Handbook of Seafood and Seafood Products Analysis

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Author: Leo M.L. Nollet | Fidel Toldra

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HANDBOOK OF

Seafood and Seafood Products Analysis

HANDBOOK OF

Seafood and Seafood Products Analysis Edited by

LEO M.L. NOLLET FIDEL TOLDRÁ

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business 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: 978-1-4200-4633-5 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, 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 seafood and seafood products analysis / editors, Leo M.L. Nollet, Fidel Toldrá. p. cm. Includes bibliographical references and index. ISBN 978-1-4200-4633-5 (hardcover : alk. paper) 1. Seafood--Analysis--Handbooks, manuals, etc. I. Nollet, Leo M. L., 1948- II. Toldrá, Fidel. III. Title. TX385.H36 2010 641.3’92--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

2009034833

Contents Preface ..................................................................................................................................ix Editors ..................................................................................................................................xi Contributors ...................................................................................................................... xiii

PART I: CHEMISTRY AND BIOCHEMISTRY 1 Introduction—Importance of Analysis in Seafood and Seafood Products, Variability and Basic Concepts.....................................................................................3 JÖRG OEHLENSCHLÄGER

2 Peptides and Proteins .................................................................................................11 TURID RUSTAD

3 Proteomics ..................................................................................................................21 HÓLMFRÍÐUR SVEINSDÓTTIR, ÁGÚSTA GUÐMUNDSDÓTTIR, AND ODDUR VILHELMSSON

4 Seafood Genomics ......................................................................................................43 ASTRID BÖHNE, DELPHINE GALIANA-ARNOUX, CHRISTINA SCHULTHEIS, FRÉDÉRIC BRUNET, AND JEAN-NICOLAS VOLFF

5 Nucleotides and Nucleosides ......................................................................................57 M. CONCEPCIÓN ARISTOY, LETICIA MORA, ALEIDA S. HERNÁNDEZ-CÁZARES, AND FIDEL TOLDRÁ

6 Lipid Compounds.......................................................................................................69 SANTIAGO P. AUBOURG

7 Lipid Oxidation ..........................................................................................................87 TURID RUSTAD

8 Volatile Aroma Compounds in Fish ...........................................................................97 GUÐRÚN ÓLAFSDÓTTIR AND RÓSA JÓNSDÓTTIR

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PART II: PROCESSING CONTROL 9 Basic Composition: Rapid Methodologies ...............................................................121 HEIDI NILSEN, KARSTEN HEIA, AND MARGRETHE ESAIASSEN

10 Microstructure .........................................................................................................139 ISABEL HERNANDO, EMPAR LLORCA, ANA PUIG, AND MARÍA-ANGELES LLUCH

11 Chemical Sensors .....................................................................................................153 CORRADO DI NATALE

12 Physical Sensors and Techniques .............................................................................169 RUTH DE LOS REYES CÁNOVAS, PEDRO JOSÉ FITO SUÑER, ANA ANDRÉS GRAU, AND PEDRO FITO-MAUPOEY

13 Methods for Freshness Quality and Deterioration...................................................189 YESIM OZOGUL

14 Analytical Methods to Differentiate Farmed from Wild Seafood ............................215 ICIAR MARTÍNEZ, INGER BEATE STANDAL, MARIT AURSAND, YUMIKO YAMASHITA, AND MICHIAKI YAMASHITA

15 Smoke Flavoring Technology in Seafood .................................................................233 VINCENT VARLET, THIERRY SEROT, AND CAROLE PROST

PART III: NUTRITIONAL QUALITY 16 Composition and Calories ........................................................................................257 EVA FALCH, INGRID OVERREIN, CHRISTEL SOLBERG, AND RASA SLIZYTE

17 Essential Amino Acids ..............................................................................................287 M. CONCEPCIÓN ARISTOY AND FIDEL TOLDRÁ

18 Antioxidants .............................................................................................................309 NICK KALOGEROPOULOS AND ANTONIA CHIOU

19 Vitamins ...................................................................................................................327 YOUNG-NAM KIM

20 Minerals and Trace Elements ...................................................................................351 JÖRG OEHLENSCHLÄGER

21 Analysis of n-3 and n-6 Fatty Acids ..........................................................................377 VITTORIO M. MORETTI AND FABIO CAPRINO

