the stabilizer mix as described above in section V.

VII. SHELF-LIFE Sour cream should have a shelf-life around 25–45 days. One study documents that, when properly stored undisturbed at 4°C, sour cream has an acceptable shelf-life for up to 6 weeks (31). In another study, Folkenberg and Skriver (7) evaluated the change of sensory properties of sour cream during storage time. As storage time approached 28 days the intensity of prickling mouthfeel, sour odor, and bitter taste increased. The samples were stored under ideal conditions, which suggest that real life distribution and storage temperature abuse would likely decrease the shelf-life of this product below 28 days. The single most important factor determining shelf-life remains cream quality. Unless the cream is of excellent quality, the sour cream quickly develops off-flavors. Two parameters that impact cream quality are 1) raw milk quality, and 2) pretreatment of milk. Good quality raw milk has a low bacterial content (low standard plate count) and comes from healthy cows (low somatic cell count). Even good quality raw milk spoils unless quickly cooled and kept at low temperatures until pasteurization. Furthermore, the time interval between milking and pasteurization should be as short as possible. Other factors to consider are proper cleaning and sanitation of all milk contact surfaces, well installed and sized pumps, and no unnecessary milk handling. Assuming that high quality cream is utilized the parameters that limit shelf-life tend to be associated with either flavor defects or surface growth of yeast and molds.

179-5

When using appropriate stabilizers the body and texture should remain adequate throughout the shelf-life. A guide on how to prevent flavor defects is included below. Yeasts and molds are controlled by improving sanitation throughout the process. As with many other dairy products sanitation trouble spots are often associated with the filler machines, which are difficult to clean.

VIII. A.

SENSORY DEFECTS IN SOUR CREAM FLAVOR

The high lipid content makes sour cream extremely vulnerable to lipid associated off flavors such as rancidity and oxidation. Other flavor defects include flat, lacks cultured flavor and high acid. Rancid: Hydrolytic rancidity or lipolysis is caused by the release of free fatty acids from the glycerol backbone of triglycerides. The reaction is catalyzed by the lipase enzyme, which can be a native milk lipoprotein lipase or can originate from bacterial sources. Triglycerides are generally protected from lipase activity as long as the milk fat globule remains intact. However, damage to the globule will lead to rapid lipolysis because lipase, which is situated on the surface of the globule, can access the triglycerides. Therefore, precautions must be taken to prevent damage to the milkfat globule until pasteurization, which denatures most types of lipase. This means that raw milk/cream must be pasteurized before or immediately after homogenization to assure denaturation of lipase. Likewise, it is strongly recommended never to recycle pasteurized milk/cream back into raw milk/cream storage, which is essentially an issue of rework handling. Cream, from poor quality raw milk, can also develop rancid offflavors during storage, as some bacterial lipases are quite heat stable and do not denature during pasteurization. Oxidized: Autoxidation of milk fat is a reaction with oxygen that proceeds through a free radical mechanism. Unsaturated fatty acids and phospholipids are the prime substrates that are broken down into smaller molecular weight compounds such as aldehydes and ketones. Oxidized cream exhibits off flavors and aromas that have been characterized as cardboardy, metallic, oily, painty, fishy, and tallowy (6). Oxidation is catalyzed by divalent cations such as iron or copper. Thus, the best prevention is to avoid contact of milk/cream with these metals. This requires attention to details, as a single fitting or pipe made of these metals can cause significant autoxidation. Lacks fine flavor/lacks cultured flavor: Both flavor defects tend to be associated with the choice of starter culture. It may be possible to improve flavor by switching to culture systems with more aroma producing capacity or to strains that retard the transfer of diacetyl into acetoin. It is also possible to add low concentrations of citric acid (below 0.1%), which is then converted to diacetyl by the aroma producing starter cultures. The defect can also result from

179-6


flavors imparted by stabilizers. Lowering the stabilizer dose or changing to another stabilizer system may be required. High acid: If the final product pH is very low (e.g. around pH 4.0) the product gets an unpleasant sour flavor. While it is possible to stop the fermentation at a higher pH, this does not necessarily solve the problem because slow fermentation continues in the cooled and packaged product. Therefore, it is often preferable to change the starter culture mixture to lower the ratio of acid producing bacteria. Bitter: Bitter off flavors are often indicators of excess proteolytic activity. Poor quality raw milk may contain heat stable proteases that remain active throughout storage. The defect is especially noticeable at the end of shelf life. Improving raw milk quality, increasing pasteurization temperature, or shortening code dates are possible solutions.

B.

BODY AND TEXTURE

As described above, texture is an essential quality parameter. Sour cream must remain highly viscous when in contact with warm food surfaces such as baked potatoes. Too firm or weak: Improper choice of stabilizers can cause over-stabilized sour cream that clings to the spoon. Alternatively, the sour cream can be weak bodied and “melt” on the hot food surface. Grainy: Grainy is primarily a mouthfeel problem, though it can be visually distracting as well in extreme cases. Grainy sour cream can be an indication of poor blending or incomplete hydration of ingredients. A different choice of stabilizers or a modification of incorporation procedure may improve the product. Another solution is to pass the product through a single stage homogenizer valve prior to packaging. Grains can also indicate that the fermentation was stopped at too high a pH and the caseins are at their isoelectric point around pH 4.6. Free whey: Whey syneresis on top of the sour cream in the package is considered a significant quality defect. There are three solutions available to solving the problem. 1) Change or increase the concentration of stabilizer. 2) Increase fat content. Higher fat sour creams have a better water binding capacity. 3) Reevaluate the entire process and eliminate points of product abuse. This would primarily include all steps following fermentation.

IX. CRÈME FRAÎCHE Crème fraîche or more correctly crème fraîche épaisse fermentée is the European counterpart to the US sour cream product. Crème fraîche has a fat content around 30–45% and has a mild, aromatic cream flavor. The differences between the two products originate in the manner of usage. The usage of sour cream is described above. Crème fraîche is used cold on desserts such as fruit or cakes, or warm as foundation in cream sauces which are

commonly used in the French cuisine. This double usage creates a unique demand for specific product attributes. The dessert utilization requires a clean, not too sour (4), cultured flavor, that doesn’t overpower flavors from other dessert components. The cultured flavor should be refreshing so that it covers the impression of fat in the product. This emphasis on flavor has led to significant research at starter culture companies and dairy processing companies to develop starter cultures that cause optimum flavor development. The body and texture should be smooth and less firm than sour cream. Crème fraîche should be “spoonable,” not “pourable,” and should spread slightly on the dessert without being a sauce. The incorporation of crème fraîche into warm sauce requires thermostability, otherwise the protein would precipitate and flocculate in the sauce. For regular crème fraîche (⬎30% fat) flocculation is rarely a problem. In contrast, low fat crème fraîche (~15% fat) is less stable when heated. Addition of stabilizers such as xanthan gum can stabilize low fat crème fraîche. However, based on European labeling legislation a crème fraîche cannot contain stabilizers and a stabilized product would therefore need to be marketed under another name. Crème fraîche is produced by a process similar to that of sour cream, with the exception that no ingredients are added. Without stabilizers, it becomes a challenge to obtain good body and texture. Each processing step requires attention to producing and maintaining high viscosity. In this case the homogenizer becomes an essential tool for building viscosity. Only single stage homogenization is utilized. The product is sometimes homogenized twice, either in subsequent runs before pasteurization, but more commonly both before and after pasteurization. Homogenization after pasteurization promotes better viscosity and, equally important, better thermostability. An additional homogenization following fermentation gives a homogeneous product with a smooth mouthfeel. Homogenization downstream from the pasteurizer (i.e. after pasteurization) should raise concerns in regard to post-pasteurization contamination. Ideally, an aseptic homogenizer should be used. However, the high price of such homogenizers makes this an unsuitable alternative. Instead, great emphasis must be placed on proper cleaning and sanitizing of the downstream homogenizer. In addition, food safety issues are normally controlled because of the high content of lactic acid bacteria and the low pH. There is some discussion as to the final pH of crème fraîche fermentée. Kosikowski et al. (25) and Kurmann et al. (32) state that the cream is fermented to pH 6.2–6.3. However, commercially it is commonly fermented to an end pH around 4.5. The mild flavor is not obtained by a higher pH but rather through selection of aroma producing starter cultures. It is the combination of aroma compounds and the high fat content that mask the sour flavor in crème fraîche.


Crème fraîche is a new product on the US market. The high fat content and small scale processing contribute to a retail price which is at least twice as expensive as traditional sour cream. Nevertheless, sales are growing. Its increasing popularity is an indication of changing culinary habits promoted by growing population diversity and exposure to European culture. While crème fraîche is far from being a mainstream product on the US market, it is an interesting addition to the dairy case and can be found in many specialty stores.

ACKNOWLEDGMENT The information in this chapter has been modified from “Sour cream and crème fraîche,” by L.M. Goddik, in Handbook of Food and Beverage Fermentation Technology, Editors: Y. H. Hui et al., Marcel Dekker, NY 2004.

REFERENCES 1. FW Bodyfelt. Cultured sour cream: Always good, always consistent. Dairy Record 82(4):84–87, 1981. 2. Code of Federal Regulations, Title 21. Section 131. U.S. Government Printing Office, Washington, DC. 3. U.S. Department of Agriculture. Economic Research Report, Agricultural Marketing Service. Washington, DC. 4. D Barnes, SJ Harper, FW Bodyfelt, MR McDaniel. Correlation of descriptive and consumer panel flavor ratings for commercial prestirred strawberry and lemon yogurts. J Dairy Sci 74:2089–2099, 1991. 5. RC Lindsay, EA Day, LA Sather. Preparation and evaluation of butter flavor concentrates. J Dairy Sci 50:25–31,1967. 6. FW Bodyfelt, J Tobias, GM Trout. The sensory evaluation of dairy products. New York: Van Nostrand Reinhold, 1988, pp. 247–251. 7. DM Folkenberg, A Skriver. Sensory properties of sour cream as affected by fermentation culture and storage time. Milchwissenschaft 56:261–264, 2001. 8. TM Cogan. History and Taxonomy of Starter Cultures. In: TM Cogan, JP Accolas. Eds. Dairy Starter Cultures. New York: VCH Publishers, 1995, pp. 1–23. 9. V Monnet, S Condon, TM Cogan, KC Gripon. Metabolism of Starter Cultures. In: TM Cogan, JP Accolas. Eds. Dairy Starter Cultures. New York: VCH Publishers. 1995, pp. 47–100. 10. SR Dessart, LR Steenson. Biotechnology of Dairy Leuconostoc. In: YH Hui, GG Khachatourians. Eds. Food Biotechnology. New York: VCH Publishers. 1995, pp. 665–702. 11. TW Keenan, RC Lindsay, EA Day. Acetaldehyde utilization by Leuconostoc species. Appl Microbiology 14:802–806, 1966. 12. RC Lindsay, EA Day, WE Sandine. Green flavor defect in lactic starter cultures. J Dairy Sci 48:863–869, 1965.

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13. EB Collins, RA Speckman. Influence of acetaldehyde on growth and acetoin production by Leuconostoc citrovorum. J Dairy Sci 57:1428–1431, 1974. 14. AY Tamime, RK Robinson. Yogurt. Science and Technology. Cambridge: CRC Press. Woodhead Publishing, 1999, pp. 432–485. 15. H Nakajima, S Toyoda, T Toba, T Itoh, T Mukal, H Kitazawa, S Adachi. A novel phosphopolysaccharide from slime-forming Lactococcus lactis subspecies cremoris SBT 0495. J Dairy Sci 73:1472–1477, 1990. 16. J Cerning, C Bouillanne, M Landon, MJ Dezmazeaud. Isolation and characterization of exopolysaccharides from slime-forming mesophilic lactic acid bacteria. J Dairy Sci 75:692–699, 1992. 17. A Adapa, KA Schmidt. Physical properties of low-fat sour cream containing exopolysaccharide-producing lactic acid. J Food Sci 63:901–903, 1998. 18. PF Fox, TP Guinee, TM Cogan, PLH McSweeney. Fundamentals of Cheese Science. Gaithersburg: Aspen 2000, pp. 363–387. 19. P Walstra, P van Vliet. The Physical chemistry of curdmaking. Netherlands Milk and Dairy J 40:241–259, 1986. 20. H Mulder, P Walstra. The Milk Fat Globule. Emulsion science as applied to milk products and comparable foods. Wageningen: Pudoc, 1974, pp. 163–192. 21. P Walstra, TJ Geurts, A Noomen, A Jellema, MAJS van Boekel. Dairy Technology. Principles of Milk Properties and Processes. New York: Marcel Dekker, 1999, pp. 245–264. 22. CC Hunt, JR Maynes. Current issues in the stabilization of cultured dairy products. J Dairy Sci 80:2639–2643, 1997. 23. RT Marshall, WS Arbuckle. Ice Cream. 5th ed. New York: Chapman & Hall, 1996, pp. 71–80. 24. E Spreer. Milk and Dairy Product Technology. New York: Marcel Dekker, 1998, pp. 157–201. 25. F Kosikowski, VV Mistry. Cheese and Fermented Milk Foods. 3rd ed. Great Falls: F.V.Kosikovski, L.L.C., 1999, pp. 6–14. 26. S Okuyama, M Uozumi, M Tomita. Effect of homogenization pressure on physical properties of sour cream. Nippon Shokuhin Gakkaishi. 41:407–412, 1994. 27. Continental Custom Ingredients, Inc. 2002. Technical bulletin regarding sour cream formulation and processing. Continental Custom Ingredients, Inc, 245 West Roosevelt Road, West Chicago, Illinois 60185. 28. FY Lee, CH White. Effect of Rennin on Stabilized Lowfat Sour Cream. Cultured Dairy Products J 28:4–13,1993. 29. AJ Kwan, A Kilara, BA Friend, KM Shahani. Comparative B-vitamin content and organoloptic qualities of cultured and acidified sour cream. J Dairy Sci 65:697–701, 1982. 30. WL Hempenius, BJ Liska, RB Harrington. Selected factors affecting consumer detection and preference of flavor levels in sour cream. J Dairy Sci 52:588–593, 1969. 31. S Warren. Influence of storage conditions on quality characteristics of sour cream. Cultured Dairy Products J 8:13–14, 16, 1987. 32. JA Kurmann, JL Rasic, M Kroger. Encyclopedia of Fermented Fresh Milk Products. New York: Van Nostrand Reinhold, 1992, pp.94–95.

180

Quality Control and Sanitation of Cheese

Søren Lillevang

Arla Foods, Innovation Centre Brabrand

CONTENTS I. Introduction ......................................................................................................................................................180-1 II. General Aspects of Cheese................................................................................................................................180-2 III. Environmental and Technological Factors ........................................................................................................180-2 A. Organic Acids and pH................................................................................................................................180-2 B. Temperature ..............................................................................................................................................180-3 C. NaCl and Water Activity............................................................................................................................180-3 D. Nitrate and Lysozyme................................................................................................................................180-3 IV. Antagonistic/Symbiotic Actions in Cheeses ....................................................................................................180-3 V. Hygienic Aspects of Equipment........................................................................................................................180-3 A. Bactofugation ............................................................................................................................................180-3 B. Microfiltration............................................................................................................................................180-3 C. Pasteurization ............................................................................................................................................180-4 D. Cheese Vats ................................................................................................................................................180-4 E. Brines ........................................................................................................................................................180-4 F . Curing and Packaging................................................................................................................................180-4 G. Distribution and Cheeses on the Market ..................................................................................................180-4 VI. Important Microorganisms ................................................................................................................................180-4 A. Clostridia ..................................................................................................................................................180-4 B. E. coli ........................................................................................................................................................180-5 C. Salmonella ................................................................................................................................................180-5 D. Listeria ......................................................................................................................................................180-5 E. Staphylococcus aureus ..............................................................................................................................180-5 F . Others ........................................................................................................................................................180-5 VII. Control Systems ................................................................................................................................................180-6 VIII. Concluding Remarks ........................................................................................................................................180-6 Acknowledgment..........................................................................................................................................................180-6 References ....................................................................................................................................................................180-6

I. INTRODUCTION Production of cheese is a process of concentrating milk by the interaction of the milk, starter cultures and in most cases rennet. Traditionally cheese has been produced in small vats, but during the last 3 to 4 decades, processing has become increasingly industrialized. Before industrialisation, little attention was directed toward the hygienic aspects of cheesemaking, partly because the batch sizes were small and partly because methods of analysis were not well developed, consumption of cheese would normally cause only a few disease cases. In the recent years,

more attention is being given to the hygienic aspects for several reasons: Methods for detecting pathogens have improved; more focus on emerging pathogens like Listeria and E. coli H7:O157; and the larger batch sizes increase risk to larger numbers of consumers, if pathogens are present. Furthermore, because of the large batches, economical losses will be substantial if the quality is not acceptable. Finally an unacceptable quality in just a few batches from a producer may lead to loss of market shares. All these reasons have led to a considerable increase in attention on the hygienic aspects of cheesemaking. These aspects include a range of factors such as 180-1

180-2


hygiene, environmental and technological factors, interactions between microorganisms, and the setting up control systems in order to prevent contamination, or at least inhibit the growth of pathogens. Complications in doing this arise from the fact that there are many different cheese varieties, such as yellow(with or without surface ripening), fresh-, blue veined-, white molded and cottage cheese, each with their own risks for the presence or growth of pathogens or spoilage microorganisms. In this chapter, the most important physical, chemical and microbiological factors required for inhibiting or avoiding pathogens or spoilage microorganisms are described. The creation of a comprehensive control system is discussed.

II. GENERAL ASPECTS OF CHEESE Cheeses comprise a huge number of varieties and thus the composition also varies. The chemical composition of a cheese results from production under either high or low acidification, depending on the type of cheese, and the starter culture, which also plays a role in formation of the metabolic profile. As an example, the starter culture is able to form lactic acid as the major component, diacetyl, ethanol, acetic acid, benzoic acid and bacteriocine (1). Depending on the type of cheese, the water content varies from very low in grana cheeses to very high in cottage cheese and the pH may vary from very low in blue veined cheeses and feta (4.6–4.8) to very high in queso fresco (6.2–6.5). As pH varies, so does the lactic acid content. The sodium chloride content varies from very high (4–6%) in some blue veined cheeses and feta to very low (0.8–1.0) in cottage cheese. The sodium chloride content, dry matter and other salts in cheese are responsible for the water activity, which is a very important growth determinant for microorganisms. Regarding growth of microorganisms on the surface of cheeses, the packaging conditions are very important because the oxygen barrier varies, depending on the packaging material. The choice of packaging material depends on the type of cheese to be packed. Curing times for some cheeses may vary from short (1–6 days) or longer (up to 2 years) time intervals. This factor challenges the hygienic conditions in the curing rooms in relation to the chemical composition of the cheeses. Another challenge in this respect is the variation in temperatures that may occur in curing rooms. The temperature may vary from very high (20–22°C), for example for some Swiss type cheese varieties, to very low (2–5°C) for fresh cheeses or special varieties. During the curing time, temperature is elevated or lowered depending on the cheese type to be produced. Finally, the addition of nitrate or lysozyme to prevent growth of primarily Clostridia is an antimicrobial factor to be considered (2).

From a hygienic point of view, the cheese process in itself is a stabilizing factor. Starter culture is added to the cheese milk at 30°C and, together with the action of rennet, the milk coagulates to form a gel. As pH drops through the formation of lactic acid from the starter culture, the water binding capacity of the proteins drops. This, together with cutting of the formed gel, separates the milk into cheese and whey. After about 90 minutes, depending on type of cheese to be produced, the pH has dropped from 6.7 in fresh milk to about 6.0. After this initial cheese process, the cheese mass is pressed and anaerobic conditions are created. After pressing, the cheeses are left to complete acidification to the minimum pH (5.2), typically requiring 24 hours. Most cheese types are then cured in curing rooms at different temperatures and they may be ripened with or without a surface ripening culture. Some cheeses are packed in different foils in the curing room. Some fresh types of cheeses, however, are packed directly, then stored at 5°C and consumed within a few weeks.

III. ENVIRONMENTAL AND TECHNOLOGICAL FACTORS A. ORGANIC ACIDS AND PH The starter culture consists of lactic acid bacteria (LAB), and within 24 hours, the minimum pH is usually achieved. The minimum pH may vary, but in most cheeses the minimum pH is about 5.2 or lower; for cheddar pH 5.0 is normal, and in feta the pH may be as low as 4.6. While the buffer capacity in the cheeses is high due to the high protein content, the amount of lactic acid formed in the cheeses is also very high, up to 1.5% for some cheese types. This amount of lactic acid and the relatively low pH, achieves inhibition of many pathogens and spoilage microorganisms, especially gram negatives. However, the gram positives will also be inhibited under these conditions. Yeast and molds are only affected a little by the low pH and high amount of lactic acid. Depending on the type of starter culture, certain amounts of other organic compounds will also be formed (1). When gas producing mesophilic LAB are used as starters, diacetyl is formed in amounts that are able to cause a little inhibition of pathogens and spoilage microorganisms. Due to the metabolism occurring in the cheeses, the starters will also form acetic acid, up to 250 ppm is normal. This amount is not enough to prevent the growth of pathogens or spoilage microorganisms, and has a little impact. Other organic compounds like benzoic acid and ethanol may also have an impact on the growth of pathogens and spoilage microorganisms. While the amounts of organic compounds formed are difficult to control, it is easy to control pH and it is important to keep it as low as possible without altering with the desirable organoleptic properties of the cheeses.


B. TEMPERATURE Temperature and curing duration are important variables from a technological and hygienic point of view. While the curing temperature and time may improve the organoleptic properties of the cheese, it may also possibly lead to microbial growth. At 1°C, given the right conditions, Listeria is able to grow (3), while others such as Clostridium tyrobutyricum are not able to grow below 8°C (4). Therefore it is important to monitor the interaction between the curing temperature and time, in relation to the growth of selected microorganisms, and the cheese’s organoleptic properties. From a hygienic point of view the temperature should be kept as low as possible.

C. NACL AND WATER ACTIVITY At a high NaCl content, and/or low water content, many microorganisms are prevented from growing (5). In such cheeses, Staphylococci, Listeria and yeast are chief concerns, as they are salt tolerant (6). In fresh cheeses of which the water activity is high and the NaCl content is about 0.8–1.0, the risk of growth is high these are physiological conditions. It is not possible to lower the NaCl amount because it originates from the milk, and, in many cases, it is not possible to elevate the amount due to changes in the organoleptic properties. In these cases, other means must be used to prevent growth of pathogens and spoilage microorganisms.

D. NITRATE AND LYSOZYME Nitrate and lysozyme are often added to cheese milk in order to prevent late blowing from Clostridium tyrobutyricum (2–7). In most cases these additives also inhibit the growth of other microorganisms. But it is worth noting that the activity of the starter may also be slightly inhibited, causing a slower decrease in pH during the fermentation process, resulting in less inhibition of pathogens and spoilage microorganisms during the acidification process.

IV. ANTAGONISTIC/SYMBIOTIC ACTIONS IN CHEESES For several years, nisin, a bacteriocin produced during fermentation has been recognized as preservative in a variety of cheeses. Nisin is produced by Lactococcus lactis subsp. lactis, one of the species used for acidification. The ability to produce nisin is strain dependant. Nisin can be added to cheese milk or processed cheese as a powder for inhibiting gram positives. Use of a living nisin producing Lactococcus in cheese production is not widespread because of inhibition of the starter culture may be a problem. Other bacteriocins are known (8–9). The starter culture used in the production of surface ripened cheeses, consists of a mixture of yeast, Brevibacterium linens, other

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coryneform bacteria, Micrococcus, Staphylococcus (primarily equorum and xylosus) and gram negatives in limited numbers (10). Bacteriocins from B. linens and Staphyloccus have been reported, and this is considered to be one way to control Listeria on surface ripened cheeses (11–13). Enterococcus sp. has also been reported to produce bacteriocins, and this production may also have an impact on the control of harmful gram positives on cheeses. Apart from producing organic inhibitors and bacteriocins, the starter culture may also inhibit other microorganisms by direct competition for substrate. The starter culture ferments lactose into lactic acid/lactate, and thus inhibits the growth of harmful lactose fermenting microorganisms like coliforms or spoilage bacteria e.g. heterofermentative lactobacilli. Other substrates converted by the starter culture during cheesemaking are citrate and protein fragments, which mean that these compounds can’t serve as substrate for pathogens. The formation of lactic acid/lactate will in turn promote the growth of lactate fermenting microorganisms (e.g. certain Clostridia). The best known is Clostridium tyrobutyricum which causes late blowing in cheeses; however, there are means available to prevent this (see Section VI.A).

V. HYGIENIC ASPECTS OF EQUIPMENT A. BACTOFUGATION Bactofugation is widely used as a mean to remove sporeformers from the milk; well functioning bactofugation removes up to 98% of the spores (14). During the autumn and winter seasons, when cows are fed with silage, the spore content of Clostridium tyrobutyricum may be as high as 4000 per liter milk; As few as 10–20 spores per liter may cause late blowing in cheese. With a removal efficincy of 98% by bactofugation, the number of spore formers remaining is about 80 per liter, thus bactofugation is not completely effective in preventing late blowing. A relatively new process involving double bactofugation, is usually enough to prevent late blowing. Normally bactofugation is able to remove about 70% of the nonsporeforming flora, but this is far from sufficient removal of the non-sporeforming microorganisms.

B. MICROFILTRATION A better, but also more expensive, way to remove bacteria in general is microfiltration. Microfiltration will remove about 99.9% or more of the microbial flora present, including spore formers. By this the quality of the cheese milk is improved and the risk of the presence of microorganisms will decrease considerably. In cases where it is crucial that special spoilage microorganisms are absent, it is appropriate to perform microfiltration prior to pasteurization.

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C. PASTEURIZATION Pasteurization is the ultimate step for removal of pathogens. Low pasteurization is defined as the combination of time and temperature and is sufficient to kill all vegetative pathogens. Still, it should be noted that not all microorganisms are killed by low pasteurisation. Spores of Bacillus and Clostridium sp. will survive along with a few spoilage microorganisms such as heterofermentative Lactobacillus. In the pasteurization process it is important to control the temperature. During pasteurization the temperature will oscillate from the set point and it is crucial that the lower temperature be above 71.8°C. Controlling this requires an accurate temperature detection system. Such a system should be able to register the temperature rapidly and with high frequency. Another issue in pasteurization is the temperature differences between the components in the pasteurization unit. For example if the differences in the regenerative system are too big, fouling may occur, leading to lower efficiency in the pasteurization unit. The operation time for the pasteurization unit is also of importance. With the demands for high production efficiency, running times tend to increase, but this is often compromised due to biofilm formation. Finally of course, the cleaning of the pasteurization unit is important considering the concentration of the cleaning agents and the temperature used.

and unprotected on shelves without surface ripening. If the cheeses are packaged, the risk of contamination and/or growth is small. In general, packed and subsequently cured cheeses, keep their characteristic low pH, which along with the packaging protects against contamination. Cheeses that are not packed, and without surface ripening, have a higher risk of contamination. For surface ripened cheeses the risk of contamination is higher than if they are also packed. The microorganisms used for surface ripening will develop into a thick layer and, hence, protect against contamination partly by producing antagonistic compounds such as methanthiol and bacteriocins, and substrate competition.

G. DISTRIBUTION AND CHEESES

ON THE

MARKET

Obviously the risk of contamination is very low when cheeses are distributed packaged and will only be contaminated by damaging the packaging. The risk is if pathogenic or spoiling microorganisms are already present in low amounts. They may grow if the cooling chain is broken. This factor is often seen in the cooling desks at the supermarkets especially, where the temperature often is as high as 15°C, 10°C above the required 5°C; precautions should be taken to keep the temperature at 5°C or below.

D. CHEESE VATS

VI. IMPORTANT MICROORGANISMS

The cheese process is normally conducted at 30°C, with a cooking temperature range from 35–55°C. These temperatures are the normal interval in which pathogens or spoilage microorganisms are able to grow or survive. It is, therefore, necessary that the cheese vats are maintained in a high hygienic condition. There should not be any dead ends in the vats and the interfaces between the cheese vat and pumps, stirring systems, etc. should be secured properly. Finally, it is important that cleaning is easy to perform either as a “cleaning in place” (CIP) system or manually.

There are many species of pathogens or spoilage microorganisms in and on cheeses to be considered, in and on cheeses. However, many microorganisms are not found in cheese or will not grow during the cheese process. Absence of other microorganisms is controlled by veterinarian authorities in the primary (at the farmhouse) production. Among other Brucella, Mycobacteria and Tuberculosis are under veterinarian control in most countries.

E. BRINES In most cases cheeses are subjected to brine with a NaCl content of about 21%. Direct salting may also be used, for example, in cheddar and cream cheeses. Due to the high salt content only a few microorganisms represent a risk; yeast (as spoilage microorganisms), S. aureus and Listeria monocytogenes are the only microorganisms of concern. It is also important to note that an infection in the brine leads only to surface contamination, as the cheese at this stage is already formed and the surface has been closed during pressing.

F. CURING AND PACKAGING There are three methods for curing cheeses: Packaged in bags or foil; unprotected on shelves with surface ripening;

A. CLOSTRIDIA Clostridia are widespread in nature and occur in raw milk. Only very few cases of illness due to C. botulinum can be attributed to cheese, thus the major concern is spoilage due to C. tyrobutyricum which causes late blowing of hard or semihard cheeses. Late blowing occurs when the number of C. tyrobutyricum in the cheese milk exceeds 10–50 spores per liter and the pH is 5.2 or higher (15). During late blowing lactate is converted into butyric acid, carbon dioxide and hydrogen; spoiling is characterized by extreme eye formation, split defects and off flavors. Prevention of spores in cheese milk can be achieved, to some extent, by bactofugation (16), but bactofugation is not adequate to prevent late blowing. Double bactofugation or microfiltration is, however sufficient to prevent late blowing. If it is not possible to bactofugate or microfiltrate, the addition of nitrate or lysozyme is an alternative, but the legal amounts


allowed of these compounds may not be sufficient to prevent late blowing. An effective alternative is to cool the cheese down below 8°C, at which the spores will not develop.

B. E.

COLI

Normally E. coli should not occur in cheeses, although 10–1000 E. coli per gram can be allowed from time to time depending on the cheese type. The major concerns are the pathogenic E. coli types. These are divided into enterohemorrhagic (EHEC), enteropathogenic (EPEC) enteroinvasive (EIEC) and enterotoxigenic (ETEC) (17). They can cause serious disease and have been reported to cause foodborne diseases in at least five outbreaks (18–20). Most concern is with the EHEC E. coli H7:O157 that was involved in an outbreak in cheese produced from raw milk. In order to prevent pathogenic E. coli it is crucial to pasteurize the cheese milk. This will assure that they are not present in the milk, although postcontamination may occur. Good manufacturing practice is normally sufficient to prevent such contamination. If postcontamination does occur, it is important to prevent its growth. The activity of the primary starter should be controlled to assure a fast pH drop to below 5.5, which will inhibit E. coli growth. If hard cheeses are produced, the water activity should be held as low as possible, because growth of pathogenic E. coli does not occur at a water activity below 0.96. Such conditions are present in some blue veined cheeses like Danish blue and roquefort.

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pasteurized milk. In the mid-1980s 34 people died due to consumption of Vacherin Mont d’Or from Switzerland (24). Listeria is difficult to control due to the relatively high heat stability (D10 at 69°C is about 15 sec). The temperature range for growth is 0–45°C, at pH 4.4–9.5, and up to 10% NaCl. In order to prevent growth it is important to pasteurize efficiently, which means ensuring that the cheese milk has been heated to at least 72°C for 15 sec. In the outbreaks, the products had characteristics that favored growth of L. monocytogenes. In the Mexican style cheese, for example only a weak acidification took place and the NaCl content was low. In the case with Vacherin Mont d’Or, insufficient hygiene, coupled with a rise in pH and a low NaCl also favored growth of L. monocytogenes (24). It is also worth noting that bacteriocin-producing starters and surface ripening cultures may inhibit the growth of L. monocytogenes (12). The infectious dose of Listeria is high compared to Salmonella and pathogenic E. coli which makes it easier to control it in the products. But on the other hand, Listeria is more likely to grow in the final product, depending on the type of cheese, because of its high resistance to low pH, NaCl and low temperature.

E. STAPHYLOCOCCUS AUREUS

As for pathogenic E. coli, only a few cheese related Salmonella outbreaks have been reported (21–23). Because Salmonella are very heat and salt sensitive, they are not likely to grow in cheese. Thus, prevention of their contamination of cheese milk is crucial. Properly pasteurized milk is sufficient to eliminate Salmonella and the same precautions as described for control of E. coli should be taken. Fast acidification and good manufacturing practices along with maintaining as low water activity as possible is usually enough to produce safe Salmonella free cheeses.

S. aureus is associated with milk due to its close association with cows. It is relatively salt tolerant but sensitive to pH. The infectious dose is high about 105 pr ml. Disease arises from heat stable toxins, which means that even though no living S. aureus may be detected, the toxins may still be present (26). Thus, it must be assured that the number of S. aureus transferred from cow to product does not exceeded 105 pr ml, which is assured by a good manufacturing practice on the farms including assuring cooling in milk tankers and raw milk silotanks. Because S. aureus does not grow below about 8°C, the holding temperature should be held below 8°C, especially if the milk is stored for a long time in a silotank. S. aureus will only grow in cheeses with low acid content or if the cheese surface pH rises during curing. Good hygiene is normally enough to assure either the absence or low numbers in or on cheeses.

D. LISTERIA

F. OTHERS

Listeria is widespread in nature and can be found in up to 50% of milk samples from raw milk silotanks, depending on geographical and seasonal variations. Listeria monocytogenes, a gram positive pathogen, has caused a few outbreaks of disease. Two of these outbreaks have caused higher rates of mortality (24,25). In 1985, 48 people died due to consumption of a Mexican style cheese (25). The reason for the contamination was a leak in the pasteurization equipment, resulting in a mixing of raw and

Other microorganisms may cause spoilage or diseases in addition to the discussed above. Yeast and heterofermentative Lactobacilli may cause gas production or off flavors in cheeses, although the problem is generally easily solved by cleaning. In both cases, spoilage is due to heavy contamination in the dairy environments, and normally it is easy to control the environments to prevent heavy contamination. There is also the possibility for mold growth, which may be a problem on the surface of cheeses.

C. SALMONELLA

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Formation of molds should be avoided partly because the damage to the product is severe and partly because formation of molds in some cases leads to formation of mycotoxins. Packaging in a modified atmosphere and in a material which creates a high oxygen barrier will prevent growth of molds. The risk from growth of molds is the formation of mycotoxins, but normally if molds become apparent, it will be destroyed. Mycotoxins are not able to penetrate the entire cheese, but are normally located in the outer rind, about 0.5–1.0 cm in depth depending on the cheese type and water activity.

VII. CONTROL SYSTEMS In building up a control system, it is important to consider in each step of cheese production from the farm to the final product the microorganisms of concern. These steps are called critical control points and by introducing hazard analysis of critical control points (HACCP) it is possible to introduce a very high safety level of the products. Critical control points are production steps where some of the physiological or chemical conditions could cause a change conductive for growth of unwanted microorganisms. This means that careful evaluation should be performed if the microorganisms of concern can survive or grow at each control point. The most important parameters to evaluate are temperature; process time at the given temperature; pH and possibility of pH to change; water activity; and addition or formation of inhibitors. There are different tools for determining whether a control point is critical. One is Predictive Modeling. Predictive Modeling is based on subjection of a large dataset on the growth of different microorganisms under different conditions into a database system. The growth data on the microorganisms derived from laboratory research, challenge tests and real product experiences are analyzed statistically and a program formed that predicts the microorganisms growth at selected temperatures, water activities, pH values, and often under other conditions such as the presence of additives. Another critical point is the cleaning system. In most dairies, cleaning in place (CIP) is used. Sodium hydroxide with a pH of about 11 and a temperature at 70–80°C is used to wash away most of the milk components from the equipment followed by flushing with water. Nitric acid at pH about 2 is then used to remove acid soluble components from the equipment. It is expected that the strength of the CIP and the temperature will drop during the long transportation distances. In both cases, the cleaning efficiency and the direct killing effect on microorganisms will be less. Thus CIP is an important critical control point. Once the critical control points are established, limits for accepted values are determined and controlled with selected intervals.

VIII. CONCLUDING REMARKS In order to control the presence and growth of pathogenic microorganisms, it is important to do whatever is possible to prevent their occurrence from farm to cheese product and to ensure that good manufacturing practice is implemented throughout the production. Implementation of HACCP is an excellent tool to control the pathogens or spoilage microorganisms. Raw milk should be of good quality, and its storage should be at low temperatures, especially if storage times are long. Pasteurization is also critical, thus the pasteurisation plant should be under careful control: The temperature must be stable and not below 72°C, the pasteurizer should be cleaned at required intervals and it must be assured that there are no dysfunctions such as mixing raw and pasteurized milk. The activity of the starter must be high; this will lead to a fast drop in pH, helping to control pathogens. If possible, the temperature should be kept as low as possible and the salt content as high as possible. During the process, hygienic precautions should include good personal hygiene, high water quality used for the production and adequate cleaning efficiency. It must be ensured that the CIP system be optimal with regard to strength of the sodium hydroxide and acid used, as well as the temperature employed during the cleaning step. It should be emphasized that foodborne outbreaks in cheese consumption seldom occur. It is encouraging that considering the huge amount of cheeses consumed worldwide, only a few outbreaks have been documented. One of the reasons for this is that cheese is a well conserved system, creating a protective chain of hurdles against pathogenic and spoilage microorganisms.

ACKNOWLEDGMENT The information in this chapter has been modified from “Quality control and sanitation of cheese,” by S. Lillevang, in Handbook of Food and Beverage Fermentation Technology, Editors: Y. H. Hui et al., Marcel Dekker, New York, 2004.

REFERENCES 1. T. M. Cogan, T. P. Beresford. Dairy Microbiology Handbook Robinson R. K. 3rd Edition, Wiley. 2002. 2. J. Stadhouders. Prevention of butyric acid fermentation by the use of nitrate. Bulletin of the IDF 251, 40–46, 1990. 3. J. R. Junttila, S.I. Niemelä, J. Hirn. Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria. J. Appl. Bacteriol. 65:321–327, 1988. 4. J. Stadhouders. Alternative methods of controling butyric acid fermentation in cheese. Bulletin of the IDF 251, 55–58, 1990.


5. T. P. Beresford, N. A. Fitzsimons, N. L. Brennan, T. M. Cogan. Recent advances in cheese microbiology. Int. Dai. J. 11 (4–7), 259–274, 2001. 6. K. M. Sorrels, D. C. Enigl. Effect of pH, acidulant, Sodium chloride, and temperature on the growth of Listeria monocytogenes. J. Food Safety 11, 31–37, 1990. 7. R. Lodi. The use of lysozyme to control butyric acid fermentation. Bulletin of the IDF 251, 51–53, 1990. 8. P. Sarantinopoulos, F. Leroy, E. Leontopoulou, M. D. Georgalaki, G. Kalantsoupoulos, E. Tsakalidou, L. deVuyst. Bacteriocin production by Enterococcus faecium FAIR-E 198 in view of its application as adjunct starter in Greek Feta cheese making. Int. J. Food Microbiol. 72, 1–2, 125–136, 2002. 9. R. K. Gupta, N. K. Noel. Antimicrobial potentials of Lactococci – a review. Microbiol. Al. Nutr. 11, 477–490, 1993. 10. W. Bockelman, U. Krusch, G. Engel, N. Klijn, G. Smit, K. J. Heller. The microbiota of Tilsit cheese. Federal Dairy Research Center, Institute of Microbiologi, Germany, Netherlands Institute for Dairy Research, 1997. 11. N. V. Stauber, H. Götz, M. Busse. Antagonistic effect of coryneform bacteria against Listeria species. Int. J. Food Microbiol. 13, 119–130, 1991. 12. N. V. Stauber, S. Scherer. Isolation and Characterization of Linocin 18, a bacteriocin produced by Brevibacterium linens. Appl. Env. Microbiol. 60 (10), 285– 294, 1994. 13. S. M. Patin, J. Richard. Activity and purification of Linescin OC2, an antibacterial substance produced by Brevibacterium linens OC2 an orange cheese coryneform bacterium. Appl. Env. Microbiol. 61 (5), 1847–1852, 1995. 14. G. Waes, A. Van Heddeghem. Prevention of butyric acid fermentation by bacterial centrifugation of the cheese milk. Bulletin of the IDF 251, 47–50, 1990. 15. J. Stadhouders. The manufactoring method for cheese and the sensitivity to butyric acid fermentation. Bulletin of the IDF 251, 37–39, 1990. 16. J. van den Berg. New technologies for semi-hard cheese. 3rd Cheese Symposium Cork, Editor: T. M Cogan, 81–89, 1992.

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17. K. A. Bettelheim. Enterohaemorrhagic Escherichia coli: A new problem, an old group of organisms. Australian Vet. J. 73 (1), 20–26, 1996. 18. R. Marier, J. G. Wells, R. C. Swansson, W. Callahan, I. J. Mehlman. An outbreak of enteropathogenic Escherichia coli foodborne disease traced to imported French cheese. Lancet 2, 1376–1378, 1973. 19. K. L. Macdonald, M. Eidson, C. Strohmayer, E. Levy, J. G. Wells, N. D. Puhr, K. Wachsmuth, N. T. Nargett, M. L. Cohen. A multistate outbreak of gastrointestinal illness caused by enterotoxigenic Escherichia coli in imported semisoft cheese. J. Infect. Diseases 151, 716–720, 1985. 20. Public Health Laboratory Service, Edinburgh. E. coli O157 phage type 28 infections in Grampian. Communicable Diseases and Environmental Health 28 (94/46) 1, 1994. 21. J. Y. D’Aoust, D. W. Warburton, A. M. Sewell. Salmonella typhimurium phage type 10 from Cheddar cheese in a major Canadian foodborne outbreak. J. Food Prot. 48, 1062–1066, 1985. 22. J. C. Desenclos, P. Bouvet, E. Benz-Lemoine, F. Grimont, H. Desqueyroux, I. Rebiere, P.A. Grimont. Large outbreak of Salmonella enterica serotype paratyphi B infection caused by a goats’ milk cheese, France 1993: A case finding and epidemiological study. British Medical J. 312, 53–58, 1996. 23. H. C. F. Maguire, M. Boyle, M. J. Lewis, J. Pankhurst, A. A. Wieneke, M. Jacob, J. Bruce, M, O’Mahony. An outbreak of Salmonella dublin infection in England and Wales associated with a soft unpasteurised cow’s milk cheese. Epidemiology and Infection 109, 389–396, 1992. 24. J. Bille. Epidemiology of human listeriosis in Europe with special reference to the Swiss outbreak. In A. J. Miller, J. L. Smith, G.A. Somkuti (Eds.), Foodborne listeriosis. Amsterdam, Elsevier, 1990. 25. M. J. Linnan, M. Mascola, X. O. Lou, V. Goulet, S. May, C. Salminen, D. W. Hird, L. Yonekura, P. Hayes, R. Weaver, A. Andurier, B. D. Pliakaytis, S. L. Fannin, A. Kleks, C. V. Broome. Epidemic listeriosis associated with Mexican-style cheese. New England J. Med. 319, 823–828, 1988. 26. P. Zangerl, W. Ginzinger. Staphylococcus aureus in käse – Eine übersicht.

181

Meat Fermentation

Fidel Toldrá

Instituto de Agroquímica y Tecnología de Alimentos (CSIC)

CONTENTS I. II. III.

Introduction........................................................................................................................................................181-1 Types of Products ..............................................................................................................................................181-2 Raw Materials ....................................................................................................................................................181-2 A. Ingredients..................................................................................................................................................181-2 B. Other Ingredients and Additives ................................................................................................................181-2 C. Starters ......................................................................................................................................................181-2 D. Casings ......................................................................................................................................................181-3 IV. Processing Technology ......................................................................................................................................181-3 A. Comminution or Chopping ........................................................................................................................181-3 B. Fermentation ..............................................................................................................................................181-3 C. Ripening and Drying..................................................................................................................................181-4 D. Smoking ....................................................................................................................................................181-4 V. Safety ................................................................................................................................................................181-5 VI. Changes During the Process ..............................................................................................................................181-5 A. Glycolysis ..................................................................................................................................................181-5 B. Proteolysis..................................................................................................................................................181-6 C. Transformation of Amino Acids ................................................................................................................181-6 D. Lipolysis ....................................................................................................................................................181-6 E. Oxidation....................................................................................................................................................181-7 VII. Development of Sensory Characteristics ..........................................................................................................181-7 A. Color ..........................................................................................................................................................181-7 B. Texture ......................................................................................................................................................181-7 C. Flavor ........................................................................................................................................................181-8 1. Generation of Taste Compounds ........................................................................................................181-8 2. Generation of Aroma Compounds......................................................................................................181-8 References ....................................................................................................................................................................181-9

I.

INTRODUCTION

Fermentation is one of the oldest preservation practices used by man and is applied to a wide variety of foods. The term fermented meat is very generic and involves a wide variety of meat products based on a mixture of minced meat and fat, with salt and/or sugar, which is stuffed into a casing, fermented and dried or smoked (1). The evolution of fermented meats has followed a traditional route, with oral transmission from generation to generation over the centuries but very empirically, with a rather poor knowledge of the process technology (2). It was just in the latest decades of the 20th century when rapid advances in the scientific

knowledge of the chemistry, biochemistry and microbiology involved in the process were reached (3). This knowledge prompted successful developments in technology and a significant progress in quality standardization. There is a wide variety of processing technologies (with important variations in the conditions for drying, ripening, smoking, etc.) as well as an important influence of the meats used as raw materials (genetic type, feed, rearing system, etc.) and microorganisms selected for the fermentation, all of this giving important variations in quality, especially in sensory characteristics. Main types of fermented meats and the most important processing technologies are described in this chapter.

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181-2


II. TYPES OF PRODUCTS

B.

Some of the most important and well-known products are listed in Table 181.1. Based on the moisture content, most of fermented meat products may be classified as dry (weight loss higher than 30%), semi-dry (weight loss lower than 20%) or just fermented sausages when no drying is applied. Some of the typical Mediterranean sausages are French saucisson, Spanish chorizo or Italian salami. On the other hand, German or Hungarian style salamis represent some of the typical northern European products. There are basic differences between both groups of products (4). So, the Mediterranean sausages, which are not smoked, undergo a slow process with nitrate addition and very mild temperatures for both fermentation and drying. On the other hand, only nitrite is used in northern European sausages with faster processes and final smoking in most cases — i.e. up to 95% of German raw sausages are smoked (5). The pH drop, reduction in water activity and drying are main factors responsible for shelflife of these products. In general, and depending on the total processing time, three main groups of fermented sausages can be established (4): Rapid (less than 7 days), regular (around 3 weeks) and slow (up to 3–4 months).

Salt constitutes the most typical curing agent. It is added within the range 2–3% and plays several important functions: It exerts a partial bacteriostatic action, contributes to an initial reduction in aw to 0.96 and to partial solubilization of the myofibrillar proteins and, finally, imparts a typical salty taste. Nitrite, and sometimes nitrate, are also added to the curing mixture. Nitrite is a well-known microbial preservative with a specific protection against pathogens, especially Cl. botulinum. Another important role of nitrite is the development of the typical cured meat color (8,9). In addition, nitrite also helps in preventing oxidation and contributes to cured meat flavor (10) although the full chemical mechanisms are not fullly understood due to the complex number of compounds in the sausage and the high reactivity of nitrite. Ascorbic and erythorbic acids or their sodium salts are used to favor nitrite reduction to nitric oxide, exert antioxidative action and inhibit the formation of nitrosamines (3). Carbohydrates are added as substrate for microbial growth and development. The choice of the carbohydrate and its amount depends on the type of desired fermentation and ripening time. The fermentation rate will be rather faster or slower depending on the type of carbohydrate. Monosaccharides are rapidly fermented while disaccharides and more complex polysacharides take longer and pH drop is thus delayed. Sometimes, glucono-delta-lactone, that hydrolyzes to gluconic acid, may be used as an alternative way for a non-microbial pH drop but the quality of the product is rather poor (11). Spices, like ground pepper, paprika, garlic, etc., are used to give a typical and characteristic flavor, and sometimes color, to the fermented meat (12). Most of them are also effective antioxidants (13).

III. A.

RAW MATERIALS INGREDIENTS

The main ingredients are chilled raw meat from skeletal muscle tissue, either porcine alone or mixed with bovine. Other species like chicken may be used. Frozen fat tissue, usually firm pork back fat, with low content of polyunsaturated fatty acids is preferred. Highly unsaturated fat may experience undesirable oxidations and result in offflavors, color oxidation and an unpleasant melting fats appearance on the cut surface. Fat kept under frozen storage for long time may also experience an intense oxidation and thus must be rejected for the same reasons. TABLE 181.1 Examples of Fermented Meats Based on the Extent of Drying (3,6,7) Extent of Drying

Weight Loss (%)

Type

No drying “ Short drying “

⬍ 10 “ ⬍ 20 “

Spreadable

Long drying

⬎ 30

Sliceable

“ “ “

“ “ “

Sliceable

Examples German teewurst Frische mettwurst Summer sausage Lebanon Bologna Saucisson d´Alsace Hungarian & Italian salami Pepperoni Spanish salchichón French saucisson

C.

OTHER INGREDIENTS AND ADDITIVES

STARTERS

Traditionally, the fermentation was held at mild temperatures with the selective growth of the indigenous flora or the inoculation of flora from a previous succesful fermentation, a technique known as back-slopping. But these practices resulted in a wide variability in both safety and quality of the final products. The use of micrbial starters had a rapid development and application in the second half of the last century. Most of these cultures are based on lactic acid bacteria (Lactobacilli or Pediococcus strains) to ensure a rapid acidulation and Micrococcaeae (Kocuria or Staphylococcus strains) to have a good sensory profile (14–18). Proteolytic and lipolytic enzyme activities are important for flavor development. Other important enzyme activity in Micrococcaceae are nitrate reductase which contributes to the reduction of nitrate to nitrite, and catalase that mediates the degradation of hydrogen peroxide (19–21). Yeast may be used as a complement in starter cultures due to its growth on the outer area of the sausage and its important deaminase/deamidase and lipolytic

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activity. Certain molds may be used as starters for the external surface of the mold-ripened sausages (22).

D. CASINGS Casings are available in many materials but all of them must exhibit a good permeability to water and air. Traditional sausages have typically used natural casings that are irregular in shape but give good elasticity, tensile strength and permeability. Collagen-based casings shrink with the sausage and have good permeability. Synthetic casings are usually based on cellulose and, although nonedible, they allow good standardization due to the uniform shape and controlled pore size (23).

IV. PROCESSING TECHNOLOGY

FIGURE 181.2 Detail of the batter after mixing in a vacuum mixer massager.

The main stages in the processing of fermented sausages are briefly described below.

A.

COMMINUTION

OR

CHOPPING

Chilled meat pieces, usually from pork and beef, and frozen fat tissues are comminuted in a meat grinder, usually in a 2/1 ratio. The size of the particles may be regulated depending on the holes of the grinder (see Figure 181.1), Then, additives (salt, nitrate/nitrite, carbohydrates, microbial starters, spices and optionally sodium ascorbate or erythorbate) are added and the whole mass is mixed for homogenization. This operation is carried out under vacuum to remove as much oxygen as possible (see Figure 181.2). Once fully homogenized, the batter is stuffed into the casings by using vacuum filling devices. A general view of stuffing machines in a fermented sausage industry is shown in Figure 181.3. Once the sausages are stuffed, they are hung in racks and placed in air-conditioned rooms with

FIGURE 181.3 General view of a sausage manufacturing plant. Stuffing machines and vacuum mixer massaging can be observed. Courtesy of Tabanera Company, Segovia, Spain.

computer control of temperature, relative humidity and air flow rate. Local traditional sausages, which are produced in an artisanal way, are stuffed in natural casings and placed in either natural or air-conditioned ripening rooms.

B.

FIGURE 181.1 Grinding of meats and fats. There are many sizes of grinder plates in accordance to the required particle size.

FERMENTATION

The main goal of the fermentation stage consists in the growth and development of the microbial flora, either naturally present in the meat or added as starter in the mixing. Simultaneously, different biochemical changes consisting in the enzymatic breakdown of carbohydrates, proteins and lipids take place. Other changes consist in the acid gelation of meat proteins as a result of pH drop, an initial moisture loss as a consequence of water release from meat proteins and a reduction in the redox potential through the combined action of the muscle and lactic acid

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bacteria enzymes (24). An example of fermentation in an industrial computer-controlled chamber is shown in Figure 181.4. The time required for fermentation depends on the type of product but is mainly a function of temperature and specific microorganism used as starter. There are two clear technologies for meat fermentation that decide the type of starter to be used as well as fermentation conditions (2). In the USA fermentation, starters such as L. plantarum or P. acidilactici are used for fermenting at high temperatures, (i.e., up to 40°C). The result is a rapid lactic acid generation that accumulates and produces a pH drop below 5.0. The spoilage microorganisms are rapidly inhibited but flavor formation is somehow restricted by low pH values due to inhibition of exopeptidases and lipolytic enzymes, most of them active at neutral pH. In Europe, milder fermentation temperatures, around 22–26°C, are used although other differences may be found between Mediterranean and northern European countries. For instance, the use of nitrate and long ripening, with no smoking, are typical of Mediterranean sausages. The shelf life mainly depends on drying and low water activity. On the other hand, northern European countries use nitrite, short ripening and smoking (25).

C.

RIPENING AND DRYING

There are two main objectives in this stage: Drying and development of sensory properties. Sausages are hung in racks and placed in either natural or air-conditioned ripening rooms. Special care must be taken with relative humidity in the chamber as an excessive dryness in the environment may result in an excessive dehydration of the sausage surface, known as case hardening. Recommended relative humidity in the environment should not exceed in more than 0.1 units the water activity value in the sausage and air speed must be kept to values as low as 0.1 m/s which are enough for environment homogenization (2). A periodic change in the circulation of the air also allows a good exchange of the air between the sausages and the fresh blown-in air (26) The length of the ripening/drying period depends on the kind of product and its diameter, ranging from 7 days to 3–4 months. The length and conditions of the process, that allow for an intense and prolonged microbial and enzymatic action, as well as the optional smoking, have a strong influence on the sensory properties. In some Mediterranean dry fermented sausages, a mold layer is grown on the outer surface, giving a particular appearance and contributing to ammonia generation and pH increase through deamination of amino acids (see Figure 181.5).

D. SMOKING Traditionally, smoking has been applied in areas with cold and humid climates for preservation, due to its bacteriostactic effect on yeasts, molds and certain bacteria. In addition to its antimicrobial and antioxidative effects, its main role has changed to the development of sensory properties that are appreciated by consumers (27). Smoking has several advantages such as giving a characteristic color

FIGURE 181.4 Example of a fermentation chamber with computer control of temperature, relative humidity and air rate. Courtesy of Tabanera Company, Segovia, Spain.

FIGURE 181.5 Example of a ripening chamber with computer control of temperature, relative humidity and air rate. Courtesy of Tabanera Company, Segovia, Spain.

Meat Fermentation

and flavor to the product, preservation due to the bactericide and bacteriostatic effect of smoke compounds and antioxidative properties due to the phenols in the smoke (28). Smoking can be applied, by controlled combustion of oak wood, before or after fermentation.

V. SAFETY Preservation of fermented sausages is achieved through a chain of succesive events known as hurdle effect (29). Nitrite added to the mass exerts its bactericidal effect, reinforced by oxygen removal during the mixing under vacuum, being aerobic bacteria inhibited by low redox potential. Lactic acid bacteria grow during fermentation and generate large amounts of lactic acid, that produce a pH drop inhibitory of acid-sensitive spoilage microorganisms, and other metabolites like acetic acid or hydrogen peroxide that contribute to preservation (30). In addition, many strains of lactic acid bacteria, associated with meat fermentation, are producers of bacteriocins, which are biological active proteins or peptides, active at micromolar concentrations (31), with a bactericidal action against other microorganisms (32,33). These bacteriocins act by adsorption to specific or non-specific receptors on the cell surface resulting in cell death (34,35). Finally, the reduction in water activity values as a consequence of dehydration during the drying/ripening stage also contributes to the stability of the product, especially in dry fermented sausages. The combination of these hurdles restrict the activity of most food-borne pathogens although strict care must be taken. For instance, Salmonella can be inhibited by pH 5.0 and aw ⬍0.95 (6,36). Staphylococcus aureus is sensitive to acid pH but its toxin, that is produced in aerobic conditions, might be produced in the elapsed time before pH drop depending on the conditions that need to be controlled (7). Clostridium botulinum is restricted by the presence of lactic acid bacteria and nitrite together with a rapid pH drop and low aw (6). The combination of low pH, specific starter cultures and aw⬍0.90 limits the growth of Listeria monocytogenes (37) and Escherichia coli (38). It is important to adopt and implement a hazard analysis and critical control points (HACCP) plan as a system of preventive controls to improve the safety of fermented meats. Hazards would include both biological and chemical contaminants. Biogenic amines constitute another group of toxic substances that can cause disease in humans. Several factors such as the presence of microorganisms with decarboxylase activity, favorable processing conditions for the growth of these microorganisms, the production of the enzyme involved in decarboxylation of amino acids and the availability of free amino acids as substrate, contribute to the generation of amines (39). Main amines are tyramine (from tyrosine), phenylethylamine (from phenyalanine), histamine (from histidine), tryptamine (from tryptophane), putrescine and cadaverine (from ornithine

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and lysine, respectively). The amine levels in different types of sausages were recently reported (40) but some variability was observed probably due to variations in the manufacturing process and the type and quality of meat used (41) but, in general, the concentrations were relatively low. Tyramine is the amine generated in larger amounts through the decarboxylation of tyrosine, activity found in strains of Lactobacillus and Enterococcus (42,43). This amine is involved in increased cardiac output and migraine (44) but, fortunately, the estimated tolerance level is higher than for other amines (45,46). Cadaverine and putrescine may appear when meats of poor hygienic quality have been used as raw materials (47). Reduced risk for amine generation implies the use of raw materials of high quality, good manufacturing practices during the whole process and the use of starter cultures with no decarboxylase activity but, if possible, competitive against amine-producing microorganisms (36). In general, the low amounts of nitrate and/or nitrite initially added to the mix and the low nitrite residual levels reduce the possibility for nitrosamines generation to negligible levels (48).

VI.

CHANGES DURING THE PROCESS

Many biochemical changes have been reported along the processing of fermented meats, being most of them as a consequence of endogenous and/or microbial enzymatic reactions. Some of these changes are restricted to the beginning of the process which is the case of nucleotide breakdown reactions or the glycolysis-related enzymes and subsequent generation of lactic acid. Proteolysis and lipolysis constitute two of the most important enzymatic phenomena, responsible for the generation of compounds with direct influence on taste and aroma (3,24) (Figure 181.6).

A.

GLYCOLYSIS

Lactic acid is the main product resulting from carbohydrate fermentation. Once the added carbohydrates (glucose, sucrose, etc.) are transported into the cell, they are metabolized via the glycolytic or Embden-Meyerhof pathway. The ratio of the enantiomers L and D lactic acid depends on the species of lactic acid bacteria present and, more specifically, on the action of the L and D lactate dehydrogenases, respectively, and the lactate racemase. There are some key enzymes in the carbohydrate metabolism like aldolases, that generates glyceraldehyde-3-phosphate, pyruvate kinase, that generates pyruvate from phosphoethanol pyruvate and lactate dehydrogenase that generates lactic acid from pyruvate (11). Glucose is mainly metabolized through a homofermentative way but some other end products like acetate, formate, ethanol and acetoin, with an impact on sausage aroma, may be produced in trace amounts from alternative heterofermentative pathways (25). The pH drops as a consequence of

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lactic acid accumulation and contributes to the preservation of the sausage by preventing the growth of undesirable microorganisms (49). The generated lactic acid also contributes directly to acid taste and indirectly to aroma, due to the formation of metabolites, and sausage consistency due to protein coagulation as pH approaches the isoelectric point of most of the myofibrillar proteins (50).

B.

PROTEOLYSIS

Proteolysis consists in the progressive degradation and breakdown of major meat proteins (sarcoplasmic and myofibrillar proteins) and the subsequent generation of peptides and free amino acids. The result is a weakening of the myofibrillar network and generation of taste compounds but its extent depends on many factors. One of the most important is the activity of endogenous muscle enzymes, which depends on the original crossbreeds (51,52) and the age of the pigs (53,54). Main muscle enzymes involved in these phenomena are cathepsins B, D and L that show a great stability in long term dry-curing processes, good activity at acid pH values and are able to act against myofibrillar proteins (55–57). Other important muscle endopeptidases like calpains exhibit poor stability and its optimal pH near 7.0 is far from that in the sausage (58). Muscle enzymes exert a combined action with microbial proteases although different enzymatic profiles may be found depending on the microorganisms used as starter cultures (59). One of the major challenges is just to establish the relative role or percentage of contribution of endogenous and microbial enzymes to proteolysis. The proteolytic system of different Lactobacillus has been studied and contains endopeptidases able to degrade sarcoplasmic and myofibrillar proteins (60–63) as well as exopeptidases like dipeptidylpeptidase (64), tripeptidase (65), dipeptidase (66) and aminopeptidases (67,68). However, some studies (40,69) revealed that protein degradation, especially myosin and actin, is initiated by cathepsin D, a muscle endopeptidase very active at pH values near 4,5 and able to degrade both proteins. Cathepsins B and L would be more restricted to actin and its degradation products. The latter stages of proteolysis would be predominantly by bacterial peptidases and exopeptidases. Other important factors are related with the processing technology. For instance, the temperature and time of ripening will determine the major or minor action of the enzymes, the amount of added salt, which is a known inhibitor of cathepsins and other proteases, will also regulate the enzyme action (70,71) and thus the proteolysis and taste (72). The generation of small peptides may be depressed by the level of salt which inhibits muscle peptidases (73–75) although intense levels of non-protein-nitrogen, up to 20% of the total nitrogen content, may be reached. Some of these peptides give characteristic tastes (76). Final

proteolysis steps by aminopeptidases, especially from microbial origin, are very important (77,78). These enzymes release free amino acids along the process and a substantial increase in the concentration of free amino acids is usually observed (79,80).

C.

TRANSFORMATION

OF

AMINO ACIDS

The released free amino acids as a consequence of proteolyisis are then subject of a number of enzymatic and/or chemical transformations that produce different compounds that will affect the sensory characteristics of the product (12). So, microbial decarboxylation of amino acids may produce biogenic amines. Transamination consists in the transference of the α-amino group of the first amino acid to the α carbon atom from an α-keto acid generating a keto acid from the first amino acid and a new amino acid. Dehydrogenases transform the amino acid in the corresponding keto acid and ammonia. Deamidation also generates ammonia (81). The microbial degradation of the amino acid side chain by liases may lead to phenol and indole formation (82). The Strecker degradation of amino acids produces branched aldehydes, like 3-methylbutanal, 2-methylbutanal and phenylacetaldehyde from leucine, isoleucine and phenylalanine, respectively, through oxidative deaminationdecarboxilation reactions (83).

D. LIPOLYSIS Lipolysis consists on the breakdown of tri-acylglycerols by lipases and phospholipids by phospholipases resulting in the generation of free fatty acids. These fatty acids may contribute directly to taste and, indirectly to the generation of aroma compounds through further oxidative reactions. Main lipolytic enzymes, located in muscle and adipose tissue, in combination with microbial lipases, are involved in these phenomena (84). Although it is difficult to establish a relative role of endogenous and microbial enzymes to lipolysis, the percentage of contribution of endogenous lipolytic enzymes to total fat hydrolysis is estimated around 60 to 80% with the rest due to microbial lipases (69). The most important lipases located in muscle are the lysosomal acid lipase and acid phospholipase while in adipose tissue are the hormone sensitive lipase and the monoacylglycerol lipase (85). These enzymes show good stability through the full process (86–88). Although their activity also depends on pH, salt concentration and water activity, the conditions found in the sausages favor their action (89). The generation rate of free fatty acids, especially oleic, linoleic, estearic and palmitic acids, increases during the process. Most of these fatty acids proceed from phospholipids degradation (3) although some of them generate volatile compounds through further oxidative reactions (90). In the case of adipose tissue, the rate of generation, especially of oleic, palmitic, linoleic, stearic, palmitoleic and myristic acids, is also high.

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Carbohydrates

Glucohydrolases

Lactate dehydrogenase

Pyruvate

Lactic acid

Deaminase

Dehydrogenase

Myofibrillar proteins

Muscle and microbial proteinases

Muscle and microbial

Peptides

exopeptidasses

Free amino acids

Amino acids

Degradation

Nitrate

Peroxides

Endogenous and microbial lipases

Nitrate reductase

Catalase

Free fatty acids

Nitrite

Oxidation

Reduction

Volatile compounds

Keto acids + ammonia

Transaminase

Decarboxylase

Triacylglycerols Phospholipids

Ammonia

Amine + CO2 Indol, phenol Sulfur compounds Methyl aldehydes

Nitric oxide

Peroxides destruction

FIGURE 181.6 Scheme showing the most important reactions by muscle and microbial enzymes affecting sensory quality of fermented meats.

E.

OXIDATION

The generated mono and polyunsaturated fatty acids are susceptible to further oxidative reations to give volatile compounds. The beginning of lipid oxidation is correlated to an adequate flavor development. On the contrary, an excess of oxidation may lead to off-flavors (91). In fact, the generation of the characteristic aroma of dry-cured meat products is in agreement with the beginning of lipid oxidation. Free radical formation is catalyzed by muscle oxidative enzymes, like peroxydases and ciclooxygenases, external light, heating and the presence of moisture and/or metallic cations. The next step in oxidation is the formation of peroxide radicals (propagation) by reaction of free radicals with oxygen. The formed hydroxyperoxides (primary oxidation products) are flavorless but very reactive giving secondary oxidation products that contribute to flavor (92). The oxidation is finished when free radicals react each other. Main products from lipid oxidation are aliphatic hydrocarbons (poor contribution to flavor), alcohols (high odor threshold), aldehydes (low odor threshold) and ketones. Alcohols may interact with free carboxylic fatty acids giving esters, especially when nitrate is not used.

VII. DEVELOPMENT OF SENSORY CHARACTERISTICS A.

COLOR

The color mainly depends on the concentration of its natural pigment myoglobin, that depends on the type of muscle and the age of the animal (93,94). For instance,

myoglobin concentration is higher in muscles with oxidative pattern and in older animals (94). The typical brightred color is due to nitrosomyoglobin, compound formed after reaction of nitric oxide with myoglobin. About 10 to 40% of total myoglobin is transformed into nitrosomyoglobin (95). Nitric oxide is produced by reduction of nitrite and is favored by ascorbic acid. Those sausages made with nitrate need its reduction to nitrite by nitrate reductase activity in Micrococcaeae. However, this bacteria is inhibited at pH values below 5.2, being necessary to control the pH drop during fermentation to ensure that nitrate reductase can reduce nitrate to nitrite (2). Nitrosomyoglobin is susceptible to oxidation, especially at low pH and redox potential, conditions found in dry fermented sausages. So, it is very important to avoid oxidants (i.e.- peroxides) and thus the convenience of the presence of antioxidants to preserve color.

B.

TEXTURE

Texture depends on several factors like the extent of drying (loss of moisture), the extent of proteolysis, especially by cathepsin D (degree of myofibrillar protein breakdown) and the content in fat and connective tissue. The lactic acid accumulation produces the coagulation of myofibrillar proteins, the release of some water and the formation of a gel. The bonds are stabilized and the matrix of the sausage is developed (96). The consistency is accelerated during the drying period. The content in fat also exerts a positive influence on some texture and appearance traits. Textural characteristics such as firmness, hardness and cohesiveness of meat particles are continuously developed during drying. Shear values

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are corelated with sausage diameter, moisture content, drying time and sometimes the initial grinding (97). In general, a good consistency is desired to facilitate the sliceability.

C. 1.

Reactions Involved Group of Compounds

FLAVOR

Oxidation

Aliphatic aldehydes

Strecker degradation

Branched-chain aldehydes Branched-chain acids Branched-chain alcohols Sulfides Ketones Alcohols Esters Hydrocarbons Dicarbonyl compounds Nitrogen compounds

Generation of Taste Compounds

Main contributors to taste are listed in Table 181.2. D and L lactic acid, especially the D enantiomer, and acetic acid are the main compounds responsible for the sour taste (43). The generation of free amino acids is the result of the combined action of muscle and microbial aminopeptidases. These enzymes are active at neutral pH, being partly inhibited at acid pH. This is the reason why those fermented meats with an intense pH drop lack significant generation of free amino acids (80). Salt is the main compound responsible for salty taste. Other compounds like glutamic and aspartic acids impart a sour taste while its sodium salts also impart a salty taste. Bitter taste is mainly associated with aromatic amino acids like phenylalanine, tryptophan and tyrosine and sweet taste with alanine, serine, proline, glycine and hydroxyproline (98,99). The generation of all these free amino acids is extremely important in fermented meats (55,79) and somehow the generation rate is affected by levels of salt as the involved enzymes (peptidases and aminopeptidases) are partly inhibited (73,74,100). Some taste enhancement may be expected from ATP-derived compounds like inosine monophosphate (IMP) and guanosin monophosphate (GMP). 2.

TABLE 181.3 Main Volatile Compounds Contributing to Aroma during Meat Fermentation

Generation of Aroma Compounds

Aroma development is a very complex process involving numerous reactions like chemical or enzymatic oxidation of unsaturated fatty acids and further interactions with proteins, peptides and free amino acids (50). In fact, a substantial number of volatile compounds have been reported in fermented sausages (101–105). Main groups of volatile compounds are listed in Table 181.3 and a scheme of flavor TABLE 181.2 Main Compounds Contributing to Taste during Meat Fermentation Reactions Involved

Group of Compounds

Proteolysis “ Lipolysis

Nitrogen compounds “ Fatty acids

ATP degradation

Nucleotides/nucleosides

Glycolysis

Acids

Addition “

Carbohydrates Inorganic compounds

Main Compounds Small peptides Free amino acids Long and short chain fatty acids Inosine monophosphate, inosine D, L lactic acid, acetic acid Glucose Salt

“ “ “ Oxidation “ Interactions Lipids autoxidation Pyruvate metabolism Deamination/ deamidation

Main Compounds Hexanal, pentanal, octanal 2- and 3-methylbutanal 2- and 3-methyl butanoic acids 2- and 3-methyl butanol Dimethyldisulfide 2-pentanone, 2-octanone Ethanol, butanol Ethyl acetate Pentane, heptane Diacetyl, acetoin, acetaldehyde Ammonia

generation routes is shown in Figure 181.7. Final flavor depends on the mixture of characteristic aromas and odor thresholds for each compound although, in general, ketones, esters, aromatic hydrocarbons and pyrazines are correlated with pleasant aromas (25). Aliphatic aldehydes, ketones, alcohols and esters are typical products of different lipid oxidation reactions. Some volatile compounds like 2-methyl propanal, 2-methyl butanal, and 3-methyl butanal arise from Strecker degradation of the amino acids valine, isoleucine and leucine, respectively (105). Branched-chain acids and alcohols are secondary products. Dimethyldisulfide proceed from the Strecker degradation of sulfur containing amino acids like methionine. Compounds like diacetyl, acetoin and acetaldehyde are typical products of pyruvate microbial metabolism (25). Some pyrazines are formed through Maillard reactions between sugars and free amino acids and, although generated in low amounts, they also impart some characteristic aromas like nutty, green, earthy, etc. The spices have an intense impact on aroma. Some sulphur compounds are derived from garlic, some terpenes from pepper, 3-hexenol in paprika, etc. (12). Ammonia is released through enzymatic deamidation and deamination reactions (81). Several hundreds of volatile compounds have been identified in the aroma of fermented meats and several techniques have been used to estimate their relative importance (105). The most important compounds contributing to aroma may be determined by comparison of a certain amount with its sensory threshold value in a similar matrix, by correlation of the respective amounts to the sensory profile of the fermented meat or by gas chromatography coupled to olfactometry (104). Some interesting correlations have been found between some volatile compounds and specific characteristics of the process. For instance, a

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Lactic acid Acetic acid

TASTE

Carbohydrates

Acetoin, diacetyl acetaldehyde

AROMA

Pyrazines

Proteins

Sulfides, indol, phenol, branched-chain aldehydes, acids and alcohols, ammonia Small peptides Free amino acids

ATP-derived compounds

Lipids

Nucleotides Nucleosides

TASTE

Free fatty acids Aldehydes, ketones Esters, hydrocarbons

Spices

Terpenes Sulfur compounds

AROMA

FIGURE 181.7 Scheme showing the contribution of fermentation to flavor compounds.

stronger and typical flavor has been reported in nitrite-containing dry-fermented sausages (106). In small diameter sausages, with mild processing conditions and short ripening time, most of the volatiles are produced by lipid autooxidation (107). Ketones, aldehydes, esters and terpenes are the volatile compounds usually found in Spanish and Italian sausages (51). Medium aged Italian Milano salami with low production of lactic acid, and thus higher pH, is preferred (108). The type of starter also has a strong influence on flavor like a lower rancidity when using S. saprophyticus, curing odor correlated with 2-pentanone, 2-hexanone and 2-heptanone when using S. carnosus in combination with either P. acidilactici, L. sakei or P. pentosaceus, or butter odor correlated with acetoin, diacetyl, 1,3-butanediol and 2,3-butanediol when using S. saprophyticus and S. warneri (109). Many aroma volatile compounds have been reported to be produced by S. xylosus (110) and S. carnosus (111). Flavor may be affected not only by time of ripening but also by packaging (112). Pre-ripening exerts a beneficial sensory effect in dry fermented sausages (113).

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182

Technologies for Jalapeño Pepper Preservation

Rosa María Galicia-Cabrera

Universidad Autónoma Metropolitana

CONTENTS I. Introduction ......................................................................................................................................................182-2 II. Processing of Jalapeño Pepper ........................................................................................................................182-2 III. Jalapeño Pepper Preservation by Fermentation or Pickling ............................................................................182-2 IV. Fermented Jalapeño Pepper..............................................................................................................................182-3 A. Preliminary Operations ............................................................................................................................182-3 V. Fermentation ....................................................................................................................................................182-3 VI. Pickled (Non-Fermented) Jalapeño Peppers ....................................................................................................182-3 A. Preliminary Operations ............................................................................................................................182-5 1. Washing ............................................................................................................................................182-5 2. Sorting and Grading ..........................................................................................................................182-6 3. Peeling ..............................................................................................................................................182-6 4. Size Reduction ..................................................................................................................................182-6 5. Spoilage Enzymes..............................................................................................................................182-6 6. Blanching ..........................................................................................................................................182-6 B. Packaging ..................................................................................................................................................182-6 C. Pickle ........................................................................................................................................................182-6 D. Blanching Vegetables ................................................................................................................................182-7 E. Filling........................................................................................................................................................182-7 F. Exhausting ................................................................................................................................................182-7 G. Sealing ......................................................................................................................................................182-7 H. Heat-Treatment ........................................................................................................................................182-7 1. Batch Processing................................................................................................................................182-7 2. Continuous Retorting ........................................................................................................................182-7 I. Marking, Labeling and Packaging............................................................................................................182-7 J. Storage ......................................................................................................................................................182-7 VII. Regulations ......................................................................................................................................................182-8 A. Mexican Specifications ............................................................................................................................182-8 B. International Specifications ......................................................................................................................182-8 1. Scope..................................................................................................................................................182-8 2. Product Definition ............................................................................................................................182-8 3. Essential Composition and Quality Factors ......................................................................................182-8 4. Weights and Measures ......................................................................................................................182-9 VIII. Ripened Jalapeño Pepper Drying (Chipotle Pepper) ......................................................................................182-9 A. Preliminary Operations ............................................................................................................................182-9 1. Sorting................................................................................................................................................182-9 2. Washing ............................................................................................................................................182-9 3. Seed Removal ....................................................................................................................................182-9

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B. Drying..........................................................................................................................................................182-9 1. Drying and Smoking ............................................................................................................................182-9 2. Hot Air Drying....................................................................................................................................182-10 3. Grading ..............................................................................................................................................182-10 C. Packaging ..................................................................................................................................................182-10 D. Storage ......................................................................................................................................................182-10 IX. Canned Chipotle Pepper in “Adobo” (Spicy Sauce) ........................................................................................182-10 A. Preliminary Operations..............................................................................................................................182-11 1. Sorting ................................................................................................................................................182-11 2. Washing ..............................................................................................................................................182-11 3. Grading ..............................................................................................................................................182-11 4. Peeling ................................................................................................................................................182-11 5. Blanching............................................................................................................................................182-11 B. Packing ......................................................................................................................................................182-12 C. Adobo (“Spicy Sauce”) ............................................................................................................................182-12 D. Blanching Vegetables ................................................................................................................................182-12 E. Filling ........................................................................................................................................................182-12 Acknowledgments ......................................................................................................................................................182-12 References ..................................................................................................................................................................182-12

I.

INTRODUCTION

Nutrition of Aztecs and other cultures living in prehispanic Mexico was based on corn, beans, hot pepper and a type of zucchini. Hot pepper (Capsicum annuum) is an excellent source of vitamins A and C; the compound responsible for irritation (“hotness”) is capsaicin located in the fruit placenta. In addition to the pungent effect, capsaicin stimulates appetite, increases saliva secretion and is considered to have beneficial effects on gastric fluid production. Ever since the Aztec empire, chili is added to a number of Mexican traditional foods. This cultivar is widely acceptance in Europe, Asia and Africa where Spanish traders took it as commercial item after the conquest of Mexico. Today, chili is distributed and consumed worldwide (1–3). The most important chili cultivars in Mexico are “ancho” (wide chili): poblano, mulatto and miahuateco; Jalapeño: classic Jalapeño, candelaria or peludo (“hairy”) and espinalteco; serrano and mirasol (known as guajillo or cascabel when dried). These cultivars represent 70 to 80% of chili national production. Sweet peppers, those with low concentration or none of capsaicin, are exported. These are mainly moron, and in a lesser extent anaheim, caribe, fresno and cherry (4). Asia was the main chili producer in 2001 (8,238,000 MT) followed by Mexico (1,961,000 MT) and the United States (885,630 MT). A high percentage of chili production undergoes processing such as freezing, canning, dehydration and pickling (5).

II. PROCESSING OF JALAPEÑO PEPPER Jalapeño pepper is the most popular in North America. Its name, jalapeño, refers to the city of Xalapa, situated in the Mexican state of Veracruz. This is a fleshy, pungent fruit; harvested when unripened, with green bright color

FIGURE 182.1 Fresh Jalapeño pepper.

(Figure 182.1). Most of the harvest (60%) is pickled and canned; about 20% is consumed raw. If the fruits are harvested when ripened (red), they are dried and smoked; this product is known as chipotle (2,4,6).

III. JALAPEÑO PEPPER PRESERVATION BY FERMENTATION OR PICKLING Pickled Jalapeño pepper is widely consumed in Mexico. This is a scalded, pasteurized product, generally merchandized in cans or glass jars, with brine to which spice and vinegar have been added. However, Jalapeño pepper shelflife extension by fermentation is carried out only at a very small industrial level. Information regarding fermented or pickled vegetables is scattered and there is no clear differentiation between pickled and fermented products (7). This section describes the processing of fermented and pickled Jalapeño pepper merchandised in cans or glass jars. Pickling and fermentation are preservation methods extending fruit and vegetable shelf lives via a simple and


inexpensive technology. The processed material undergoes transformation resulting in a highly acceptable food to the consumer. Pederson (8) pointed out the various methods for fruit and vegetable preservation: 1. 2. 3. 4.

Pickling without undergoing fermentation Fermentation in a low concentrated brine Fermentation in a highly concentrated brine Preservation by drying and salting at low salt concentration

However, there is a controversy regarding whether the terms “pickling” and “fermentation” are equivalent. According to Pederson and Luh (9) pickled products are those added with edible acids, either lactic or acetic (vinegar); on the other hand, fermented products are such that acid was produced from sugars by bacterial metabolism. Both pickled and fermented vegetables are mainly preserved by the action of acid, also improving sensory characteristics and possibly increasing its nutritive value. According to these definitions, Jalapeño pepper can be either fermented or pickled. Undesirable microbial growth is inhibited by acid as well as by salt concentration (10). In addition of reducing populations of spoilage microorganisms shelf-life extension of fermented or pickled vegetables also depends on inhibition of plant enzymatic activity involved in the ripening process. Control of both spoilage mechanisms, microbial and enzymatic, in Jalapeño peppers is achieved by pickling and fermentation.

IV. FERMENTED JALAPEÑO PEPPER Acid is produced by fermenting sugars through the action of lactic acid bacteria, such as Lactobacillum plantarum, although the presence of Leuconostoc mesenteroides also has a marked effect upon fermentation and product quality (11,12). In addition to lactic acid bacteria activity, other fermentative bacteria, such as acetic acid-producing microorganisms also carry out vegetable fermentation enhancing shelf life and sensory characteristics (12). Undesirable microorganisms are inhibited by various mechanisms. Salt addition allows the growth of naturally present lactic acid bacteria, but the combined salt and acid action allows the selection of microflora associated with vegetable preservation. In some cases, sugars are added to enhance the fermentation process (10). At the same time, fermentation reduces carbohydrate concentration and increases acid production (13). The most important conditions for an adequate vegetable fermentation are: anaerobiosis, salt concentration, temperature, and the used of suitable starters. Lactic acid bacteria can be present as native microflora in the pepper, but to assure a uniform fermentation, selected starters are usually added. To obtain the best fermented jalapeño pepper quality, the raw material (Capsicum annuum) cv. Jalapeño must be

182-3

recently harvested, still green, and without wounds or peduncle. Figure 182.2 shows the general flow diagram of fermented Jalapeño pepper processing (14).

A.

PRELIMINARY OPERATIONS

Raw Jalapeño peppers are selected according to their size and quality. They are washed, and small incisions are made in order to facilitate brine diffusion to the central part, and to eliminate gas formed during fermentation. Washing and blanching diminishes hot pepper fermentative ability; therefore it is necessary to add a starter culture (Lactobacillum plantarum).

V. FERMENTATION Fermentation is carried out by facultative anaerobic homofermentative strains such as Lactobacillum plantarum and Pediococcus cereviseae. L. plantarum produces acetic acids as well as ethanol and gas (CO2 and H2). Peppers are immersed in 10% brine for 4 to 6 weeks, and 0.5 to 1% sucrose is added although pepper cell fluid contains carbohydrates as well as nitrogen compounds and minerals. The fruit cell fluid, however, tends to dilute the brine. For this reason, it is also necessary to add 1% salt daily during the first week, and three times a week during the rest of the immersion time in order to keep the desired brine concentration (18–20%). The peppers must be completely covered by the brine at all times. Fermentation takes place in 4 to 6 weeks. It is carried out in closed tanks, with a vent to allow the gas formed during the process to dissipate. At the end of the fermentation period, the peppers, originally bright green, turn into olive green. The plant tissue also changes, taking a translucent aspect. Acid concentration increases from 0.8 to 1.5% (expressed as lactic acid) promoting a decrease in pH. The peppers are then washed to eliminate salt excess, classified according to their size, placed in glass jars or plastic bags, mixed with other vegetables, usually carrots and onions, and covered with vinegar. Fermented Jalapeño peppers are highly perishable if the vinegar has less than 3% acetic acid. In this case, pasteurization is necessary. It is carried out over 30 min at 71°C (for glass jars containing 280 g of product). Finally, the product is labeled, packaged and stored in a similar way as for pickled (non-fermented) Jalapeño peppers.

VI. PICKLED (NON-FERMENTED) JALAPEÑO PEPPERS The most widely merchandized Jalapeño peppers in producing countries such as Mexico, are pickled nonfermented products. They are sold in different can sizes and consist of whole cut peppers, mixed with scalded onions, carrots, mushrooms and vinegar to which spices have been added (Figures 182.3 and 182.4).

182-4


Jalapeño pepper

Storage

Sorting and grading

Lactic fermentation: 10% brine, 0.5 to 1% sugar Inoculum: Lactobacillus plantarum

Fermented Jalapeño peppers (1 to 5% lactic acid)

Immersion in brine for 4 to 6 weeks

Brine separation and washing with water

Filling with vinegar (1000°C. As vegetables pass, the outmost layer and fine roots are burnt and eliminated by high pressure water spraying. 4.

Size Reduction

inactivating the enzymes. Scalding efficiency in vegetables is measured by inactivation of two enzymes: catalase and peroxidase. Jalapeño scalding in water is carried out for 8 to 10 min at 95°C; carrots are scalded for 6 to 8 min, water at 95°C (15). 6.

Blanching

It is applied prior to processing in order to inhibit enzymatic activity and to decrease microbial populations. Blanching can be combined with other operations such as peeling or cleaning (17–19). Efficient enzyme inactivation is carried out by heating at given temperature-time conditions, and further fast cooling to room temperature. The two blanching methods commonly used are: applying saturated steam and immersion in hot water. At industrial levels, steam blanching is the most widely applied (17), it consists in applying steam to the vegetables placed on a conveyor moving through a steam tunnel. Varying the conveyor speed controls the residence time in the tunnel. In some cases water spray is applied at the start and end of the conveyor in order to condense excess steam. During hot water blanching vegetables are held for a given time at 70–100°C, with a further draining-cooling period.

B.

PACKAGING

The aim of this operation is to keep the product, from processing to the consumer, in the same hygienic and quality conditions. Cans are made from three-piece tin sheets, coated on the inside with an epoxyphenolic enamel (Figure 182.6). The lids are also made of tinfoil and coated with the same material used in the can. The lid also has two or three circular expansion rings, providing resistance against deformation due to an increase in the internal pressure (20).

C.

PICKLE

According to Mexican regulations (21), pickle is a mixture of vinegar, vegetable oil, onion, carrots, laurel, garlic,

In this operation the average size of a solid food material is reduced by the application of external forces such as impact, compression or abrasion (17). In the case of Jalapeño peppers they are cut lengthwise into four parts and the peduncle and seeds eliminated. Cutters consist of a series of rotating blades, and centrifugal force holds the product against the blades. 5.

Spoilage Enzymes

Enzymes, endocelular, exocelular or microbial, assume an active role in food deterioration. Microbial enzymes are also able to act on the food substrate even when the microbial cell is inactivated or dead (19). Insufficient scalding can result in an increase in food spoilage as heat applied can disrupt the tissues, liberating the substrate but not

FIGURE 182.6 Three-sheet tin can, with internal porcelain enamel covering.


salt, sugar and optional spices. Vinegar has 2% acetic acid and 5% sodium chloride.

D. BLANCHING VEGETABLES Cut peppers, carrots and onions must not be less than 60% total product weight, peppers must be included in a higher proportion.

E.

FILLING

Vegetable mix is first added to the can, previously washed with hot water; the brine (pickle) is then added at 82 to 86°C. Filling must be carefully controlled in order to assure that the correct amount of vegetable mix and pickle is added, and to fulfill specifications. Headspace must be 10% of total can volume. Filling is done when transported by the conveyors, which carry the cans to the vegetable mix filler and then to the liquid one.

F. EXHAUSTING When air is evacuated from the headspace before sealing internal pressure is decreased during sterilization. At the same time oxygen evacuation prevents tin corrosion and oxidation. During this operation air is replaced by vapor promoting a partial vacuum in the headspace after condensing. Exhaustion is carried out in tunnels (or exhausters), as shown in Figure 182.7. Another way to promote exhaustion is by using steaming equipment, which inject steam to the headspace before closing the cans (20).

G.