PART IV: SENSORY QUALITY 22 Quality Assessment of Fish and Fishery Products by Color Measurement ..............395 REINHARD SCHUBRING

23 Instrumental Texture ...............................................................................................425 ISABEL SÁNCHEZ-ALONSO, MARTA BARROSO, AND MERCEDES CARECHE

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24 Aroma .......................................................................................................................439 JOHN STEPHEN ELMORE

25 Quality Index Methods ............................................................................................463 GRETHE HYLDIG, EMILÍA MARTINSDÓTTIR, KOLBRÚN SVEINSDÓTTIR, RIAN SCHELVIS, AND ALLAN BREMNER

26 Sensory Descriptors ..................................................................................................481 GRETHE HYLDIG

27 Sensory Aspects of Heat-Treated Seafood.................................................................499 GRETHE HYLDIG

PART V: SAFETY 28 Assessment of Seafood Spoilage and the Microorganisms Involved.........................515 ROBERT E. LEVIN

29 Detection of Fish Spoilage........................................................................................537 GEORGE-JOHN E. NYCHAS AND E.H. DROSINOS

30 Detection of the Principal Foodborne Pathogens in Seafoods and Seafood-Related Environments ................................................................................557 DAVID RODRÍGUEZ-LÁZARO AND MARTA HERNANDEZ

31 Parasites....................................................................................................................579 JUAN ANTONIO BALBUENA AND JUAN ANTONIO RAGA

32 Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases .................603 CARLOS PEREIRA DOPAZO AND ISABEL BANDÍN

33 Marine Toxins ..........................................................................................................649 CARA EMPEY CAMPORA AND YOSHITSUGI HOKAMA

34 Detection of Adulterations: Addition of Foreign Proteins .......................................675 VÉRONIQUE VERREZ-BAGNIS

35 Detection of Adulterations: Identification of Seafood Species .................................687 ANTONIO PUYET AND JOSÉ M. BAUTISTA

36 Veterinary Drugs ......................................................................................................713 ANTON KAUFMANN

37 Differentiation of Fresh and Frozen–Thawed Fish ...................................................735 MUSLEH UDDIN

38 Spectrochemical Methods for the Determination of Metals in Seafood .....................................................................................................................751 JOSEPH SNEDDON AND CHAD A. THIBODEAUX

39 Food Irradiation and Its Detection ..........................................................................773 YIU CHUNG WONG, DELLA WAI MEI SIN, AND WAI YIN YAO

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40 Analysis of Dioxins in Seafood and Seafood Products .............................................797 LUISA RAMOS BORDAJANDI, BELÉN GÓMARA, AND MARÍA JOSÉ GONZÁLEZ

41 Environmental Contaminants: Persistent Organic Pollutants .................................817 MONIA PERUGINI

42 Biogenic Amines in Seafood Products......................................................................833 CLAUDIA RUIZ-CAPILLAS AND FRANCISCO JIMÉNEZ-COLMENERO

43 Residues of Food Contact Materials .........................................................................851 EMMA L. BRADLEY AND LAURENCE CASTLE

44 Detection of GM Ingredients in Fish Feed ...............................................................871 KATHY MESSENS, NICOLAS GRYSON, KRIS AUDENAERT, AND MIA EECKHOUT

Index .................................................................................................................................889