SEALING

Sealing is carried out in steaming machine. According to the design and speed of the operation, the basic stages of the operations are: (a) folding edges; (b) pressing the folded tin to form an hermetic seal impermeable to air (22).

182-7

H.

HEAT-TREATMENT

Cans or glass jars are subjected to heat treatment to sterilize or pasteurize their contents. It can be done in batches or by continuous retorting. Cans are heated at a time-temperature condition in vapor or hot water. Pasteurization of pickled Jalapeño peppers destroys microorganisms resistant to high acetic acid concentrations, able to promote product alteration. Heat treatment also inhibits vegetable or microbial enzymes (23). Heat treatment of 93.3°C and 10 min are recommended for acid pickles (pH 4.3 to 4.5). However, a time-temperature process depends on type of container, volume, and heat processing equipment. 1.

Batch Processing

During this operation retorts are saturated with vapor and containers are placed in baskets. Retorts can be horizontal or vertical, and the cans can be still or rotating during the process. Can rotation promotes heat transfer, so that processing time is reduced and higher temperatures can be achieved. 2.

Continuous Retorting

This type of equipment is fitted with hydrostatic closings before and after the pressurized sections. Processing can be also carried out by can rotation, where the cans move in and out of the pressurized section through hydrostatic column seals, equilibrating the internal pressure. A variation of this equipment is the flame retort, operated at atmospheric pressure throughout the process. Flame retort equipment is fitted with direct heating, applied to the rotating retort. An advantage of this type of retorting is a high product quality due to mild heating conditions. In all heat treatments the final part is can cooling with water to reach final temperatures not less than 38°C. Because the cans are not completely cooled down, water is eliminated from the outside, avoiding corrosion.

I.

MARKING, LABELING AND PACKAGING

Once the containers undergo heat treatment, each can or jar is marked with a code, a production date, a batch number and a plant code. The label includes the product name, the commercial name, the drained and net weight, the ingredients and other specifications required by the country’s regulations (21). Packing is automatically carried out in cardboard boxes or high-density polyethylene bags, or other suitable packaging material with enough resistance to protect the product and containers.

J.

FIGURE 182.7 Vapor tunnel or exhauster.

STORAGE

Heat-treated Jalapeño peppers keep their quality characteristics at 18 to 21°C. At a higher temperature, acid products in cans without inner coating consume oxygen in the

182-8


headspace faster than in coated cans. The result is a considerable loss in ascorbic acid content and fast product oxidation (23,24). On the other hand, canned Jalapeño peppers have a longer shelf life if stored at 0 to 5°C (23).

VII. A.

REGULATIONS MEXICAN SPECIFICATIONS

NMX-F-121-1982, 21 is Mexico’s quality bylaw (Norma Mexicana) regulation for pickled Jalapeño or serrano peppers. This regulation includes six consumer presentations or styles and two quality levels. The presentations are: whole peppers, peppers without seeds, peppers in halves, peppers cut lengthwise, pepper cut in rings and chopped peppers. There are two quality classifications for whole peppers only; for the rest of the presentations there is one quality classification. Table 182.1 shows physical and chemical specifications. These specifications also include microbial characteristics, chemical contaminants, optional ingredients, sampling and specificity of quality degrees, labeling, containers and packaging. Among optional ingredients are: garlic, pepper, cinnamon, cloves, ginger, laurel, marjoram, thyme and nutmeg. In defining the Mexican official specifications the main Jalapeño pepper processing industries took part, such as Productos Del Monte, La Costeña, Hérdez, Conservas San Miguel, Conservas Guajardo and Elías Pando.

B.

product nature, expressed by several member countries, with respect to the edible characteristics of the covering medium or if it should be eliminated, as well as pH, salt concentration and processing conditions (scalding, lactic fermentation, heat treatment before or after container filling, etc.) (25). The Draft Codex Standard for Pickled Products (26) includes the following specifications: 1.

This standard applies to edible fruits, vegetables, cereals, legumes, spices and condiments which have been cured, treated or processed to produce an acid product and which are offered for direct consumption in oil, brine or acidic media. 2.

TABLE 182.1 Specification of Jalapeño Peppers (Mexican Legislation NMX-F-121-1982, 21) Specification Acidity (as acetic acid) (%) Chloride (as sodium chloride)(%) pH Filling (%) Headspace (%) Vacuum (mm Hg)

Minimum

Maximum

0.75 2.0

2.0 7.0 4.3

90 10 76.2

Product Definition

Due to the wide variety of pickled or fermented products, this chapter deals with a general definition. Pickled products are: a) Prepared from sound, clean and edible fruits, vegetables, cereals, legumes, spices and condiments. b) Subjected to curing and processing with ingredients appropriate to the type in order to ensure preservation of the product and its quality. c) Processed in an appropriate manner in order to ensure the quality and proper preservation of the product d) Preserved in an appropriate manner in a suitable packing medium with ingredients appropriate to the type and variety of pickled product.

INTERNATIONAL SPECIFICATIONS

The processed fruit and vegetable Committee of the Codex Alimentarius Commission FAO/OMS, has elaborated a General Specification project for pickled products. At present, this project is at the sixth stage, that is revision by all member countries. However, the project does not include pickled cucumbers or kimchi (25). During the 21st session of the Codex Committee on Processed Fruits and Vegetables, held in San Antonio, Texas on 2002, it was agreed to stop the draft Codex standard on the sixth stage of Codex Alimentarius normalization procedure. This decision was taken on the basis of the

Scope

3.

Essential Composition and Quality Factors

This section includes basic ingredients such as edible fruits, vegetables, cereals, legumes, spices and condiments in a liquid medium in a combination with one or more of the optional ingredients. The optional ingredients are nutritive sweeteners, unrefined nutritive sweeteners, edible vegetables oils, vinegar, citrus juice, dried fruits, malt extract, salt, brine, chilies, seasoning (of plant origin and animal origin). Some specific requirements are: Pickled products in edible oils: Oil must not be less than 10% by weight. Pickled products in brine: Salt in the covering liquid must not be less than 10% by weight, if salt is used as the main preservation agent. Pickled products in acidic media: Acid must not be less than 2% by weight, expressed as acetic acid. Accepted food additives are shown in Table 182.2.


TABLE 182.2 Authorized Food Additives (Draft Codex Standard for Pickled Products, 26) Preservatives 220 Sulphur dioxide 221 Sodium sulphite 222 Sodium hydrogen sulphite 223 Sodium metabisulphite 224 Potassium metabisulphite 211 Sodium benzoate 212 Potassium benzoate 200 Sorbic acid 202 Potassium sorbate

182-9

Whole ripened Jalapeño peppers

4.

Drying and smoking 30 mg/kg (as sulphur dioxide)

Grading

250 mg/kg (as benzoic acid) 1000 mg/kg as sorbate

Storage Limited by GMP

Weights and Measures

Pickled products in edible oil, in brine and in acid media: basic ingredient in the final product (drained weight) must not be less than 60% by weight. This draft standard also includes other chapters on contaminants, hygiene, labeling and methods of analysis and sampling, referring to relevant legislations of other related Codex Committees.

VIII. RIPENED JALAPEÑO PEPPER DRYING (CHIPOTLE PEPPER) Jalapeño pepper is a conic fruit of approximately 6 cm long, 4 cm width, tasty and consistently, in general the surface is scorched. This is a highly acceptable characteristic as it prevents cuticle removal during pickling. However, if it is excessive, the fruits are directed to chipotle fabrication (2,3). A flow diagram depicting this process is shown in Figure 182.8.

A. 1.

PRELIMINARY OPERATIONS Sorting

Jalapeño peppers, if directed to dehydration and smoking, must be healthy, ripened and showing intense red color. 2.

Washing

Fruits are washed by immersion in tanks added with detergent or chlorine to reduce microbial loads. 3.

Washing

Maximum Level

Acidity Regulator 260 Acetic acid (glacial)

Sorting

Seed Removal

The fruits can be processed complete, with seeds, or once the seeds are removed, in this case the product has a higher commercial value. In addition, seeds are used for

Packaging

FIGURE 182.8 Drying of ripened Jalapeño pepper.

the next seeding season. Seed extraction is carried out before smoking; chilies without seeds are called “capones” (capons) (3).

B.

DRYING

This operation removes through evaporation by heating procedures most of the water present in fruits and vegetables. The main objective is to extend the shelf life by reducing water activity. Microbial growth and enzymatic activity is considerably reduced due to the reduction of available water. Drying also reduced food weight and volume; therefore transportation and storage costs are also reduced (17). Drying and smoking, or applying hot air carries out dehydration of Jalapeño peppers. 1.

Drying and Smoking

It consists in applying hot smoke in ovens, located close to the producing area. In small processing plants, the oven consists of two brick chambers linked together with a tunnel; in one chamber smoke is generated by burning sawdust or wood, chilies are placed in a second chamber on a 1.5 to 2.5 cm thick, 2 ⫻ 2 m long rack made generally of bamboo, with 20 cm ⫻ 2 m wood supports (27). Chilies on the rack in 18 to 20 cm thick layers are in direct contact with the smoke; they are constantly turn over using a 1.5 m long shovel to reach homogeneous drying and smoking of the product. The combined effect of heat and smoke is an efficient drying. Process duration varies depending on the desired final moisture and smoke content, average duration is 3 to 6 days obtaining a dark brown product with smoke-like flavor taste (Figure 182.9) (2,27). In large processing plants ovens can held 800 a 900 kg of raw peppers, producing a 60 to 70 kg dehydrated product. If more than 900 kg raw chilies are processed, fruits can be damaged or broken, reducing product quality (3).

182-10


D. STORAGE If the product has been properly packed and protected against oxygen humidity and light, it can reach 1 or 2 years shelf life (22). Oxygen presence deteriorates carotenoides present in the skin. Pigment oxidation increases if other extrinsic factors are present such as high storage temperature, light, metal ions, oxygen or peroxidases (28). These factors affect color, aroma and composition, and, consequently, considerably reduce the commercial quality.

FIGURE 182.9 Chipotles (dried and smoked Jalapeño peppers).

The final product quality (chipotle pepper) depends on ripening stage of the harvested raw material as well as drying conditions. 2.

Hot Air Drying

In this method the fruits are dried in hot air tunnels with isolated walls, allowing continuous operations and high production capacity (18). Jalapeño peppers are placed in trays stocked in moving racks, with enough separation to allow drying air to circulate around them. The racks are introduced in the drying tunnel at suitable intervals. When one rack is introduced in the “wet” end of the tunnel, another is removed from the “dry” end. Air is forced by fans though heaters, producing forced convection; this sir is then horizontally fed into the tunnel and trays, although some turbulence is also produced by air circulating between trays. Air is applied at rates between 2.5 to 6 m/s; each rack contains 15 trays with a total of 350 to 400 kg raw pepper, following a 25 m total tunnel length with a rectangular or square 2 ⫻ 2 m transversal section and capacity for 22 moving racks; air counter currently fed. After 8 hours drying, the tunnel is at 55 to 60°C; drying of the first racks fed is achieved after 36 hours; after this time 2 to 3 racks are dried every 4 hours (18,27). Dried fruits should have 6% to 10% moisture content (2,17,23). 3.

IX. CANNED CHIPOTLE PEPPER IN “ADOBO” (SPICY SAUCE) Canning, still as the main food preserving method, is based on the premise of microbial destruction by heat, and on recontamination prevention. With the exception of certain heat-tolerant bacteria, lethality starts at about 46–49°C. In conventional canning, food is placed inside containers, air is removed and cans are hermetically sealed and placed in a retort to be sterilized with steam. The rate at which heat penetrates into the canned product must be measured from the slowest-heating part of the can, or cold point. The basic heat penetration processes are convection and conduction, and a combination of the two (29). The quality of canned fruits and vegetables is affected not only by the heat process but also by the method used to prepare the food material. Such preparation involves washing, trimming, sorting, blanching, filling into containers, and maintenance of the headspace in the can upon vacuum closing. Canned chipotle peppers are of great demand in Mexico; they are consumed directly from the can or used as meat or sauce seasoning. Cans are merchandized in different sizes and formats; chipotle peppers are usually canned in a spicy sauce, or “adobo,” made of other peppers (anchos, or “wide,” and mulattos) tomato, garlic, onion, cumin, oregano, salt, sugar and vinegar (Figure 182.10).

Grading

The dried product is classified into three categories: First quality: Peppers of largest size, uniform color, without any deterioration or breakage Second quality: Peppers of the same size as previous ones, but non-uniform color. Third quality: Broken or damaged peppers, nonuniform color.

C.

PACKAGING

In general, dried peppers are packaged in 55 to 60 kg sacks.

FIGURE 182.10 Adobo (“spicy sauce”) chipotle peppers.


A.

182-11

PRELIMINARY OPERATIONS

4.

Canned chipotle pepper in “adobo” is shown in Figure 182.11. Whole dried chipotle peppers, as well as dried ancho and mulatto are transported to the processing plant in 50 to 60 kg sacks. 1.

Washing

Pepper and other vegetables washing is carried out by immersion in stirred tanks, or by water spraying. 3.

Garlic and onion peeling is carried out applying the flame method already described for non-fermented pickled peppers. Chipotle peppers peduncles are removed; seeds, veins and peduncles are also removed from ancho and mulatto peppers.

Sorting

Dried peppers are sorted in order to eliminate broken peppers, seeds or leaves. Tomatoes, onions, garlic are also sorted to remove any material, unsuitable for processing. 2.

Peeling

Grading

It is manually carried out.

Whole chipotle peppers

5.

Blanching

Tomato blanching is carried out in water during 1 to 2 min at 95°C; blanching and peeling is carried out at the same time. Fast cooling at room temperature improves the efficiency of both operations. Dried pepper scaling softens the disuse, reduces pungency, removes seeds, improves color and decreases microbial loads. Chipotle pepper is scalded in water during 10 to 15 min at 95°C; ancho and mulatto peppers are scalded in water at 95°C for 15 to 20 min. At the industrial level, scalding with steam is generally applied.

Sorting

Washing

Grading

Peeling garlic and onions

Blanching chipotle peppers, ancho peppers, mulato peppers and tomatoes

Packaging in cans

“Adobo” (sauce of ancho pepper, mulato pepper, tomato, garlic, onion, cumin, oregano and vinegar)

Labeling and packaging

Heat treatment

Storage

FIGURE 182.11 Canning of adobo (“spicy sauce”) chipotle peppers.

Exhausting and sealing

182-12

B.


PACKING

Packaging is carried out in 3-piece tin cans with epoxy phenol enamel; lids are made of the same material, fitted with an easy-opening ring and three expansion moldings (Figure 182.12).

C.

ADOBO (“SPICY SAUCE”)

To date, there is no Mexican standard (NMX) for chipotle peppers in adobo. Therefore, adobo formulation varies from one food processor to other, mainly regarding the amount of mulatto pepper sometimes using other pepper (guajillo) as well as amount and type of spices included. Several industries buy the adobo from specialized plants that formulate and dry this sauce.

D. BLANCHING VEGETABLES Whole chipotle peppers, as well as sliced onion must be 60% minimum weight of total net weight, although chipotle peppers must be the dominant vegetable present. The covering medium is adobo sauce.

E.

FILLING

Chipotle peppers and onion slices are placed in previously washed cans; adobo is then added at 82–86°C. Headspace must be 10% total can volume. Filled cans are exhausted, closed, pasteurized, marked, labeled, packed and stored in a similar way as with non-fermented pickled Jalapeño peppers.

FIGURE 182.12 Three-piece easy-opening can.

ACKNOWLEDGMENTS ●

●

The author thanks Dr. Isabel Guerrero Legarreta for the manuscript revision. The information in this chapter has been modified from “Jalapeño pepper preservation by fermentation or pickling,” by R. M. Galicia-Cabrera, in Handbook of Food and Beverage Fermentation Technology, Editors: Y. H. Hui et al., Marcel Dekker, New York, 2004.

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23. Arthey, D. and Dennis, C. Vegetable Processing, Bishopbriggs, Glasgow: Blackie, 1991, pp 163–186. 24. Fuselli, S.R., Echeverria, M. C., Casales, M. R., Fritz, R. and Yeannes, M. L. Selección del proceso óptimo en la elaboración de Ají (Capsicum annum) en vinagre, Alimentaria, 232: 57–61, 1992. 25. Codex Alimentarius Commission. Report of the 21th Session of Codex Commitee on Processed Fruit and Vegetables, 23 to 27 September, 2002, San Antonio, Texas, United States of America. 26. Codex Alimentarius Commission. Draft Codex Standard for Pickled Products. In Report of the 20th Session of Codex Commitee on Processed Fruit and Vegetables, 11 to 15 September, 2000, Washington, D.C., United States of America. 27. Salabarria, A. R. Retos y oportunidades del sistema agroindustrial chile Jalapeño (Capsicum annuum) en el estado de Quintana Roo. Tesis profesional. Universidad Autónoma Chapingo, México City, 1999. 28. Lease, J. G. and Lease, E. J. Effect of drying conditions on inicial color, color retention and pungency of red pepper. Food Technol. 16: 104–106, 1962. 29. Luh, B. S. and Kean, C. E. Canning of vegetables. In: Luh, B.S. and Woodroof, J. G. Commercial vegetable processing. 2nd ed. Westport AVI, 1988, pp 195–206.

183

Sourdough Bread

Åse Hansen

Department of Food Science, Royal Veterinary and Agricultural University

CONTENTS I. Introduction..........................................................................................................................................................183-1 A. Wheat Sourdough ........................................................................................................................................183-2 B. Rye Sourdough ............................................................................................................................................183-2 C. Why Is Sourdough Used?............................................................................................................................183-2 II. Characterization of Sourdough............................................................................................................................183-3 A. Definition of Sourdough ..............................................................................................................................183-3 B. Types of Sourdoughs ..................................................................................................................................183-3 1. Spontaneous Fermentation....................................................................................................................183-3 2. Mature Sourdough ................................................................................................................................183-3 3. Starter Cultures of Pure Strains of LAB ..............................................................................................183-4 C. Sourdough Parameters ................................................................................................................................183-4 III. Microbiology of Sourdough ................................................................................................................................183-4 A. Lactic Acid Bacteria ....................................................................................................................................183-9 1. Identification ........................................................................................................................................183-9 2. Occurrence ............................................................................................................................................183-9 B. Yeast in Sourdoughs ..................................................................................................................................183-11 C. Microbial Interactions................................................................................................................................183-12 IV. Technological Aspects ......................................................................................................................................183-12 A. Production of Sourdough ..........................................................................................................................183-12 B. Flour Type..................................................................................................................................................183-12 1. Extraction Rate....................................................................................................................................183-13 C. Water Content ............................................................................................................................................183-13 D. Temperature ..............................................................................................................................................183-13 E. Amount of Mother Sponge........................................................................................................................183-13 V. Dough Properties and Bread Quality ................................................................................................................183-14 A. Dough Properties and Bread Texture ........................................................................................................183-14 1. Wheat Dough and Bread ....................................................................................................................183-14 2. Rye Dough and Bread ........................................................................................................................183-14 B. Flavor and Taste ........................................................................................................................................183-14 C. Longer Shelf Life ......................................................................................................................................183-15 1. Anti-Mold Activity of Sourdough Bread ............................................................................................183-15 2. Prevention of Rope Spoilage ..............................................................................................................183-15 3. Bread Firmness and Staling Rate........................................................................................................183-16 VI. Nutritional Value................................................................................................................................................183-16 A. Reduced Phytate Content by Sourdough ..................................................................................................183-16 B. Reduced Glycemic Response with Sourdough Bread ..............................................................................183-16 Acknowledgment........................................................................................................................................................183-16 References ..................................................................................................................................................................183-16

I. INTRODUCTION Sourdough is used as an essential ingredient in the production of wheat and rye bread and mixtures hereof.

Sourdough has been used for leavening of bread dough for several hundreds of years, and sourdough bread was made in Egypt as early as 3000 BC [1]. The sourdough was a piece of dough from the previous baking which was kept 183-1

183-2


until the next baking, where it was mixed with flour, salt and water to make the bread dough. The intervals between baking could be from one day in bakeries to one month in home baking. If the time between baking was long, salt could be added to the surface of the sourdough to avoid wrong fermentation. While this piece of dough was saved, lactic acid fermentation took place due to multiplication and metabolic activity of lactic acid bacteria (LAB) originally present in the flour. During this fermentation, selection and multiplication of yeasts from the flour also occurred. The natural content of LAB and yeasts from the sourdough was responsible for the leavening capacity of the bread dough primarily due to their production of carbon dioxide. Yeast from beer or wine production could also be added to the dough to increase the leavening capacity until production of commercial baker’s yeast began during the 19th century [2]. The sourdough still holds a place of honour in many households throughout the world, and small portions are passed on to the daughters at marriage [2].

A. WHEAT SOURDOUGH Sourdough is used as an important ingredient in the production of wheat bread [3,4] as well as crackers and the Italian sweet baked products as Pandoro, Colomba and Panatone [5,6]. The tradition of making wheat bread with the addition of sourdough is widely used in the Mediterranean area such as Italy [5,7], Greece [8,9], Spain [10], Egypt [11] and Morocco [12]. The tradition is also known from The Netherlands [13], Iran [14] and the San Francisco bay in the US [15,16]. The cereal intake in the traditional diet of Greece is mostly in the form of sourdough bread rather than pasta [8]. In Italy, sourdough is used in more than 30% of bakery products, which include more that 200 different types of sourdough bread. In some regions of southern Italy, most of the bread including sourdough bread is made from durum wheat instead of common bread wheat [7]. In Morocco, commercial bakeries supply only part of the population with bread, while most people eat homemade bread made with traditional sourdough, which has been carefully kept in every family. Addition of baker’s yeast is used mainly in towns and villages where refrigeration can be employed [12].

B.

RYE SOURDOUGH

Sourdough is essential in rye bread making and the tradition of rye sourdough fermentation correspond to the rye-growing areas in the north, central and eastern European countries including the Baltic states, where rye bread constitutes a considerable amount of the bread consumption. Rye sourdoughs have been characterised from Finland [17], Sweden [18] and Denmark [19,20], Germany [21–23], Austria [24], Poland [25], Czechoslovakia [26], Russia [27] and Portugal [28].

Bread made from mixed wheat and rye is very common in many European countries, and sourdough should be used to enhance the sensory properties of the bread and prolong the microbial shelf life if more than 20% of the flour is from rye [29]. One of the most famous rye sourdough bread still produced today is the pumpernickel named after the Swiss baker Pumper Nickel. The bread originates from 1443, where there was a significant scarcity of wheat in Europe [2]. The tradition of production of rye bread without the addition of baker’s yeast has continued even in large-scale bakeries until today, and the leavening capacity of the sourdough is still very important in rye bread production. In the sixties and seventies, the time between baking and consumption of bread increased due to changes in the society, and in some bakeries, preservative compounds such as vinegar, propionic acid or sorbic acid were added to the dough for the prevention of moulds. However, the natural content of yeasts from the sourdough is also inhibited by those preservatives, resulting in decreased leavening capacity, and it was necessary to add baker’s yeast to increase the bread volume. The use of propionic acid as a preservative in bread is prohibited in many countries today. Stringent hygiene in bakeries makes it possible to produce bread with long shelf life without added preservatives, if sourdough is added.

C. WHY IS SOURDOUGH USED? The advantages of using sourdough for bread production include the possibility of leavening bread dough with little or no baker’s yeast added, improved dough properties, and the achievement of a better and more aromatic bread flavor and texture compared to bread only leavened by bakers yeast (Table 183.1). Sourdough flavor is developed by a long fermentation process that requires 12–24 hours, while fermentation by baker’s yeast has to be finished within 1–2 hours. The addition of sourdough can also extend the shelf-life of bread by several days by increasing the mold-free period of bread and retarding the development of rope. The nutritional value of sourdough bread made from high extraction flour is enhanced TABLE 183.1 The Advantages of Using Sourdough in Bread Making Leavening of dough Improved dough properties - inhibition of α-amylase Increased flavor and taste of bread Improved nutritional value of sourdough bread - higher bioavailability of minerals - lower glycemic index Extended shelf life of sourdough bread - longer mold-free period - prevention of rope in bread - antistaling

Sourdough Bread

183-3

compared to bread made without sourdough due to a higher content of free minerals, which are separated from phytic acid during the long fermentation processes. Interest in using sourdough in bread production has increased considerably in many European countries during recent decades [4,30,31]. Today, a larger part of the consumers prefer healthy bread with aromatic taste, good texture, and long shelf-life without the addition of artificial preservatives. The demand for organic food is also on the rise, and a larger part of the bread made from organically grown cereals is made with sourdough due to its higher quality and better image. More consumers are also interested in food with history, and sourdough bread is related to traditional and original food.

II. CHARACTERIZATION OF SOURDOUGH High quality sourdough bread is dependent on a consistent and microbial stable sourdough. Good fermentation capacity of the sourdough is influenced by the microbial flora (lactic acid bacteria and yeasts) in the sourdough, flour type (wheat/rye, flour extraction rate, activity of enzymes), flour/water ratio (dough yield), and the process parameters. The process parameters such as temperature, initial pH, quantity of added sourdough starter, time of fermentation, and type of production system (batch/continuous) have to be strictly controlled.

A. DEFINITION

OF

SOURDOUGH

Sourdough is a mixture of flour and water, in which LAB have caused a lactic acid fermentation to occur. It is in general accepted that the LAB should still be able to produce acids, when flour and water are added (metabolically active). The sourdough also has a natural content of sourdough yeasts, which are important for the leavening capacity of the dough. However, no official definition of sourdough exists, but it should include all different types of sourdough products with “living” LAB and exclude artificial sourdough products. According to Lönner [32], a sourdough should contain more than 5 ⫻ 108 metabolically active LAB/g, and have a pH value below 4.5.

B. TYPES

OF

tendency to use defined starter cultures with specific fermentation patterns. This tendency increases as these cultures become commercially available. 1. Spontaneous Fermentation When dough made from flour and water is left for one to two days at ambient temperature, a spontaneous fermentation will take place due to the naturally occurring microorganisms in the flour. The dough will become acidified due to lactic acid fermentation. During the fermentation there is a successive favoring of the Gram-positive LAB from the flour at the expense of the Gram-negative bacteria, which dominate the microflora of the flour [21,32]. The microflora of some spontaneously fermented rye sourdoughs were dominated by a homofermentative Lactobacillus spp. and Pediococcus spp. [32,33]. The level of LAB in sourdoughs was up to 3 ⫻ 109 colony forming units (CFU)/g and the number of yeasts about 106 to 107 CFU/g. However, spontaneous sourdoughs do not always succeed and may result in products with off-flavor. 2. Mature Sourdough Sourdoughs used by artisan bakers and in bakeries have traditionally been based on spontaneous fermentation, during which the sourdough has been kept metabolically active and probably microbial stable for decades by the addition of flour and water daily, the so called “freshening” of the dough based on “back-sloping” (Figure 183.1). The fermented sourdough is used for bread production, but part of it is used as starter by initiating a new sourdough. The terminology for sourdough and starter in different countries is listed in Table 183.2. In commercial rye bread baking, the bakeries can use their own adapted sourdough or, if they have quality Sourdough preparation Flour, water + Mother sponge

a) during spontaneous fermentation b) by adding a piece of mature sourdough (mother sponge) c) by adding a defined starter culture Most sourdoughs used in both wheat and rye bread baking are still initiated by adding a piece of mature or ripe sourdough also called mother sponge, but there is a

10−20 hours Sourdough

SOURDOUGHS

Sourdoughs can be started as follows:

Unfermented sourdough

Mixing

Flour, water, salt

Bread dough

Resting, proofing, baking Bread

FIGURE 183.1 A schedule for production of sourdough.

183-4


TABLE 183.2 Terminology for Sourdough in Different Countries Sourdough for bread production Sourdough used as starter for a new sourdough

English

German

French

Spanish

Italian

Sourdough Leaven Mother sponge Starter

Sauerteig

Levain natural

Anstellgut Reinzuchtsauer

Le chef

Masa madre (Masa agria) Pie

Lievito naturale (impasto acido) Madre, Capolievieto

Modified after [5].

problems due to unstable process control, they can add a commercial sourdough as a starter. Most bakeries in Germany and Denmark regularly add commercial sourdoughs composed of a well-adapted microflora derived from natural sourdough fermentation. Examples of commercial sourdoughs are the Sanfrancisco sour for wheat bread production [34] and the Böcker-Reinzucht-Sauer® for rye bread production. Some products sold as sourdough have no living microorganisms, and these products will not contribute to a natural acidification and development of flavor compounds in the dough. 3. Starter Cultures of Pure Strains of LAB Starter cultures for sourdough fermentation are pure cultures of dried or freeze-dried LAB, or a mixture of LAB and sourdough yeast. They should be mixed with flour and water, and kept for several hours for multiplication and fermentation of the microflora. This fermented dough can then be used as a sourdough. The microorganisms have been selected due to their ability to acidify dough in a short time and result in acceptable bread flavor when used in bread baking. Cultures containing Lactobacillus sanfranciscensis, L. plantarum, L. brevis and L. fructivorans or L. brevis, L. pontis and Saccharomyces cervisiae are available [35]. Use of defined starter cultures with specific properties gives rise to new interesting opportunities for controlling and regulating of the sourdough fermentation. The term “starter culture” is sometimes used in the literature for a mature sourdough which has to be mixed with flour and water to ferment, or for commercial sourdoughs.

C. SOURDOUGH PARAMETERS A sourdough can be characterized by the chemical parameters pH, content of total titratable acids (TTA), content of lactic and acetic acid, and the microbial parameters such as number and species of LAB and yeast. The microbial parameters are described in the following section. The final pH of a mature sourdough is 3.5–3.8 in most rye and wheat sourdoughs [3,36,37]. Sourdough pH values show less variation and differences than TTA values. The TTA values in sourdoughs are dependent on the fermentation temperature, extraction rate of the flour, and the water content. In wheat sourdoughs, TTA has been found

to vary between 8 and 11 in sourdoughs made from low extraction flour and 16 to 22 in wholemeal sourdoughs [3,38]. Rye sourdoughs are often made from flours with higher extraction rate than wheat flour and TTA values vary between 15 and 26 [21,36]. The content of lactic and acetic acid in sourdoughs is very important for the taste and flavor of the sourdough bread [31,39]. The fermentation quotient (FQ), the molar ratio between lactic and acetic acid, is used as a measure in German studies of sourdoughs for the balance in production of those acids. The FQ should be around 4 in sourdough to result in a harmonic taste of bread. A low content of acetic acid results in a high FQ with a too little flavor, whereas a low FQ results in too strong an acid flavor [21]. However, acetic acid has a more efficient antimicrobial effect against mold- and rope-producing bacteria compared to lactic acid [40].

III. MICROBIOLOGY OF SOURDOUGH The microflora of the sourdoughs includes adapted LAB and yeasts that have optimal conditions for growth and fermentation similar to the conditions for the sourdough (temperature, water content, pH), and which probably produce antimicrobial compounds [41]. The microflora in bakery sourdoughs remains remarkably stable in spite of the use of non-aseptic fermentation conditions [20,23,34,42]. The LAB and yeasts isolated and identified from wheat and rye sourdoughs are listed in Tables 183.3 and 183.4, respectively. Early systematic studies of the microflora responsible for the sourdough fermentation were made on sourdoughs from Germany by Hollinger in 1902, from Russia by Seliber in 1939 (cited in [43]), and from Denmark by Knudsen in 1924 [19]. Spicher and co-workers have carried out many profound investigations concerning the identification of the microflora from different types of sourdoughs, both adapted sourdoughs from bakeries and in commercial starter cultures for sourdough [4,21]. Recent investigations on sourdough fermentation have mainly dealt with interactions between sourdough microorganisms, identification of new species, inhibitory substances of sourdough LAB, and induced specific enzymatic activities. LAB are mainly responsible for the acidification of the sourdough, whereas the sourdough yeasts are very

Streptobacterium plantarum (L. plantarum) Str. plantarum L. delbrueckii L. plantarum L. leichmanii L. alimentarius L. plantarum L. acidophilus L. casei L. farciminis

Russia

L. acidophilus L. plantarum

Finland

Sweden

L. acidophilus L. plantarum

L. plantarum (firm) L. plantarum (liquid) L. leichmanii L. casei var. casei L. delbruckii (48–52°C)

Russia

Finland

L. alimentarius L. casei L. rhamnosus

Austria

Germany

Germany

Czechoslovakia Germany

Homoferm.

Country

Lactic Acid Bacteria

L. brevis L. brevis ssp. lindneri

L. büchnerii L. cellobiosus L. viridescens

L. brevis ssp. lindneri L. büchnerii L. fermentum L. fructivorans L. brevis L. fermenti L. brevis L. büchnerii

L. brevis L. brevis ssp. lindneri L. plantarum L. fermentum L. fructivorans

L. brevis L. fermenti non-identified species L. brevis L. fermenti

Heteroferm.

TABLE 183.3 Lactic Acid Bacteria and Yeasts Isolated from Rye Sourdoughs

Torulopsis holmii S. cerevisiae T. unisporus T. stellata Endomycopsis fibuliger Hansenula anomala

S. minor/S. exiguus S. minor S. exiguus

S. cerevisiae Pichia saitoi T. holmii S. cerevisiae C. krusei

C. krusei

S. cerevisiae

Non-identified species

Yeasts

Spicher, Lönner

Salovaara, Savolainen

Salovaara, Katunpäa

Kazanskaya, Afanasyeva, Patt

Foramitti, Mar

Spicher, Schröder, Schoellhammer

Spicher, Schröder

Pokorny Spicher

Seliber

Authors

1985

1984

1984

1983

1982

1979

1978

1955 1959

1939

Year

(Continued)

[18]

[57]

[17]

[27]

[24]

[120]

[119]

Cit. from [43] [43]

Cit. from [43]

Reference

Sourdough Bread 183-5

L., Lactobacillus; Str., Streptobacterium; S., Saccharomyces, C., Candida; T., Torulopsis.

Germany

L. frumenti sp. nov. L. panis

Germany Denmark

L. sanfrancisco L. fermentum L. fructivorans L. pontis sp. nov.

L. sanfrancisco Non-identified species closely related to L. fermentum and L. reuteri L. brevis

L. panis sp. nov.

L. acidophilus (liquid) L. amylovorus L. mindensis sp. nov.

L. fermentum L. viridescens

L. farciminis L. casei rhamnosus L. delbruckii (53°C)

L. brevis L. sanfrancisco L. curvatus

Heteroferm.


Homoferm.

(Continued)

Germany Finland

Portugal (rye and maize)

Germany

Germany

Germany

Poland

Country

TABLE 183.3

S. cerevisiae

C. milleri S. cerevisiae S. exiguus

S. cerevisiae Torulaspora delbrueckii Issatchenkia orientalis Pichia anomala P. membranaefaciens

S. cerevisiae

S. cerevisiae S. exiguus T. candida C. krusei

Yeasts

Ehrmann et al.

Müller, Ehrmann, Vogel Rosenquist, Hansen

Wiese et al. Mäntynen et al.

Almeida, Pais

2003

2000 2000

1996 1999

1996

1994

1992

1992

Okada, Ishikawa, Yoshida, Uchimura, Ohara, Kozaki

Strohmar, Diekmann Vogel, Böcker, Stolz, Ehrmann, Fanta, Ludwig, Pot, Kersters, Schleifer, Hammes

1990

1986

Year

Böcker, Hammes

Wlodarczyk

Authors

[53]

[52] [20]

[50] [55]

[58]

[49]

[42,123]

[122]

[121]

[25]

Reference


bread

Pannettone, bread

Wheat bread

Wheat bread

Swiss panettone/cake Swiss bread Panettone, brioches, wheat bread, crackers

L. farciminis

L. plantarum L. delbrueckii Lactococcus casei L. plantarum

Pediococcus L. plantarum Lc. mesenteroides

L. plantarum

L. plantarum L. farciminis L. casei

Wheat bread

L. brevis var. lindneri

L. brevis

L. sanfrancisco L. fermentum Lc. mesenteroides L. brevis L. cellobiosus

L. brevis var. lindneri L. sanfrancisco L. brevis var. lindneri L. brevis var. lindneri L. brevis var. lindneri

S. exiguus C. krusei S. cerevisiae S. exiguus C. krusei Pichia norvegensis

S. cerevisiae

S. cerevisiae C. stellata C. milleri S. cerevisiae C. boidinii C. guilliermondii Rhodutorula glutinis Pichia polymorpha Tricocporon margaritiferum C. milleri S. cerevisiae

S. exiguus

S. exiguus

L. plantarum

Wheat bread

Wheat bread

T. candida

Lc. mesenteroides L. brevis L. brevis

T. collucolasa

L. brevis

L. plantarum

Balady bread Sangak bread

S. exiguus S. inusitas

Yeasts

L. sanfrancisco

Heteroferm.


Homoferm.

San Francisco Bread

Product

TABLE 183.4 Lactic Acid Bacteria and Yeasts Isolated from Wheat Sourdoughs

Rossi

Gobbetti, Corsetti, Rossi, Rosa, Vincenzi-S-de

Boraam et al.

Barber, Baguena

Galli et al.

Spicher Spicher

Nout, Creemer-Molenaar

Spicher

Spicher, Lönner

Kline, Sugihara, McCready Sugihara et al. Abd-el-Malek, El-Leithy, Awad Azar, Ter Sarkissian, Ghavifek, Ferguson, Ghassemi

Authors

1996

1994

1993

1988

1988

1987 1987

1987

1987

1985

1977

1971 1971 1974

Year

(Continued)

[59]

[127]

[126]

[10]

[125]

[124] [124]

[13]

[124]

[18]

[14]

[15] [34] [11]

Reference

Sourdough Bread 183-7

L. sanfranciscensis L. brevis Lc. citreum L. fermentum L. sanfranciscensis

L. brevis

L. alimentarius

L. plantarum

L. paralimentarius

Weissella cibaria

L. delbrueckii

L. sanfranciscensis L. brevis L. sanfranciscensis

L. brevis

L., Lactobacillus; S., Saccharomyces, C., Candida; T., Torulopsis.

Wheat bread

Bread: durum wheat and bread wheat

Cakes: panettone, colomba, brioche

Wheat bread

L. delbrueckii L. curvatus L. plantarum L. lactis spp. Lactis L. paralimentarius sp. nov

Maize bread

Heteroferm.


Homoferm.

(Continued)

Product

TABLE 183.4

S. cerevisiae

S. cerevisiae

C. holmii

Rocha and Malcata

Hansenula anomala S. cerevisiae

De Vuyst, Schrijvers, Paramithiotis, Hoste, Vancanneyt, Swings, Kalantzopoulos, Tsakalidou, Messens

Corsetti, Lavermicocca, Morea, Baruzzi, Tosti, Gobbetti

Foschino, Terraneo, Mora, Galli

Cai, Okada, Mori, Benno, Nakase

Authors

Yeasts

2003

2001

1999

1999

1999

Year

[9]

[7]

[60]

[51]

[28]

Reference


Sourdough Bread

important for the production of flavor compounds and for a harmonic bread flavor in combination with the acids. The levels of LAB in sourdoughs are 108–109 cfu/g and yeasts are 106–107 cfu/g, respectively [4]. The LAB: yeast ratio in sourdoughs is generally 100:1.

A. LACTIC ACID BACTERIA 1. Identification LAB are a group of Gram-positive bacteria, which are catalase-negative, non-motile nonspore-forming rods or cocci which produce lactic acid as the major end product during the fermentation of carbohydrates. They are strictly fermentative, aero-tolerant or micro-aerofile, acidophilic, salt-tolerant and have complex nutritional requirements for carbohydrates, amino acids, peptides, fatty acids, salts, nucleic acids derivates and vitamins [44,45]. The LAB have traditionally been classified taxonomically into different genera based on colony and cell morphology, sugar fermentation, growth at different temperatures, configuration of lactic acid produced, ability to grow at high salt concentration, acid tolerance or cell wall analyses [46]. Genera of LAB identified from sourdoughs are Lactobacillus, Leuconostoc, Pediococcus and Streptococcus, and the majority of the sourdough LAB belongs to the genus Lactobacillus. The taxonomy of LAB is still under revision. Lactobacillus have been divided into three groups according to their carbohydrate fermentation patterns [46]: ●

●

●

Obligately homofermentative LAB: Hexoses are almost completely fermented to lactic acid (⬎85%) by the Embden-Meyerhof-Parnas (EMP) pathway. Fructose is also fermented, but neither gluconate nor pentoses are fermented. Facultatively heterofermentative LAB: Hexoses are almost completely fermented to lactic acid by the EMP pathway. Pentoses are fermented to lactic acid and acetic acid by an inducible phosphoketolase. Obligately heterofermentative LAB: Hexoses are fermented to lactic acid, acetic acid (ethanol) and CO2. Pentoses are fermented to lactic and acetic acid. In general, both pathways involve phosphoketolase.

Lactobacillus isolated from sourdoughs are divided into the three groups shown in Table 183.5. When the LAB are only divided as homofermentative or heterofermentative LAB, the facultative heterofermentative LAB are grouped as homofermentative due to the fermentation pathway of glucose (Tables 183.3 and 183.4). In the presence of oxygen or other oxidants increased amounts of acetate may be produced at the expense of lactate or ethanol [45]. Various compounds such as

183-9

citrate, malate, tartrate, quinolate and nitrate may also be metabolized and used as energy sources or electron acceptors [44,45]. The techniques used for classification of LAB are not reliable for many LAB, and they have often led to misidentification [47]. Some organisms badly grow on laboratory media and may escape isolation and can therefore not be identified by standard procedures. For rapid classification, a set of molecular probes was developed. These include hybridization- and PCR based techniques as well as recognizing specific sequences in the ribosomal genes [47]. Nevertheless, the use of probes at the species level is restricted, as their specificity may be lost during discovery of new species sharing the same part of an RNA sequence [48]. Alternatively, the taxonomic method Random Amplified Polymorphic DNA (RAPD) allows elucidation of strain biodiversity below the species level, and the resulting electrophoretic patterns can be clustered and compared to a database [48]. The consequent application of 16S rRNA sequence analysis and DNA-DNA hybridization experiments have led to identification of many new species. L. pontis [49] and L. panis [50] were isolated from rye sourdoughs, and L. paralimentarius [51] was isolated from wheat sourdough. Recently described species isolated from sourdoughs are L. frumenti [52] and L. mindensis sp. nov [53]. 2. Occurrence Heterofermentative LAB play a major role in sourdough fermentation compared to other fermented food systems. L. sanfranciscensis (former names L. brevis var. lindneri and L. sanfrancisco) is by far the most dominant LAB in both wheat and rye sourdoughs (Tables 183.3 and 183.4). L. brevis and L. plantarum also occur frequently in both types of sourdoughs. Some strains initially classified as L. brevis were renamed as L. pontis [49]. Several other Lactobacilli have been identified from rye sourdoughs e.g., the homofermentative L. acidophilus, L. alimentarius, L. amylovorus, L. casei, L. delbrueckii, L. farciminis, L. leichmanii, L. rhamnosus, and recently L. mindensis, and the heterofermentative L. büchnerii, L. cellobiosus, L. curvatus, L. fructivorans, L. fermentum, L. viridescens including the new identified species L. panis, L. frumenti and L. pontis. Strains of L. pontis utilize only a very limited number of carbohydrates and they are found in close association with L. sanfranciscensis, from which they are difficult to separate physically. Fewer different Lactobacilli have been identified from wheat sourdoughs, such as L. alimentarius, L. casei, L. cellobiosus, L. curvatus L. delbrueckii, L. farciminis, L. fermentum L. lactis and the recently identified L. paralimentarius (Table 183.4). However, the homofermentative Pediococcus and Weissella and the heterofermentative Leuconostoc have also been isolated from wheat sourdoughs.

when fermented; b FDP fructose-1,6-diphosphate; c inducible by pentoses.

Former names of some of the bacteria: d L. brevis spp. lindneri and L. sanfrancisco.