Preface There are several seafood and seafood products, which represent some of the most important foods in almost all types of societies, including those in developed and developing countries. The intensive production of fish and shellfish has raised some concerns related to the nutritional and sensory qualities of cultured fish in comparison to their wild-catch counterparts. In addition, there are several processing and preservation technologies, from traditional drying or curing to high-pressure processing, and different methods of storage. This increase of variability in products attending the consumers’ demands necessitates the use of adequate analytical methodologies as presented in this book. These analyses will be focused on the chemistry and biochemistry of postmortem seafood; the technological, nutritional, and sensory qualities; as well as the safety aspects related to processing and preservation. This book contains 44 chapters. Part I—Chemistry and Biochemistry (Chapters 1 through 8)—focuses on the analysis of the main chemical and biochemical compounds of seafood. Chapter 1 provides a general introduction to the topics covered in this book. Part II—Processing Control (Chapters 9 through 15)—describes the analysis of technological quality and the use of some nondestructive techniques. Various methods to differentiate between farmed and wild seafood, to check freshness, and to evaluate smoke flavoring are discussed in these chapters. Part III—Nutritional Quality (Chapters 16 through 21)—deals with the analysis of nutrients in muscle foods such as essential amino acids, omega fatty acids, antioxidants, vitamins, minerals, and trace elements. Part IV—Sensory Quality (Chapters 22 through 27)—covers the sensory quality and the main analytical tools to determine the color texture, the flavor and off-flavor, etc. Sensory descriptors and sensory aspects of heat-treated seafood are also discussed. Finally, Part V—Safety (Chapters 28 through 44)—is concerned with safety, especially related to analytical tools, for the detection of pathogens, parasites, viruses, marine toxins, antibiotics, adulterations, and chemical toxic compounds from the environment generated during processing, or intentionally added, that can be found in either cultured or wild-catch seafood. The last chapter also deals with the analysis of genetically modified ingredients in fish feed. This book provides an overview of the analytical tools available for the analysis of seafood, either cultured fish or their wild-catch counterparts, and its derived products. It also provides an extensive description of techniques and methodologies for quality assurance, and describes analytical methodologies for safety control. In summary, this handbook deals with the main types of analytical techniques available worldwide, and the methodologies for the analysis of seafood and seafood products. ix

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We would like to thank all the contributors for their excellent work. Their hard work and dedication have resulted in this comprehensive and prized handbook. We wish them all the very best in their academic and/or scientific careers. Leo M.L. Nollet Fidel Toldrá

Editors Dr. Leo M.L. Nollet is the editor and associate editor of several books. He edited for Marcel Dekker, New York—now CRC Press of Taylor & Francis Group—the first and second editions of Food Analysis by HPLC and the Handbook of Food Analysis. The Handbook of Food Analysis is a three-volume book. He also edited the third edition of the Handbook of Water Analysis, Chromatographic Analysis of the Environment (CRC Press) and the second edition of the Handbook of Water Analysis (CRC Press) in 2007. He coedited two books with F. Toldrá that were published in 2006: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing). He also coedited Radionuclide Concentrations in Foods and the Environment with M. Pöschl in 2006 (CRC Press). Nollet has coedited several books with Y.H. Hui and other colleagues: the Handbook of Food Product Manufacturing (Wiley, 2007); the Handbook of Food Science, Technology and Engineering (CRC Press, 2005); and Food Biochemistry and Food Processing (Blackwell Publishing, 2005). Finally, he also edited the Handbook of Meat, Poultry and Seafood Quality (Blackwell Publishing, 2007). He has worked on the following five books on analysis methodologies with F. Toldrá for foods of animal origin, all to be published by CRC Press: Handbook of Muscle Foods Analysis Handbook of Processed Meats and Poultry Analysis Handbook of Seafood and Seafood Products Analysis Handbook of Dairy Foods Analysis Handbook of Analysis of Edible Animal By-Products Handbook of Analysis of Active Compounds in Functional Foods He has worked with Professor H. Rathore on the Handbook of Pesticides: Methods of Pesticides Residues Analysis, which was published by CRC Press in 2009. Dr. Fidel Toldrá is a research professor in the Department of Food Science at the Instituto de Agroquímica y Tecnología de Alimentos (CSIC) and serves as the European editor of Trends in Food Science & Technology, the editor-in-chief of Current Nutrition & Food Science, and as a member of the Flavorings and Enzymes Panel at the European Food Safety Authority. In recent years, he has served as an editor or associate editor of several books. He was the editor of Research Advances in the Quality of Meat and Meat Products (Research Signpost, 2002) and the associate editor of the Handbook of Food and Beverage Fermentation Technology and the Handbook of Food Science, xi

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Editors

Technology and Engineering published in 2004 and 2006, respectively, by CRC Press. He coedited two books with L. Nollet that were published in 2006: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing). Both he and Nollet are also associate editors of the Handbook of Food Product Manufacturing published by John Wiley & Sons in 2007. Professor Toldrá has edited Safety of Meat and Processed Meat (Springer, 2009) and has also authored Dry-Cured Meat Products (Food & Nutrition Press—now Wiley-Blackwell, 2002). He has worked on the following five books on analysis methodologies with L. Nollet for foods of animal origin, all to be published by CRC Press: Handbook of Muscle Foods Analysis Handbook of Processed Meats and Poultry Analysis Handbook of Seafood and Seafood Products Analysis Handbook of Dairy Foods Analysis Handbook of Analysis of Edible Animal By-Products Handbook of Analysis of Active Compounds in Functional Foods Toldrá was awarded the 2002 International Prize for Meat Science and Technology by the International Meat Secretariat. He was elected as a fellow of the International Academy of Food Science & Technology in 2008 and as a fellow of the Institute of Food Technologists in 2009.