L. alimentarius L. casei L. curvatus L. paralimentarius L. plantarum L. rhamnosus

⫹ ⫺ ⫹ ⫺ ⫹a ⫹ ⫹c

⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ L. acidophilus L. amylovorus L. delbrueckii spp. bulgaricus L. delbrueckii spp. delbrueckii L. farciminis L. helviticus L. leichmanni L. mindensis

Facultatively Heterofermentative

Obligately Homofermentative

The table is modified after Kandler and Weiss,1986 [46].

a

Lactobacillus

Growth at 15°C 45°C Pentose fermentation CO2 from glucose CO2 from gluconate FDP b aldolase present Phosphoketolase present

Characteristics

TABLE 183.5 Groups of Lactobacillus Isolated from Sourdoughs

L. brevis L. buchneri L. fermentum L. fructivorans L. frumenti L. panis L. pontis L. reuteri L. sanfranciscensis d L. viridescens

⫹/⫺ ⫹/⫺ ⫹ ⫹ ⫹a ⫺ ⫹

Obligately Heterofermentative


Sourdough Bread

The variation in the composition of the microflora depends on the fermentation conditions such as flour type, extraction rate, water content, fermentation temperature, fermentations time and how the sourdough is refreshed. Most sourdoughs are fermented about 30°C, but L. delbrueckii has been isolated from rye sourdoughs with a fermentation temperature above 50°C [18,27]. Hammes, Stolz, and Gänzle [35] found that the most predominant LAB in firm sourdoughs with fermentation temperature between 23 and 30°C are L. sanfranciscensis and L. pontis. However, L. fructivorans, L. fermentum and L. brevis were also identified from this type of sourdough. Some industrial sourdoughs are characterized by high water content to fluid conditions (suitable for pumping), elevated fermentation temperature (⬎30°C), and shorter fermentation time (15–20 hours). Fluid sourdoughs can be produced in large volumes — often by continuous fermentation systems, and they can be cooled for storing in silos up to one week. The microflora in this type of sourdough is dominated by L. panis [50], L. reuteri, L. sonfranciscensis and L. pontis [35]. The development of sourdough yeast is poor in fluid sourdoughs and consequently it is necessary to add baker’s yeast is to the bread dough. Sourdoughs kept at ambient temperature will continue acidification. The LAB are sensitive to low pH in longer time and the LAB will thus die off. Therefore dried sourdough preparations are preferred for commercial sourdough samples. However, LAB are rather sensitive to preservation by drying, and LAB present in commercial sourdoughs must survive drying. L. plantarum, L. brevis, Pediococcus pentosaceus have been identified from dried commercial sourdough preparations, and dried starter cultures containing strains of L. sanfranciscensis have only recently become commercially available [35].

B. YEAST

IN

SOURDOUGHS

Several species of yeasts have been isolated from bakery and commercial sourdoughs. However, the taxonomy of yeasts has been gradually changed since the 1970s, and various synonyms have been used (Table 183.6). The traditional systematization and identification of yeasts have been based on biochemical tests as well as morphological and physiological criteria [54], but imperfect fungi cannot be studied using traditional genetics. New molecular characteristics have defined and changed the taxonomy of yeasts [55]. The physiological features of industrial yeasts have been shown to alter when changes occur in growth conditions, and species of Saccharomyces cerevisiae, S. exiguous and Torulopsis delbrueckii have been found to intermix genetically with each other [55]. The most frequently isolated yeast species from rye and wheat sourdoughs are S. cerevisiae (Tables 183.3 and 183.4). Other yeast species often isolated from sourdoughs are S. exiguus, Candida milleri (C. holmii), C. krusei

183-11

TABLE 183.6 Yeasts Isolated from Sourdoughs and Their Synonyms Perfect Fungi

Imperfect Fungi

Saccharomyces cerevisiae S. exiguus

Candida holmii

Candida milleri S. delbrueckii S. uvarum Issatchenkia orientalis

C. krusei

Pichia anomala

C. peliculosa

P. membrafaciens P. norvegensis P. polymorpha P. satoi Endomycopsis fibuligera

C. valida

Synonyms

Torulopsis holmii Torula holmii S. rosei Torulopsis holmii Torulaspora delbrueckii S. inusitatus S. krusei Endomyces krusei Hansenula anomala

S. fibuliger

C., Candida; P., Pichia; S., Saccharomyces. The table is modified after Kurtzman and Fell [128] and Barnett, Payne and Yarrow [129].

(Issatchenkia orientalis). The yeast species Pichia saitoi, P. norvegensis and Hansenula anomala and some Saccharomyces spp. have occasionally been isolated from sourdoughs. Candida spp. are members of Deuteromycetes (fungi imperfect), because they have lost their ability to undergo sexual development. C. milleri is a non-sporulating form of S. exiguous and was first described by Yarrow in 1978 [56]. C. milleri is physiological similar to C. holmii, but is different according to DNA identification. Some strains identified as Torulopsis holmii in the literature before 1978 have subsequently been assigned to C. milleri [55]. T. holmii and S. cerevisiae were the dominating yeasts in bakery rye sourdoughs from Finland, whereas S. cerevisiae dominated in rye sourdoughs used for home baking [57]. A later study showed that the yeasts isolated from rye bakery sourdoughs in Finland were similar to C. milleri [55]. Wlodarczyk [25] found that S. cerevisiae accounted for 99% of all yeasts found in starters from three industrial rye bread bakeries in Poland, whereas the sourdoughs from the smaller bakeries contained a wider range of yeast strains. Traditional Portuguese sourdoughs prepared from maize and rye were dominated by S. cerevisiae and Torulaspora delbrueckii [58], and S. cerevisiae and C. pelliculosa [28], respectively. S. cerevisiae was also the

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dominating yeast in wheat sourdoughs from Italy, followed by S. exiguous, C. krusei, P. norvegensis and Hansenula anomala [59]. Addition of baker’s yeast is widely used in some Italian wheat sourdoughs [7], whereas many sourdoughs are also prepared without sourdoughs [60]. The yeasts present in sourdoughs are generally acidtolerant. Strains of baker’s yeast S. cerevisiae have poor tolerance of acetic acid in sourdoughs [61], whereas strains of S. cerevisiae isolated from sourdoughs can grow on MYGP broth acidified with acetic acid to pH 3.5 [37].

C. MICROBIAL INTERACTIONS The high stability of sourdoughs used for a longer period might be caused by production of inhibitory substances [41], but also microbial interaction between the LAB and the sourdough yeasts are of importance. Several sourdough LAB produce inhibitory substances against spoiling microorganisms. These compounds are organic acids in particular acetic acid, carbon dioxide, ethanol, hydrogen peroxide, and diacetyl [62]. The inhibition, however, can also be caused by bacteriocins that are low moleculemass peptides, or proteins, with a bactericidal or bacteriostatic mode of action, in particular against closely related species [41]. Microbial interaction was demonstrated early for the Sanfrancisco sourdough. The sourdough yeast T. holmii (C. millery) does not assimilate maltose [34,63], whereas L. sanfrancisco hydrolyses maltose and excretes one of the glucose molecules to be used for the sourdough yeast [64]. The glucose uptake of the yeast cell can induce an outflow of amino acids, and this liberation of amino acids has made growth of L. sanfranciscensis possible even in a medium initially deficient in essential amino acids [65]. Several LAB increase the acidification of sourdoughs when the sourdough are added to the sourdough yeasts T. holmii or S. cerevisiae [63,66]. However, LAB might also multiply more slowly and decrease the production of acids in mixtures with yeasts [67]. A real risk of bacteriophage contamination of sourdoughs exists as bacteriophages with activity against L. fermentum have been isolated from an Italian sourdough [68].

IV. TECHNOLOGICAL ASPECTS A. PRODUCTION

OF

SOURDOUGH

Sourdough can be made with variations in the following parameters: flour type — wheat/rye, flour extraction rate, flour/water ratio, temperature, time and amount of starter. Sourdough can also be made in one to three steps. The one-stage process is the basic way to make a sourdough and is widely used. Two- and three-step sourdoughs have traditionally been used in rye bread production in many

German bakeries [21]. Industrialization in bakeries has also included the sourdough production, where the time consuming multiple-stage processes have changed to the work-saving one-stage process. Traditional rye sourdoughs have often been based on firm sourdoughs, but in automated large scale bakeries firm sourdoughs are difficult to handle, and they have been replaced by pumpable semi-fluid to fluid sourdoughs which are suitable for automated fermentation systems. Today, continuous fermentation plants are used in many bakeries in Europe. The following deals with how sourdough fermentation can be influenced by the flour type, flour extraction rate, fermentation temperature, water content in sourdough, and by the amount of added ripe sourdough.

B. FLOUR TYPE The flour in the sourdough is the substrate for the fermenting microorganisms. Wheat and rye flour are mostly used for sourdough making, but maize flour can also be used [28,69]. The amount of fermentable carbohydrates in the flour varies with the type of cereal, but in particular with the activity of endogenous enzymes in the flour. The activities of amylases, xylanases and peptidases are important for liberation of the fermentable low molecular weight carbohydrates and amino acids. On dough stage, the αamylase can not degrade intact starch granules, but some granules are damaged during the milling process and may be partly degraded in the dough. Starch is generally not degraded by LAB, and the content of fermentable mono- and disaccharides in rye flour is up to 5% with maltose (3%) as the main part [70]. Savola found that this content of free sugars decreased by 3% during sourdough fermentation. However, Henry and Saini [71] found only small amounts of low molecular weight sugars in rye (0.7% sucrose and ⬍ 0.1% of glucose, fructose, raffinose and stachylose). The content of pentosans (arabinoxylans) in rye flour is high (6.5 to 12.2%) [72] compared to wheat flour (2–3%) [73], and they can be degraded to the pentoses xylose and arabinose by the corresponding enzymes during the bread making processes [74]. The content of fermentable carbohydrates in wheat flour is 1–2% [67,75]. The content of maltose increased during the sourdough fermentation from 1.5 to 2.4%, and the content of fructose from 0.05 to 0.45% in a sourdough fermented with Lc. mesenteroides [75]. The content of glucose was unchanged at the level of 0.17% as a result of a balance between bacterial consumption and hydrolysis by the enzymatic activity. No sucrose was detected in the samples, so the increase in fructose could not be caused by yeast invertase. Most Lactobacillus isolated from sourdoughs are nonamylolytic, but amylolytic strains have been isolated from African fermented cereal products made from maize such as ogi, mawé and kunu-zakki [76].

Sourdough Bread

1. Extraction Rate The extraction rate of the flour is one of the most important factors for determining the character of sourdough [77,78]. With a high extraction rate (80–100%), the content of nutrients such as B-vitamins and minerals increases compared to low extraction rate flour (65–75%), as does the buffering capacity of the flour primarily due to the phytic acid from the aleurone layer of the cereals. These factors can stimulate the growth and biochemical activity of the microflora in the sourdough followed by a higher production of acids and flavor compounds. Rye flours have a generally higher extraction rate than wheat flours. A linear relationship between ash content and TTA was found in wheat sourdough. The final TTA in sourdoughs made from wholemeal flour (ash 1.5%) was almost double the value compared to sourdoughs made from straight-grade flour (ash 0.55%), and the final pH was reached in less time in sourdoughs made from the low extraction flours [3].

C. WATER CONTENT The water content in the sourdough determines the firmness of sourdoughs, and it can be expressed as the dough yield (DY), which is the amount of sourdough per kg per 100 kg flour. DY varies from 150 in firm sourdoughs to 300 in fluid sourdoughs. The development in TTA is lower in fluid rye sourdoughs compared to firm sourdoughs, but if the acidity is measured per gram dry matter it will be lower in firm sourdoughs [21,36]. This indicates that the nutrients are better used by the LAB in fluid sourdoughs compared to firm sourdoughs. The production of lactic acid is not influenced by the DY, whereas the production of acetic acid is generally lower in fluid sourdoughs [21,36]. The water content in sourdoughs influences the acidification of the dough more than the temperature [79]. The content of LAB was not influenced by the firmness of rye sourdoughs, whereas the yeast propagation was low in the firm sourdoughs with levels below103 cfu/g in six of the seven sourdoughs [36]. However, a surface layer of yeast cells was seen on the firm sourdough fermented with L. plantarum.

D. TEMPERATURE The temperature of the sourdough is influenced by the temperature of the flour, the water and the mother sponge, and it is often adjusted and regulated by the water temperature. In practice, the temperature increases 6 to 8°C during industrial fermentation, if the temperature is not thermostatically regulated. So it is important that the temperature of the water is not too high. The temperature of the sourdough greatly influences the microbial propagation and production of acids, as the optimal temperature for growth and acidification varies for the different species

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of LAB. Spicher [80] found that the lowest generation time was 20 min for L. brevis at 35°C and L. plantarum at 40°C, and 60 min for L. fructivorans at 30°C and L. fermentum at 40°C. Changes in the fermentation temperature from the optimal conditions increased the generation time considerably, and the generation time for L. fermentum was prolonged to 120 min at 40°C and 140 min at 25°C. The optimum temperature for growth of the LAB is close to the optimal temperature for acid production, and most LAB have temperature optima between 30 and 35°C [21]. In general, the final pH is reached more quickly at higher temperatures (30–35°C) compared to lower temperatures (20–25°C) [21,79,81]. Some species, mostly heterofermentative, can grow below 15°C, such as L. farciminis, L. plantarum, L. rhamnosus, L. brevis, L. fructivorans, Lb. sanfranciscensi. The highest temperature for growth is between 45 and 55°C and most species which can tolerate high temperatures are homofermentative, such as L. acidophilus, L. amylovorus, L. delbrueckii. However, also the heterofermentative species of L. pontis, L. rhamnosus, L. fermentum and L. reuteri can grow above 45°C [45]. The optimum temperature for growth of sourdough yeasts has not been intensively investigated, but it seems to be lower than for the LAB. The optimum temperature for growth of C. milleri was determined to be 27°C [44], while C. milleri and S. exiguous do not grow at temperatures above 35°C [56]. The minimum temperatures for growth of LAB and yeast are important when sourdoughs are stored by cooling, as the sourdough should not develop during the storage. The minimum temperature for growth of most sourdough yeasts has been found to be 8°C [55]. The content of acids produced in sourdough increases with increased fermentation temperature due to higher production of lactic acid, whereas the production of acetic acid is only negligible influenced by the temperature [21,81]. This confirms the general rule that the relative content of acetic acid is higher in cold sourdoughs compared to warmer sourdoughs [21]. Investigation of the influence of the fermentation temperature on the production of flavor compounds in rye sourdoughs showed that the starter cultures themselves produced few volatile compounds, whereas the production of iso-alcohols and ethyl acetate increased considerably with higher temperature in sourdoughs fermented with homofermentative LAB due to activity by the propagating yeasts [81].

E. AMOUNT

OF

MOTHER SPONGE

The amount of mother sponge to be mixed with flour and water for a new sourdough should be so high that the content of LAB in the sourdough is able quickly to decrease the pH to inhibit the growth of the gram-negative bacteria in the flour. The amount of mother sponge influences the pH-lowering capacity in a sourdough, as low pH is

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reached more quickly when the amount of added mother sponge is high [79]. However, higher levels of acids are produced when a lower amount of mother sponge is added, as the fermentation time is longer before the pH drops to the critical pH level [20]. The recommended amount of mother sponge is generally 10–20% for both rye and wheat sourdoughs [21,79]. The Sanfrancisco sourdough is rebuilt every eight hours or at least two to three times a day, seven days a week. The amount of mother sponge used in preparing a new sourdough is 25–40% of the sourdough [15]. This high amount of mother sponge makes the sourdough very stable, and this sourdough has been continued for more than a century.

V. DOUGH PROPERTIES AND BREAD QUALITY A. DOUGH PROPERTIES AND BREAD TEXTURE 1. Wheat Dough and Bread Incorporation of sourdoughs in wheat bread making influences the gluten proteins and the viscoelastic behavior of doughs due to the drop in pH value caused by the organic acids produced. Several investigations have shown that the addition of acid to wheat dough decreased the dough stability during mixing, and the acidified doughs became considerably softer than a non-acidified control dough [82–84]. Dough stability was also decreased when it was prepared with the addition of sourdough [85] [86]. The dough consistency was unchanged when the sourdough was fermented by a heterofermentative culture and softer if a homofermentative culture was used [86]. Proteolytic breakdown of proteins was enhanced at low pH during fermentation of wheat dough, and major effects were attributed to changes in pH rather than to microbial proteolytic activity from the sourdough [87]. In spite of the decreased stability in doughs with added sourdough increased bread volumes are reported for bread containing up to 20% sourdough [31,86]. The crumb structure of bread containing up to 20% sourdough has been comparable to standard bread without sourdough, whereas inferior crumb structure was observed in bread containing 40% sourdough [88]. 2. Rye Dough and Bread The main component of rye and wheat is starch, and its content has a crucial influence on the bread texture. It becomes sticky and pasty if the starch is degraded during the bread making due to too high activity of amylases. This problem is greater in rye bread making than for wheat bread, as the activity of the sprout-induced enzyme α-amylase is highest in rye [89]. This is caused by rainy summers in the rye-growing area. Furthermore, the period from harvest to possible sprouting is extremely short for

rye; it can even sprout in the fields [89]. One of the main functions of sourdough in rye bread making is inactivation of the α-amylase activity, and a general rule in bakeries is to add a larger amount of sourdough when the activity of enzymes in the flour is high. Bread with a rye content of more than 20% normally require the addition of sourdough to prevent degradation of starch [88,90]. Rye starch begin to swell as low as 52°C and subsequently the αamylase can degrade the starch until it will be heat-inactivated at 80°C [91] (s.169, 171). Rye α-amylase has pH-optimum at pH 5.5 [92] and the activity is totally inactivated in sourdough at pH below 4. Wassermann and Dörfner [93] found that the viscosity of rye doughs (rye flour and water) was lowest at pH 5. The activity of α-amylase is not only reduced considerably in the sourdough, but also in the rye dough with added sourdough. The activity of α-amylase was totally inactivated in an imitated sourdough acidified to pH 3.5 (TTA 32) by lactic and acetic acid [94]. The activity of αamylase in the bread dough after resting (pH 4.5), with 20% sourdough added, was about half the activity in the flour. Pentosans (arabinoxylans) play a key role in the viscosity of rye doughs due to high water-binding capacity. The viscosity of sourdoughs decreases during the sourdough fermentation due to the activity of the pentosan-degrading enzymes at the beginning of the fermentation. However, those enzymes are inactivated in the fermented sourdough [94]. Rye proteins are different from wheat proteins, as they do not form gluten structure. Kratochvil and Holas [95] found that proteolytic activity in rye sourdough was caused by enzymes from the flour.

B. FLAVOR AND TASTE The flavor of bread crumb depends mainly upon the flour type and the enzymatic reactions taking place due to yeast and sourdough fermentations, whereas the flavor of bread crust is more influenced by the thermal reactions during the baking process. Including sourdough in the bread recipe is recommended for a more aromatic bread flavour [31,96] and sourdough bread has higher content of volatile compounds [31,39,97–99] and higher scores in sensory tests [31,100,101]. The content of volatile compounds produced during sourdough fermentation depends on the flour type (wheat, rye, maize), the extraction rate of the flour, the fermentation temperature, the water content in the sourdough and the microorganisms in the sourdough. Generally, the LAB in the sourdough are mostly responsible for the acidification of the dough, and the sourdough yeasts for the production of flavor compounds. Factors that favor the propagation of yeasts will also result in higher content of yeast fermentation products. The extraction rate of the flour and the water content in the sourdough mostly influences the acidification of the sourdough. Higher extraction rate of the flour results in

Sourdough Bread

higher production of lactic and acetic acid [38,102], however, sourdoughs fermented with heterofermentative cultures have much higher content of ethyl acetate [38]. The production of acids calculated per gram dry matter is higher in fluid sourdoughs than in firm sourdoughs. Higher water content in the sourdough and increased fermentation temperature result in higher propagation of yeasts and in higher content of iso-alcohols [36,81,102]. Sourdoughs fermented with heterofermentative LAB have, aside from much higher content of acetic acid and ethanol, a higher content of ethylacetate and ethyl-hexanoate compared to sourdoughs fermented with homofermentative LAB, which have higher contents of diacetyl and some other carbonyls [36,81,102,103]. The production of acetic acid in sourdoughs can be increased in heterofermentative cultures with the addition of fructose as a hydrogen acceptor [102,104]. When sourdough yeasts are added in the preparation of the sourdough, the production of ethanol, iso-alcohols, esters and diacetyl increase considerably [37] [103]. In sourdough bread, the content of esters are very low compared to the corresponding sourdoughs [31,39]. Sensory evaluation of rye bread crumb shows that the most intense and bread-like flavor is associated with 2-propanone, 3methyl-butanal, benzylalcohol and 2-phenylethanol [39]. However, vanillin, 2,3-butandione, 3-hydroxy-4,5-dimethylfuranone and methylbutanoic acids also contribute to the overall crumb flavor [98]. The perceived taste of salt is enhanced in sourdough rye bread compared to wheat bread, so less salt can be added in sourdough rye bread [105]. Sensory evaluation of wheat bread crumb showed that bread made with sourdough fermented with the heterofermentative L. sanfranciscensis had a pleasant mild, sour odor and taste. Bread fermented with L. plantarum had an unpleasant metallic sour taste, but when the sourdough was also supplemented with the sourdough yeast S. cerevisiae, the bread acquired a more aromatic bread flavor. That bread had a higher content of methyl-butanol, methylpropanoic acids and 2-phenylethanol which may, in part, cause the more aromatic flavor [31]. Mixed cultures with both Lab and yeast are recommended for an aromatic and pleasant sourdough bread flavor [31,103,106]. A lexicon for description of the flavor of wheat sourdough bread has been developed [107].

C. LONGER SHELF LIFE During storage of bread, several different physical and microbiological changes occur, lowering the quality of bread. The bread crumb becomes hard, the bread crust changes from crispy to leathery, and the characteristic and favorable bread flavor disappears. All these changes are characterized for the staling process. Within few days the bread might be spoiled due to contamination and growth of molds on the surface or development of rope in the bread crumb caused by Bacillus spp. Addition of sourdough in the

183-15

bread recipe can be used to retard the staling process of the bread, prevent the bread against ropiness and prolong the mold free period. Sourdough addition is the most promising procedure to preserve bread from spoilage, since it is in agreement with the consumer demand for natural and additive free food products. 1. Anti-Mold Activity of Sourdough Bread Mold is the most frequent cause of bread spoilage. Addition of sourdough in the bread recipe increases the mould-free period both for rye bread [21,32] and wheat bread [78,108]. The length of mold-free period was prolonged from 4 days in wheat bread to 6 to 8 days in sourdough bread [78], or from 9 days in wheat bread supplemented with a prefermented dough to 20 days in bread supplemented with 20% sourdough [108]. No correlation was found between pH and bread shelf life. The mold-free period was prolonged 1 to 3 days in slices of sourdough rye bread inoculated with Aspergillus glaucus when the sourdough was fermented with heterofermentative LAB compared to homofermentative LAB, or bread without addition of sourdough [32]. The antimicrobial effect of the heterofermentative LAB was supposed to be their production of acetic acid. Two hundred and thirty two strains of sourdough LAB belonging to nine different species were screened for production of anti-mold substances against Aspergillus niger, Fusarium graminearum, Penicillium expansum and Monilia sitophila using agar-well-diffusion assay [109]. The anti-mold activity varied very much among the strains and was mainly detected within obligately heterofermentative LAB. L. sanfrancisensis had the largest spectrum of anti-mold activity. Not only the acetic acid had inhibitory effect, but the LAB produced also formic, propionic, butyric, n-valeric and caproic acid, and a mixture hereof was responsible for the anti-mold effect. 2. Prevention of Rope Spoilage Ropiness is spoilage of wheat bread noticed as an unpleasant odor similar to that of over-ripe melons, followed by the occurrence of a discolored sticky bread crumb and sticky threads, that can be pulled from the crumb. This bread spoilage is caused by heat-resistant strains of Bacillus and occurs particularly in summer when the climate favors growth of the bacteria. It is mainly caused by Bacillus subtilis, formerly referred to as B. mesentericus, because the heat resistant spores can survive the baking process, sporulate and multiply in the baked bread. The rope symptoms can be recognized when the level of Bacillus in bread crumb is 108 bacillus/g [110]. Its incidence has increased during the last decade, presumably because most bread is now produced without preservatives and often with the addition of raw materials such as oat products, wheat bran and sunflower seed with a high contamination level of Bacillus

183-16


spores [110]. Even a low level of the heat-resistant spores (101–102 bacillus/g) in raw materials resulted in a level of 107 bacillus/g bread in two days. One potential way to prevent development of rope is to include sourdough in the bread recipe. Addition of 10% sourdough inhibited the natural Bacillus contaminants in wheat dough, but it was insufficient to inhibit the Bacillus strains inoculated at a level of 106 spores/g [40]. Addition of 15% sourdough was more efficient as the strains of rope producing Bacillus were effectively inhibited by sourdough fermented by strains of L. sanfranciscensis, L. brevis, L. maltaromicus or by three different strains of L. plantarum. In this investigation B. subtilis tended to be inhibited, if the TTA value in the sourdough was more than 10 and when the pH of the bread crumb was below 4.8. Röcken [29] demonstrated that sourdough effectively decreased the heat resistance (D97-value) of a rope-producing strain of Bacillus. He found that the heat resistance was reduced from 143 min without the addition of sourdough to 5.9 min and 6.9 min with the addition of 10% and 20% sourdough, respectively.

B. REDUCED GLYCEMIC RESPONSE WITH SOURDOUGH BREAD

3. Bread Firmness and Staling Rate Bread becomes firmer during storage, and retrogradation of starch towards a more crystalline form is considered to be the primary cause of this bread staling. Several sourdoughs have been investigated for their potential effect on delaying the development of bread firmness and staling rate of wheat bread, but most investigations did not find any influence on staling rate by the sourdough compared to yeast- and sponge-leavened bread [108,111]. However, delayed staling rate has been observed in sourdough bread [112]. The rate of starch retrogradation was not influenced if the acidification was rather low, whereas a standard sourdough (L. sanfranciscensis 57, L. plantarum 13, S. cerevisiae 141) was able to retard the staling rate. The staling rate was mostly influenced if the starter culture had amylolytic activity (L. amylovorus or a genetic modified strain L. sanfranciscensis CB1 Amy). In some investigations the addition of sourdough resulted in lower bread firming. However, sourdough wheat bread has higher bread volume [31,86,88] and the measured resistance will thus be lower.

VI. NUTRITIONAL VALUE The addition of sourdough to the bread recipe has a positive influence on the nutritive value of the bread, as the minerals become bioavailable [113], and the blood glucose and insulin responses are lowered after eating sourdough bread compared to wheat bread [114].

A. REDUCED PHYTATE CONTENT

BY

is poor for those minerals which are stored as phytate, an insoluble complex with phytic acid (myoinositol hexa-phosphoric acid, IP6). The content of phytate is 6 mg/g rye grain [115], 3–4 mg/g in flour of soft wheat and 9 mg/g in hard wheat flour [116]. Phytate accounts for more than 70% of the total phosphorus in cereals, and it can be degraded during the bread making process due to the activity of endogenous phytase and thus liberate the bound minerals when the ester-bound phosphoric acids are hydrolyzed. The pH-optimum of rye phytase is found to be at pH 6.0 [115]. Sourdough fermentation has been shown to be more efficient than yeast fermentation in reducing the phytate content in whole bread (⫺62% and ⫺38% respectively) [113]. The prolonged fermentation with sourdough enhanced the acidification and led to increased solubility of Mg and P. Five different strains of LAB isolated from sourdoughs have been tested for their ability to degrade phytic acid, but no difference was observed among the strains in the levels of phytic acid hydrolyses [117].

SOURDOUGH

Whole meal cereals are good sources of minerals such as K, P, Mg, Fe and Zn, but without treatment the bioavailability

Conventional wheat bread products are rapidly digested and absorbed, thus giving rise to high blood glucose and insulin responses. Eating wholemeal sourdough bread resulted in both lowered blood glucose and insulin response compared to wholemeal bread made without sourdough [118]. This nutritional positive effect was possibly due to a reduced gastric emptying rate caused by the lactic acid produced during the sourdough fermentation [114].

ACKNOWLEDGMENT The information in this chapter has been modified from “Sourdough bread,” by A. S. Hansen, in Handbook of Food and Beverage Fermentation Technology, Editors: Y. H. Hui et al., Marcel Dekker, NY 2004.

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66. G Spicher, E Rabe, R Sommer, and H Stephan, The Microflora of Sourdough.15. Communication - on the Behavior of Heterofermentative Sourdough Bacteria and Yeasts in Mixed Culture, Zeitschrift fur LebensmittelUntersuchung Und-Forschung 174:222–227, 1982. 67. C Collar, Biochemical and technological assessment of the metabolism of pure and mixed cultures of yeast and lactic acid bacteria in breadmaking applications, Food Science and Technology International 2:349–367, 1996. 68. R Foschino, F Perrone, and A Galli, Characterization of two virulent Lactobacillus fermentum bacteriophages isolated from sour dough, Journal of Applied Bacteriology 79:677–683, 1995. 69. AI Sanni, AA Onilude, and MO Fatungase, Production of sour maize bread using starter-cultures, World Journal of Microbiology & Biotechnology 14:101–106, 1998. 70. P Savola, H Salovaara, and J Enqvist, Concentrations of carbohydrate fractions in the sour dough rye bread process, Progress in cereal chemistry and technology: Development in food science, 5A., J Holas and J Kratochvil, eds., Elsevier Science Publishers, Amsterdam, 1983, pp 465–470. 71. RJ Henry and HS Saini, Characterization of Cereal Sugars and Oligosaccharides, Cereal Chem. 66:362–365, 1989. 72. M Nilsson, P Aman, H Harkonen, G Hallmans, KEB Knudsen, W Mazur, and H Adlercreutz, Content of nutrients and lignans in roller milled fractions of rye, J. Sci. Food. Agric. 73:143–148, 1997. 73. F Meuser and P Suckow, Non-starch polysaccharides, Chemistry and physics of baking, J Blanshard, Frazier PJ, and MG Ganzle, eds., Royal Society of Chemistry, London, 1986, pp 42–61. 74. HB Hansen, MF Andreasen, MM Nielsen, LM Larsen, KEB Knudsen, AS Meyer, LP Christensen, and A Hansen, Changes in dietary fibre, phenolic acids and activity of endogenous enzymes during rye bread-making, European Food Research and Technology 214:33–42, 2002. 75. D Lefebvre, V Gabriel, Y Vayssier, and C FontagneFaucher, Simultaneous HPLC determination of sugars, organic acids and ethanol in sourdough process, Lebensmittel-Wissenschaft Und-Technologie-Food Science and Technology 35:407–414, 2002. 76. AI Sanni, J Morlon-Guyot, and JP Guyot, New efficient amylase-producing strains of Lactobacillus plantarum and L-fermentum isolated from different Nigerian traditional fermented foods, International Journal of Food Microbiology 72:53–62, 2002. 77. MA Martinez-Anaya, dB Benito, and CC Esteve, Effect of processing conditions on acidification properties of wheat sour doughs, International Journal of Food Microbiology 22:249–255, 1994. 78. H Salovaara and T Valjakka, The effect of fermentation temperature, flour type, and starter on the properties of sour wheat bread, International Journal of Food Science & Technology 22:591–597, 1987. 79. JM Brummer and K Lorenz, European Developments in Wheat Sourdoughs, Cereal Foods World 36:310–&, 1991. 80. G Spicher, [Various new biological aspects of sourdough fermentation.] Einige neue Aspekte der Biologie der Sauerteiggaerung, Getreide 36:12–16, 1982.

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81. A Hansen, B Lund, and MJ Lewis, Flavour production and acidification of sourdoughs in relation to starter culture and fermentation temperature, Lebensmittel Wissenschaft und Technologie 22:145–149, 1989. 82. AM Galal, ME Varriano, and JA Johnson, Rheological dough properties as affected by organic acids and salt, Cereal Chem. 55:683–691, 1978. 83. K Wehrle, H Grau, and EK Arendt, Effects of lactic acid, acetic acid, and table salt on fundamental rheological properties of wheat dough, Cereal Chem. 74:739–744, 1997. 84. RC Hoseney and RA Brown, Mixograph Studies.5. Effect of Ph, Cereal Chem. 60:124–126, 1983. 85. K Wehrle and EK Arendt, Rheological changes in wheat sourdough during controlled and spontaneous fermentation, Cereal Chem. 75:882–886, 1998. 86. EC Collar, dB Benedito, and MA Martinez-Anaya, Microbial sour doughs influence acidification properties and breadmaking potential of wheat dough, J. Food Sci. 59:629–633, 1994. 87. C Thiele, MG Ganzle, and R Vogel, Fluorescence Labeling of Wheat Proteins for Determination of Gluten Hydrolysis and Depolymerization during Dough Processing and Sourdough Fermentation, J. Agric. Food Chem. ; ASAP Web Release Date: 27-Mar-2003; DOI: 10.1021/jf020897e: 2003. 88. P Crowley, TJ Schober, CI Clarke, and EK Arendt, The effect of storage time on textural and crumb grain characteristics of sourdough wheat bread, European Food Research and Technology 214:489–496, 2002. 89. W Seibel and D Weipert, Bread making and other food uses around the world, Rye Production, Chemistry and Technology, Bushuk W, ed., American Association of Cereal Chemists, USA, St. Paul, 2001, pp 147–211. 90. P Stolz and G Boecker, Technology, properties and applications of sourdough products, Advances in Food Sciences 18:234–236, 1996. 91. W Seibel and D Weipert, Bread making and other food uses around the world, Rye, production, chemistry and technology, Bushuk W, ed., American Association of cereal Chemists, St. Paul, 2001, pp 147–211. 92. R Gabor, A Taufel, U Behnke, and J Heckel, Studies on the germination specific alpha-amylase and its inhibitor of rye (Secale-Cereale).1. Isolation and characterization of the enzyme, Zeitschrift fur Lebensmittel-Untersuchung Und-Forschung 192:230–233, 1991. 93. L Wassermann and Dörfner H.H., Die viskosen Eigenschaften von Roggenteiges unterschiedlicher Zusammensetzung, Getreide, Mehl und Brot 31:95–97, 1977. 94. HH Boskov, MF Andreasen, MM Nielsen, LM Larsen, KE Bach-Knudsen, AS Meyer, LP Christensen, and A Hansen, Changes in dietary fibre, phenolic acids and activity of endogenous enzymes during rye bread-making, European Food Research and Technology 214:33–42, 2002. 95. J Kratochvil and J Holas, [Studies on proteolysis in rye sour dough.] Untersuchungen ueber proteolytische Vorgaenge in Roggensauerteig, Getreide 38:330–332, 1984.

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96. C Thiele, MG Ganzle, and RF Vogel, Contribution of sourdough lactobacilli, yeast, and cereal enzymes to the generation of amino acids in dough relevant for bread flavor, Cereal Chem. 79:45–51, 2002. 97. LM Seitz, OK Chung, and R Rengarajan, Volatiles in selected commercial breads, Cereal Chem. 75:847–853, 1998. 98. E Kirchhoff and P Schieberle, Determination of key aroma compounds in the crumb of a three- stage sourdough rye bread by stable isotope dilution assays and sensory studies, J. Agric. Food Chem. 49:4304–4311, 2001. 99. E Kirchhoff and P Schieberle, Quantitation of odoractive compounds in rye flour and rye sourdough using stable isotope dilution assays, J. Agric. Food Chem. 50:5378–5385, 2002. 100. L Varapha, Chambers-E-IV, and DH Chambers, Determination of the sensory attributes of wheat sourdough bread, Journal of Sensory Studies 15:309–326, 2000. 101. JM Murray, D Cox, K Easton, and VS Mialon, Descriptive sensory analysis of Australian breads, Food Australia 54:247–252, 2002. 102. M Gobbetti, MS Simonetti, A Corsetti, F Santinelli, J Rossi, and P Damiani, Volatile compound and organic acid productions by mixed wheat sour dough starters: Influence of fermentation parameters and dynamics during baking, Food Microbiology 12:497–507, 1995. 103. P Damiani, M Gobbetti, L Cossignani, A Corsetti, MS Simonetti, and J Rossi, The sourdough microflora. Characterization of hetero- and homofermentative lactic acid bacteria, yeasts and their interactions on the basis of the volatile compounds produced, Food Science and Technology-Lebensmittel-Wissenschaft & Technologie 29:63–70, 1996. 104. W Rocken, M Rick, and M Reinkemeier, Controlled Production of Acetic-Acid in Wheat Sour Doughs, Zeitschrift fur Lebensmittel-Untersuchung UndForschung 195:259–263, 1992. 105. H Salovaara, Hellemann U., and Kurkula R., Effect of Salt on Bread Flavour, Lebensmittel-Wissenschaft & Technologie 15:270–274, 1982. 106. S Barber, MJ Torner, MA Martinezanaya, and CB Debarber, Microflora of the Sour Dough of Wheat-Flour Bread .9. Biochemical Characteristics and Baking Performance of Wheat Doughs Elaborated with Mixtures of Pure Microorganisms, Zeitschrift fur Lebensmittel-Untersuchung Und-Forschung 189:6–11, 1989. 107. V Lotong, E Chambers, and DH Chambers, Determination of the sensory attributes of wheat sourdough bread, Journal of Sensory Studies 15:309–326, 2000. 108. B Barber, C Ortola, S Barber, and F Fernandez, Storage of Packaged White Bread .3. Effects of Sour Dough and Addition of Acids on Bread Characteristics, Zeitschrift fur Lebensmittel-Untersuchung Und-Forschung 194:442–449, 1992. 109. A Corsetti, M Gobbetti, J Rossi, and P Damiani, Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by

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Part U Food Microbiology

184

Food Microbiology and Safety: Basic Requirements

James S. Dickson

Department of Animal Science, Iowa State University

Douglas L. Marshall

Department of Food Science Nutrition, and Health Promotion, Mississippi State University

CONTENTS I. Introduction ........................................................................................................................................................184-1 II. Administrative Regulation ................................................................................................................................184-2 A. U.S. Department of Agriculture ................................................................................................................184-2 B. U.S. Food and Drug Administration ..........................................................................................................184-3 C. Milk Sanitation ..........................................................................................................................................184-3 D. International Administration ......................................................................................................................184-3 III. Pre-Requisite Programs......................................................................................................................................184-3 A. Good Manufacturing Practices ..................................................................................................................184-3 B. Training and Personal Hygiene ..................................................................................................................184-4 C. Pest Control ................................................................................................................................................184-4 IV. Sanitation............................................................................................................................................................184-4 A. Sanitary Facility Design ............................................................................................................................184-4 B. Sanitary Equipment Design........................................................................................................................184-5 C. Cleaning and Sanitizing Procedures ..........................................................................................................184-5 V. Hazard Analysis Critical Control Point System (HACCP)................................................................................184-6 VI. HACCP Plan Development................................................................................................................................184-6 VII. Summary ............................................................................................................................................................184-8 References ....................................................................................................................................................................184-8

I.

INTRODUCTION

The objective of food processing and preparation is to provide safe, wholesome, and nutritious food to the consumer. The responsibilities for accomplishing this objective lie with every step in the food chain; beginning with food production on the farms, and continuing through processing, storage, distribution, retail sale, and consumption. Producing safe food is a continuum, where each party has certain obligations to meet and certain reasonable expectations of the other parties involved in the process. No single group is solely responsible for producing safe food, and no single group is without obligations in assuring the safety of food. Food producers have a reasonable expectation that the food he or she produces will be processed in such a manner

that further contamination is minimized. Food producers are an integral part of the food production system, but are not solely responsible for food safety. It is not practical to deliver fresh unprocessed food that is completely free of microorganisms, whether the food in question is of animal or plant origin. The environment in which the food is produced precludes the possibility that uncontaminated food can be grown or produced. However, appropriate methods can be utilized to reduce, to the extent possible, this level of background contamination. These methods are referred to as “Good Agricultural Practices” (GAPs) (1). Alternately, producers have an obligation to use these same reasonable practices to prevent hazards from entering the food chain. As an example, when dairy cattle are treated with antibiotics for mastitis, producers have an obligation to withhold milk from those animals from the normal production lot.

184-1

184-2


Milk from these animals must be withheld for the specified withdrawal time, so that antibiotic residues will not occur in milk delivered to dairies. In contrast, production of salmonellae-free poultry in the United States has been an elusive goal for poultry producers. While it is not a reasonable expectation for producers to deliver salmonellae-free birds to poultry processors, it is reasonable to expect producers to use good livestock management practices to minimize the incidence of Salmonella within a flock. Food processors have reasonable expectations that raw materials delivered to the processing facility are of reasonable quality and not contaminated with violative levels of any drugs or pesticides. In addition, processors have a reasonable expectation that processed food will be properly handled through the distribution and retail chain, and that it will be properly prepared by the consumer. The latter is particularly important, as processors have responsibility for products because they are labeled with the processor’s name, even though the food is no longer under processor control once it leaves the processing facility. Processor obligations are to process raw foods in a manner that minimizes growth of existing microorganisms as well as minimizes additional contamination during processing. These obligations extend from general facility maintenance to the use of the best available methods and technologies to process a given food. Clearly, consumers have an important role in the microbiological safety of foods. However, it is not reasonable to expect every consumer to have a college degree in food science or microbiology. Consumers have an expectation that foods they purchase have been produced and processed under hygienic conditions. They also have a reasonable expectation that foods have not been held under unsanitary conditions, or that foods have not been adulterated by the addition of any biological, chemical, or physical hazards. In addition, consumers have an expectation that foods will be appropriately labeled, so that the consumer has information available on both composition and nutritional aspects of products. These expectations are enforced by regulations that govern production, processing, distribution, and retailing of foods in the U.S. The vast majority of foods meets or exceeds these expectations, and the average consumer has relatively little to be concerned with regarding the food they consume. Some consumers have advocated additional expectations, which may or may not be reasonable. For example, some would argue that raw foods should be free of infectious microorganisms. Initially, this would appear to be reasonable; however, in many cases technologies or processes do not exist in a legal or practical form to assure that raw foods are not contaminated with infectious agents. Two recent examples are the outbreaks of Cyclospora epidemiologically linked to imported raspberries and Escherichia coli O157:H7 in raw ground beef.

With the exception of irradiation, technologies do not exist to assure that either of these foods would be absolutely free of infectious agents while still retaining desirable characteristics associated with raw food. Therefore, in some cases, the expectation that raw foods should be free of infectious agents may not be reasonable. Consumers have several obligations regarding food safety. As part of the food production to consumption chain, consumers have similar obligations to food processors. Namely, not holding foods under unsanitary conditions prior to consumption and not adulterating foods with the addition of biological, chemical, or physical agents. Improper food handling can increase foodborne illness risks by allowing infectious bacteria to increase in numbers or by allowing for cross contamination between raw and cooked foods. In addition, consumers have an obligation to use reasonable care preparing foods for consumption, as do personnel in food service operations. As an example, consumers should cook poultry until it is “done” (internal temperature at or above 68°C) to eliminate any concerns with salmonellae. Consumer education on the basics of food safety in the home should be a priority. Every consumer should understand that food is not sterile, and the way food is handled in the kitchen may affect the health of individuals consuming it. Although our long-term goal is to reduce or eliminate foodborne disease hazards, in the near term we need to remind consumers of what some of the potential risks are and how consumers can avoid them. In the end, it is the consumer who decides what they will or will not consume.

II.

ADMINISTRATIVE REGULATION

Several regulatory groups are involved in the regulation of food safety and quality standards, from local and state agencies to international agencies. Since there is tremendous variation within and between local and state agencies, this discussion will be confined to the national and international agencies that regulate food. At the national level, two federal agencies regulate the vast majority of food produced and consumed in the United States; namely, the U.S. Department of Agriculture (USDA) (2) and the Food and Drug Administration (FDA) (3).

A.