Contributors M. Concepción Aristoy Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain Santiago P. Aubourg Instituto de Investigaciones Marinas Consejo Superior de Investigaciones Científicas Vigo, Spain Kris Audenaert Department of Plant Production Faculty of Biosciences and Landscape Architecture University College Ghent Ghent, Belgium Marit Aursand SINTEF Fisheries and Aquaculture Trondheim, Norway Juan Antonio Balbuena Cavanilles Institute of Biodiversity and Evolutionary Biology University of Valencia Valencia, Spain

Isabel Bandín Departamento de Microbiología y Parasitología Instituto de Acuicultura Universidad de Santiago de Compostela Santiago de Compostela, Spain Marta Barroso Instituto del Frío Consejo Superior de Investigaciones Científicas Madrid, Spain José M. Bautista Faculty of Veterinary Sciences Department of Biochemistry and Molecular Biology IV Universidad Complutense de Madrid Ciudad Universitaria Madrid, Spain Astrid Böhne Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Luisa Ramos Bordajandi Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute Consejo Superior de Investigaciones Científicas Madrid, Spain

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Emma L. Bradley Food and Environment Research Agency York, United Kingdom Allan Bremner Allan Bremner and Associates Mount Coolum, Queensland, Australia Frédéric Brunet Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Cara Empey Campora Department of Pathology John A. Burns School of Medicine University of Hawaii Honolulu, Hawaii Fabio Caprino Dipartimento de Scienze e Technologie Veterinari per la Sicurezza Alimentare Università degli Studi di Milano Milan, Italy Mercedes Careche Instituto del Frío Consejo Superior de Investigaciones Científicas Madrid, Spain Laurence Castle Food and Environment Research Agency York, United Kingdom

Corrado Di Natale Department of Electronic Engineering University of Rome Tor Vergata Rome, Italy Carlos Pereira Dopazo Departamento de Microbiología y Parasitología Instituto de Acuicultura Universidad de Santiago de Compostela Santiago de Compostela, Spain E.H. Drosinos Laboratory of Food Quality Control and Hygiene Department of Food Science & Technology Agricultural University of Athens Athens, Greece Mia Eeckhout Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Ghent University Association Ghent, Belgium John Stephen Elmore Department of Food Biosciences University of Reading Reading, United Kingdom Margrethe Esaiassen Nofima Marked Tromsø, Norway

Antonia Chiou Department of Science of Dietetics-Nutrition Harokopio University Athens, Greece

Eva Falch Mills DA Trondheim, Norway

Ruth De los Reyes Cánovas Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain

Pedro Fito-Maupoey Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain

Contributors

Delphine Galiana-Arnoux Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Belén Gómara Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute Consejo Superior de Investigaciones Científicas Madrid, Spain María José González Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute Consejo Superior de Investigaciones Científicas Madrid, Spain Ana Andrés Grau Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain Nicolas Gryson Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Ghent University Association Ghent, Belgium Ágústa Guðmundsdóttir Department of Food Science and Nutrition School of Health Sciences Science Institute University of Iceland Reykjavik, Iceland Karsten Heia Nofima Marine Tromsø, Norway

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Marta Hernandez Molecular Biology and Microbiology Laboratory Instituto Tecnologico Agrario de Castilla y León Valladolid, Spain Aleida S. Hernández-Cázares Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain Isabel Hernando Department of Food Technology Universidad Polite′cnica de Valencia Valencia, Spain Yoshitsugi Hokama Department of Pathology John A. Burns School of Medicine University of Hawaii Honolulu, Hawaii Grethe Hyldig Aquatic Process and Product Technology National Institute of Aquatic Resources (DTU Aqua) Technical University of Denmark Kongens Lyngby, Denmark Francisco Jiménez-Colmenero Department of Meat and Fish Science and Technology Instituto del Frío Consejo Superior de Investigaciones Científicas Ciudad Universitaria Madrid, Spain Rósa Jónsdóttir Matís Icelandic Food Research Reykjavik, Iceland Nick Kalogeropoulos Department of Science of Dietetics-Nutrition Harokopio University Athens, Greece

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Anton Kaufmann Kantonales Labor Zurich Zurich, Switzerland Young-Nam Kim Department of Nutrition and Health Sciences Duksung Women’s University Seoul, South Korea Robert E. Levin Department of Food Science University of Massachusetts Amherst, Massachusetts Empar Llorca Departamento de Tecnología de Alimentos Universidad Politécnica de Valencia Valencia, Spain María-Angeles Lluch Department of Food Technology Universidad Politécnica de Valencia Valencia, Spain Iciar Martínez Instituto de Investigaciones Marinas (CSIC) Consejo Superior de Investigaciones Científicas Vigo, Spain Emilía Martinsdóttir Matís Iceland Food Research Reykjavík, Iceland Kathy Messens Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Ghent University Association Ghent, Belgium Leticia Mora Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain

Vittorio M. Moretti Dipartimento de Scienze e Technologie Veterinari per la Sicurezza Alimentare Università degli Studi di Milano Milan, Italy Heidi Nilsen Nofima Marine Tromsø, Norway George-John E. Nychas Laboratory of Microbiology and Biotechnology of Foods Department of Food Science and Technology Agricultural University of Athens Athens, Greece Jörg Oehlenschläger Max Rubner-Institute Federal Research Centre for Nutrition and Food Hamburg, Germany Guðrún Ólafsdóttir Syni Laboratory Services and University of Iceland Reykjavik, Iceland Ingrid Overrein SINTEF Fisheries and Aquaculture and Department of Biotechnology Norwegian University of Science and Technology Trondheim, Norway Yesim Ozogul Department of Seafood Processing Technology Faculty of Fisheries Cukurova University Adana, Turkey Monia Perugini Department of Food Science University of Teramo Teramo, Italy

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Carole Prost Food Aroma Quality Group LBAI—ENITIAA Rue de la Géraudière Nantes, France

Isabel Sánchez-Alonso Instituto del Frío Consejo Superior de Investigaciones Científicas Madrid, Spain

Ana Puig Department of Food Technology Universidad Politécnica de Valencia Valencia, Spain

Rian Schelvis Wageningen IMARES Institute for Marine Resources & Ecosytem Studies IJmuiden, the Netherlands

Antonio Puyet Faculty of Veterinary Sciences Department of Biochemistry and Molecular Biology IV Universidad Complutense de Madrid Ciudad Universitaria Madrid, Spain Juan Antonio Raga Cavanilles Institute of Biodiversity and Evolutionary Biology University of Valencia Valencia, Spain David Rodríguez-Lázaro Food Safety and Technology Research Group Instituto Tecnologico Agrario de Castilla y León Valladolid, Spain Claudia Ruiz-Capillas Department of Meat and Fish Science and Technology Instituto del Frío Consejo Superior de Investigaciones Científicas Ciudad Universitaria Madrid, Spain Turid Rustad Department of Biotechnology Norwegian University of Science and Technology Trondheim, Norway

Reinhard Schubring Department of Safety and Quality of Milk and Fish Products Federal Research Institute for Nutrition and Food Max Rubner-Institut Hamburg, Germany Christina Schultheis Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Thierry Serot Food Aroma Quality Group LBAI—ENITIAA Rue de la Géraudière Nantes, France Della Wai Mei Sin Analytical and Advisory Services Division Government Laboratory Hong Kong, People’s Republic of China Rasa Slizyte SINTEF Fisheries and Aquaculture Trondheim, Norway Joseph Sneddon Department of Chemistry McNeese State University Lake Charles, Louisiana

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Christel Solberg Faculty of Biosciences and Aquaculture Bodø University College Bodø, Norway Inger Beate Standal SINTEF Fisheries and Aquaculture Trondheim, Norway

Vincent Varlet Food Aroma Quality Group LBAI—ENITIAA Rue de la Géraudière Nantes, France Véronique Verrez-Bagnis Ifremer Nantes, France