U.S. DEPARTMENT

OF

AGRICULTURE

USDA has responsibility for certification, grading, and inspection of all agricultural products. All federally inspected meat and meat products, including animals, facilities, and procedures, are covered under a series of meat inspection laws that began in 1906 and have been modified on several different occasions, culminating in the latest revisions in 1996 (4). These laws cover only meat that is in interstate commerce, leaving the legal jurisdiction of intrastate meats to individual states. In the states


that do have state inspected meats, in addition to federally inspected meats, the regulations require that the state inspection program be “equivalent” to the federal program. Key elements in meat inspection are examination of live animals for obvious signs of clinical illness and examination of gross pathology of carcasses and viscera for evidence of transmissible diseases. The newest regulations also require the implementation of a HACCP system and microbiological testing of carcasses after chilling. Eggs and egg products are also covered by USDA inspection under the Egg Products Inspection Act of 1970 (5). This act mandates inspection of egg products at all phases of production and processing. USDA inspection of meat processing is continuous; that is, products cannot be processed without an inspector or inspectors present to verify the operation.

B.

D. INTERNATIONAL ADMINISTRATION The Codex Alimentarius Commission, created by the Food and Agriculture Organization and the World Health Organization, has the daunting task of implementing food standards on an international scale (7). These standards apply to both general and specific food categories and also set limits for pesticide residues in foods. Acceptance of these standards is voluntary and at the discretion of individual governments, but acceptance of the standards requires that the country apply them equally to both domestically produced and imported products. The importance of international standards is growing daily as international trade in food expands. Many countries find that they are both importing and exporting foods, and a common set of standards is critical in establishing trade without the presence of non-tariff trade barriers.

U.S. FOOD AND DRUG ADMINISTRATION

FDA has responsibility for ensuring that foods are wholesome, safe, and have been stored under sanitary conditions, as outlined by the Food Drug and Cosmetic Act of 1938. This act has been amended to include food additives, packaging, and labeling. The last two issues relate not only to product safety and wholesomeness, but also to nutritional labeling and economic fraud. FDA is also empowered to act if pesticide residues exceed tolerances set by the U.S. Environmental Protection Agency. Unlike USDA inspection, FDA inspection is discontinuous, with food processing plants being required to maintain their own quality control records while inspectors themselves make random visits to facilities.

C.

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MILK SANITATION

Perhaps one of the greatest public health success stories of the 20th century has been the pasteurization of milk. The U.S. Public Health Service drafted a model milk ordinance in 1924, which has been adopted by most local and state regulatory authorities and has become known as the Grade A PMO (Pasteurized Grade A Milk Ordinance) (6). This ordinance covers all phases of milk production, including but not limited to animal health, design and construction of milk processing facilities, equipment, and most importantly, the pasteurization process itself. The PMO sets quality standards for both raw and processed milk, in the form of cooling requirements and bacteriological populations. The PMO also standardizes the pasteurization requirements for fluid milk, which ensures that bacteria of public health significance will not survive in the finished product. From a historical perspective, it is interesting to note that neither the public nor the industry initially embraced pasteurization, but that constant pressure from public health officials finally succeeded in making this important advance in public health almost universal.

III.

PRE-REQUISITE PROGRAMS

In order to achieve the goal of producing a safe food product, food processors should have in place a variety of fundamental programs covering the general operation of the process and the processing facility. These programs are considered “pre-requisites,” as without these basic programs in place, it is impossible to produce safe and wholesome foods, irrespective of the available technology, inspection process or microbiological testing. These pre-requisite programs fall generally under the term “good manufacturing practices” (GMPs), but also include sanitation, equipment and facility design, personal hygiene issues, and pest control.

A.

GOOD MANUFACTURING PRACTICES (GMPS)

GMPs cover a broad range of activities with the food-processing establishment. Although there is general guidance in the Code of Federal Regulations (8), GMPs are established by the food processor, and are specific to their own operation. There is also general guidance on GMPs available from a variety of organizations representing specific commodities or trades. Specific applications of GMPs are discussed in the following sections, but GMPs also apply activities that affect not only the safety of the product, but also the quality. As an example, a refrigerated holding or storage temperature may be set by a GMP at a point below that which is actually required for product safety, but is set at that point for product quality reasons. Conversely, if a raw material or partially manufactured product, which under normal circumstances would be kept refrigerated, were subsequently found to be at a higher temperature, it would be deemed to be out of compliance with the GMP. GMPs may also focus on the actual production processes and controls within those processes. GMPs may be viewed as rules that assure fitness of raw materials and

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ingredients, rules that maintain the integrity of processed foods, and rules to protect the finished product (foods) from deterioration during storage and distribution. Other GMPs may address the presence of foreign materials in the processing area, such as tramp metal from equipment maintenance or broken glass from a shattered light bulb. These GMPs are established to provide employees with specific guidance as to the company’s procedures for addressing certain uncommon but unavoidable issues. While GMPs by their nature cover broad areas of operation, the individual GMP is usually quite specific, presenting complete information in a logical, step-wise fashion. An employee should be able to retrieve a written GMP from a file, and should be able to perform the required GMP function with little or no interpretation of the written material.

B.

TRAINING AND PERSONAL HYGIENE

Personnel who are actually involved in food processing operations should also understand the necessity for proper cleaning and sanitation, and not simply rely on the sanitation crew to take care of all issues. In addition, all employees must be aware of basic issues of personal hygiene, especially when they are in direct contact with food or food processing equipment. Some key elements, such as hand washing and clean clothing and gloves, should be reemphasized on a periodic basis. An important aspect of this is an emphasis on no “bare handed” contact with the edible product, using utensils or gloves to prevent this from occurring. This information has been outlined by the U.S. Food and Drug Administration in the Good Manufacturing Practices section of the Code of Federal Regulations (8).

C.

PEST CONTROL

Pests, such as insects and rodents, present both physical as well as biological hazards (10). While the consumer would undoubtedly object to the proverbial “fly in the soup,” the concerns with the introduction of biological hazards into the foods by pests are even greater. Integrated Pest Management (IPM) includes the physical and mechanical methods of controlling pests within the food processing environment and the surrounding premises. At a minimum, the processing environment and the area surrounding the processing plant should be evaluated by a competent inspector for both the types of pests likely to be present, and the potential harborages for such pests. A comprehensive program should be established that addresses flying insects, crawling insects, and rodents, the objective of which being to prevent access to the processing environment. Given that it is impossible to completely deny pest access to the processing environment, internal measures should be taken to reduce the numbers of any pests that enter the processing area. Since it is undesirable

to have poisonous chemicals in areas surrounding actual food production, active pest reduction methods should be mechanical in nature (traps, insect electrocuters, etc.). Record keeping is an important aspect of pest management. Documentation of pest management activities should include maps and maintenance schedules for rodent stations, bait stations, insect electrocutors, an inventory of pesticides on the premises, and reports of inspections and corrective actions. There should be standard operating procedures for applying pesticides, and they should only be applied by properly trained individuals. Many food-processing establishments contract with external pest control operators to address their pest control needs.

IV. SANITATION Sanitation is the fundamental program for all food processing operations, irrespective of whether they are converting raw products into processed food or preparing food for final consumption. Sanitation impacts all attributes of processed foods, from organoleptic properties of the food to the safety and quality of the food itself. From a food processors perspective, an effective sanitation program is essential to producing quality foods with reasonable shelf lives. Without an effective program, even the best operational management and technology will ultimately fail to deliver the quality product that consumers demand. Sanitation programs are all encompassing, focusing not only on the details of soil types and chemicals, but the broader environmental issues of equipment and processing plant design. Many foodborne microorganisms, both spoilage organisms and bacteria of public health significance, can be transferred from the plant environment to the food itself (11). Perhaps one of the most serious of these microorganisms came to national and international attention in the mid-1980s, when Listeria monocytogenes was found in processed dairy products. Listeria was considered to be a relatively minor veterinary pathogen until that time, and not even considered a potential foodborne agent. However, subsequent research demonstrated that Listeria monocytogenes was a serious human health concern, and more importantly was found to be widely distributed in nature. In many food processing plants, Listeria were found to be in the general plant environment, and subsequently efforts have been made to improve plant sanitation, through facility and equipment design as well as focusing more attention on basic cleaning and sanitation.

A.

SANITARY FACILITY DESIGN

Some of the basic considerations of food processing facility design include the physical separation of raw and processed products, adequate storage areas for nonfood items (such as packaging materials), and a physical layout that minimizes employee traffic between raw and


processed areas. While these considerations are easily addressed in newly constructed facilities, they may present challenges in older facilities that have been renovated or added on to. Exposed surfaces, such as floors, walls, and ceilings, in the processing area should be constructed of material that allows for thorough cleaning. Although these surfaces are not direct food contact surfaces, they contribute to overall environmental contamination in the processing area. These surfaces are particularly important in areas where food is open to the environment, and the potential for contamination is greater when temperature differences in the environment result in condensation (12). As an example, a large open cooking kettle will generate some steam that may condense on surfaces above the kettle. This condensate may, without proper design and sanitation, drip back down into the product carrying any dirt and dust from overhead surfaces back into the food. Other obvious considerations are basic facility maintenance as well as insect and rodent control programs, as all of these factors may contribute to contamination of food.

B.

SANITARY EQUIPMENT DESIGN

Many of the same considerations for sanitary plant design also apply to the design of food processing equipment. Irrespective of its function, processing equipment must protect food from external contamination and from undue conditions that will allow existing bacteria to grow. The issue of condensate as a form of external contamination has already been raised. Opportunities for existing bacteria to reproduce may be found in the so-called “dead spaces” within some equipment. These areas can allow food to accumulate over time under conditions that allow bacteria to grow. These areas then become a constant inoculation source for additional product as it moves through the equipment, increasing the bacteriological population within the food. Other considerations of food equipment design include avoiding construction techniques that may allow product to become trapped within small areas of the equipment, creating the same situation that occurs in the larger dead spaces within the equipment. As an example, lap seams that are tack welded provide ample space for product to become trapped. Not only does this create a location for bacteria to grow and contaminate the food product, it also creates a point on the equipment that is difficult if not impossible to clean.

C.

CLEANING AND SANITIZING PROCEDURES

Cleaning and sanitizing processes can be generically divided into five separate steps that apply to any sanitation task (13). The first step is removal of residual food, waste materials, and debris. This is frequently referred to as a “dry” clean up. The dry clean up is followed by a rinse with warm (48° to 55°C) water, to remove material that is only loosely attached to surfaces and to hydrate material

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that is more firmly attached to surfaces. Actual cleaning follows the warm water rinse, which usually involves the application of cleaning chemicals and some form of scrubbing force, either with mechanical brushes or with highpressure hoses. The nature of the residual food material will determine the type of cleaning compound applied. After this, surfaces are rinsed and inspected for visual cleanliness. At this point, the cleaning process is repeated on any areas that require further attention. Carbohydrates and lipids can generally be removed with warm to hot water and sufficient mechanical scrubbing. Proteins require the use of alkaline cleaners, while mineral deposits can be removed with acid cleaners. Commercially available cleaning compounds generally contain materials to clean the specific type of food residue of concern, as well as surfactants and, as necessary, sequesterants that allow cleaners to function more effectively in hard water (14). When surfaces are visually clean, a sanitizer is applied to reduce or eliminate remaining bacteriological contamination. Inadequately cleaned equipment cannot be sanitized, as the residual food material will protect bacteria from the sanitizer. One of the most common sanitizing agents widely used in small and medium sized processing facilities, is hot water. Most regulatory agencies require that when hot water is used as the sole method of sanitization, the temperature must be at or above 85°C. While heat sanitization in effective, it is not as economical as chemical sanitizers because of the energy costs required to maintain the appropriate temperature. Chlorine containing sanitizers are economical and effective against a wide range of bacterial species, and are widely used in the food industry (15). Typically, the concentrations of chlorine applied to equipment and surfaces are in the 150 to 200 parts per million range. Chlorine sanitizers are corrosive and can, if improperly handled, release chlorine gas into the environment. Iodine containing sanitizers are less corrosive than chlorine sanitizers, but are also somewhat less effective. These sanitizers must be used at slightly acidic pH values to allow for the release of free iodine. The amber color of iodine sanitizers can give an approximate indication of concentration, but can also leave residual stains on treated surfaces. Quaternary ammonium compounds (QACs) are noncorrosive and demonstrate effective bactericidal action against a wide range of microorganisms. These sanitizers are generally more costly and not as effective as chlorine compounds, but they are stable and provide residual antimicrobial activity on sanitized surfaces. Food processing plants will frequently alternate between chlorine and QAC sanitizers to prevent development of resistant bacterial populations or will use chlorine sanitizers on regular production days and then apply QACs during periods when the facility is not operating (for example, over a weekend). Another element in food plant sanitation programs is the personnel who perform the sanitation operations as well as the employees who work in the processing area.

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Sanitation personnel should be adequately trained to understand the importance of their function in the overall processing operation in addition to the training necessary to properly use the chemicals and equipment necessary for them to perform their duties.

V. HAZARD ANALYSIS CRITICAL CONTROL POINT SYSTEM (HACCP) The basic concept of HACCP was developed in the late 1950s and early 1960s as a joint effort to produce food for the manned space program. The U.S. Air Force Space Laboratory Project Group, the U.S. Army Natick Laboratories, and the National Aeronautics and Space Administration contributed to the development of the process, as did the Pillsbury Company, which had a major role in developing and producing the actual food products. Since that time, the HACCP system has evolved and been refined, but still focuses on the original goal of producing food that is safe for consumption (16). Since development, HACCP principles have been used in many different ways. However, recent interest in the system has been driven by changes in the regulatory agencies, specifically the U.S. Department of Agriculture– Food Safety and Inspection Service, and the U.S. Food and Drug Administration. USDA-FSIS recently revised the regulations that govern meat inspection to move all federally inspected meat plants to a HACCP-based system of production and inspection (4). FDA has also changed the regulations for fish and seafood, again moving this to a HACCP-based system for production (17). It is likely, given current trends by federal agencies, that most commercially produced foods will be produced under HACCP systems within the next ten years. The goal of a HACCP system is to produce foods that are free of biological, chemical, and physical hazards (18). HACCP is a preventative system, designed to prevent problems before they occur, rather than trying to fix problems after they occur. Biological hazards fall into two distinct categories, those that can potentially cause infection and those that can potentially cause intoxications. Infectious agents require the presence of viable organisms in the food and may not, depending on the organisms and the circumstances, require that the organism actually reproduce in the food. As an example, Escherichia coli O157:H7 has an extremely low infectious dose for humans (possibly less than 100 viable cells), and as such the mere presence of the bacterium in foods is a cause for concern. In contrast, organisms involved in intoxications usually require higher numbers of the organism in the food to produce sufficient amounts of toxin to cause clinical illness in humans. However, some of the toxins involved in foodborne diseases are heat stable, so that absence of viable organisms in the food is not necessarily an indication of the relative safety of the food.

Staphylococcus aureus is a good example, where it typically requires greater than 1,000,000 to 10,000,000 cells per gram of food to produce sufficient toxin to cause illness in humans (19). However, because the toxin itself is extremely heat stable, cooking the food will eliminate the bacterium but not the toxin, and the food can still potentially cause an outbreak of foodborne illness. Chemical hazards include chemicals that are specifically prohibited in foods, such as cleaning agents, as well as food additives that are allowed in foods but only at regulated concentrations. Foods containing prohibited chemicals or food additives in levels higher than allowed are considered adulterated. Adulterated foods are not allowed for human consumption and are subject to regulatory action by the appropriate agency (USDA or FDA). Chemical hazards can be minimized by assuring that raw materials (foods and packaging materials) are acquired from reliable sources that provide written assurances that the products do not contain illegal chemical contaminants or additives. During processing, adequate process controls should be in place to minimize the possibility that an approved additive will be used at levels not exceeding maximum legal limits for both the additive and the food product. Other process controls and GMPs should also insure that industrial chemicals, such as cleaners or lubricants, will not contaminate food during production or storage (8). Physical hazards are extraneous material or foreign objects that are not normally found in foods. For example, wood, glass or metal fragments are extraneous materials that are not normally found in foods. Physical hazards typically affect only a single individual or a very small group of individuals, but because they are easily recognized by the consumer, are sources of many complaints. Physical hazards can originate from food processing equipment, packaging materials, the environment, and from employees. Physical contaminants can be minimized by complying with good manufacturing practices and by employee training. While some physical hazards can be detected during food processing (e.g., metal by the use of metal detectors), many non-ferrous materials are virtually impossible to detect by any means and so control often resides with employees.

VI.

HACCP PLAN DEVELOPMENT

Prior to the implementation of HACCP, a review should be conducted of all existing pre-requisite programs. Deficiencies ion these programs should be addressed prior to the implementation of HACCP, because a HACCP plan presumes that these basic programs are fully functional and effective. Development of a HACCP plan begins with the formation of a HACCP team (20). Individuals on this team should represent diverse sections within a given operation, from purchasing to sanitation. The team is then responsible for development of the plan. Initial tasks that the team must accomplish are to identify the food and


method of distribution, and to identify the consumer and intended use of the food. Having done this, the HACCP team should construct a flow diagram of the process and verify that this diagram is accurate. The development of a HACCP plan is based on seven principles or steps in logical order (21). With the flow diagram as a reference point, the first principle or step is to conduct a hazard analysis of the process. The HACCP team identifies all biological, chemical, and physical hazards that may occur at each step during the process. Once the list is completed, it is reviewed to determine the relative risk of each potential hazard, which helps identify significant hazards. Risk is the interaction of “likelihood of occurrence” with “severity of occurrence.” As an extreme example, a sudden structural failure in the building could potentially contaminate any exposed food with foreign material. However, likelihood of the occurrence of such an event is small. In contrast, if exposed food is held directly below surfaces that are frequently covered with condensate, then the likelihood of condensate dripping on exposed food is considerably higher. An important point in the determination of significant hazards is a written explanation by the HACCP team regarding how the determination of “significant” was made. This documentation can provide a valuable reference in the future, when processing methods change or when new equipment is added to the production line. The second principle in the development of a HACCP plan is the identification of critical control points (CCPs) within the system. A CCP is a point, step, or procedure where control can be applied and a food safety hazard can be prevented, eliminated, or reduced to acceptable levels (18). An example of a CCP is the terminal heat process applied to canned foods after cans have been filled and sealed. This process, when properly conducted according to FDA guidelines, effectively eliminates a potential food safety hazard, Clostridium botulinum. Once CCPs have been identified, the third principle in the development of a HACCP plan is to establish critical limits for each CCP. These limits are not necessarily the ideal processing parameters, but the minimum acceptable levels required to maintain the safety of the product. Again, in the example of a canned food, the critical limit is the minimum time and temperature relationship to insure that each can has met the appropriate standards required by FDA. The fourth principle, following in logical order, is to establish appropriate monitoring requirements for each critical control point. The intent of monitoring is to ensure that critical limits are being met at each critical control point. Monitoring may be on a continuous or discontinuous basis. Presence of a physical hazard, such as metal, can be monitored continuously by passing all of the food produced through a metal detector. Alternately, presence of foreign material can be monitored on a continuous basis by visual inspection. Discontinuous inspection may

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involve taking analytical measurements, such as temperature or pH, at designated intervals during the production day. Some analytical measurements can be made on a continuous basis by the use of data recording equipment, but it is essential that continuous measures be checked periodically by production personnel. The fifth principle in the development of a HACCP plan is to establish appropriate corrective actions for occasions when critical limits are not met. Corrective actions must address the necessary steps to correct the process that is out of control (such as increasing the temperature on an oven) as well as addressing disposition of the product that was made while the process was out of control. A literal interpretation of the HACCP system and a CCP is that when a CCP fails to meet the critical limits, then the food product is potentially unsafe for human consumption. As a result, food produced while the CCP was not under control cannot be put into the normal distribution chain without corrective actions being taken to that product. Typically this means that the product must be either re-worked or destroyed, depending on the nature of the process and the volume of product that was produced while the CCP was out of control. This argues for frequent monitoring, so that the actual volume of product produced during each monitoring interval is relatively small. The sixth principle in the development of a HACCP plan is verification. Verification can take many forms. Microbiological tests of finished products can be performed to evaluate the effectiveness of a HACCP plan. Alternately, external auditors can be used to evaluate all parts of the HACCP plan, to ensure that the stated goals and objectives are being met. A HACCP plan must also be periodically reviewed and updated, to reflect changes in production methods and use of different equipment. Another critical aspect of verification is education of new employees on the HACCP plan itself. As HACCP is phased in to many food-processing environments, many employees who are unfamiliar with the concepts and goals of HACCP will have to be educated on the necessity of following the plan. In one sense, USDA-FSIS regulations have guaranteed that meat processors will follow HACCP plans, as the penalty for not following the HACCP plan can be as severe as the loss of inspection at an establishment. However, HACCP is an excellent system for monitoring and improving production of food products, and many food processors will discover that HACCP plans offer many benefits, well above and beyond the legal requirements of the regulatory agencies. The seventh principle in the development of a HACCP plan is the establishment of effective record keeping procedures. In many respects, a HACCP plan is an elaborate record-keeping program. Records should document what was monitored, when it was monitored and by whom, and what was done in the event of a deviation. Reliable records are essential from both a business

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and regulatory perspective. From the business perspective, HACCP records allow a processor to develop an accurate longitudinal record of production practices and deviations. Reviewing HACCP records may provide insight on a variety of issues, from an individual raw material supplier whose product frequently results in production deviations, to an indication of an equipment or environmental problem within a processing plant. From a regulatory perspective, records allow inspectors to determine if a food processor has been fulfilling commitments made in the HACCP plan. If a processor has designated a particular step in the process as a CCP, then they should have records to indicate that the CCP has been monitored on a frequent basis and should also indicate corrective actions taken in the event of a deviation.

VII. SUMMARY The intent of food processing is to deliver safe and wholesome products to the consumer. Basic food safety programs, including GMPs and sanitation, are the minimum requirements to achieve this goal. HACCP is a logical extension of these programs, and focuses on the prevention of hazards before they occur, rather than waiting for a failure to occur, and then addressing the problem. HACCP provides the most comprehensive approach to food safety in the processing environment, but is not foolproof. Perhaps the most challenging aspect is that, even with the best designed and implemented HACCP plan, it may not always be possible to “prevent, eliminate or reduce to acceptable levels” the pathogen of concern. This is particularly true with foods that are purchased by the consumer in their raw state, and then cooked. A specific example is Escherichia coli O157:H7 in ground beef. Irrespective of the preventative efforts of the processor, it is not possible to assure that the product is free of the bacterium, and there is no “acceptable level” of this organism in ground beef.

REFERENCES 1. Food and Drug Administration. Guide to minimize microbial food safety hazards for fresh fruits and vegetables. Available via the Internet at http://www. cfsan.fda.gov/~dms/prodguid.html. 1998. 2. Department of Agriculture, Food Safety and Inspection Service, Agency Mission and Organization. Code of Federal Regulations, Title 9, Animals and Animal Products, Part 300. 2003. 3. Food and Drug Administration, Department of Health and Human Services, Product Jurisdiction. Code of Federal Regulations, Title 21, Food and Drugs, Part 3. 2003. 4. Department of Agriculture, Food Safety and Inspection Service: Pathogen reduction; hazard analysis and critical control point (HACCP) systems; action: Final rule. 9 CFR Parts 304, 308, 310, 320, 327, 381, 416, and 417. Federal Register: Volume 61, Number 144, 38805, July 25, 1996.

5. Inspection of eggs and egg products (Egg Products Inspection Act). Food Safety and Inspection Service, Code of Federal Regulations, Title 9, Animals and Animal Products, Part 590. 2003. 6. U.S. Food and Drug Administration. Grade “A” Pasteurized Milk Ordinance 2001 Revision. Accessed from the U.S. Food and Drug Administration web page, http://vm.cfsan.fda.gov/~ear/pmo01toc.html/. 2002. 7. Food and Agriculture Organization. Understanding the Codex Alimetarius. Accessed from the Codex Alimetarius Commission web page, http://www. fao.org/docrep/w9114e/w9114e00.htm. 2003. 8. Food and Drug Administration, Department of Health and Human Services, Current Good Manufacturing Practice in manufacturing, packing, or holding human food. Code of Federal Regulations, Title 21, Food and Drugs, Part 110. 2003. 9. NG Marriott. Personal hygiene and sanitary food handling. In: NG Marriott. ed. Principles of Food Sanitation, 4th ed. Gaithersburg, MD: Aspen, 1999, pp 60–74. 10. NG Marriott. Pest control. In: NG Marriott. ed. Essentials of Food Sanitation, New York: Chapman and Hall, 1997, pp 129–149. 11. FDA/MIF/IICA. Recommended guidelines for controlling environmental contamination in dairy plants. Dairy Food Environ Sanitation 8:52–56, 1988. 12. D Gabis, RE Faust. Controlling microbial growth in food processing environments. Food Technol 42(12):81–83, 1988. 13. SC Ingham, BH Ingham, DR Buege. Sanitation Programs and Standard Operating procedures for Meat and Poultry Plants. Elizabethtown, PA: American Association of Meat Processors, 1996. 14. NG Marriott. Cleaning compounds. In: NG Marriott. ed. Principles of Food Sanitation, 4th ed. Gaithersburg, MD: Aspen, 1999, pp 114–138. 15. NG Marriott. Sanitizers. In: NG Marriott. ed. Principles of Food Sanitation, 4th ed. Gaithersburg, MD: Aspen, 1999, pp 139–157. 16. MD Pierson, DA Corlett. HACCP; Principles and Applications. New York: Chapman and Hall, 1992. 17. Department of Health and Human Services, Food and Drug Administration. Procedures for the safe and sanitary processing and importing of fish and fishery products; final rule. 21 CFR Parts 123 and 1240. Federal Register: Vol 60, No. 242, 65096, December 18, 1995. 18. KE Stevenson, DT Bernard. HACCP: Establishing Hazard Analysis Critical Control Point Programs. Washington, D.C.: The Food Processors Institute, 1995. 19. AL Noleto, MS Bergdoll. Production of enterotoxin by a Staphylococcus aureus strain that produces three identifiable enterotoxins. J Food Prot 45:1096–1097, 1982. 20. American Meat Institute Foundation: HACCP. The Hazard Analysis Critical Control Point System in the Meat and Poultry Industry. Washington, D.C.: American Meat Institute Foundation, 1994. 21. National Advisory Committee on Microbiological Criteria for Foods. Hazard Analysis and Critical Control Point Principles and Applications Guidelines. J Food Prot 61:1246–1259, 1998.

185

Conventional Microbial Testing Methods and Microscopy Techniques

Keith R. Schneider

Food Science and Human Nutrition Department, University of Florida

Mickey E. Parish

Lake Alfred, CREC, University of Florida

Jennifer Joy


CONTENTS I. Introduction..........................................................................................................................................................185-2 II. Microorganisms ..................................................................................................................................................185-2 A. Bacteria ........................................................................................................................................................185-2 B. Yeasts ..........................................................................................................................................................185-3 C. Molds ..........................................................................................................................................................185-3 III. Microbial Growth Factors....................................................................................................................................185-3 A. Nutrient Requirements ................................................................................................................................185-3 B. Moisture Content ........................................................................................................................................185-3 C. pH ................................................................................................................................................................185-3 D. Redox Potential (Eh) ..................................................................................................................................185-3 E. Temperature ................................................................................................................................................185-4 F. Relative Humidity........................................................................................................................................185-4 G. Atmosphere..................................................................................................................................................185-4 H. Biological Structures ..................................................................................................................................185-4 I. Antimicrobial Constituents..........................................................................................................................185-4 IV. Sampling Plans ....................................................................................................................................................185-5 A. Attribute Plans ............................................................................................................................................185-5 B. Variable Plans ..............................................................................................................................................185-5 V. Microscopy ..........................................................................................................................................................185-5 A. Light Microscopy ........................................................................................................................................185-5 B. Phase Contrast Microscopy ........................................................................................................................185-5 C. Epifluorescence Microscopy ......................................................................................................................185-5 D. Transmission Electron Microscopy ............................................................................................................185-5 E. Scanning Electron Microscopy....................................................................................................................185-6 F. Low-Voltage Field-Emission Scanning Electron Microscopy ....................................................................185-6 G. Variable-Pressure Scanning Electron Microscopy ......................................................................................185-6 H. Cryo-Scanning Electron Microscopy ..........................................................................................................185-6 I. Environmental Scanning Electron Microscopy ..........................................................................................185-6 J. Confocal Laser Scanning Microscopy ........................................................................................................185-6 VI. Detection and Identification ................................................................................................................................185-6 A. Indicator Organisms ....................................................................................................................................185-7

185-1

185-2


B.

Culture Media ..........................................................................................................................................185-7 1. Selective Media ................................................................................................................................185-7 2. Differential Media ............................................................................................................................185-7 3. Synthetic Media................................................................................................................................185-7 4. Complex Media ................................................................................................................................185-7 5. Liquid Media ....................................................................................................................................185-7 6. Enrichments ......................................................................................................................................185-7 7. Commercially Available Agars ........................................................................................................185-8 C. Culturing Methods....................................................................................................................................185-8 1. AOAC ..............................................................................................................................................185-8 2. American Public Health Association Standard Methods ................................................................185-8 3. Food and Drug Administration’s Bacteriological Analytical Manual..............................................185-8 4. United States Department of Agriculture, Food Safety Inspection Service’s Microbiology Laboratory Guidebook......................................................................................................................185-9 VII. Enumeration of Viable Cells ............................................................................................................................185-9 A. Direct Enumeration ..................................................................................................................................185-9 B. Indirect Enumeration................................................................................................................................185-9 1. Plate Counts......................................................................................................................................185-9 2. Most Probable Number (MPN) ........................................................................................................185-9 VIII. Metabolic Activity Measurement ..................................................................................................................185-10 A. VITEK ....................................................................................................................................................185-10 B. API Strips ..............................................................................................................................................185-10 C. BBL Crystal............................................................................................................................................185-11 D. Impedance ..............................................................................................................................................185-11 E. BioSys ....................................................................................................................................................185-11 F. Bioluminescence ....................................................................................................................................185-11 G. ATP Test Kits..........................................................................................................................................185-12 IX. Other Developments in Microbial Testing ....................................................................................................185-12 X. Summary ........................................................................................................................................................185-13 XI. Non-Endorsement of Commercial Products and Services ............................................................................185-13 Acknowledgment........................................................................................................................................................185-13 References ..................................................................................................................................................................185-13

I.

INTRODUCTION

Microbial testing has been a concern since it was determined that microbes play an integral role in food quality and safety. One of the earliest volumes that discussed standardized testing methods was Food Adulteration and its Detection (1), which was followed by numerous other texts. Today many similar volumes are in use by a variety of regulatory agencies, industries and scientific groups. These include, but are not limited to the Food and Drug Administration’s (FDA) Bacterial Analytical Manual (BAM), U.S. Department of Agriculture’s (USDA) Microbiological Laboratory Guidebook (MLG), AOAC’s Official Methods of Analysis and the American Public Health Association’s (APHA) Compendium of Methods for the Microbiological Examination of Foods. This chapter will briefly review some of the basic concepts surrounding food microbiology, microscopy, and testing. It will touch upon a variety of microscopy- and media-based methodologies, but will exclude antibody or enzyme linked immunosorbent assays (ELISA) and genetic-based testing.

II.

MICROORGANISMS

Microorganisms are single to multi-celled microscopic living units that are generally categorized as bacteria, viruses, yeasts, molds, algae, and protozoa. Microorganisms are ubiquitous, including but not limited to humans, animals, plants, soil and water. They first appeared on earth over 3 billion years ago and have coexisted with humans providing both beneficial and detrimental consequences on our food supply (2). Of most concern for food quality and safety have been bacteria, yeasts, and molds. While other microorganisms are still significant, this chapter will focus on the aforementioned three.

A.

BACTERIA

Although bacteria are extremely diverse with great differences in their structural and biochemical components, they share a basic cellular organization. Bacteria are the smallest and fastest growing of all living cells. They have physical shapes that are generally classified as cocci (round) or rods. Rods can be straight, spiral, or curved in


appearance. Often, the types of morphological forms observed depend upon environmental conditions in which the bacterial cells are cultured. Depending on the conditions present, cells may differentiate into spores, a dormant state, or form actively growing cells referred to as vegetative (3). Spores, commonly referred to as endospores, are produced as a bacterial survival strategy. Some vegetative cells form spores during periods of environmental stress such as depletion of nutrients or moisture needed for growth. Spores exhibit no metabolism and are capable of withstanding adverse conditions such as heat, radiation, disinfectants, desiccation, and ultraviolet light. When optimum environmental conditions are introduced, spores germinate and form a single vegetative bacterial cell. Common spore-formers that are important to the food industry are Gram-positive members of the Bacillus and Clostridium genera (4).

B.

YEASTS

Yeasts are members of a higher group of microorganisms called fungi. They are single-celled organisms of spherical, elliptical, or cylindrical shape. Their size varies greatly but is generally larger than bacterial cells. Yeasts may be divided generally into two groups according to their method of reproduction; those that reproduce by budding only (asporogenous), and those that reproduce by both budding and spore formation. Unlike bacterial spores, yeasts form spores as a method of reproduction (3).

C.

MOLDS

Molds are filamentous, multi–celled fungi with an average size larger than both bacteria and yeasts (10 ⫻ 40 µm). Each filament is referred to as a hypha. Mats of hyphae that spread over a food substrate are called mycelia. Molds may reproduce either asexually or sexually, sometimes both within the same species (3). Some mold species produce only one type of asexual spore, while others are capable of producing several types. Most mold species produce asexual spores called conidia. Some molds reproduce asexually through a process of fragmentation, in which the hyphae separate into individual cells called arthrospores. Other mold asexual spore states include zoospores or chlamydospores among others. Asexual spores may be produced in the tip of fruiting hyphae, along the length of the hyphae, or in swollen structures called sporangia. When reproducing sexually, sexual spores are produced by nuclear fission in times of unfavorable conditions to ensure survival.

III.

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limited to the following: nutrient content; water activity; pH; reduction-oxidation (redox) potential; temperature; humidity; available oxygen; biological structures; and antimicrobial constituents. These factors all play a role in the survival, growth and subsequent recovery of microorganisms (3).

A.

NUTRIENT REQUIREMENTS

Microbial growth is achieved through the synthesis of cellular components and energy from the catabolism of nutrients. All of the nutrients necessary for growth are obtained through the immediate environment of the microbial cell. Essential nutrients required for microbial growth include a carbon source such as sugar, or compounds with sugar moieties, a nitrogen source such as ammonia, nitrates or other compounds, and growth factors such as vitamins, minerals, and water. From these building blocks, microorganisms can synthesize carbohydrates, proteins, and lipids needed to produce complex cellular structures like the cell wall, membranes and organelles (4).

B.

MOISTURE CONTENT

Water is one of the most important factors involved in the degradation of food. Water activity (aw) is a measure of free unbound water and is defined as the ratio of the vapor pressure of water in a food, P, to the vapor pressure of pure water, P0, at the same temperature. Most fresh, raw food products have aw levels of 0.98 or higher, which will support growth of most microorganisms. Generally, Gramnegative bacteria have the highest aw requirement ranging from 0.99–0.88, while some yeasts and molds are capable of growing at much lower aw than bacteria (4). There is a correlation between aw, temperature, and nutrition. At any given temperature, microorganisms have a reduced ability to grow when the aw is lowered. Also, when nutrients are present, there is an increase in the range of aw in which microorganisms are able to survive (3).

C.

PH

The term pH is defined as the logarithm of the reciprocal hydrogen ion concentration in solution. The measure of the hydrogen ion concentration, or pH, generally corresponds to the degree of acidity in a sample (5). The majority of microorganisms thrive in a neutral pH (6.6–7.5), although some microorganisms are capable of growing below a pH of 4.0 (3). Other bacteria, such as Alicyclobacillus spp., are known as acidophiles and grow only at lower pH levels found in acidic foods such as fruit juices.

MICROBIAL GROWTH FACTORS

There are a number of factors that affect the survival and growth of microorganisms in food and in culture media. Intrinsic factors inherent to the food include but are not

D. REDOX POTENTIAL (EH) The reduction–oxidation or redox potential (Eh) measures the potential difference generated by a coupled reaction in

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which one substance is oxidized and the second substance is reduced simultaneously. The reduced substance gains electrons while the oxidized substance loses electrons. When electrons are transferred, there is a potential difference created which can be measured electrometrically with most pH meters (measured in millivolts or mV). Microorganisms can be grouped according to the Eh range capable of supporting growth. Aerobes grow between the range of ⫹500 and ⫹300 mV, facultative anaerobes grow best between ⫹300 and ⫺100 mV, and anaerobes are most capable of growing between ⫹100 and ⫺250 mV and less (2). Redox potentials are dependant on pH and are useful in determining the degree of anaerobiosis. Eh can also be estimated by using common redox dyes such as methylene blue and resazurin (5). The redox potential of a specific food can be influenced by several factors: the characteristic redox potential of the original food, the poising capacity, the oxygen tension of the atmosphere around the food and the access of the atmosphere to the food (3).

E.

TEMPERATURE

Microorganisms are capable of growing over a wide range of temperatures. The simplest way of organizing these organisms is placing them in three categories: psychrophiles, mesophiles and thermophiles. Psychrophiles have an optimum growth temperature ranging from 10°C to 15°C, but can also grow well at or below 7°C and may grow at sub 0°C temperatures. These bacteria may produce spoilage or safety issues in refrigerated foods. Mesophiles grow well between 20°C and 45°C with an optimum between 30°C and 40°C. Although mesophiles are often found on foods held under refrigeration temperatures, they are usually not capable of proliferation at those temperatures. Those mesophiles that can grow under refrigeration are called psychrotrophs. Their growth is best at mesophilic temperature and very slow under refrigeration. Thermophiles require high temperatures for growth with an optimum range between 55°C and 65°C. The bacteria included in this category are of special interest in the canning industry (3). It should be noted that some microorganisms of importance to food spoilage are true psychrophiles (“cold loving”) and will only grow under refrigeration conditions. An example, Mrakia frigida, is a fermentative yeast isolated from some spoiled, chilled fruit juices.

F. RELATIVE HUMIDITY Relative humidity is important with respect to the aw within foods and the growth of microorganisms at the surface of foods. Relative humidity and temperature have a relationship that should be carefully considered when storing foods. Generally, an inverse relationship exists in that the higher the temperature, the lower the relative humidity, and vice versa. It is important that foods under storage conditions are exposed to an appropriate relative humidity that inhibits microbial growth. The best way to

prevent surface spoilage of foods such as chicken and beef is to store them at a low relative humidity (3).

G.

ATMOSPHERE

It is possible to classify microorganisms by oxygen requirements that facilitate their growth and survival. Obligate aerobes require oxygen, while facultative organisms are capable of growing in the presence or absence of oxygen. Microaerophilic organisms grow best at very low levels of oxygen. Aerotolerant anaerobes do not require oxygen for growth but are not harmed if oxygen is present. Obligate anaerobes can only grow in complete absence of oxygen and if present oxygen can be lethal (6). There are two important atmospheric gases that exhibit antimicrobial properties. The most important is carbon dioxide (CO2), which is commonly used as a food preservative. It has been known since 1882 that raising the CO2 concentration in the immediate environment of fresh meat will increase the shelf life of the product (3). In a study utilizing modified atmosphere packaging with chilled storage temperatures, growth of Aeromonas hydrophila on fresh turkey and pork slices was strongly inhibited at 1°C, especially in those meats packaged with CO2. No growth of A. hydrophila was observed on the pork or turkey at 40/60 CO2/O2 concentrations (7). Ozone (O3) is another important atmospheric gas that displays antimicrobial properties. This gas has been shown to be effective against many microorganisms. Since ozone is a strong oxidizing agent, it should not be used on foods that have high lipid contents due to the likelihood of rancidity development from the oxidized fats. In 1997, ozone was granted GRAS (generally recognized as safe) status in the United States for food use (3). While Escherichia coli O157:H7 can be destroyed within 20 to 50 minutes upon exposure to ozone at 3 to 18 ppm in culture media (8), its usefulness in foods is not fully realized. One reason for this may be that ozone concentration in the processing plant environment and subsequent employee exposure to the compound must be carefully monitored and controlled to ensure employee safety.

H.

BIOLOGICAL STRUCTURES

There are many foods that have natural exterior barriers that provide protection from spoilage microorganisms. Some examples are the shells of nuts and eggs, the hides of animals, and the outer coverings of fruits and vegetables. Once these protective barriers have been compromised, the interior substances are generally susceptible to the invasion of microorganisms (3).

I.

ANTIMICROBIAL CONSTITUENTS

As part of the natural protection against microorganisms, many foods contain constituents such as essential oils that may have antimicrobial properties. Some examples of these


oils are eugenol in cloves and allicin in garlic. Cow’s milk contains antimicrobial substances such as lactoferrin, conglutinin, and the lactoperoxidase system (3). Lactoperoxidase is an enzyme that is naturally present in raw milk, saliva, colostrums, and other natural secretions. An antimicrobial compound is formed when this enzyme reacts with thiocyanate in the presence of hydrogen peroxide. This reaction is commonly referred to as the lactoperoxidase system (4). Ground beef studies have demonstrated that the lactoperoxidase system is effective against food-borne pathogens. A broad range of food products may be treated with the lactoperoxidase system in order to prevent the growth of pathogenic microorganisms (9).

IV. SAMPLING PLANS Since it is not practical to examine an entire lot of food for the presence or absence of microorganisms, an appropriate sampling plan must be employed. Statistical methods of population probability must be utilized to determine the number and size of sample units needed to produce statistically valid analytical results (4).

A.

ATTRIBUTE PLANS

When shipments of food are received, very limited information may accompany the shipment about the processing methods. Attribute plans are most appropriate for this type of situation. One of the simplest ways to choose whether to accept or reject a food lot can be based upon the results from a microbiological test performed on several sample units (n). Attribute plans test for the presence or absence of a microorganism. Concentrations of microorganisms can be allocated to a specific attribute class by observing whether they are above or below a preset concentration (10).

B.

VARIABLE PLANS

Unlike attribute plans, variable plans are utilized when the distribution of microorganisms is known or can be estimated. Variable plans can be considered more useful than attribute plans because they determine microbial counts instead of assigning counts to categories or ranges. The greatest advantage of variable plans is that fewer samples are required, which in turn results in a lower cost to obtain the same protection as a single attribute plan. The most significant disadvantage includes the numerous calculations involved in evaluating a lot, the multiple calculations needed for each variable, and the requirement that a probability distribution must be known or assumed for each measurement (10).

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prepared stained isolates. The following summarizes the common types of microscopy used by researchers and the food industry.

A.

LIGHT MICROSCOPY

The invention of the light microscope is first accredited to Antony van Leeuwenhoek in 1668. It consisted of a single lens that was moved by a screw mechanism. Today there are an enormous variety of microscopes that utilize numerous light microscopy techniques to study microbes and their environment. The light microscope has many limitations, but this form of microscopy is usually the first employed for microbiological evaluation of food and water. A light microscope consists of three essential components, the eyepiece, the objective, and an illumination source, with the last two components having the greatest influence on image quality (11). A major disadvantage of the microscopic examination of foods is that particulate matter often interferes with the observation.

B.

PHASE CONTRAST MICROSCOPY

Presently, there are small and easily useable phase contrast microscopes that are capable of quickly identifying microbial characteristics such as morphology, motility, and spores. A limiting factor for this type of microscopy is that there must be a fairly high bacterial count, greater than 105 CFU/mL, before the cells would be visible. Utilizing a Petroff-Hauser cell counter or other suitable counting device, it is possible to conduct a direct microscopic count to estimate the cell density viewed with a phase contrast microscope (2).

C.