Pedro José Fito Suñer Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain

Oddur Vilhelmsson Department of Science University of Akureyri Akureyri, Iceland

Hólmfríður Sveinsdóttir Division of Biotechnology and Biomolecules Matís Iceland Food Research SauđárkrÓkur, Iceland

Jean-Nicolas Volff Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France

Kolbrún Sveinsdóttir Matís Iceland Food Research Reykjavik, Iceland

Yiu Chung Wong Analytical and Advisory Services Division Government Laboratory Hong Kong, People’s Republic of China

Chad A. Thibodeaux Department of Chemistry McNeese State University Lake Charles, Louisiana

Michiaki Yamashita Food Biotechnology Section National Research Institute of Fisheries Science Yokohama, Japan

Fidel Toldrá Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain

Yumiko Yamashita Food Biotechnology Section National Research Institute of Fisheries Science Yokohama, Japan

Musleh Uddin Corporate Quality Assurance Albion Fisheries Ltd. Vancouver, British Columbia, Canada

Wai Yin Yao Analytical and Advisory Services Division Government Laboratory Hong Kong, People’s Republic of China

CHEMISTRY AND BIOCHEMISTRY

I

Chapter 1

Introduction—Importance of Analysis in Seafood and Seafood Products, Variability and Basic Concepts Jörg Oehlenschläger Contents 1.1 World Catch and Harvest .................................................................................................. 3 1.2 Variability of Aquatic Animals ........................................................................................... 5 1.3 Special Problems with Aquatic Animals ............................................................................. 5 1.4 Benefits and Risks .............................................................................................................. 6 1.5 Sampling ............................................................................................................................ 6 1.6 Analytical Methodologies................................................................................................... 7 1.7 Analytical Problems ........................................................................................................... 8 1.8 Trends and Outlook ........................................................................................................... 9 References ..................................................................................................................................10

1.1

World Catch and Harvest

Seafood has by far the greatest variety of all animal-based foods. Whereas the species consumed as warm-blooded mammals (beef, pork, lamb, goat, and donkey) or poultry (hen, turkey, geese, and duck) are represented by very few species, fishes and other aquatic animals show an abundant 3

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Handbook of Seafood and Seafood Products Analysis

number of species and variability. The fish group alone is represented by 25,000–35,000 species. However, only a little proportion of this large number of about 5% is present in the world’s oceans in amounts huge enough to allow an economical use (catch and following processing). Further, only some of these 5% have the desired sensory properties and give a good or satisfying fillet yield that catching and processing them can be justified. Another difference compared with land-living animals is the fact that the quality (size, state of maturity, nutritional status, infestation with parasites, burden of pollutants, etc.) of aquatic animals when captured by fishing techniques is—with few exceptions—completely unknown. Although land-based animals are today tailor made according to industry’s and consumer’s wishes in weight, body composition, appearance, and sensory properties, in the case of captured seafood we have to accept what we find in the trawl despite modern advanced technology of sonar and echo sounders. Further, fish and other seafood are highly perishable products when stored without chilling. They deteriorate at ambient temperature in a few days, and only correct storage of wet fish in melting ice or of certain products at chilled temperatures can prolong the shelf life up to weeks or months. The total world seafood supply for 2007 amounted to 143 million tons. The world’s aquaculture provided 52 million tons (36%), and the captured fish, 91 million tons (64%) of the total supply. Although the amount of captured fish is almost constant at a level around 90 million tons/ year since 1990 after a continuous growth for more than 40 years, aquaculture is dramatically growing (1960: 2 million tons, 1970: 4 million tons, 1980: 7 million tons, 1990: 16 million tons, 2000: 40 million tons, including plants) [1]. The stagnation of captured fish is mainly due to fully exploited or partially overfished stocks. The most important primary product producing countries of marine and inland (freshwater) fisheries in 2005 were China (17.1 million tons), Peru (9.4 million tons), the United States (4.9 million tons), Indonesia (4.4 million tons), Chile (4.3 million tons), Japan (4.1 million tons), India (3.5 million tons), Russia (3.2 million tons), Thailand (2.6 million tons), and Norway (2.4 million tons). The top 10 species being caught in huge amounts in 2005 were Anchoveta (10.2 million tons), Alaska Pollock (2.8 million tons), Atlantic herring (2.3 million tons), Skipjack tuna (2.3 million tons), Blue whiting (2.1 million tons), Chub mackerel (2.0 million tons), Chilean jack mackerel (1.7 million tons), Japanese anchovy (1.6 million tons), Largehead hairtail (1.4 million tons), and Yellowfin tuna (1.3 million tons). Most fish was caught in the Pacific Ocean (Northeast and Southeast) followed by Northeast Atlantic Ocean. The major aquaculture (excluding plants) producers (>1 million tons) in 2005 were China (32.4 million tons, whereof the major part are cyprinids like carp), India (2.8 million tons), Vietnam (1.4 million tons, mostly Pangasius species), Indonesia (1.1 million tons), and Thailand (1.1 million tons). By major groupings, fish is the top group in aquaculture at 47.4% by quantity. Aquatic plants that are popular in Southeast Asia are second in quantity at 23.4%, whereas crustaceans are fourth by quantity at 6.2% but second by value at 20.4%. Mollusks (bivalves and cephalopods) are the third most important group both by quantity and by value at 22.3% and 14.2%, respectively. About 75% of the world’s total seafood supply is used for human consumption, 25% is converted into fishmeal and other nonfood products, 40% is consumed as wet fish without any further technological processing or preservation, about 20% is converted into deep frozen products, 8% is transformed into cured products, and another 8% into canned products.