EPIFLUORESCENCE MICROSCOPY

Epifluorescence microscopy or EFM is a visual set-up for a fluorescence microscope where the objective lens is used to focus ultraviolet light on the specimen and collect fluorescent light. This form of microscopy has a greater efficiency than transmitted fluorescence, in which a separate lens or condenser is utilized to focus ultraviolet light on the sample. Epifluorescence allows for fluorescence microscopy to be merged with another type of microscopy in the same device (12). A recent study comparing the effectiveness of virus enumeration using an epifluorescence microscope, transmission electron microscope, and flow cytometry demonstrated that the epifluorescence microscope displayed the greatest accuracy and precision. The epifluorescence microscope deviated less than 5% from the true and relative errors. It was also shown that an EFM is considerably more time and cost efficient than a transmission electron microscope (13).

V. MICROSCOPY One of the first steps in the identification of microorganisms is the direct examination of the isolated organism using microscopy. The organisms can be viewed live or as

D. TRANSMISSION ELECTRON MICROSCOPY A transmission electron microscope (TEM) consists of an electron gun, which generates electrons, a series of

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condenser lenses that focuses the electron beam onto the specimen, a goniometer stage that manipulates the specimen under the electron beam, another series of lenses that produces a magnified image, and a phosphor screen on which the image of the specimen being observed is projected. There are several disadvantages to TEM that restrict its usefulness. These include specimen stability in vacuum, the need for very thin samples to produce an image, and damaging effects on the specimen produced by the high current density of the electron beam (14).

E.

SCANNING ELECTRON MICROSCOPY

The scanning electron microscope (SEM) uses a thinly focused electron beam to scan the surface of a sample and produce high quality images with magnification up to 100,000X. SEM also has a large depth of field that enables the entire surface of the specimen to be focused (14). With the use of TEM and SEM, it is possible to observe the effects of various technologies to inactivate microorganisms present in foods. With these advanced microscopes, changes in the cell cytoplasm and cell membrane can be clearly observed (15).

F. LOW-VOLTAGE FIELD-EMISSION SCANNING ELECTRON MICROSCOPY Field-emission scanning electron microscopes (FESEM) are capable of producing clear sharp images of superior resolution to conventional SEM. One distinct advantage to FESEM is that low kinetic energy electrons are able to probe close to the specimen surface to produce high quality, low voltage images with minor electrical charging of samples. With FESEM, there is no need to introduce conducting coatings on insulating materials (16). Testing procedures for bacterial microfiltration membranes often utilize FESEM to observe entrapment of bacteria in the membrane matrix (17).

G. VARIABLE-PRESSURE SCANNING ELECTRON MICROSCOPY A scanning electron microscope functions in high vacuum mode by scanning a focused beam of high-energy electrons over the surface of a sample. The advantage of variable pressure modes is that they allow for the microscopy of wet, oily and nonconductive specimens in their natural state without the addition of conventional sample preparation and coating (18).

H.

CRYO-SCANNING ELECTRON MICROSCOPY

The most direct approach for electron microscopy of organic material is through the imaging of fast-frozen samples. This method does not require chemical fixation and drying of artifacts that might influence results. One

advantage to this method is that charged artifacts and the effects of beam damage are greatly decreased. This method is particularly suitable for high-pressure frozen samples (19).

I. ENVIRONMENTAL SCANNING ELECTRON MICROSCOPY A major advantage of ESEM is that it is capable of being operated with a low vacuum in the specimen chamber. Conventional scanning electron microscopy requires a moderately high vacuum in the specimen chamber in order to avoid atmospheric interference with primary or secondary electrons. This method is termed “wet mode” imaging. This method allows the specimen chamber to be isolated from the rest of the vacuum system by valves, pressure-limiting apertures, and a large-diameter bypass tube. The most commonly used imaging gas is water vapor and is controlled by a separate vacuum pump in the specimen chamber. The electron beam of ESEM consists of primary electrons and ejects secondary electrons from the surface of the sample. As a result, the secondary electrons collide with water molecules, functioning as a cascade amplifier to deliver the secondary electron signal to the positively biased gaseous secondary electron detector (GSED). Due to the loss of electrons in this exchange, the water molecules become positively ionized and are attracted to the specimen which may be nonconductive and uncoated and acts to neutralize the negative charge produced by the primary electron beam (20).

J.

CONFOCAL LASER SCANNING MICROSCOPY

Confocal laser scanning microscopy (CLSM) has many advantages over light microscopy in that there is improved contrast at high resolution, improved resolution in fluorescent specimens, and improved depth resolution which allows for optical sectioning of the specimen being observed. Compared to transmission electron microscopy, CLSM has poorer resolution but requires less specimen preparation and is capable of relaying three-dimensional information about internal structures with greater convenience. Regarding biological applications, CLSM was mainly developed with fluorescent staining and especially for localization with fluorescent markers used to derive three-dimensional images (21). In a recent CSLM study, viable and nonviable cells of E. coli O157:H7 that were labeled with a fluorescent antibody were observed on fresh fruits and vegetables (22).

VI.

DETECTION AND IDENTIFICATION

There are numerous methods that can be utilized for the evaluation or detection of microorganisms. They are broadly grouped as quantitative or qualitative methods. The quantitative methods are used to enumerate or to


directly or indirectly estimate the microbial load in a test sample. Some examples of quantitative methods used are aerobic plate counts, coliform counts, yeast and mold counts, direct microscopic counts, and most probable number protocols. Qualitative methods are intended to determine if a sample contains a specific microbial species amidst the total microbial population based upon a presence/absence determination. These methods are commonly used to detect the presence of certain foodborne pathogens including Salmonella, E. coli O157:H7, and Clostridium botulinum (2).

A.

INDICATOR ORGANISMS

Groups of microorganisms, such as Enterobacteriaceae, total coliforms, thermotolerant coliforms, and E. coli, are sometimes utilized to indicate potential contamination of food and water by enteric pathogens. Testing for thermotolerant coliforms is often used as a presumptive test for E. coli, which often indicates fecal contamination. Although testing for total coliforms is sometimes used as an indicator for the possible presence of E. coli, the usefulness of this test method is questionable since there are many coliforms that are not of fecal origin. Standard methods for the enumeration of indicator organisms rely on the use of specific microbiological media and protocols that isolate and enumerate viable cells in the sample (23).

B.

CULTURE MEDIA

Bacteria are differentiated using several major characteristics, including cultural requirements of each microorganism. The nutrients that each organism requires for optimal growth can be used to identify it from closely related organisms. 1.

Selective Media

Many media contain selective components that prevent the growth of non-target microorganisms. Selective media are useful in the isolation of specific microorganisms from mixed populations. In many media, compounds are included as sole sources of carbon or nitrogen so that only a few types of microorganisms can proliferate. Selective toxic compounds are also frequently added to select for the cultivation of particular microorganisms. These toxic compounds are incorporated into media to suppress the growth of the background microbiota while allowing for the cultivation of the target microbe. Examples of toxic chemicals are bile salts, azide, sodium lauryl sulfate, and various dyes such as crystal violet and methylene blue (24). 2.

Differential Media

The use of certain chemicals, food sources, and reagents can result in a pattern of growth or change in expression

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that can be used to differentiate between different types of microorganisms (23). An example of this type of media is blood agar plates. Some bacteria can hemolyze the red blood cell, resulting in a clear zone on the agar. These bacteria can easily be distinguished from microorganisms that do not produce hemolysins. In this example, the blood-agar plates serve both as a differential medium and as a source for enrichment. 3.

Synthetic Media

A synthetic medium is one that is composed completely of chemically defined nutrients. Most synthetic media contain a mineral base, which provides the inorganic nutrients necessary for microbial growth. This base can then be supplemented, as required, with a carbon source, a nitrogen source, and any required growth factors. These supplements will vary with the nutritional properties of the particular organism being cultured (25). 4.

Complex Media

Complex media is one that contains ingredients of unknown chemical composition. An example would be potato dextrose agar, which contains a potato extract of unknown composition. Complex media are useful for the cultivation of a wide range of microorganisms, including those whose precise growth-factor requirements are unknown. Even when the growth-factor requirements of a microorganism have been precisely determined, it is often more convenient to grow that organism in a complex medium, especially if the growth-factor requirements are numerous (25). 5.

Liquid Media

Many larger-celled bacteria, protozoa, and algae are not capable of growth on solid media. Often, these microorganisms are easily isolated by the use of liquid media. The simplest procedure of isolation in liquid media is the dilution method. The sample is serially diluted into a sterile medium, with the goal being to inoculate a series of tubes with a microbial suspension so dilute that the probability of introducing even one individual into a given tube is very small. From this, if a tube shows any growth, there is a high probability that the growth resulted from the introduction of a single organism (25). 6.

Enrichments

When a mixed microbial population is introduced into a liquid selective medium, competition for nutrients among that population will arise. Liquid enrichment media will select the microorganism of highest growth rate among all the members of microbial population. The resulting growth from enrichment can be greatly modified by variation of

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other factors such as temperature, pH, aeration or source of inoculum (25). 7.

Commercially Available Agars

Several companies market selective and differential agars targeting common pathogens. These have been used for the rapid screening of pathogens found on food. ALOA™ is a prepared selective and differential medium for the isolation of Listeria spp. from foodstuffs and other samples and for the presumptive identification of Listeria monocytogenes (26). The selectivity of the medium is due to lithium chloride and the addition of an antimicrobial mixture. The differential activity is due to the presence in the medium of the chromogenic compound X-glucoside as a substrate for the detection of beta-glucosidase enzyme, common to all Listeria species. The specificity is obtained by detecting the metabolism of a substrate by an enzyme (phospholipase), which is only present in the L. monocytogenes species. The combination of both substrates allows the differentiation of non-monocytogenes Listeria spp., which develop blue colonies, from Listeria monocytogenes, which develops blue colonies surrounded by an opaque halo. ASAP™ is a selective medium for the isolation of Salmonella from foodstuffs, clinical, and environment samples. The activity of the C8-esterase, which is found in all Salmonella species, is detected using a chromogenic substrate. The enzymatic activity of Salmonella is visualized by the pink to purple coloration of their colonies. Biolog, Inc. manufactures several types of differential media such as Rainbow® Agar O157 and Rainbow® Agar Salmonella. The selective and chromogenic properties of Rainbow® Agar O157 make it particularly useful for isolating pathogenic E. coli strains. The medium contains chromogenic substrates that are specific for two E. coli-associated enzymes: β-galactosidase and β-glucuronidase. Rainbow® Agar Salmonella utilizes a formulation designed to take advantage of H2S production common among Salmonella spp. Black colonies are formed by even weak H2S-producing strains. In addition, other compounds increase the recovery rate of Salmonella while inhibiting the growth of other microorganisms and inhibiting H2S production by Citrobacter and other H2S positive species (27). Chromagenic Shigella spp. Plating Medium (CSPM) (28) is a selective medium (bile salts, antibiotic supplementation) that offers an alternative to differentiation methods that are based on lactose fermentation. Instead, differentiation on CSPM is based on proprietary agents consisting of select carbohydrates, pH indicators, and chromagens. Shigella spp., which are negative for the select carbohydrates and the chromagens, produce white to clear colonies on CSPM. Colonies of Enterobacter spp., Klebsiella spp., and Acenitobacter spp. are blue on CSPM, while colonies of Citrobacter spp. are green.

C.

CULTURING METHODS

Almost all measurements in microbiology are method dependent. When selecting the methods to be used in the laboratory, it is vital to utilize published standard methods. Sources of standard culture methods that are commonly used by food microbiologists include the Official Methods of Analysis of AOAC International, Compendium of Methods for the Microbiological Examination of Foods published by APHA, FDA’s Bacteriological Analytical Manual, and USDA FSIS’s Microbiological Laboratory Guidebook. 1.

AOAC

The Association of Analytical Communities (AOAC) International is a worldwide provider and facilitator in the development and use of validated analytical methods and laboratory quality assurance programs and services. Primarily, the AOAC focuses on the validation of chemical and microbiological analytical methods. AOAC International also acts as the primary source for knowledge exchange, networking, and high-quality laboratory information for its members. In order to establish these goals, AOAC International has three method validation programs: AOAC® Official MethodsSM Program®, PeerVerified MethodsSM Program, and AOAC® Performance Tested MethodsSM Program. Methods validated by AOAC International are utilized worldwide by various governments and industries for the analysis for a variety of products, especially those related to food, agriculture, public health and safety, and the environment (29). 2. American Public Health Association Standard Methods The Compendium of Methods for the Microbiological Examination of Foods is widely utilized by food microbiologists as a collection of standard methods for microbiological detection and enumeration techniques. It is published by the American Public Health Association and is a comprehensive and all-inclusive reference for protocols related to specific food products. The Compendium presents information on standard enumeration/detection procedures, sampling plans and analysis preparations, indicator organisms, microbiological aspects of specific food products, and methods related to specific spoilage and safety-related microorganisms. 3. Food and Drug Administration’s Bacteriological Analytical Manual FDA’s Bacteriological Analytical Manual or BAM is a compilation of methods and techniques preferred by analysts in the U.S. Food and Drug Administration laboratories for the detection of pathogens in food and cosmetic


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products. Methods included in the BAM have been analyzed and peer reviewed by FDA scientists and other researchers (30). BAM methods have been organized into an accessible spreadsheet format and are complimentary in the hopes of increasing awareness of microbial safety of food and water (31). These methods are available on line at http://www.cfsan.fda.gov/~ebam/bam-mm.html.

and nonviable cells. DMC is most efficient when there are a large number of microorganisms in the sample (greater than 105 CFU/mL). Due to the fact that DMC cannot differentiate between live and dead cells and requires a large number of cells in the test sample, its use for food analyses is limited for quality issues in which a gross, highly variable enumeration is adequate (4).

4. United States Department of Agriculture, Food Safety Inspection Service’s Microbiology Laboratory Guidebook

B.

The Microbiology Laboratory Guidebook (MLG) is a manual created by the USDA agency FSIS to aid in the microbiological analysis of meat, poultry, and egg products. The MLG contains techniques that FSIS prefers to employ for the analysis of these foods. Because USDA does not endorse or approve these techniques for use in the food industry, inclusion of a specific method in the MLG should not be interpreted in this manner (32). These methods are available on line at http://www.fsis.usda.gov/ophs/microlab/mlgbook.htm.

VII. ENUMERATION OF VIABLE CELLS Standard plate counts, Most Probable Number (MPN), membrane filtration, plate loop methods, and spiral plating are techniques that allow for the estimation of viable cells. These methods can be successfully utilized in the food industry to enumerate fermentation, spoilage, pathogenic, and indicator organisms (6). Several pathogens of interest such as Salmonella, Campylobacter spp., pathogenic E. coli and Vibrio spp. are not capable of sporulation; however, they may exist in a viable but non-culturable (VNC) state in which they cannot be cultured using normal microbiological techniques (4). VNC cells are typically severely injured and incapable of reproduction under standard protocols. Pathogenic Vibrio parahaemolyticus is known to demonstrate a VNC state when subjected to low incubation temperatures and starvation. Previously, it was unknown whether this microorganism was capable of resuscitation or if regrowth of a few remaining culturable cells occurred. Recent studies have shown that the VNC cells are capable of being resuscitated after plating onto agar containing H2O2-degrading compounds such as catalase or sodium pyruvate (33). It is unknown whether VNC pathogens are capable of causing illness, but there is concern that this physiological state could result in disease outbreaks from foods that have yielded false negative detection results for specific pathogens.

A.

DIRECT ENUMERATION

The direct microscopic count or DMC is utilized to obtain a gross estimation of cell density that includes both viable

INDIRECT ENUMERATION

Unlike direct enumeration of microbes by microscopy, indirect enumeration can be accomplished by plate counts or statistical estimation. The plate count technique involves spreading a sample on a nutrient agar surface or incorporation of the sample within the agar. If organisms are present and if they are plated on a suitable medium, each will grow a viable unit called a colony. Each colonyforming unit (CFU) can be counted and thus theoretically relates to the viable number of microorganisms in the sample. Statistical estimations, such as the Most Probable Number assay, are useful when the microorganisms in question need an enriched environment to grow or are in very low numbers. 1.

Plate Counts

Aerobic plate counts (APC) indicate the level of microorganisms in a product. Aerobic plate counts generally do not relate to food safety hazards, but sometimes can be useful to indicate quality, shelflife and post-heat-processing contamination. The plating medium used in an APC can affect the number and types of bacteria isolated because of the variation in nutrient and salt requirements of various microorganisms (34). The commercially available 3M Petrifilm™ plate decreases labor by eliminating the need for media preparation. Another benefit of this product is it delivers consistent easy-to-read results, creating a lesser chance for error than with conventional agar methods. There are numerous formulations that allow for several common types of microbial testing, including coliform, aerobic, E. coli, Staphylococcus aureus, and yeast and mold counts. 3M Petrifilm™ plates enable food processors to test products and equipment easily, which can assist in rapid detection and resolution of problem areas. These sample-ready microbial testing products reduce the possibility of human error in test preparation to produce consistent results (35). They have been particularly useful in field application due to the reduced need for incubator space. One disadvantage of Petrifilm™ is that samples with numerous particles may be difficult to read. 2.

Most Probable Number (MPN)

The Most Probable Number or MPN is a broth dilution technique that is especially useful when establishing low

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concentrations of microrganisms, specifically less than 100 per gram. The method as described in the classic paper by Oblinger and Koburger (36) has several advantages over other enumeration procedures. One advantage is that the sample is prepared to ensure that bacteria are distributed randomly and do not clump together thereby giving an equal opportunity for any one sample to contain viable cells if they are present in the food. Also, culture medium and incubation conditions are carefully chosen to encourage even one viable organism to generate detectable growth. The amount of food sample that produces growth at each dilution will suggest an estimate of the original, undiluted concentration of bacteria in the sample (30). Isogrid is a hydrophobic grid membrane filter method utilized for the detection and quantification of target microorganisms such as Salmonella spp., Listeria spp., total E. coli and E. coli O157:H7, coliforms, S. aureus, yeast and mold, and total bacteria count. A sample’s total bacterial count is enumerated through a membrane filter containing a grid of 1,600 squares. Diluted samples are filtered through a 5 µm stainless steel prefilter to eliminate any food particles. Samples are then filtered through a hydrophobic membrane, and the membrane is placed on Tryptic Soy Fast Green Agar (TSFGA). TSFGA is specially formulated to provide a total bacterial count. After incubation the membrane filter is examined, and all squares containing one or more green or blue colonies are counted. The total number of positive squares is converted to the corresponding Most Probable Number (MPN) determined by the Isogrid manual (37). As with Petrifilm™, samples that contain large numbers of particles may obscure the results. This system has been used for milk, meat, black pepper, flour, mushrooms, seafood, and oysters (38–41). The SimPlate™ system includes specific formulations of media and a patented plating device. The SimPlate™ device has a broad counting range to minimize the number of dilutions needed for accurate counts. This unique combination of media and plating device offers advantages over some other techniques, including greater accuracy, ease of use, and faster time to obtain results. SimPlate™ tests are available for the quantification of total plate counts (TPC), total coliforms and E. coli, yeast and mold, and Campylobacter. Another benefit of the SimPlate™ is that positive and negative results can be easily distinguished by simply counting the number of positive wells and referring to the SimPlate™ Conversion table provided with the kit. SimPlate™ results are usually available in 24 hours, with the exception of Campylobacter and yeast and mold, which takes 48–72 hours, still days faster than other methods. SimPlate™ media comes pre-measured and ready to hydrate for single or multiple tests. While agar plate and film counting ranges may be limited to 300 CFU or less, SimPlate™ devices are available in two sizes with maximum counting ranges of 738 or 1659 CFU per plate.

The number of dilutions and reruns are minimized, saving labor, time, and material costs (42).

VIII. METABOLIC ACTIVITY MEASUREMENT Dye reduction tests, acid production, electrical impedance, and batteries of other metabolic assays are all used to determine cell population sizes and to identify specific isolates. The level of bacterial activity can be used to assess the quality and freshness of food products. Toxin levels can also be measured to indicate the presence of toxin producing pathogens (6). Measurable undesirable metabolites produced from the action of specific microorganisms on certain food products include histamine in canned tuna, lactic acid in canned vegetables, cadaverine and putrescine in vacuum-packaged beef, and mycotoxins in various cereal and fruit based products (4). Specific substrate utilization can be used to determine the type of microorganism in question. The following sections will discuss common tests used for this purpose. The AOAC has developed a comprehensive list of test kits that are available to measure or detect specific chemicals or microorganisms commonly referred to as analytes. The performance tested certified kits have been extensively reviewed by the AOAC Research Institute, a subsidiary of the AOAC International. All performance tested certified kits have been evaluated for accuracy, precision, detection limits, false positive/negative rates, stability and compared to an already existing method. There are several test kit analyte categories consisting of potential biological and chemical agents, microbiological, antibiotic, toxin, hormones, chemical, biochemical, and genetically modified organisms. The biochemical test kits include analytes such as ATP, sugars, enzymes, DNA, and many others (43).

A.

VITEK

VITEK by bioMérieux is an automated instrument that offers rapid results within 2 to 6 hours, random or batch processing, and a quality control module. This instrument is used for bacterial and yeast identification, antimicrobial susceptibility testing, and urine screening. Since the VITEK is an automated system, this allows for greater safety and eliminates repetitive manual operations to quicken response time and provide faster results (44).

B.

API STRIPS

Another reliable product used for the rapid identification of microbes is API Strips, which generally contain 20 miniature biochemical tests and databases. To date, there are 16 identification products covering most bacterial groups and more than 550 different species. There are test


kits to identify Gram-positive and negative bacteria and also yeasts. API Strips are economical when compared to the cost of preparing numerous biochemical media, are generally user-friendly, and have a long shelf-life (45).

C. BBL CRYSTAL There are many different BBL Crystal miniature identification kits available. Those utilized most for the identification of microorganisms are the Gram-Positive ID Kit, Rapid Gram-Positive ID Kit, and the Anaerobe ID Kit. The Gram-Positive ID kit is the most comprehensive Gram-Positive ID System available. Both the GramPositive ID System and the Rapid Gram-Positive ID System utilize modified conventional, fluorogenic, and chromogenic substrates. These kits are capable of identifying 121 Gram-positive bacteria including cocci and bacilli. The Gram-Positive ID System can be completed in 18 hours while the Rapid Gram-Positive ID System can be completed in 4 hours. The Anaerobe ID kit is a miniaturized 4 hour identification method for clinically significant anaerobes (46).

D. IMPEDANCE Impedance is described as the resistance to the flow of a sinusoidal alternating current through a conducting material. Detection of cells by electrical impedance is determined from changes in the growth medium associated with the increase in biomass of metabolically active microorganisms. The components of growth media such as proteins, carbohydrates, and lipids are uncharged or weakly charged substrates. As microorganisms metabolize these compounds, they are converted into more highly charged molecules such as amino acids, lactate, and acetate. Consequently, these metabolic products increase the conductivity of the growth medium. Because this increase is small, a microbial level of at least 106 CFU/mL must be attained before significant changes can be observed. There are also disadvantages to electrical impedance method. The most critical disadvantage is that this method cannot function under high salt concentrations that are often found in particular growth media (5). In a recent study to determine the efficiency of a modified impedance method, numerous samples of ground beef were inoculated with 12 different serotypes of Salmonella. The method proved to be a reliable way for rapid detection of different Salmonella serotypes in fresh meat (47). There are several commercial systems available that utilize a color system to monitor levels of contamination. Red generally depicts samples that are “highly contaminated”; yellow signifies “caution zones” and green signifies “acceptable” levels. Common impedance-monitoring devices are the Bactometer, the RABIT, the Malthus and the BacTrac (5).

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The Bactometer by bioMérieux can be utilized to test raw materials and finished products. It is used to make important quality control decisions quickly, prevents unnecessary delays in production, reduces testing costs, and helps protect companies’ reputations. The Bactometer consists of a processing unit (BPU), computer, and printer. The Bactometer analyzes samples that are simultaneously incubated and read by the BPU every six minutes. The computer continuously monitors operations and interprets the results. The terminal allows all results to be displayed quickly at any time. The Bactometer can be used to assay for total microbial counts, coliforms, yeast and mold, lactic acid bacteria, shelf-life testing, and environmental monitoring. Typical testing times can be reduced to less than 48 hours while conventional methods can take up to 5 days. Additional benefits associated with the Bactometer are a 512 sample capacity, performing different tests simultaneously, and working with a wide temperature range, all of which increase laboratory efficiency (50).

E.

BIOSYS

The BioSys system is a computerized instrument designed to rapidly detect microbial contamination in industrial samples. It can be applied to foods, beverages, dairy, wine, cosmetics, toiletries, and nutraceuticals (48,49). The system has applications for the detection of the presence of various groups of microorganisms in food samples or swabs (e.g., Total Viable Count, Enterobacteriaceae, Coliforms, Yeast, Lactic Acid Bacteria, E. coli, etc.). Other tests performed by the system are the detection of spoilage microorganisms, shelflife assessment, microbial limits and preservative challenge test, and environmental sponges and swabs for the presence of certain organisms such as Listeria spp. BioSys system uses vials consisting of a nutrient broth with an agar plug at the bottom. The system measures microbial growth by monitoring changes in pH or other biochemical reactions, thereby resulting in a color change as microorganisms grow and metabolize. Color changes in the agar mirror the color change in the broth, without letting the sample particles or turbidity influence the measurements. Light from light emitting diodes passes though the agar and a photo diode on the other side of the vial reads the color change as microbial growth occurs. A measurement is taken every six minutes. As soon as a color change is detected, the time of such detection is recorded. Detection times (DT) are inversely related to the number of organisms in the sample.

F. BIOLUMINESCENCE Bioluminescence is defined as the production of light by living organisms. Common bioluminescent organisms

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include certain fungi and bacteria that continuously emit light. The production of bioluminescent light is formed as a result of the conversion of chemical energy to light energy (51). Adenosine triphosphate (ATP) is the main source of energy in all living organisms. Two hours after cell death ATP completely disappears. One of the simplest techniques that measures ATP is the firefly luciferinluciferase test. When ATP is present, luciferase produces light that is measured with a liquid scintillation spectrometer or a luminometer. The amount of light that is produced by the luciferase is directly proportional to the amount of ATP. The luciferase method is an example of a rapid and sensitive test that indicates the presence of bacterial cells (3). A rapid system has been developed using bioluminescence to detect bacteria with minimal cultivation. Food samples are cultivated to produce a sufficient amount of ATP and are sprayed with luminescent reagents. Bioluminescent cells appear as spots to indicate the presence of bacteria in the sample. This method of detection is fast and is useful in hygiene testing for food, beverages, and water (52). In a recent study, ground beef containing E. coli O157:H7 was analyzed using a luciferin-luciferase assay. It was determined that in the presence of glucose, the cellular ATP of the E. coli increased causing significant luciferin-luciferase bioluminescence in beef hamburger. This method, coupled with an immunomagnetic capture technique allowed for rapid detection of viable E. coli O157:H7 cells in certain food samples (53).

G.

ATP TEST KITS

There are many different ATP test kits available that are used as a measure of total hygiene after sanitation or are used to detect presence/absence of microorganisms in samples. Bev-Trace™ is an example of an ATP test specially designed for the rapid detection of microbial contamination in beverages, such as beer, wine, and soft drinks (54). Results from ATP tests can be obtained faster than traditional techniques, saving time and money. The luminometers that are commonly used with this type of test are usually lightweight, easy to operate, and might be able to provide printed results. Some key features of these tests are earlier detection of contamination, decreased chance of recall, brand protection, and reduced stock holding requirements. Another type of ATP test kit is used as a sanitation check on equipment surfaces. An example is the PocketSwab (55). These types of test kits are generally self contained, single service hygiene tests that use the firefly luciferin-luciferase enzyme system to emit light when unclean surfaces are detected. Tests are capable of verifying within intervals as short as 30 seconds if a surface is clean enough for production. ATP from food residues, microorganisms, biofilms, and human contact

are detected to provide a quick check for cleaning effectiveness that enhances overall food safety.

IX. OTHER DEVELOPMENTS IN MICROBIAL TESTING Rapid methods for microbiological analysis of foods also include improvements in sample preparation. One of the more useful instruments developed for sample preparation is the Seward/Tekmar Stomacher™ (Norfolk, UK) and similar devices (56). After transferring a food sample to a sterile plastic bag that contains appropriate diluents, the sample is placed within the stomacher where two paddles massage the bag for 1 to 5 minutes or longer using alternate strokes. The massage action simulates activity of the human stomach to mix the sample and dislodge microorganisms for further microbiological analysis, such as plate counting of viable cells. Conventional viable cell count, or standard plate count, is tedious and time consuming. As a result, alternative methods to enumerate colony forming units within a food sample have been developed. One particular instrument, the spiral plater has gained wide acceptance and has become an essential element in many food microbiology laboratories (56). This instrument spreads a liquid sample onto an agar surface in an Archimedes spiral pattern. The sample is applied such that there is a concentration gradient starting from the center and decreasing as the spiral progresses outward on the plate. After the liquid containing the microorganism is spread, the plate is incubated to allow colonies to develop. The colonies are then counted either manually or electronically. The sample gradient is spread across the agar surface in such a manner that the equivalent of three standard dilutions can be read from one plate. This substantially decreases sample preparation and plating time and reduces supplies needed to enumerate microorganisms in a sample. Major advancements in spiral platers include automation and plate readers. Automated spiral platers require little more than the presentation of a liquid sample followed by the press of a button. This activates the plater to apply an appropriate size sample to an agar surface after which the instrument goes through a clean and sanitation cycle. Despite the benefit of automation, there have been complications involving clogging of the dispensing stylus by large food particles. This problem was largely eliminated with the use of sterile sample preparation bags with filters that separate large food particles from the liquid sample (56). Automated plate readers are designed to allow realtime enumeration of colonies on a plate. Scanners originally used with the early spiral platers were based on laser technology. Automatic readers today often utilize camera imaging coupled with sophisticated software to detect and count colonies. These readers can be utilized with any type of plating method; pour, spread, or spiral.


X.

SUMMARY

No review can completely cover all the methods currently available, especially when considering that products continuously enter and leave the marketplace. It is critically important for safety and quality reasons to conduct microbiological analyses of food samples. Some microorganisms that are occasionally found in food products can cause illness or death, while other microorganisms produce spoilage and economic losses. Numerous variables are considered during the selection of a method to detect or enumerate microorganisms. The analyst must decide if a qualitative or quantitative test is required, and which microorganisms are of importance for the sample. If regulatory issues are involved, an analyst might want to consider the type of sampling plan used, and other factors such as speed, specificity, cost, and ease of use. Relatively rapid methods include direct microscopic observations, ATP assays, and test kits to detect or enumerate specific microorganisms or groups of microorganisms. Conventional methods of enumeration (plate counts or MPNs) generally consume more time and supplies than rapid methods, but are often less expensive and can yield more accurate data.

XI. NON-ENDORSEMENT OF COMMERCIAL PRODUCTS AND SERVICES References, hypertext links and images to all products and services are provided for information only and do not constitute endorsement or warranty, express or implied, by the authors and/or their employers or the publishers of this work, as to their suitability, content, usefulness, functioning, completeness, or accuracy.

ACKNOWLEDGMENT This is Florida Agricultural Experiment Station Journal Series number R-09807.

REFERENCES 1. JP Battershall. Food Adulteration and Its Detection. New York, NY: E. & F.N. SPON, 1887. 2. B Ray. Fundamental Food Microbiology. 2nd ed. Boca Raton, FL: CRC Press, 2001. 3. J Jay. Modern Food Microbiology. 6th ed. New York: Aspen Publishers, Inc., 2000. 4. M Doyle, L Beuchat, TJ Montville. Food Microbiology: Fundamentals and Frontiers. Washington, D.C.: American Society for Microbiology, 1997. 5. FP Downes, K Ito. Compendium of Methods for the Microbiological Examination of Foods. 4th ed. Washington, D.C.: American Public Health Association, 2001.

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6. D Goff. Dairy Microbiology. Retrieved July 18, 2003 from http://www.foodsci.uoguelph.ca/dairyedu/ micro.html 7. SB Mano, JA Ordonez, GD Fernando. Growth/survival of natural flora and Aeromonas hydrophila on refrigerated uncooked pork and turkey packaged in modified atmospheres. Food Microbiol 17:657–669, 2000. 8. MW Byun, OJ Kwon, HS Yook, KS Kim. Gamma irradiation and ozone treatment for inactivation of Escherichia coli O157:H7 in culture media. J Food Prot 61:728–730, 1998. 9. M Kennedy, A O’Rourke, J McLay, R Simmonds. Use of a ground beef model to assess the effect of the lactoperoxidase system on the growth of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus in red meat. Int J Food Microbiol 57:147–158, 2000. 10. ICMSF (International Commission on Microbiological Specifications for Foods). Microorganisms in Foods 7: Microbiological Testing in Food Safety Management. New York: Kluwer Academic/Plenum Publishers, 2002. 11. RW Lovitt, CJ Wright. Microscopy: light microscope. In: RK Robinson, CA Batt, PD Patel. ed. Encyclopedia of Food Microbiology. San Diego, CA: Academic Press, 1999, pp 1379–1388. 12. C Freudenrich. How Light Microscopes Work. How Stuff Works. Retrieved 20 July 2003 from http://science.howstuffworks.com/light-microscope.htm/printable 13. MM Ferris, CL Stoffel, TT Maurer, KL Rowlen. Quantitative intercomparison of transmission electron microscopy, flow cytometry, and epifluorescence microscopy for nanometric particle analysis. Anal Biochem 304:249–256, 2002. 14. UJ Potter, G Love. Microscopy: scanning electron microscopy. In: RK Robinson, CA Batt, PD Patel. ed. Encyclopedia of Food Microbiology. San Diego, CA: Academic Press, 1999, pp 1397–1406. 15. N Dutreux, S Notermans, T Wijtzesa, MM GóngoraNietob, GV Barbosa-Cánovas, BG Swanson. Pulsed electric fields inactivation of attached and free-living Escherichia coli and Listeria innocua under several conditions. Int J Food Microbiol 54:91–98, 2000. 16. PhotoMetrics, Inc. FESEM: Field Emission Scanning Electron Microscopy. Retrieved 4 August 2003 from http://www.photometrics.net/fesem.html 17. SB Sadr-Ghayenia, PJ Beatsona, AJ Fanea, RP Schneider. Bacterial passage through microfiltration membranes in wastewater applications. J Membrane Sci 153:71–82, 1999. 18. N Leddy. Hitachi S-3500N Variable Pressure Scanning Electron Microscope. Retrieved 1 July 2003 and 4 August 2003 from http://cma.tcd.ie/html/s-3500n.html 19. P Walther, M Muller. Double-layer coating for fieldemission cryo-scanning electron microscopy-present state and applications. Scanning 19:343–348, 1997. 20. S Robinson. Environmental Scanning Electron Microscope. Retrieved 5 August 2003 from http://www. itg.uiuc. edu/ms/equipment/microscopes/ esem/ 21. DF Lewis. Microscopy: confocal laser scanning microscopy. In: RK Robinson, CA Batt, PD Patel. ed. Encyclopedia of Food Microbiology. San Diego, CA: Academic Press, 1999, pp 1389–1396.

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22. Y Han, RH Linton, SS Nielsen, PE Nelson. Inactivation of Escherichia coli O157:H7 on surface-uninjured and injured green pepper (Capsicum annuum L.) by chlorine dioxide gas as demonstrated by confocal laser scanning microscopy. Food Microbiol 17:643–655, 2000. 23. MJ Pelczar Jr, RD Reid, ECS Chan. Microbiology. 4th ed. New York: McGraw-Hill Inc, 1977. 24. RM Atlas. Handbook of Microbiological Media. 2nd ed. Boca Raton, FL: CRC Press, Inc., 1997. 25. RY Stanier, JL Ingraham, ML Wheelis, PR Painter. The Microbial World. 5th ed. Englewood Cliffs, NJ: Prentice-Hall, 1986. 26. Microbiology International. ASAP Culture Medium – Product Information. Retrieved from http://www. 800ezmicro.com/ product Details.asp?mb=02&ez=62, 2003. 27. Biolog, Inc. Product Information. Retrieved from http://www.biolog.com/ download_center2.html, 2003. 28. BR Warren. Comparison of conventional culture methods and the polymerase chain reaction for the detection of Shigella on tomato surfaces. Master’s Thesis, University of Florida, Gainesville, FL, 2003. 29. P Cunniff. Official Methods of Analysis of AOAC International. 16th ed. Gaithersburg, MD: AOAC International, 1996. 30. Food and Drug Administration Bacteriological Analytical Manual Online. Retrieved 5 August 2003 from http://vm.cfsan.fda.gov/~ebam/bam-mm.html 31. WE Garthright, RJ Blodgett. FDA’s preferred MPN methods for standard, large or unusual tests, with a spreadsheet. Food Microbiol 20:439–445, 2003. 32. BP Dey, CP Lattuada. Microbiology Laboratory Guidebook. 3rd ed. Retrieved 5 August 2003 from http://www.fsis.usda.gov/ophs/microlab/mlgbook.pdf 33. Y Mizunoe, SN Wai, T Ishikawa, A Takade, S Yoshida. Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation. FEMS Microbiol Lett 186:115–120, 2000. 34. RJ Price. Aerobic Plate Count. Retrieved 6 August 2003 from http://www-seafood.ucdavis.edu/haccp/compendium/chapt09.htm 35. 3M Worldwide. 3M Petrifilm™ Plates. 3M Innovative Solutions Catalogs. Retrieved 29 September 2003 from http://products3.3m.com/catalog/us/en001/government/innovative_solutions/node_GS64S6K84Pbe/root_ GS3RBW6QFVgv/vroot_31S2JJ7584ge/gvel_3FBK2 XCFWDgl/command_AbcPageHandler/theme_us_inno vativesolutions_3_0 36. JL Oblinger, JA Koburger. Understanding and teaching the most probable number technique. J Milk Food Technol 38:540–545, 1975. 37. Neogen Corporation. Foodborne Bacteria Tests. Retrieved 26 September 2003 from http://www. neogen.com/bacteria3.htm 38. P Entis. Enumeration of coliforms in nonfat dry milk and canned custard by hydrophobic grid membrane filter method: Collaborative study. J Assoc Off Anal Chem 66:897, 1982.

39. P Entis. Enumeration of total coliforms and Escherischia coli in foods by hydrophobic grid membrane filters collaborative study. J Assoc Off Anal Chem 67:812, 1984. 40. P Entis, MH Brodsky, AN Sharpe. Effect of prefiltration and enzyme treatment on membrane filtration of foods. J Food Prot 45:812, 1982. 41. AW Sharpe, PI Peterkin. Membrane Filter Food Microbiology. Research Studies Press, Letchworth, UK, 1988. 42. BioControl. SimPlate™. Retrieved 26 September 2003 from http://www.rapidmethods.com/pdf/ SimPlate™_Brochure.pdf 43. AOAC International. Rapid Test Kits. Retrieved 26 September 2003 from http://www.aoac.org/testkits/ Tkdata1.html 44. bioMérieux, Inc. VITEK. Retrieved 26 September 2003 from http://www.bioMérieux-usa.com/clinical/microbiology/vitek/index.htm 45. bioMérieux, Inc. API. Retrieved 26 September 2003 from http://industry.bioMérieux-usa.com/industry/cosmetic/ api/ 46. BD. Clinical Products. Becton, Dickinson and Company. Retrieved 26 September 2003 from http://www.bd.com/ clinical/products /idsus/crystal.asp 47. G Di-Falco, V Giaccone, GP Amerio, E Parisi. 1993. A modified impedance method to detect Salmonella spp. in fresh meat. Food Microbiol 10:421–427, 1993. 48. JA Odumeru, J Belvedere. Validation of the MicroFoss system for enumeration of total viable organisms, coliform, and E. coli in ground beef. J Microbiol Meth 50:33–38, 2002. 49. RF Eden-Firstenberg, D Foti, ST McDougal, J Baker. Optical instrument for the rapid detection of microorganisms in dairy products. Int Dairy J 12:225–232, 2002. 50. bioMérieux, Inc. Bactometer. Retrieved 26 September 2003 from http://industry.bioMérieux-usa.com/industry/ food/bactometer/ 51. Columbia Electronic Encyclopedia. Bioluminescence. 1Up Info. Retrieved 5 August 2003 from http://www.1upinfo.com/encyclopedia/B/biolumin.html 52. H Tanaka, T Shinji, K Sawada, Y Monji, S Seto, M Yajima, O Yagi. Development and application of a bioluminescence ATP assay method for rapid detection of coliform bacteria. Water Res 31:1913–1918, 1997. 53. S Tu, D Patterson, J Uknalis, P Irwin. Detection of Escherichia coli O157:H7 using immunomagnetic capture and luciferin-luciferase ATP measurement. Food Res Int 33:375–380, 2000. 54. Biotrace International. Products: Bev–trace. Retrieved 26 September 2003 from http://www.biotrace.com/content.php?hID=2&nhID=48&pID=3 55. Charm Sciences Inc. ATP Hygiene. Retrieved on 26 September 2003 from http://world.std.com/~charm1/ pocktech.htm 56. HA Konuma, H Kurata. Improved Stomacher 400 bag applicable to the spiral plate system for counting bacteria. Appl Environ Microbiol 44:765, 1982.

186

Immuno-Based Methods for the Detection of Bacterial Pathogens

Keith R. Schneider and Minh Lam


Mickey E. Parish

Lake Alfred, CREC, University of Florida

CONTENTS I. Introduction ......................................................................................................................................................186-1 II. Immunological Methods ..................................................................................................................................186-2 III. Enzyme-Linked Immunosorbent Assay (ELISA) Methods..............................................................................186-2 A. Microtiter Plate Format ............................................................................................................................186-2 IV. Immunoprecipitation ........................................................................................................................................186-3 A. Lateral Flow Devices ................................................................................................................................186-4 B. Dip Stick Assays ......................................................................................................................................186-4 C. Immunodiffusion ......................................................................................................................................186-4 V. Immunomagnetic Separation ............................................................................................................................186-4 VI. Latex Agglutination ..........................................................................................................................................186-6 VII. Immunostaining ................................................................................................................................................186-6 VIII. Biosensors ........................................................................................................................................................186-6 IX. Automated Systems ..........................................................................................................................................186-7 X. Summary ..........................................................................................................................................................186-7 XI. Non-Endorsement of Commercial Products and Services ..............................................................................186-7 Acknowledgment..........................................................................................................................................................186-8 References ....................................................................................................................................................................186-8

I.

INTRODUCTION

According to the Centers for Disease Control and Prevention (CDC), there are over 250 known different foodborne diseases (1). These diseases are caused by bacteria, viruses, chemicals, toxins, and fungi. In the United States, where the food supply is one of the safest in the world, the number of food related illnesses is estimated to result in 76 million sick individuals, and nearly 5,000 deaths yearly. Many of these pathogens, such as Campylobacter jejuni, Escherichia coli O157:H7, and Listeria monocytogenes, were not recognized as major causes of foodborne illness until recently (2). One of the main reasons for the emergence of foodborne pathogens is the increased complexity of food products and processes. Each year, hundreds of thousands

of new food products are introduced into the market place, and as a result different and more complex food matrices are produced. Other reasons that foodborne disease outbreaks appear to be increasing are: increasing consumer demand for fresh, unprocessed, and minimally processed foods that are inherently less safe than pasteurized or processed counterparts; public health officials have established national surveillance programs that are more sensitive at outbreak detection; there are increasing imports of foods from countries that may not have programs that would decrease contamination levels; and, innovative packaging and processing techniques may allow for much longer shelflife of sensitive foods, thereby allowing a very small pathogen population to proliferate to infective levels. Despite efforts by the government and the food industry to curtail illnesses, such as through the

186-1

186-2


use of programs like Hazard Analysis and Critical Control Point (HACCP) and Good Manufacturing Practices (GMPs) there is still a need for rapid and efficient microbiological testing. There have been many improvements in the last few decades in both the conventional and newer microbiological techniques. Driven by food safety issues and economics, advanced pathogen detection and identification protocols make use of rapid and automated microbiological analyses. This includes the use of microbiological, biochemical, immunological, and serological methods for improving the isolation, early detection, characterization, and enumeration of microorganisms and their products. Whether these methods were originally developed for a clinical, industrial, or environmental setting, such innovative technology has been adopted by the food industry over the past decade.

II.

IMMUNOLOGICAL METHODS

Immunological methods rely on the specific binding of an antibody to an antigen. An antigen is a substance that is capable of eliciting the production of antibodies in a living organism (the host). Figure 186.1 illustrates the Y-shaped structure of an antibody, with the antigen binding sites on the arms of the Y structure. It is these binding sites that account for the specificity of the antibody, particularly the regions termed the light and heavy chains. The suitability of the antibodies for food application toward a particular microbiological target depends on their specificity, including whether they are monoclonal or polyclonal. Polyclonal antibodies contain an assortment of antibodies, each with different specificities for specific antigens. Monoclonal antibodies react with only one antigen. Improvements in

Antigen-binding sites

H

ea

vy

ch

ai

n

Li

gh

Variable region

tc

ha

in

Fc fragment

Constant region

FIGURE 186.1 Antibody Structure. The immunoglobulin molecule consists of two identical light and heavy chains. Binding occurs at the variable regions.

monoclonal antibody production have led to better supplies of these potentially powerful diagnostic tools (3). Typically, monoclonal kits are portrayed as having less variability than polyclonal-based kits. Commonly used immunological techniques include enzyme-linked immunosorbent assay (ELISA) (sometimes referred to commercially as an enzyme immunoassay, or EIA), immunomagnetic separation (IMS), latex agglutination, precipitation assays, immunostaining, biosensors and automated systems. Numerous commercial products to simplify immunodiagnostics are based on these detection systems.

III. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) METHODS In an enzyme-linked immunosorbent assay (ELISA), an enzyme is used as a label on an antigen or antibody, which will then bind to the antigen (or antibody) of interest (the analyte). After binding, the enzyme portion can be assayed, which allows for the detection of an immune reaction and the estimation of the analyte (4). Occasionally, the term enzyme immunoassay (EIA) will be encountered, rather than the term ELISA. EIA is a nonspecific term used to refer to all ELISA-like assays, including those designed to detect nucleic acids as well as antigens or antibodies. Thus, the terms EIA and ELISA are oftentimes used interchangeably. There are two general forms of enzyme immunoassays, the heterogeneous and the homogeneous, typically differentiated by the use of an incubation period and a wash step. The most commonly used form, and the one most often associated with the term ELISA, is the heterogeneous enzyme immunoassay method. In heterogeneous ELISA, the antibody or antigen is bound either covalently or noncovalently to the solid matrix. The unreacted antigen or antibody in the heterogeneous method is removed by washing or centrifugation. Unlike the heterogeneous assay, the homogeneous ELISA has no separation of the immune complex and the free reactants via a wash step. Heterogeneous ELISA can be simple or complex. In simple ELISA, there is just binding of the labeled antibody to the antigen, followed by a detection step. The more complex sandwich ELISA, in which a primary antibody is “sandwiched” between a bound antigen and a second labeled antibody, is commonly used to detect bacterial antigens in foods. Figure 186.2 is a schematic diagram of the sandwich ELISA and the components involved in the assay. A list of ELISA test kit manufacturers can be seen in Table 186.1.

A.

MICROTITER PLATE FORMAT

These assays are usually performed on plastic microtiter plates. These are trays containing a fixed number of wells,


186-3

TABLE 186.1 Partial List of Commercially Available, ELISA-Based Assays for the Detection of Foodborne Pathogens Organism/Toxin (A)

Campylobacter

EHECc O157:H7

(B)

Listeria

Pseudomonas Salmonella (C)

FIGURE 186.2 The double antibody sandwich methods for the detection of specific antibodies. A) Antibody specific for target antigen(s) is bonded to surface (e.g., plate, bead, paper, etc.). Sample is added and antigen binds with antibody. B) Enzymelinked antibody specific for the target antigen(s) is added and binds to antigen. C) Enzyme substrate is added and reaction produces a visible color change. (Reprinted by permission from Tecra Diagnostic, Ltd.)

typically 24, 48, or 96. When the antibody (or antigen) is added to the well, it then binds to the inside surface of the well. This binding is due to the hydrophobic interaction between the hydrophobic residues on the protein and the nonpolar plastic surface. Once it is bound, the protein cannot be easily washed from the surface of the well. After application of the sample and subsequent binding of labeled antigen or antibody, the response, either enzymatic or isotopic, is then read via the plate reader. One commercially available ELISA, manufactured by BioControl Systems, Inc. (Bellevue, WA) uses a mixture of monoclonal antibodies specific for Salmonella detection (BioControl). Another manufacturer, TECRA Diagnostic Ltd. (NSW, Australia) uses polyclonal antibodies rather than monoclonal antibodies for the detection of pathogens (5,6). Figure 186.3 shows an

Staphylococcus aureus

Trade Name VIDAS ALERT VIA Assurance Gold EIAd Transia Plate Assurance EIAd VIAd ALERT Transia Plate VIDAS VIAd Assurance EIAd Transia Plate ListerTest Pathalert Listeria-TEKd VIDASd VIDAS (monocytogenes II) VIA VIAd ULTIMA ALERT Assurance EIAd Assurance Gold EIAd Transia Bioline VIDASd Salmonella-TEKd VIA

Assay Formata

Manufacturer

b

ELFA ELISA ELISA ELISA

bioMérieux Neogen TECRA BioControl

ELISA ELISA ELISA ELISA ELISA ELFAb ELISA ELISA ELISA ELISA ELISA ELISA ELFAb ELFAb

Transia BioControl TECRA Neogen Transia bioMérieux TECRA BioControl Transia Vicam Merck bioMérieux bioMérieux bioMérieux

ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELFAb ELISA ELISA

Tecra TECRA TECRA Neogen BioControl BioControl Transia Bioline bioMérieux bioMérieux TECRA

a

Abbreviations: ELISA, enzyme linked immunosorbent assay; ELFA, enzyme linked fluorescent assay. b Automated System. c EHEC - Enterohemorrhagic E. coli. d Adopted AOAC Official First or Final Action. This table has been adapted from the FDA Bacterial Analytical Manual.

example of a standard ELISA test kit and Table 186.1 gives a list of the various manufacturers and/or distributors of ELISA based detection systems.

IV. IMMUNOPRECIPITATION Immunoprecipitation, also called immunochromatography, is another method that is antibody-based. These assays use the technology originally developed for home pregnancy tests. It utilizes the same “sandwich” technology described in the section on ELISA assays. The main

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FIGURE 186.3 Typical ELISA kits for the detection of a bacterial pathogen. In addition to detecting pathogens such as E. coli O157:H7, Salmonella and Listeria monocytogenes, some test kits will test for the presences of bacterial toxins (e.g., Staphylococcus aureus). (Reprinted by permission from Tecra Diagnostic, Ltd.)

difference between the two systems is that instead of enzyme conjugates, the detection antibody is bound to latex beads or to colloidal gold, which produces a color change. The sample is placed on the device and is wicked across media. The antibodies for the specific components in a sample are then bound to an antibody. It is this binding of the antibody/antigen complex which results in a visibly detectable line known as a precipitation band (7).

A.

LATERAL FLOW DEVICES

These assays combine the recognition ability of an immunoassay with the separation power of chromatography. In the lateral flow device (LFD) an extracted sample is introduced onto one end of a membrane strip, usually encased in a plastic holder. The sample is drawn through to a reagent zone containing labeled antibodies specific for the target analyte. A positive reaction occurs when the analyte in the sample extract combines with the labeled antibody where it stops at a line of anchored antibodies. This zone also has antibodies specific for the target analyte. Here they form an antibody-antigen-antibody “sandwich” that is visualized as a line. A second “control” zone also wicks from the reagent zone and forms a second line further along the device (Figure 186.4). One benefit of this class of tests is that they can be read visually. Strategic Diagnostics, Inc. (Newark, DE) has developed rapid screening tests for E. coli O157 (including O157:H7) and for Salmonella via their RapidChekTM kits. The RapidChek™ E. coli O157 system has been approved by the AOAC for use in ground beef, boneless beef, and apple cider (7). Neogen Corporation (Lansing, MI) has also developed commercially available lateral flow devices for the detection of Salmonella, Salmonella enteritidis, E. coli

FIGURE 186.4 The RapidChek™ Lateral Flow Assay is an immunoassay which employs a combination of anti-pathogen antibodies and colloidal gold conjugate coated on the surface of a membrane encased within a plastic cassette. (Reprinted by permission from Strategic Diagnostics, Inc.)

O157:H7, as well as Listeria in their line of Reveal® kits (8,9). BioControl Systems, Inc. (Bellevue, WA) produces the VIP® test kit for Salmonella, Listeria, and E. coli O157:H7 (10). These as well as other commercially available lateral flow kits are listed in Table 186.2.

B.

DIP STICK ASSAYS

Dip stick assays, such as Tecra Diagnostics Ltd.’s E. coli 0157 Immunocapture and UNIQUE™ test kits, offer yet another iteration of the ELISA methodology. These assays use an antibody-coated dipstick to capture target antigens from an enriched sample. All steps take place within a self-contained module that contains all necessary reagents and eliminates media preparation (11). These and other commercially available dip stick assays are listed in Table 186.2.

C.

IMMUNODIFFUSION

BioControl Systems, in addition to its VIP™ line of immunoprecipitation tests also produces the 1–2 test used for detection of motile Salmonella in food. The method, sometimes referred to as an immunoimmobilization assay (12), utilizes a unique two-chamber unit. One unit contains the enrichment media while the other is used for the immunoimmobilization process. Anti-Salmonella antibodies are added to one chamber as the motile Salmonella migrate in the other. At the point where the diffusing antibodies contact the advancing microorganisms, a visual “immunoband” is formed when a positive result is present.

V. IMMUNOMAGNETIC SEPARATION Immunomagnetic separation is the selective concentration of a target organism using antibody-coated magnetic beads or other devices to selectively trap the target


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TABLE 186.2 Partial List of Immuno-Based (Non-ELISA) Assays for the Detection of Foodborne Pathogens Organism/Toxin Campylobacter

EHECc O157:H7

Listeria

Salmonella

Shigella Staphylococcus aureus

Trade Name Campyslide Latex Campylobacter Prolex Wellcolex O157 VIPe Reveal Immunocapture™ Reveal Transia Card Dry Spot Dynabeads Latex Listertest Dynabeads VIPe Reveal UNIQUE™ UNIQUE PLUS™ Immunocapture UNIQUE™ UNIQUE PLUS™ VIPe Reveal Reveal (enteritidis) Capture-TEK Transia Card Latex Salmonella Latex Wellcolex Dynabeads Screen/Verify Screen/SE Verify 1–2 Testd UNIQUE PLUS™ Wellcolex Staphyloslide Staphaurex Staph Latex Dry Spot Prolex

Assay Formata LA LA LA LA LA LFD LFD DS LFD LFD LA Ab-beads LA Ab-beads Ab-beads LFD LFD DS DSb DS DS DSb LFD LFD LFD Ab-beads LFD LA LA LA Ab-beads Ab-beads Ab-beads Diffusion DSb LA LA LA LA LA LA

Manufacturer Becton Dickinson Microgen Oxoid PRO-LAB Murex BioControl Neogen Tecra Neogen Transia Oxoid Dynal Microgen VICAM Dynal BioControl Neogen Tecra Tecra Tecra Tecra Tecra BioControl Neogen Neogen bioMérieux Transia Microgen Oxoid Murex Dynal VICAM VICAM BioControl Tecra Murex Becton Dickinson Remel Wampole Labs Oxoid PRO-LAB

a

Abbreviations: RPLA, reverse passive latex agglutination; LA, latex agglutination; ab-beads, immunomagnetic; Ab-ppt, immunoprecipitation; DS, dip stick; LFD, lateral flow device. b Automated System. c EHEC - Enterohemorrhagic E. coli. d Adopted AOAC Official First or Final Action. This table has been adapted from the FDA Bacterial Analytical Manual.

microorganism (13). The beads are typically uniform polymeric particles coated with a polystyrene shell, providing a smooth hydrophobic surface (15). This allows for the facilitated absorption of the immunoglobulin

molecules. It can be effectively used to reduce background flora and eliminate interfering food particles, but is sometimes labor intensive and therefore not well suited for high-volume users. Both VICAM (Watertown, MA)

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and Dynal Biotech (Oslo, Norway) have successfully developed magnetic beads coated with various antibodies for the detection of specific pathogens, such as Salmonella (Salmonella Screen/Salmonella Verify™) (15) and enteropathogenic E. coli (Dynabeads®) (16). VICAM also produces beads for Listeria detection under the name of ListerTest®. Immunomagnetic bead separation technology can rule out the possible presence of pathogens in less than 24 hours, with similar or better sensitivities as conventional methods. Upon use of this method, further microbiological procedures, such as direct plating or ELISA tests, can be performed on the charged beads. This is quite useful for foods with very low numbers of target pathogens such as Listeria and Salmonella (5). Magnetic bead separation has been used for detection of pathogens in many food and environmental matrices, such as Cryptosporidium species in water samples, Bacillus spores in food and environmental samples, and Staphylococcus species in milk (17–20). Commercially available immunomagnetic kits are listed in Table 186.2.

VI.

LATEX AGGLUTINATION

Another relatively fast and simple immunological method for food pathogen detection involves latex agglutination. This method, sometimes referred to as the slide test, involves the reaction of latex particles coated with a specific antibody to the corresponding pathogenic antigen (21). A positive test yields agglutination, or visible clumping of the test reagents, while a negative control is indicated by the absence of clumped particles. Latex agglutination has been developed for many foodborne pathogens as well as their toxins; commercial products are available for Salmonella, pathogenic E. coli, Staphylococcus aureus, and Clostridium. These test kits have similar or better sensitivities than conventional or ELISA methods (21–25). Oxoid Ltd. (Scotland) produces various commercial latex agglutination kits for microorganisms such as Bacillus cereus (BCETRPLA), E. coli and Vibrio cholerae (VET-RPLA). Other latex agglutination products that are readily available can be found in Table 186.2.

VII.

IMMUNOSTAINING

Immunostaining, also known as immunoblotting, is the transfer of antigenic material from one surface onto a nitrocellulose membrane, and is often used in conjunction with ELISA. Once antigens are transferred to the membrane, antibodies specific to the antigen are added. The resulting membrane containing the antigens and antibodies is then assayed as with the ELISA method (12). Few commercial immunostaining kits are currently in use for food pathogen detection. 3M Corporation had developed a system for the detection of E. coli O157:H7, which involved the inoculation of a Petrifilm™ Test

Kit-HEC plate. If the target bacteria were present, colonies would form on the plate. Colony antigens were then transferred from the plate to a reactive disc. If the E. coli O157:H7 antigens were present, they would be transferred to the disc and would capture the enzyme-labeled antibody, which was added later. The antibody-antigen complex was then visually detected via the presence of a black spot on the plate. This system is no longer available and is only mentioned to provide historical background, although this technique is still used as a research tool for the detection of bacteria in food matrices (12).

VIII.

BIOSENSORS

A biosensor is a compact analytical device incorporating a biological or biologically derived sensing element. These can include enzymes, antibodies, or DNA, either integrated within or associated with a physiochemical transducer (27). The biological compounds can be used to detect changes in the environment, such as the presence of microorganisms, and can vary from simple temperature sensitive paint, to very complex DNA-RNA probes (28). Biosensors provide a means for production of very sensitive, miniaturized systems that can be used to detect microbial activity or the presence of biological compounds. There are two commonly used definitions for the term biosensors. The first definition refers to any device or instrument consisting of a biological sensing element combined or attached to a transducer (29,30). Thus enzymes, antibodies, cells, DNA, and tissues are considered the sensing element while typical transducers for these molecules can consist of electrochemical, calorimetric, optical, acoustical, or even mechanical means. The second definition for biosensors refers to a self-contained analytical system that is capable of responding both directly and selectively to biological species (29,30). Enzymes are the most commonly used biological elements. Biosensors using microorganisms are called microbial biosensors and they exploit the metabolic functions of living microorganisms to effect detection and measurements of analytes (31). Immunosensors are biosensors that use antigens or antibodies as sensing elements, and are analytical devices based on the affinity and specificity of the antigen-antibody reaction. At Georgia Institute of Technology, a biosensor has been developed that operates with three primary components – integrated optics, immunoassay techniques, and surface chemistry tests (32). It indirectly detects pathogens by combining immunoassays with a chemical sensing scheme. In the immunoassay, a series of antibodies selectively recognizes the target bacteria. An antibody, termed the “capture antibody” is bound to the biosensor and captures the target bacterium as it passes nearby. Then a set of “reporter” antibodies, which bind with the same target pathogen, contain the enzyme (urease), which breaks down


186-7

urea that is then added and produces ammonia. The chemical sensor detects the ammonia, affecting the optical properties of the sensor and signaling changes in transmitted laser light. Biosensor development has been accelerated by improvements in materials research and miniaturized technologies. Biosensors’ specificity for the biological binding reaction is derived from numerous types of interactions and affinities (33,34). Some common interactions that have been studied for biosensor applications include antigen/antibody, enzyme/substrate/cofactor, receptor/ligand, energy transducer systems, synthetic chemical interactions, and nucleic acid hybridization. Funded by the United States Department of Agriculture, a group at the University of Rhode Island’s Fiber Optic and Biosensor Research lab has developed sensors that use vibrating quartz crystal or fiber optic probes in conjunction with antibodies for the detection of Salmonella (35). Another group at Cornell University has used nucleic acid sequences to detect pathogens (36). In this system, the biosensor consists of disposable microchannels with areas for capture and detection. DNA probes complimentary to the target pathogen RNA serves as the biorecognition element. To obtain a signal, two other probes are used, one coupled to dye encapsulated liposomes, and the other coupled to superparamagnetic beads for target capture. The probe then hybridizes to the target RNA and the liposometarget bead complex is captured on a magnet. Biosensor techniques are still commercially limited, but hold promise with applications in the poultry, beef, and seafood industries (32).

performing all stages of the analysis, such that all that is required for analysis is the addition of the sample to the analyzer. VIDAS® (bioMérieux) and mini VIDAS® are two currently available autoimmuno-analyzers that use enzyme linked fluorescent assays (37). VIDAS® is capable of analyzing four different modules concurrently, thus allowing it to run 240 tests per hour. The mini VIDAS® is a smaller version of the VIDAS® and contains a built in computer, keyboard, and printer. It has two independent sections capable of analyzing a total of 12 samples simultaneously. This system has been used for the detection of E. coli O157 in cheese, and Listeria in milk (38,39). The Tecra Diagnostics, Ltd. UNIQUE PLUS™ system automates the steps required for the UNIQUE™ dip stick test kit. The system currently supports assays for Salmonella and Listeria (11). Figure 186.5 shows the UNIQUE PLUS™ automated system by Tecra Diagnostics, Ltd.

IX.

XI. NON-ENDORSEMENT OF COMMERCIAL PRODUCTS AND SERVICES

AUTOMATED SYSTEMS

The latest immunological detection methods involve the development of automated systems. These allow for the rapid testing of multiple samples concurrently. Typically, these automated systems are comprised of an analytical module, a computer, and a recording device such as a printer. The analytical module is capable of automatically

X. SUMMARY Immunological assay methods, particularly ELISA and latex agglutination, are routinely used for the detection and analysis of pathogens in food products. The use of automated systems and biosensors is not as common, but should increase in popularity as sensitivity increases and costs decline with these systems. Most developments in immunological assays have come about within the last few decades and future improvements are likely to continue at the same pace, as immuno-techniques have potential for superior accuracy and can provide rapid analytical results.

References, hypertext links and images to all products and services are provided for information only and do not constitute endorsement or warranty, express or implied, by the authors and/or their employers or the publishers of this

FIGURE 186.5 The UNIQUE PLUS™ utilizes the UNIQUE™ ELISA format for pathogen screening. Modules are plugged into UNIQUE PLUS™ and results collected. The automated system performs all the steps of UNIQUE™ ELISA which are normally done by a technician. (Reprinted by permission from Tecra Diagnostic, Ltd.)

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work, as to their suitability, content, usefulness, functioning, completeness, or accuracy. 14.

ACKNOWLEDGMENT The authors would like to thank the following individuals for providing product literature and art that was instrumental in the preparation of this manuscript: Tony Vagnino, Strategic Diagnostics and Laura Gleeson, TECRA Diagnostics Limited. This is Florida Agricultural Experiment Station Journal Series number R-09806.

REFERENCES 1. Centers for Disease Control and Prevention. CDC Fact Book 2000/2001. 2. PS Mead, S Laurence, V Dietz, LF McCaig, JS Bresee, C Shapiro, PM Griffin, and RV Tauxe. Food-related illness and death in the United States. Emerg Infect Dis 5:607–625, 1999. 3. WM Barbour, G Tice. Genetic and immunologic techniques for detecting foodborne pathogens and toxins. In: MP Doyle, LR Beuchat, TJ Montville. ed. Food Microbiology: Fundamental and Frontiers. ASM Press, Washington, D.C., 1997, pp 710–727. 4. A Sharma. Enzyme immunoassays: overview. In: RK Robinson, CA Batt, PD Patel. ed. Encyclopedia of Food Microbiology. San Diego, CA: Academic Press, 1999, pp 625–633. 5. DYC Fung. Overview of Rapid Methods of Microbiological Analysis. In: ML Tortorello, SM Gendel. ed. Food Microbiological Analysis: New Technologies. New York: Marcel Dekker, pp 1–25, 1997. 6. CE Park, M Akhtar, MK Rayman. Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods. Appl Environ Microbiol 58:2509–2512, 1992. 7. Strategic Diagnostics Inc. Product Literature. Retrieved on 03 Oct 2003 from http://www.sdix.com/Product Specs.asp?nProductID=15 8. Neogen Corporation. Product Literature. Retrieved on 03 Oct 2003 from http://www.neogen.com/ bacteria3.htm 9. ADGEN Ltd. Product Literature. Retrieved on 03 Oct 2003 from http://www.adgen.co.uk/foodfeed_technology.php 10. BioControl Systems, Inc. Product Literature. Retrieved on 03 Oct 2003 from http://www.rapidmethods.com/ products/index.html. 11. Tecra Diagnostics, Ltd. Product Literature. Retrieved on 03 Oct 2003 from http://www.tecra.net/launcher.asp? action=products 12. BJ Robison. Immunodiagnostics in the detection of foodborne pathogens. In: ML Tortorello, SM Gendel. ed. Food Microbiological Analysis: New Technologies. New York: Marcel Dekker, 1997, pp 77–89. 13. S Brunelle. Electroimmunoassay technology for foodborne pathogen detection. Retrieved on 07 Oct 2003

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from http://www.devicelink.com/ivdt/archive/01/06/ 003.html ECY Wang. Sorting of human peripheral blood T-cell subsets using immunomagnetic beads. In: Methods in Molecular Biology, 80. Totowa, NJ: Humana Press, 1998, pp 365–376. Vicam. Product literature. Retrieved 03 Oct 2003 from http://www.vicam.com/products/microbiological.html Dynal Biotech. Product literature. Retrieved 03 Oct 2003 from http://www.dynalbiotech.com SP Yazdankah, AL Hellemann, K Ronningen, E Olsen. Rapid and sensitive detection of Staphylococcus species in milk by ELISA based on monodisperse magnetic particles. Vet Microbiol 62:16–26, 1998. CJ Lowery, JE Moore, BC Millar, DP Burke, KAJ. McCorry, E Crothers, JSG Dooley. Detection and speciation of Cryptosporidium spp. in environmental water samples by immunomagnetic separation, PCR and endonuclease restriction. J Med Microbiol 47:779–785, 2000. MR Blake, BC Weimer. Immunomagnetic detection of Bacillus stearothermophilus spores in food and environmental samples. Appl Environ Microbiol 63:1643–1646, 1997. GD Sturbaum, PT Klonicki, MM Marshall, BH Jost, BL Clay, CR Sterling. Immunomagnetic separation (IMS)fluorescent antibody detection and IMS-PCR detection of seeded Cryptosporidium parvum oocysts in natural waters and their limitations. Appl Environ Microbiol 68:2991–2996, 2002. PL Lim. A one-step two-particle latex immunoassay for the detection of Salmonella typhi endotoxin. J Immunol Meth 135:257–261, 1990. Novamed, Ltd. Technical information sheet. E. coli-Stat Latex agglutination test for presumptive identification of E. coli O157:H7. 1999. TC Chung, SH Huang. Efficacy of a latex agglutination test for rapid identification of Staphylococcus aureus: a collaborative study. J AOAC Int 79:661–669, 1996. MM Brett, LC Rodhouse, TJ Donovan, GM Tebbutt, DN Hutchinson. Detection of Clostridium perfringens and its enterotoxin in cases of sporadic diarrhea. J Clin Pathol 45:609–611, 1992. SB March, S Ratnam. Latex agglutination test for detection of Escherichia coli serotype O157. J Clin Microbiol 27:1675–1677, 1989. BE Rice, C Lamichhane, SW Joseph, DM Rollins. Development of a rapid and specific colony-lift immunoassay for detection and enumeration of Campylobacter jejuni, C. coli, and C. lari. Clin Diag Lab Immun 3(6):669–667, 1996. APF Turner, I Karube, GS Wilson. Biosensors Fundamentals and Applications. Oxford, England: Oxford University Press, 1987. ER Richter. Biosensors: Applications for dairy food industry. J Dairy Sci 76:3114–3117, 1993. PR Coulet. What is a biosensor? In: LJ Blum, PR Coulet. ed. Biosensor Principles and Applications. New York: Marcel Dekker, 1991, pp 1–6.


30. WH Mullen, PM Vadgama. Microbial enzymes as biosensors. J Appl Bacteriol 61:181, 1986. 31. M Mascini. What is a biosensor? Retrieved 03 Oct 2003 from http://srv.chim.unifi.it/ana/biosen.htm. 32. J Sanders. Food safety: Biosensor that detects pathogens in poultry and other foods to be tested in metro Atlanta processing plant. Georgia Tech Research News, 1999. Retrieved 03 Oct 2003 from http://gtresearchnews.gatech.edu/newsrelease/SENSOR.html. 33. RH Hall. Biosensor technologies for detection of microbiological foodborne hazards. Microbes Infect 4:425–432, 2002. 34. KR Rogers. Principles of affinity-based biosensors. Mol Biotechnol 14: 109–129, 2000. 35. T McLeish. New biosensor makes detection of food pathogens quick, easy. Retrieved on 03 Oct 2003 from http://advance.uri.edu/pacer/october2000/ story15.htm.

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36. S. Kwakye, A. Baeumner. Micro-System for nucleic acid based pathogen detection. Retrieved on 03 Oct 2003 from http://eqs.syr.edu/htm/_research/MicroSystem%20for%20Nucleic%20Acid%20Based%20Pat hogen%20Detection.pdf. 37. bioMérieux, Inc. Product Literature. Retrieved on 26 September 2003 from http://industry.biomerieuxusa.com/industry/food/index.htm. 38. AE Cohen, KF Kerdahi. Evaluation of a rapid and automated enzyme-linked fluorescent immunoassay for detecting Escherichia coli serogroup O157 in cheese. J AOAC Int 79:858–860, 1996. 39. F Allerberger, DM Dierich, G Petranyi, M Lalic, A Bubert. Nonhemolytic strains of Listeria monocytogenes detected in milk products using VIDAS immunoassay kit. Zentralbl Hyg Umweltmed 200:189–195, 1997.

187

Genetic-Based Methods for Detection of Bacterial Pathogens

John L. McKillip

Department of Biology, Ball State University

MaryAnne Drake

Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University


Introduction ........................................................................................................................................................187-1 Background on Traditional Microbiological Methods in Food ..........................................................................187-2 Pulsed Field Gel Electrophoresis ........................................................................................................................187-2 Polymerase Chain Reaction (PCR) ....................................................................................................................187-3 A. General Principles of PCR-Based Detection ..............................................................................................187-3 B. Multiplex and Nested PCR ........................................................................................................................187-4 C. RAPD-PCR and Rep-PCR for DNA Fingerprinting ..................................................................................187-4 D. Quantitative-Competitive (Qc-) PCR ..........................................................................................................187-5 E. Real-Time PCR ..........................................................................................................................................187-5 1. Nonspecific Real-Time Chemistries ....................................................................................................187-6 2. Specific Real-Time Detection Chemistries ..........................................................................................187-6 V. RNA Assays––Monitoring Virulence Gene Expression in Food Pathogens ......................................................187-9 A. The VBNC Dilemma ..................................................................................................................................187-9 B. Nucleic Acid Sequence-Based Amplification (NASBA) ............................................................................187-9 C. Microarrays ..............................................................................................................................................187-10 VI. Summary ..........................................................................................................................................................187-10 References ..................................................................................................................................................................187-11

I.

INTRODUCTION

The food industry is witnessing a tug-of-war as processors make a slow transition from the use of traditional microbiological methods for quality control/quality assurance of foods, which are essentially designed around the recovery and enumeration of viable bacteria in the food matrix, to miniaturized rapid methods and molecular tools that achieve the same purpose with greater sensitivity, specificity, and in less time. In order to successfully counter increasing consumer demand for high quality food, to remain abreast of emerging food-associated pathogenic bacteria, and in keeping with Hazard Analysis and Critical Control Point (HACCP) implementation, significant resources are being invested in monitoring food during and

immediately following processing to preclude spoilage and/or pathogenic bacteria from contaminating food, or detect them prior to shipment for retail sale (1). Novel means of detecting and enumerating bacteria of interest are continually being reported. Some of these strategies still rely heavily on traditional, relatively inexpensive microbiological methods, but the majority of new assays in the literature entail a molecular component that affords rapid (8–48 hour), sensitive, and specific results for detection of particular target microbes (or their products). Such techniques may offer the food processor higher sample throughput, greater assay versatility, and speed compared to traditional (albeit more widely accepted) manual methods (2,3). This chapter will present a comparative overview of many of the commonly used

187-1

187-2


approaches in food microbiology for enumeration and/or detection of bacteria in foods, including many of the emerging molecular-based technologies currently flooding the literature.

II. BACKGROUND ON TRADITIONAL MICROBIOLOGICAL METHODS IN FOOD Of the more than 200 known diseases transmitted through food in the United States yearly, at least 5,000 deaths are recorded on average from over 75 million reported cases. Surveillance and detection of food-associated pathogenic bacteria is complicated by emerging strains not routinely encountered, and the unclear route of transmission of many bacteria, which may be problematic in being spread by water or direct contact, in addition to contaminating specific foods (4). Routine screening of an increasingly diverse array of fresh and processed foods obligates pre- and postharvest food safety measures be dynamic, sensitive, specific, as well as versatile and cost-effective for large sample numbers. In order to assess the microbiological quality of foods, detection of viable bacteria is traditionally performed by implementing a means of culturing/measuring growth of individual microorganisms. Hundreds of commonly used bacteriological media used in the food industry are met with unique ways of applying them to best monitor for spoilage and/or pathogenic bacteria in food (5). The use of routine nonselective media such as trypticase soy agar or standard methods agar, known as the aerobic plate count (APC) or standard plate count (SPC), offer low cost and ease of use. However, these approaches are not sensitive below levels of approximately 10 2 viable cfu/ml or gram of suspect food, require extended incubation times, and do not adequately address the presence of key virulence determinants in specific food-associated pathogens that may or may not be present in target bacteria. The widespread use of these traditional approaches is being superseded by molecular tools that are protein- and nucleic acid-based. The latter, in particular, lend themselves well to not only sensitive and specific detection of spoilage or pathogenic bacteria, but DNA typing/fingerprinting, quantitation, and differentiation of viable from dead microorganisms, frequently in real-time depending on the format of the assay. Following are descriptions of some of the most common nucleic acid-based methods used in quality control/quality assurance within the food industry, as well as some specialized DNA and RNA technologies that have demonstrated potential for application in food safety.

III.

PULSED FIELD GEL ELECTROPHORESIS

Nucleic acid-based analysis of food-associated spoilage and pathogenic bacteria encompasses a diverse array of

methods, many of which have had their origins in the clinical arena. One such method dates back to the early 1980s and is now the basis for the PulseNet molecular subtyping network of bacterial foodborne disease surveillance. Pulsed field gel electrophoresis (PFGE) is a fundamental method in molecular biology for separation of high molecular weight DNA for typing bacterial strains and tracing foodborne disease outbreaks through standardized protocols and data sharing. In 1984, Schwartz and Cantor (6) described PFGE and demonstrated the ability of this technique to resolve yeast chromosomal DNA fragments and in doing so, raised the upper limit on the size of nucleic acids able to be separated electrophoretically. Following this initial high-profile study, a battery of subsequent papers reported on the utility of PFGE in genetic analyses of other organisms, as well as improvements on the protocol itself (7,8,9,10). In principle, PFGE is based on the physics of high molecular weight DNA fragments (i.e., chromosomal DNA or bacterial genomic DNA) not being resolved when exposed to constant voltage, but if the DNA is forced to change through periodic polarity inversion during electrophoresis, the mobility of large molecular weight DNA is altered and separation as distinct bands may be obtained (11). Various instruments and protocols are commercially available that reorient the DNA at unique angles depending on the specific experimental objectives, in an effort to obtain optimal separation within particular size ranges, but the separation principle is essentially the same (12,13,14). In an effort to standardize protocols for molecular subtyping of bacterial food pathogens, PFGE has been developed as the tool of choice for characterizing and epidemiologically tracing isolates associated with foodborne illness outbreaks. In 1996, the Centers for Disease Control and Prevention in Atlanta, GA, and several state health departments established PulseNet with just 10 laboratories focusing on a single pathogen – Escherichia coli O157:H7 – following an outbreak of hemorrhagic colitis from contaminated ground beef consumed in a fast food restaurant (15). PulseNet now encompasses nearly every state in the US, and several provincial Canadian laboratories in an effort to meet the growing frequency of documented foodborne illness outbreaks with information on strain designations, tracing outbreak clusters, and sharing improvements on DNA extraction and end point analysis methods (16,17,18,19). PulseNet is also established overseas, and implements the same PFGE technology in the molecular subtyping of bacteria associated with foodborne illness outbreaks in Japan (20). In terms of applicability to food safety, PFGE has been useful for typing Listeria monocytogenes (21,22), Staphylococcus aureus (23,24), Shigella flexneri (25), Campylobacter jejuni (26), Salmonella spp. (27), Clostridium perfringens (28), and pathogenic E. coli (29,30).


IV. POLYMERASE CHAIN REACTION (PCR) A. GENERAL PRINCIPLES DETECTION

OF

PCR-BASED

Ideally, the development of a commercially viable detection assay for spoilage or pathogenic bacteria would be supplemented by a molecular approach with the potential for extreme sensitivity and specificity, while still maintaining low cost per assay. The polymerase chain reaction (PCR) has been in use in the food microbiology arena for over 10 years, with many variations of the common theme of this technique (Figure 187.1), some of these manifesting themselves as commercial assays (31,32) (Table 187.1). PCR has the potential to significantly reduce the necessary time for detection and screening of foods for pathogenic or spoilage bacteria, with a myriad of offshoot technologies that afford real-time, fingerprinting, quantitative, and/or RNA-based virulence gene expression assessment in a variety of data generation and collection formats (33,34). The specificity of PCR usually relies on DNA sequence-specific oligonucleotide primers that initiate repetitive rounds of in vitro replication of a target gene fragment through denaturation, primer annealing, and new strand synthesis (35) (Figure 187.1). The end product, or amplicon, is traditionally analyzed by agarose gel electrophoresis, and visual confirmation establishes that

5′ 3′

3′ 5′

5′

3′

3′

5′

5′ 3′

Target DNA Separate strands, anneal primers 3′ Extend primers 5′

Separate strands, anneal primers Extend primers

FIGURE 187.1 Process of polymerase chain reaction (PCR), showing template strands, sequence-specific primers (usually) employed that anneal to complementary base sequences, and extension (new strand synthesis) using Taq DNA polymerase. Each cycle of PCR theoretically doubles the amount of DNA in the reaction tube, but only within the region spanned by the forward and reverse primers.

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TABLE 187.1 Commercially Available Nucleic Acid-Based Rapid Methods for Pathogen Detection in Food Nucleic Acid Assays Organism Clostridium botulinum Campylobacter Escherichia coli E. coli O157:H7 Listeria

Salmonella

Staphylococcus aureus Yersinia enterocolitica Any (NASBA) assay

Trade Name

Manufacturer

Probelia AccuProbe GENE-TRAK GENE-TRAK BAX Probelia GENE-TRAKb AccuProbe BAX Probelia GENE-TRAKb BAX BINDa Probelia AccuProbe GENE-TRAK GENE-TRAK Nuclisens

BioControl GEN-PROBE GENE-TRAK GENE-TRAK Qualicon BioControl GENE-TRAK GEN-PROBE Qualicon BioControl GENE-TRAK Qualicon BioControl BioControl GEN-PROBE GENE-TRAK GENE-TRAK Organon Teknika

the expected size fragment has been amplified from DNA extracted from contaminated food. The general PCR technique has been used in many applications for pathogen detection in food, enough to be previously well reviewed in a variety of sources (36–46). The composition of the food medium directly impacts PCR assay sensitivity, however, and thus no universal DNA extraction procedure exists; rather, each food matrix presents its own set of challenges according to composition and must be addressed on a case-by-case basis (47,48). In fact, many factors affect efficiency of DNA template purification from food matrices, subsequent PCR amplification robustness, or both. For example, in dairy products and meats lipids, proteases, divalent cations, carbohydrates, or a host of undefined organic material may drastically interfere with PCR assay detection sensitivity (49–53). Rarely is one able to apply a PCR-based detection assay that lacks sample processing and template cleanup prior to setting up reactions (54). In virtually all foods under scrutiny by PCR-based analyses, debris and other inhibitory components may be at least partially sequestered or minimized using specialty buffer or detergent cocktails, solvent extractions, PCR additives, or a combination of these (55–61). Several means have been described for removing the target spoilage or pathogenic bacterium from the food medium prior to DNA extraction as an additional efficient way to obtain PCR template of higher quality than solvent-extracted DNA. Immunomagnetic separation (IMS)

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is perhaps the best described method, and has been used to concentrate E. coli, Salmonella, Campylobacter spp., Bacillus spp., and other food-associated pathogenic bacteria from a variety of foods, including fruit juices, dairy products eggs, seafood, ethnic foods, chicken, and meat homogenates (62–85). Additionally, insoluble metal hydroxides have been employed with great success for bacteria and spore immobilization in food suspensions for subsequent removal, resuspension, and DNA purification in a cleaner environment more conducive to high template yields (68,86–88). Metal hydroxides, essentially a supersaturated suspension of zirconium, titanous, or hafnium chloride in pH-adjusted ammonium hydroxide and sterile saline bind to the negatively-charged bacterial surface and sequester the cells from polluted suspensions such as food slurries or other contaminated liquid matrix. Once the cell-free supernatant is decanted following a low speed centrifugation, the cells may be rinsed, plated, or subjected to DNA extraction (89). The various factors that may antagonize DNA extraction and yield, template purity, or PCR amplification efficiency have been comprehensively reported (90) for clinical, food, and environmental DNA template purification applications.

B.

MULTIPLEX AND NESTED PCR

In order to balance PCR assay versatility with optimal levels of detection sensitivity and specificity under varied contaminated food matrices, multiple primer sets may be employed to simultaneously detect two or more target bacterial DNA sequences followed by agarose gel electrophoresis (91). This approach, called multiplex PCR, reduces the incidence of false-negative results. If one or more of the DNA fragments are visible, the sample is presumably positive. In most cases, multiplex PCR targets two gene sequences at once, although several target sequences may be possible (92–94). The notion of multiplex PCR may lend itself well to high throughput sample processing because once experimental conditions are optimized for a specific food/bacteria system, reactions may be in large part prepared ahead of time and stored in bulk, frozen until needed (95). When designing primers for multiplex PCR reactions, one needs to ensure that the primers will have minimal tendency to form primer dimers or secondary structure elements when placed together in the same tube. Primary sequence homology and GC content of each primer should be analyzed individually and in concert with the other primers to be used in the assay to confirm that the Tm of each is within a few degrees for optimal annealing efficiency. Additionally, the multiplex amplicons should be different enough in size to resolve using agarose gel electrophoresis (91). Multiplex PCR affords increased assay versatility, but sacrifices assay sensitivity. Multiplex detection of Salmonella and Vibrio spp. in shellfish and

mussels (93,96), and enterotoxigenic Staphylococcus aureus in skim milk and cheddar cheese (97) have been documented. The use of multiplex PCR for detection of food-associated bacteria is quite prevalent in the literature, and in many cases this tool as a rapid method is partially negated by the need for a selective or nonselective enrichment from the food matrix that may add up to 20 hours to the assay (53,98–102) while other multiplex regimes follow confirmation plating steps on selective media (103), or are not demonstrated in a food system, limiting their potential applicability in a food processing environment and with a diverse array of foods (101). While multiplex PCR techniques decrease the likelihood of obtaining false negative results, assay sensitivity may be improved with a nested PCR approach. This method utilizes sequence-specific primers for an initial round of amplification that when analyzed using agarose gel electrophoresis, may not yield visible amplicon bands. By using some of the PCR product in a second round of reactions with primers internal in annealing position with respect to the first set, a reamplification is done, with the goal of obtaining a visible amplicon (albeit one smaller than the original) on the gel (33). Although nested PCR has largely been supplanted by faster and more sophisticated real-time methods, the basic technique has merit in terms of sensitivity, being used for detecting Listeria monocytogenes and Yersinia enterocolitica in raw milk (105–107), Campylobacter spp. (108), verotoxigenic E. coli in ground beef (109), and Vibrio vulnificus in fish (110), to cite but a few representative studies. As in standard PCR detection assays applied in food systems, nested PCR protocols generally implement an enrichment step and typically yield sensitivity on the order of 101–102 cfu per m contaminated food following such steps.

C. RAPD-PCR AND REP-PCR DNA FINGERPRINTING

FOR

The application of random amplified polymorphic DNA (RAPD) analysis for typing of food-associated spoilage and pathogenic bacteria is widely reported in the literature. This technique employs relatively short (~10 bases) arbitrarily designed primers (one or two) in PCR reactions having a much lower annealing temperature than standard sequence-specific amplifications. Such conditions allow for the generation of PCR amplicons that represent more or less a DNA fingerprint of particular bacterial strains under that set of defined conditions (111). Although potentially useful as a rapid screening tool for tracking contaminants by the RAPD banding pattern on gel electrophoresis, this technique has limitations, including difficulty in obtaining reproducible amplicon banding profiles within replicates. The issue of variable results due to


random primer design and low annealing temperatures used, negate the utility of the technique as a feasible quality assurance tool in the food industry. Nevertheless, RAPD-based PCR detection assays have been reported in the detection of food-associated bacteria ranging from arcobacters in poultry (112) to assessment of Bacillus cereus ecology and contamination in processing facilities and commercial dairy powders, (113–115). RAPD analysis has also been reported for the typing pathogens such as E. coli O157:H7 (116), Salmonella (117–119), Vibrio spp. (120,121), Campylobacter spp. (122–125), and L. monocytogenes (126–129) in food matrices as diverse as cheese, seafood, pork, and beef. A unique PCR-based approach to characterizing pathogens, repetitive element palindromic-based PCR (rep-PCR), has been applied in clinical settings to differentiate the genetic diversity of bacterial pathogens from hospitals (130). Rep-PCR is used to amplify repetitive, noncoding DNA sequences interspersed within bacterial genomes using primers specific to the repeated elements (Figure 187.2). Differences in the resulting banding profile are used to categorize new isolates, or identify strains based on known DNA banding patterns, or fingerprints (131–135) (Figure 187.2). Rep-PCR has been recently employed in typing Bacillus sporothermodurans and other Bacillus spp. isolated from milk (136,137), and to differentiate Bacillus anthracis strains (138,139) and enterotoxigenic Bacillus spp. from nonenterotoxigenic strains in contaminated milk (140). Rep-PCR shows broader species applicability and better discriminatory power than biochemical profiling and RAPD analysis and allows consistent pattern formation and storage of strain typing information in a database as a digitized image. Because this technique is sequence-specific, rep-PCR fingerprints are highly reproducible, unlike RAPD analyses. Unknown strains characterized by rep-PCR can be compared against the stored databases across laboratories for identification purposes and to monitor changes in microbial populations (141), similar to the PulseNet infrastructure using pulsed-field gel electrophoresis (PFGE) as the means of generating strain typing data.

D. QUANTITATIVE-COMPETITIVE (QC-) PCR For detection and enumeration of target genome equivalents or bacterial numbers in foods, including viable-butnonculturable (VBNC) state cells, QC-PCR may be used (142). Enumeration of cells is possible through the coamplification of the target sequence with a shorter fragment (the competicon) containing the same primer annealing regions, allowing amplification of both target and competicon to occur with equal efficiency (143). By assembling QC-PCR reactions using titrations of competicon DNA concentrations but constant levels of target DNA, a series of doublet bands result following electrophoresis,

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Repetitive sequences are distributed throughout bacterial DNA

Taq DNA polymerase rep-PCR primers and Taq bind to repetitive sequences

rep-PCR primer

FIGURE 187.2 Repetitive element palindromic polymerase chain reaction (rep-PCR). Primers in rep-PCR are specific for conserved noncoding DNA elements found throughout the genome of bacteria, and as such generate muliple, but reproducible amplicon banding patterns useful for molecular subtyping following electrophoretic separation.

with band intensity of competicon fragments decreasing with inversely increasing band intensities of the slightly larger target DNA sequence. The concentration of competicon that is equal in band intensity to that of the target fragment is calculated by scanning densitometry of the gel image and/or generation of a regression plot. Genome equivalents are determined and converted to a value for cell number in the suspect food sample. QC-PCR has been applied to a few food-based systems, such as quantitation of E. coli O157:H7 (142), and for GMO screening in grains (144). Because of the logistical difficulty in optimizing QC-PCR assays, the approach has limited potential for large-scale applications in the food industry as a rapid method, particularly in light of the many real-time chemistries in use.

E.

REAL-TIME PCR

Despite the specific advantages as a sensitive tool for detection and/or screening suspect foods for the presence

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of spoilage or pathogenic bacteria, PCR and the variations discussed above all have one caveat in common –– the need to analyze the data using traditional end point analysis (i.e., agarose gel electrophoresis). While this technique is well-understood by many and does not require expensive equipment, analysis of amplicon band intensity in a gel following a cycle run adds 1–2 hours to the assay, may not be quantitative, and possesses a narrow dynamic range when attempting to detect differences in amplification efficiency among multiple samples. Moreover, post-PCR processing is necessary if one wishes to confirm amplicon identity by restriction enzyme digest analysis or a hybridization assay, either of which would completely negate the effect of using PCR as a rapid method for pathogen detection by stretching the protocol from hours to days. The nature of PCR chemistry dictates that the exponential phase of amplification is the most accurate stage for quantification of products, rather than the plateau phase when reaction conditions are suboptimal and the relative amplicon band intensities of a set of templates that were at varying concentrations prior to PCR are now essentially equivalent (33). PCR assays that measure the reaction progress during each amplification, rather than after reaching a plateau, represent an attractive means of obtaining real-time quantitative data for rapid and sensitive detection using uniplex, multiplex, nested, or fingerprinting-based variations on the common theme of PCR to detect DNA (or RNA) (145,146). Currently, a number of real-time chemistries are commercially available for use in PCR. These can be divided into those that are not sequence specific––such as DNA minor groove binding dyes, and those approaches that are sequence-specific and may even afford simultaneous detection and confirmation of target amplicon during the PCR reaction. 1.

Nonspecific Real-Time Chemistries

The standard method for nonspecific real-time detection of PCR amplicons is use of fluorescent double-stranded (ds) DNA intercalating dyes such as SYBR Green™ I or SYBR Gold™. Both of these commercial dyes are DNA minor groove binding dyes that fluoresce after interacting with dsDNA (Figure 187.3). Most real-time PCR instruments are programmed to read near the emission and excitation wavelength spectrum of SYBR Green™ (495 and 537 nm, respectively). This dye is very light sensitive, degrading quickly following dilution to working concentrations, but when fully active, affords the user the ability to obtain realtime fluorescence emission data (relative fluorescence units on the y-axis of a plot) as a function of cycle number on the x-axis. Since relative fluorescence units for each sample are plotted during the exponential phase of amplification, results are quantitative and thus useful for determining copy number and genome equivalents from

Intercalation

Fluorescence

FIGURE 187.3 Interaction of SYBR Green™ I intercalating dye with double-stranded DNA and subsequent fluorescence under appropriate wavelength. The interaction is not sequencespecific.

template DNA purified from food. SYBR Green™ I has been used as an alternative to ethidium bromide for staining DNA in agarose gels, but is also useful for real-time PCR detection assays in food systems, such as quantification of enterotoxigenic S. aureus in cheese (147), E. coli O157:H7 in a multiplex design (148), and for GMO screening in grains (144). Due to the logistical difficulty in optimizing QC-PCR assays, the approach has limited potential for large-scale applications, particularly in light of many of the real-time chemistries. In addition to simply quantitative detection of target pathogenic or spoilage bacteria in foods, intercalating dyes such as SYBR Green™ I allow one to discriminate among amplicons in a multiplex PCR reaction by using melt curve analysis. This approach subjects the PCR reactions to slow and continual heating to 95°C while monitoring fluorescence over time. Since each amplicon of a varying length and/or GC content will melt at a slightly different temperature, fluorescence will decrease incrementally according to the population of products in the reaction tube. Once conditions are optimized, the negative derivative of the fluorescence vs. temperature line will allow for small sequence differences, and certainly differences in length of products to become apparent (33) (Figure 187.4). Melt curve analysis has been applied primarily for mutation screening in specific clinical pathogens, but also may be useful for food pathogen detection. 2.

Specific Real-Time Detection Chemistries

A diverse array of fluorescently labeled probes are in use clinically and industrially for sequence-specific detection of target DNA or RNA, and many of these have been applied in food analysis. The primary category of these


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Fluorescence vs. temperature (raw data) 95.7 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 1.2

104 copies 10 copies 0 copies

65 66 68

70

72

74

76

78

80

82

84

86

88

90

92 94 95

FIGURE 187.4 Melt curves of typical multiplex PCR amplicons showing the typical patterns that may be generated as fluorescence decreases with increasing temperature of samples. Melt curve profiles are useful in distinguishing amplicons in multiplex PCR reactions, or for mutation screening.

involves fluorescence resonance energy transfer (FRET) between a specific fluorophore and a quencher group. Perhaps the most widely used FRET conjugate pair for real-time PCR assays includes the fluorophore FAM (fluorescein) and the quencher TAMRA. The resonance energy from the fluorophore is passed to the appropriate quenching moiety, and if in close proximity (as described below for specific primer and probe regimes), generates low levels, if any, detectable fluorescence as measured by a PCR cycler with fluorimeter capabilities. If separated or alone in solution, the fluorophore will not be quenched and the resonance energy will be emitted as a detectable fluorescent signal at the appropriate wavelength. Depending on the format of the PCR assay, the signal generated will be directly correlated with the amount of target DNA present or amplicon concentration (Figure 187.5). Regardless of the specific means in which the fluorophore/quenching pair is applied, the basis remains the

40 1000 Cells

35

Fluorescence

30

100 Cells

25

10 Cells

20 15

1 Cell

10 5

Negative control

0 0

4

8

12

16 20 24 Cycle number

28

32

36

40

FIGURE 187.5 Real-time fluorescence plot of multiple samples at varying target cell densities analyzed using FRET-labeled probes. Relative fluorescence units are plotted as a function of time or cycle number on the x-axis.

same, and includes the added advantage of sequence specificity that dsDNA intercalating dyes do not offer. One of the earliest uses for the FRET-based probe approach was the 5ⴕ-nuclease (TaqMan) assay, first described as a radioisotopic system, but soon modified to be based on fluorogenics (149). The 5ⴕ-nuclease activity incorporates a target gene-specific primer set and a dual-labeled probe that will hybridize to a region on one of the template strands within the primer annealing sites (Figure 187.6). During the extension phase of a PCR cycle, the 5ⴕ-3ⴕ exonuclease activity of Taq polymerase will cleave the 5ⴕ fluorophore from the terminal end of the hybridized probe, separating it from the quenching moiety, eliciting fluorescence at a specific wavelength (150) (Figure 187.5). Depending on the instrument being used for real-time detection, the investigator may choose to use multiple TaqMan primer and probe combinations in the same reaction tube for multiplexing, with each being detected in a unique optical channel at the respective wavelength. Regardless, TaqMan is a specific and sensitive assay for detection of pathogenic and/or spoilage bacteria in food. In recent years, the TaqMan approach has been reported for E. coli O157:H7 in raw milk and other foods (151,152), Salmonella spp. in meat and seafood (153,154), Campylobacter jejuni from poultry, shellfish, and other commodities (155,156), Vibrio cholerae in raw oysters (157), Yersinia enterocolitica in raw meats and tofu (158), Clostridium botulinum in MAP-packaged Japanese mackerel (159), enterotoxigenic Bacillus cereus from nonfat dry milk (160), and L. monocytogenes in dairy foods (161,162). These representative studies illustrate the versatility of the TaqMan assay for a very diverse array of foods to detect pathogens to levels as low as 101 cfu per ml, although frequently following several hours of preenrichment. Though not as prevalent in the literature for applications in foods, another interesting variation on the use of double dye FRET-based probes for real-time PCR is the use of molecular beacons. Molecular beacons, first described

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Polymerization Forward primer

PCR product-specific nucleotides R

+

Target

Q

5′ 3′ 5′

Molecular beacon

5′ 3′ 5′ Quencher dye

Fluorescent reporter dye

Reverse primer

Hybrid

Strand displacement R (A)

R Q

3′ 5′

5′ 3′

Cleavage R

Q

3′ 5′

5′ 3′

Polymerization completed

R Q

5′ 3′

3′ 5′

5′ 3′

3′ 5′

FIGURE 187.6 Mechanism of TaqMan 5ⴕ nuclease assay for real-time detection of PCR products using FRET-labeled probe internal to the sequence-specific primers. R denotes the reporter dye while Q represents the quenching moiety.

by Tyagi and Kramer (163), are short ssDNA probe molecules that are complimentary to target DNA sequences within the gene (or transcript) under study (Figure 187.7). Beacons are comprised of a loop region (the probe sequence) flanked by stem sequences 4–6 bases in length. The loop is comprised of bases with complete complementarity to the target DNA or RNA, and must match with perfect identity to the nucleic acid sequence being detected. The stem portions are designed to be complementary to each other and frequently are comprised of a majority guanine and cytosine bases. A fluorophore reporter dye is conjugated to one end of the molecular beacon and a quencher is attached to the other end (164). When the labeled probe is in solution alone, the beacon assumes the secondary structure conformation by forming intramolecular base

Q

Fluorophore

Quencher

(B)

FIGURE 187.7 (A) Molecular beacon stem-loop conformation that forms by intramolecular base pairing when in solution without the presence of complementary target nucleic acid. (B) When in the presence of target DNA or RNA, the molecular beacon unfolds because the bases comprising the loop (probe) region form more numerous and more stable base pairs than those allowing the stem-loop secondary structure to form. A single base mismatch between the target nucleic acid and the probe portion drastically decreases stability of molecular beacon interaction and may preclude it altogether.

pairs involving the stem portion. In this state, the beacon does not fluoresce, or fluoresces at baseline levels. When in the presence of the target nucleic acid, the loop attraction to the target sequence is stronger than the C/G bonds holding the beacon as a stem-loop, resulting in an unfolding of the probe, separating the quencher and reporter dye and emitting detectable fluorescence (163,165). Although not widely employed in foods to date, molecular beacon technology offers many advantages, including simultaneous detection and confirmation of target nucleic acid when incorporated in PCR reactions flanked with sequence-specific primers. The stability of the stem structure helps to ensure that unfolding and hybridization will only occur in the presence of perfectly complementary base pairs, making the use of molecular beacons essentially a solution-based fluorimetric Southern blot. FRET-labeled beacon probes have been used as a clever means of assessment for ribonuclease H activity in vitro (166), but have recently been demonstrated on pathogens relevant to the food industry, including Salmonella and E. coli O157:H7 (167–169). Although extremely specific and capable of multiplexing, molecular beacons are still fairly cost-prohibitive, a feature likely to delay extended use as a means of rapid pathogen detection in foods. Specific variations of the FRET chemistries exist commercially, such as the Scorpion® primer (Eurogentec, Belgium) approach that relies on a quenched hairpin loop-based PCR primer that unfolds following the extension step and elicits fluorescence (170). Although not widely used yet in the food industry, such proprietary spin-off technologies offer great utility beyond the clinical arena. Regardless of the real-time chemistry selected, a number of commercial real-time instruments are


available such as the ABI Prism® 7000 (Applied Biosystems), RotorGene (Corbett Research), Cepheid’s Smart Cycler® II System, and the BioRad iCycler iQ RealTime Detection System. Most of these offer 2–4 optical channels to allow for multiplex capabilities, as well as interactive software for user-friendly data analyses.

V. RNA ASSAYS––MONITORING VIRULENCE GENE EXPRESSION IN FOOD PATHOGENS A.

THE VBNC DILEMMA

Although DNA (i.e., virulence determinant gene sequence) is the most frequent choice of target molecule when designing a PCR-based detection assay for foodborne pathogens, differentiation of living from dead bacteria is not possible, as DNA may be quite persistent in dead cells (171–175). Moreover, traditional culture-based approaches for enumeration of sublethally injured and/or viable-but-nonculturable (VBNC) bacteria are not accurate, as the selective media employed prevents many such bacteria from growing to visible, countable colonies. Specialized approaches utilizing viability dyes that interact specifically with DNA from dead cells and prevent it from being amplified by PCR may be augmented using additional fluorescent tags that allow for quantitation with confocal laser scanning microscopy are not practical for high-throughput sample analyses (176). Therefore, in order to accurately detect and monitor pathogenic foodborne bacteria (particularly VBNC cells) as well as virulence gene expression, RNA-based methods must generally be used. When selecting RNA as a determinant of cell physiological state (177), one must bear in mind that ribosomal RNA (rRNA) is not an appropriate target, as bacterial ribosomes are stable for at least 48 hours after cell death (174). Ultimately, only mRNA is ideal to use as an indicator of either the metabolic status of bacteria or assessment of VBNC pathogens that must be assumed to still pose a threat if ingested by the consumer in contaminated food (178,179). Initial studies reporting the detection and measurement of gene expression in foodborne pathogenic bacteria implemented reverse transcriptase PCR (RTPCR) as the means to the qualitative end of mRNA analysis. This labor-intensive protocol involves total RNA extraction from enrichment cultures, DNase-I treatment to eliminate genomic DNA (to preclude the incidence of false positive results), reprecipitation of RNA, and a reverse transcriptase step to enzymatically convert target transcripts to cDNA using a sequence–specific primer. The product is eventually converted to dsDNA in traditional PCR cycling using a second (forward) primer flanking the region of interest. In addition to samples, one needs to prepare a no reverse transcriptase control reaction

187-9

to confirm the absence of gDNA carryover, as well as a no template control as a contamination screen (both of which should never yield an amplicon on the gel). In practice, mRNA amplification using RT-PCR has been used to monitor cell viability in bacteria of relevance to the food industry (172,173,179–181). However, because of the inherent sensitivity issue (owing to the laborious process of RNA recovery), as well as moderate sample-to-sample variations in yield, RT-PCR is not a feasible means of high throughput gene expression analysis for the food industry, even with the onset of new commercial products such as single step RT protocols designed to streamline the process.

B. NUCLEIC ACID SEQUENCE-BASED AMPLIFICATION (NASBA) A more rapid means of RNA analysis has been applied in studies of virulence gene expression in bacteria and viruses for clinical microbiology, and also lends itself particularly well for viable cell determination (182,183). First described by Compton (184) and Fahy et al. (185), nucleic acid sequence-based amplification (NASBA) is an isothermic cyclical series of reactions utilizing RNA as template (either purified using acidic/phenol or ‘whole cell’ NASBA starting template) combined with an enzyme cocktail (Figure 187.8). NASBA begins with first-strand cDNA synthesis catalyzed by AMV reverse transcriptase using a transcript-specific forward primer. RNase H activity digests only the RNA half in the RNA-DNA heteroduplex, leaving ssDNA. Second-strand DNA synthesis then occurs by way of a second sequence-specific (reverse) primer containing a T7 RNA polymerase promoter sequence engineered on the 5ⴕ end, extended with the DNA polymerase activity of the AMV reverse transcriptase. Double-stranded

RNA RNA amplification

Reverse 1st strand cDNA

RNase H digest

ssDNA

in vitro transcription

Reverse transcriptase

FIGURE 187.8 Nucleic acid sequence-based amplification (NASBA) for RNA amplification (i.e., virulence gene expression studies). Details of this isothermic process are explained in the text.

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DNA results, allowing in vitro transcription to occur using T7 RNA polymerase (interacting at the promotor site on the second primer, incorporated into the dsDNA extension products), generating many mRNA copies of the original transcript template. A typical 90 minute NASBA reaction series is performed at 42°C and may amplify mRNA some 1015-fold as in vitro transcription products serve as template for subsequent rounds of NASBA. The amplified RNA may be visualized via subsequent RT-PCR, or any of a number of real-time chemistries with appropriate fluorophores. Because of the promotor-containing reverse primer utilized in NASBA assays, this procedure may be performed in a DNA background, unlike RT-PCR. Therefore, NASBA is more rapid than traditional methods for RNA detection, and if linked with real-time detection chemistry such as FRET probes (i.e., molecular beacons), has the ability to detect virulence gene expression in any pathogen relevant to the food industry. NASBA has recently been applied in the study of Campylobacter jejuni in foods (186,187) and to monitor enterotoxin gene (hblC) expression in three strains of toxigenic Bacillus spp. in contaminated milk (188). Although seemingly a very specialized technology at first glance, the speed and versatility of NASBA to be modified with a variety of uniplex or multiplex real-time chemistries make this method an attractive option when one wishes to assess virulence gene expression in target foodborne pathogens. To date, only one commercial supplier (Organon Teknika, Durham, NC, USA & Markham, Ontario, Canada) manufactures a NASBA assay in kit form (Nuclisens®), although the individual enzymes are available from virtually any supplier of molecular biology reagents for individual optimization sample-to-sample.

C.

MICROARRAYS

It is widely believed that thousands of genes and their products (i.e., RNA and proteins) in a given living organism function in a complicated and orchestrated way that creates the mystery of life. However, traditional methods in molecular biology generally work on a one gene in one experiment basis, which means that the throughput is very limited and the whole picture of gene function is hard to obtain. In the past several years, a new technology, called DNA microarray, has attracted tremendous interests among biologists and offers much in the way of high throughput analysis of virulence gene expression in foodassociated pathogenic bacteria (189). This technology promises to monitor the whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously. An array is an orderly arrangement of samples. It provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of

identifying the unknowns. An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample (189,190). In general, arrays are described as macroarrays or microarrays, the difference being the size of the sample spots. Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarrays are typically less than 200 microns in diameter and these arrays usually contains thousands of spots. Microarrays require specialized robotics and imaging equipment that generally are not commercially available as a complete system (Figure 187.9). There are two major application forms for DNA microarray technology: 1) Identification of sequence (gene/gene mutation); and 2) Determination of expression level (abundance) of genes. In the former, a cDNA probe (500–5,000 bases long) is immobilized to a solid surface such as glass using robot spotting/lithography and exposed to a set of targets either separately or in a mixture. This method, traditionally called DNA microarray, is widely considered as developed at Stanford University (191,192). The second method is likely to prove more directly useful in the food industry over the next several years as a means of global gene expression analysis. In this variation, an array of oligonucleotides (20–80-mer oligos) are synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined using quantitative instrumentation (Figure 187.9). DNA microarray technology has been a most powerful technique in areas of clinical and environmental microbiology since its inception, and will likely demonstrate great potential in the food industry as a sensitive means of detecting gene expression in a battery of target pathogens.

VI.

SUMMARY

In an effort to stay abreast of heightened public awareness of food safety, and in light of contemporary concerns of food bioterrorism, research and development of nucleic acid-based molecular tools for pathogen detection, enumeration, and subtyping must expand. Such DNA- and RNAbased assays offer versatility in reducing the incidences of false negative quality assurance screening measures, sensitivity in processing heterogenous food matrices, specificity in differentiating among closely related target bacterial strains, and speed, as in the case of many of the real-time fluorescent chemistries flooding the market (3). Although some of these nucleic acid-based technologies are unlikely to supersede conventional culture, biochemical, or antibody based testing regimes completely, most offer significant


187-11

A. RNA isolation Sample A

Sample B

E. Imaging

Sample A > B Sample B > A

Sample A = B

B. cDNA generation C. Labeling of probe Reverse transcriptase Fluorescent tags

+

D. Hybridization to array

FIGURE 187.9 DNA microarray showing the steps in preparing oligonucleotide fragments that are subsequently probed using complementary sequences for quantitative large-scale, high throughput screening of gene expression using fluorescence. Details are explained in the text.

potential for high throughput and reliability, advantages that offset the cost of initial equipment needs and ongoing maintenance. The notion of quality control by performing a PCR assay and sample analysis by agarose gel electrophoresis is obsolescent. The need to perform an enrichment step in order to increase cell numbers prior to the appropriate assay will likely be replaced with molecular approaches that are powerful enough to elicit reliable data in the midst of carbohydrates, lipids, and cellular debris from varied food matrices. Whether this will involve a spin-off of one of the existing approaches discussed on the previous pages remains to be seen (193), but in a time when the public demands both fresh ready-to-eat foods and a wide safety margin, detection of existing and emerging bacterial foodborne pathogens, and quantitation of spoilage microbes for accurate shelf life prediction is vital to meet an ever-increasing demand for a growing spectrum of convenience as well as fresh, ready-to-eat foods. The food industry’s proverbial tug-of-war between conventional microbiological techniques for ensuring food safety and

the implementation of molecular approaches must be carefully staged and stringently refereed.

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148. N Jothikumar, MW Griffiths. Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays. Appl Environ Microbiol 68:3169–3171, 2002. 149. PM Holland, RD Abramson, R Watson, DH Gelfand. Detection of specific polymerase chain reaction product by utilization of the 5ⴕ→3ⴕ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Nat Acad Sci USA 88:7276–7280, 1991. 150. CA Batt. Molecular diagnostics for dairy-borne pathogens. J Dairy Sci 80:220–229, 1997. 151. LK Bohra, RD Oberst, RK Phebus, MP Hays, RL Green, JM Sargeant. Evaluation of 5ⴕ nuclease based detection assays to detect Escherichia coli O157:H7 from food products. J Rapid Meth Automation Microbiol 9:143–160, 2001. 152. C Buerk, IGB Braumiller, H Becker, E Maertlbauer. Nuclease fluorescence assay for the detection of verotoxin genes in raw milk. Lett Appl Microbiol 35:153–156, 2002. 153. S Chen, A Yee, M Griffiths, C Larkin, CT Yamashiro, R Behari, C Paszki-Kolva, K Rahn, SA De Grandis. The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food. Int J Food Microbiol 35:239–250, 1997. 154. B Kimura, S Kawasaki, T Fugii, J Kusunoki, T Itoh, SJA Flood. Evaluation of TaqMan PCR assay for detecting Salmonella in raw meat and shrimp. J Food Prot 62:329–335, 1999. 155. P Padungtod, R Hanson, DL Wilson, J Bell, JE Linz, JB Kaneene. Identification of Campylobacter jejuni isolates from cloacal and carcass swabs of chickens in Thailand by a 5ⴕ nuclease fluorogenic polymerase chain reaction assay. J Food Prot 65:1712–1718, 2002. 156. AD Sails, AJ Fox, FJ Bolton, DRA Wareing, DLA Greenway. A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture. Appl Environ Microbiol 69:1383–1390, 2003. 157. WJ Lyon. TaqMan PCR for detection of Vibrio cholerae O1, O139, non-O1, and non-O139 in pure cultures, raw oysters, and synthetic seawater. Appl Environ Microbiol 67:4685–4693, 2001. 158. A Vishnubhatla, RD Obersh, DYC Fung, W Wonglumsom, MP Hays, TG Nagaraja. Evaluation of a 5ⴕ-nuclease (TaqMan) assay for the detection of virulent strains of Yersinia enterocolitica in raw meat and tofu samples. J Food Prot 64:355–360, 2001. 159. B Kimura, S Kawasaki, H Nakano, T Fujii. Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish. Appl Environ Microbiol 67:206–216, 2001. 160. Y-R Kim, J Czaijka, CA Batt. Development of a fluorogenic probe-based PCR assay for detection of Bacillus cereus in nonfat dry milk. Appl Environ Microbiol 66:1453–1459, 2000. 161. T Cox, C Frazier, J Tuttle, S Flood, L Yagi, CT Yamashiro, R Behari, C Paszko, R Cano. Rapid detection of Listeria monocytogenes in dairy samples utilizing a PCR-based fluorogenic 5ⴕ nuclease assay. J Ind Microbiol Biotechnol 21:167–174, 1998.

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188

Methods for the Detection of Viral and Parasitic Protozoan Pathogens in Foods

Doris H. D’Souza, Julie Jean, and Lee-Ann Jaykus

Department of Food Science, College of Agriculture and Life Sciences, North Carolina State University


Introduction ......................................................................................................................................................188-1 General Detection Considerations....................................................................................................................188-3 Sampling ..........................................................................................................................................................188-3 Pathogen Concentration....................................................................................................................................188-3 A. Principles of Virus Concentration in Foods..............................................................................................188-3 1. Virus Concentration Methods for Shellfish ......................................................................................188-5 2. Virus Concentration Methods for Other Foods ................................................................................188-6 B. Principles of Parasitic Protozoa Concentration in Foods ........................................................................188-6 1. Parasitic Protozoa Concentration Methods for Foods ......................................................................188-7 V. Nucleic Acid Extraction ..................................................................................................................................188-9 A. Nucleic Acid Extraction of Food Concentrates — Viruses......................................................................188-9 B. Nucleic Acid Extraction of Food Concentrates — Parasitic Protozoa ..................................................188-10 VI. Detection ........................................................................................................................................................188-10 A. RT-PCR Detection of Viruses in Foods ..................................................................................................188-11 B. PCR Detection of Parasitic Protozoa in Foods ......................................................................................188-14 C. Alternative Nucleic Acid Amplification Methods ..................................................................................188-14 VII. Confirmation ..................................................................................................................................................188-14 A. Real-Time Detection ..............................................................................................................................188-15 VIII. Detection of Viruses and Parasitic Protozoa in Field and Foodborne Disease Outbreak Specimens Using Molecular Methods ..............................................................................................................................188-15 IX. Discussion and Conclusions ..........................................................................................................................188-16 Acknowledgments ......................................................................................................................................................188-17 References ..................................................................................................................................................................188-17

I. INTRODUCTION Both the human enteric viruses and the parasitic protozoa are now recognized as significant causes of human disease, perhaps being responsible for as much as 68% and 3% of all foodborne illness in the U.S., respectively (1). Although they have been recognized for years, the human enteric viruses and parasitic protozoa could be considered “emerging” agents of foodborne disease, largely because scientists have only recently been able to detect these pathogens. In fact, prior to the advent of molecular

biological techniques, epidemiological criteria were the primary means by which cases of enteric viral and parasitic illness were recognized. Unfortunately, epidemiology had several limitations including the fact that the diseases caused by most gastrointestinal viruses and parasites were (and are) not reportable in the U.S.; only the largest, most severe, and/or most widespread outbreaks were (and are) investigated, leaving smaller outbreaks and sporadic disease underestimated; and early detection capabilities, even for clinical (fecal and blood) specimens, were severely limited. These early detection methods, which sought to 188-1

188-2


directly detect either the virus particle or parasite cyst or oocyst, were based mostly on some form of microscopy. Later methods relied on detection of antigen (enzyme immunoassay) in the stool, or alternatively, on seroconversion, i.e., a rise in specific antibody titer against the pathogen. The microscopic methods had poor sensitivity, and the reagents necessary for the serological methods were not always available to clinical laboratories. Among other factors, the absence of dependable detection methods contributed to an underestimate of the true scope and significance of foodborne viral and parasitic protozoan infections. The human enteric viruses replicate in the intestines of infected human hosts, are excreted in the feces, and are therefore transmitted by the fecal-oral route through contact with human fecal pollution. In some instances, the parasitic protozoa are less species specific and can therefore be transmitted by the fecal oral-route through contact with either human and animal feces. Both viruses and parasites can also be spread by person-to-person contact, a phenomenon which is frequently responsible for the propagation of primary foodborne outbreaks. Contamination may occur directly, through poor personal hygiene practices of infected food handlers, or indirectly, via contact with fecally contaminated waters or soils. Since both types of agents must survive the pH variations and enzymes present in the human gastrointestinal tract, they are regarded as highly environmentally stable, allowing virtually any food to serve as a vehicle for their transmission and enabling them to withstand a wide variety of food processing and storage conditions. Neither viruses nor parasitic protozoa are able to replicate in contaminated foods. Furthermore, when found in foods, they are likely to be present in low numbers, but since their infectious doses are presumed to be low, any level of contamination may pose a public health threat. The human enteric viruses of primary epidemiological significance include the hepatitis A virus (HAV) and the Noroviruses, formerly known as the Norwalk-like viruses (NLVs) and before that, as the small round structured viruses (SRSVs) (reviewed in ref. 2). The Sapoviruses (previously called Sapporo-like viruses) which are genetically related to the Noroviruses, have also caused cases of viral gastroenteritis in humans. Both the Noroviruses and the Sapoviruses are members of the Calicivirideae family, an antigenically and genetically diverse group of gastrointestinal viruses. The other viruses that can cause food and waterborne disease include the astroviruses, the human enteroviruses (polioviruses, echoviruses, groups A and B coxsackieviruses), hepatitis E virus, parvoviruses, and other relatively uncharacterized small round viruses. The rotaviruses, which are the leading cause of infantile diarrhea worldwide, are transmitted primarily by contaminated water but can on occasion be foodborne. The parasitic protozoa of primary foodborne significance include Cyptosporidium parvum, Giardia

lamblia, Cyclospora cayetanensis, and Toxoplasma gondii (reviewed in refs. 3–6). The former three organisms cause predominantly gastrointestinal manifestations, while the latter organism is associated with severe birth defects in infants whose mothers become infected during pregnancy. All of the parasitic agents can cause serious disease in immunocompromised hosts. Like the viruses, parasitic protozoa are obligate intracellular parasites that produce environmentally stable forms that serve as the vehicle for infection. Both Cryptosporidium and Giardia are primarily transmitted by waterborne routes, but foodborne infections have been reported. Toxoplasmosis has long been recognized as an uncommon but nonetheless severe foodborne infection that can be transmitted by the consumption of contaminated meats, offal, or unpasteurized milk, as well as by waterborne routes and contact with cat feces (5). Cyclospora cayetanensis, which to date has been almost exclusively foodborne, has been associated with the consumption of contaminated imported produce items (7). Historically, the detection of human enteric viruses from food concentrates has been based on virus infectivity assays using susceptible, live laboratory hosts. Host systems employed were mainly mammalian cell cultures of primate origin, particularly primary and secondary human embryonic kidney and monkey kidney cell cultures. However, it is critical to note that to a large degree, the epidemiologically important human enteric viruses, including the Noroviruses and wild-type hepatitis A virus, cannot be propagated in mammalian cell culture systems and so these are not viable detection options. For the parasitic protozoa, many of the same considerations exist. For instance, cell culture and animal models for the propagation of C. cayatanensis are in developmental phases only (3). Although C. parvum can be assayed for infectivity using either cell culture or the mouse bioassay, neither method is very practical for the routine detection of this pathogen. Likewise, immunological and DNA hybridizationbased assays are not practical approaches for the detection of viruses and parasitic protozoa in foods. For viruses, this is due in part to the unavailability of immunological reagents, particularly for the antigenically diverse Noroviruses. While acid fast and immunologically based fluorescent staining techniques (or autofluorescence for Cyclospora) may be considered the “gold standard” for the detection of parasitic protozoa in environmental samples, and many effective kits exist for their detection in clinical specimens (8), these methods are laborious, require highly trained personnel, and are subject to interpretive problems when sample matrix components interfere with the assay. Furthermore, immunological methods tend to have relatively poor assay detection limits (⬎103–105 detection units/sample) which restricts their applicability to food samples, which are likely to be contaminated with small numbers of pathogens. Much the same can be said for DNA hybridization methods.

Methods for the Detection of Viral and Parasitic Protozoan Pathogens in Foods

Without question, nucleic acid amplification methods have emerged as a promising approach when it comes to methods to detect enteric viruses and parasitic protozoa in foods. Methods such as the polymerase chain reaction (PCR) have the theoretical ability to replace standard cultural enrichment methods with faster nucleic acid enrichment. For the detection of viruses and parasitic protozoa, where cultural enrichment methods are virtually not feasible, this is a tremendous improvement. The purpose of this chapter is to discuss recent developments in molecular detection methodology that are enabling scientists to begin detecting viruses and parasitic protozoa in foods, and to identify research needs that must be effectively addressed before this effort can become a routine reality.

II. GENERAL DETECTION CONSIDERATIONS There are significant impediments to the development of effective virus and parasitic protozoan detection methods as applied to food commodities. Similar to bacterial pathogens, these agents are likely to be present at low levels in contaminated foods. However, unlike bacterial pathogens, both viruses and protozoa require live mammalian cells in order to replicate, so the traditional food microbiological techniques of cultural enrichment and selective plating cannot be used. The general concept, then, is to separate and concentrate the agents from the food matrix prior to detection. In summary, in order to effectively detect viruses and parasitic protozoa from foods, one must consider the following restrictions: (i) the agents are inert in the food; (ii) they are likely to be present in very low numbers or intermittently in the product; (iii) because of (ii) above, it is necessary to process relatively large sample volumes to assure representation and promote detection; and (iv) the food matrix possesses inhibitory substances or interferences that can later compromise detection. In general, molecular detection schemes for viruses and parasitic protozoa in foods rely on five sequential steps, which can be designated (i) sampling; (ii) pathogen concentration and purification; (iii) nucleic acid extraction; (iv) detection; and (v) confirmation. These will be discussed in detail below.

III.

SAMPLING

For effective sampling, a large and representative sample size is needed. A sample size of 25⫺100 g is usually recommended, which is large enough to provide adequate representation yet small enough to work with in the laboratory. To further increase the chances for detection, the analyst may choose to obtain multiple samples of the suspected or implicated product. For complex food products such as sandwiches, it may be easier to divide the product into its component parts, processing each part separately for pathogen concentration and detection. For instance, some

188-3

investigators have processed dissected digestive tracts in an effort to improve virus recovery efficiency from contaminated raw molluscan shellfish. Although the viruses and parasites may be relatively stable in the food matrix, food samples should nonetheless be refrigerated upon collection and processed immediately for virus recovery upon receipt by the testing facility. Freezing may inactivate parasitic protozoa and should hence be avoided if attempting detection of these pathogens (9).

IV. PATHOGEN CONCENTRATION The most common molecular amplification method applied for the detection of pathogens, including the parasitic protozoa, is the polymerase chain reaction (PCR). Since the nucleic acid for the enteric viruses is RNA, the PCR must be preceded by a reverse transcription step, producing cDNA which can then be readily amplified in a subsequent PCR, hence the designation RT-PCR. When using PCR or RT-PCR for the detection of viruses or parasitic protozoa in foods, one must consider pathogen concentration as a prerequisite to detection. In actuality, appropriate sample preparation prior to detection is even more important when applying molecular methods because of the small sample volumes (⬍10 µl) used in nucleic acid amplification reactions, as compared to 0.5–1.0 ml volumes used for cell culture infectivity assays or ELISA methods. Therefore, the pathogens and/or their nucleic acids must be concentrated and purified from food matrices before applying detection methods such as PCR or RT-PCR. The challenges of high sample volumes, low levels of contamination, and the presence of residual food components that can act as enzymatic inhibitors (10–14) must all be considered in designing these assays.

A. PRINCIPLES FOODS

OF

VIRUS CONCENTRATION

IN

The purpose of virus concentration methods is to reduce sample volume and remove matrix-associated interfering substances, all the while recovering most of the viruses that are present in the food sample. Sample manipulations undertaken during concentration utilize the property of the viruses to behave as proteins in solutions, and their ability to remain infectious at extremes of pH or in the presence of organic solvents such as chloroform, trichloro-trifluoro ethane (Freon), and the more environmentally friendly solvent Vertrel (DuPont Chemical Company). Almost all of the early work in virus concentration and purification from foods was limited to bivalve molluscan shellfish, largely because of their frequent association with viral foodborne disease outbreaks. More recent efforts have targeted a wider variety of at-risk foods. As applied to shellfish, two general schemes for virus concentration have been reported, designated

188-4


extraction-concentration and adsorption-elution-concentration (15). These methods were developed in the decades between 1970 and 1980 and produced concentrates that could be assayed for virus infectivity using mammalian cell culture techniques. Both schemes employ conditions that favor the separation of viruses from shellfish tissues, primarily through the use of filtration, centrifugation, adsorption, elution, solvent extraction, precipitation, and organic flocculation. All protocols begin with sample blending in some type of buffer, usually containing amino acids and an elevated pH. Further processing may sometimes be preceded by a crude filtration step, through a mesh material such as cheesecloth, to remove particularly large sample particulates. A general theme in all of these scenarios is that viruses, since they are so small, do not sediment unaided, even at high centrifugation speeds (10,000 ⫻ g). This means that that routine centrifugation can be used without substantial virus loss as long as the virus-containing supernatant is recovered in the process. By pH manipulation or the addition of precipitation agents, conditions can be created such that viruses adsorb to the shellfish tissues, and when followed by centrifugation, the adsorbed viruses will sediment with the tissues and residual fluids are discarded with the supernatant. This is usually followed by an elution step, whereby virus desorption from the tissues is facilitated by further pH and/or ionic manipulations and subsequent centrifugation, discarding the precipitated tissue in the process. In this case, a large proportion of the food matrix can be disposed of while retaining a relatively clear solution that contains most of the recovered viruses and smaller amounts of matrix-associated organic material. Other sample manipulations are designed to further remove matrix-associated organic materials and reduce sample volume, all the while optimizing recovery of viruses. For instance, a variety of organic solvents can be used to remove lipid materials, capitalizing on the fact that virus infectivity remains intact even after exposure to organic solvents. Virus precipitation can be accomplished using pH reduction, called acid precipitation, or else through the use of polyethylene glycol (PEG). Both methods are based on the fact that viruses behave as proteins in solution; by reducing the pH to that approximating the virus isoelectric point, the virus will precipitate, along with other matrix-associated proteins. Polyethylene glycol essentially removes water, allowing proteins to fall out of solution. A related precipitation method is organic flocculation. Used extensively in water treatment, flocculating agents interact with organic material in the matrix, causing the formation of a gelatinous “floc” to which the viruses adsorb. In the case of acid and PEG precipitation, and in organic flocculation, the viruscontaining solid materials can be readily harvested by centrifugation. Finally, methods such as ultrafiltration can further reduce same volumes. The same general

principles of filtration, centrifugation, adsorption, elution, solvent extraction, precipitation, and organic flocculation are used when extracting and concentrating viruses from food commodities other than shellfish. For purposes of illustration, a candidate virus concentration and detection protocol is illustrated in Figure 188.1. Note that this is only one of literally hundreds of iterations of the basic techniques used for the concentration of viruses from foods. With the advent of molecular biology methods and their use in detection, additional virus concentration methods have been reported. For instance, alternative 50-g Food sample Dilute 1:10 in elution buffer (elevated pH) Homogenize (total volume 400−500 ml)

Filter Cheesecloth

Solvent extraction 1:1 Freon or chloroform Centrifuge and retain supernatant (total volume 300−400 ml)

1° precipitation PEG or acid Centrifuge and retain precipitate (total volume 30−40 ml)

2° precipitation PEG or acid Centrifuge and retain precipitate (total volume 3−4 ml)

RNA extraction SDS/proteinase K/phenol-chloroform or GITC (total volume 2000 MW 30 to 60% None None None None None near 100% near 100% near 100% 80 to 150 psi

50 to 70% 50 to 70% nearly 100% most >200 MW 90⫹% 35 to 75% 35 to 60% 50 to 95⫹% 70 to 95⫹% 20 to 35% near 100% near 100% near 100% 100 to 200 psi

95 to 98% 95 to 98% nearly 100% most >100 MW 90⫹% 90 to 99% 90 to 99% 90 to 99% 90 to 99% 90 to 95% near 100% near 100% near 100% 200 to 450 psi

Actual performance is system-specific. Source: Adapted from Brittan (1997).

3.

Nanofiltration

For removal at the level of dissolved inorganic salts, nanofiltration and reverse osmosis are the only two feasible options. Both are very similar technologies, so similar, in fact, that nanofiltration used to sometimes be referred to as “reduced pressure reverse osmosis.” Two major differences are that the percent rejection of nanofiltration membranes (30 to 70%, in general) is much lower than that of reverse osmosis membranes, and the operating pressures for nanofiltration systems, which typically range from 100 to 200 psi, are generally lower than for reverse osmosis systems. Nanofiltration is an excellent technology where inorganics (salts) removal needs are less important (approx. 50% removal) than removing organics and microorganisms. There is less concentration of salts into the concentrated waste-water effluent which, in some cases, can be critical (e.g. for regulatory compliance). As there are so many similarities between nanofiltration and reverse osmosis, the discussion below in relation to mechanisms of how reverse osmosis works and membrane fouling is also applicable to nanofiltration systems. 4.

Reverse Osmosis

Reverse osmosis, or in some countries termed inverse osmosis, can afford removal of from 95 to greater than 99% of many dissolved salts, resulting in a treated water exiting the system with a total dissolved solids concentration often below 10 mg/L. Significantly, it reduces inorganics not reduced by coagulation, such as sodium, chloride, sulfate and nitrate. It reduces large organic molecules and microorganisms (bacteria, mold, viruses and

water-borne parasites) at efficiency of greater than 99%. Typical operating pressures range from 200 to 450 psi. The major disadvantage of reverse osmosis is the volume of concentrated waste-water that is produced (typically 20–25%). This can be costly on both ends i.e. where cost of source water is high and where sewer surcharges are high for concentrate disposal. Membrane materials of construction include cellulose acetate, polyamide and thin film composite, the latter having become predominant in the food industry in recent years. A comparison of the attributes of each type of membrane is provided in Table 191.4. The mechanism of operation of reverse osmosis systems can become complicated, but the principle is fairly straightforward. In normal osmosis, water flows from a less concentrated salt solution through a semi-permeable membrane into a more concentrated salt solution. By applying pressure in excess of the osmotic pressure, this process is reversed, and water will flow through the membrane, leaving most of the salts, organics and microbial life to remain in the high salts solution (or concentrate). The purified water (or permeate) then goes on to be used for production. Several mathematic models exist which describe the movement of water and its components across the reverse osmosis membrane, but the key point is to design the system in such a way so as to maximize the water flux (flow through the membrane per unit of surface area in a given time). Water flux will naturally decrease as the membrane ages, but the key to any pre-treatment operations is to minimize membrane fouling, thereby minimizing the flux reduction and maximizing the useful life of the membrane. The pre-treatment processes will normally include the in-line dosing of acid, antiscalant, or both. These steps help prevent a loss of membrane

191-10


TABLE 191.4 Comparison of Cellulose Acetate, Polyamide and Thin Film Composite Membranes Parameter Operating pH range Langelier index, preferred Chlorine tolerance, free, mg/l Bacterial resistance Required silt density index, % Overall rejection, % Turbidity, NTU Temperature (operating), °C Life expectancy, years Membrane cost

Cellulose Acetate

Polyamide

Thin Film Composite

4–8 Slight negative 0.2–1.0 Very low

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