Introduction ◾ 5

1.2 Variability of Aquatic Animals The variability of aquatic animals can be described and explained in many different ways. Based on taxonomic criteria, we have different groups such as bony and cartilaginous fishes, crustaceans, and mollusks, which are very different from each other in appearance, composition, and nutritive properties. When concentrating on fish as the major group contributing to the world’s fish supply, we arrange them in order according to their shape into round fish, flat fishes, eellike fishes, and so forth, or according to their occurrence in the ocean’s water column into pelagic fish, bottom fish, demersal fish, and ground fish. We can also group them according to their fat content into three groups: lean fish species (1% to 10% fat). However, these are all very rough classifications. In addition, the main difficulty in the analysis of fish and other seafood is that there is not only a big variation between groups of species and species but also within a given species. Not only weight and length are varying with age but also other factors such as proximate composition, mineral, and trace element content, which are subject to variations based on state of maturity, fishing area, season, pollution of water, and so on. This means that each fish can be different and unique, and before analyzing fish, a careful consideration has to be made if the variation is important and if it is worth or essential knowing (leading to analysis of individuals) or if a more general impression about the target component is sufficient (pooled samples). A drastic example illustrating the variability in fish is the Atlantic mackerel. The prespawning fish can have a fat content in fillet up to 35%, and the spawned fish can exhibit fillet fat contents of down to 5%. Mackerel is a typical pelagic swarm fish occurring in big schools. When captured during the spawning season, in one haul specimen of 5% fat and 35% fat are present. This can lead to extreme problems not only in processing but also in analysis, since parallel with fat content, other parameters such as organic pollutant concentrations vary. Also within the fish body, a certain degree of variability is found. Components like water, fat, and protein are not even distributed in the edible part and also trace element concentrations vary from head to tail or back to belly. With all these variations in the raw seafood material before the analysis of any components, decisions must be made where the results should be used and how detailed an analysis must be.

1.3 Special Problems with Aquatic Animals The main problem with aquatic animals is the fact that from the moment that they are caught or harvested, a change in properties starts, which continues until a state of spoilage is reached. After catch and harvest, not only spoilage and freshness parameters are changing due to metabolic (autolytic) and microbiological processes but also the microbial flora is changing. Besides this more general aspect, some groups offer special problems to which a lot of attention has to be given: aquatic animals may contain parasites (e.g., nematodes, cestodes) that can be harmful to humans when they enter live and intact into the human body. Predatory fish species such as sharks, which are at the end of the marine food web, can accumulate mercury during their long life span to quantities that exceed legal limits. Toxins from dinoflagellates can accumulate in bivalve mollusks, leading to several diseases such as diarrhetic shellfi sh poisoning (DSP), paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), and

6

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amnesic shellfish poisoning (ASP), and in fish, leading to ciguatera or maitotoxin poisoning. In the digestive glands of mollusks (hepatopancreas) such as cephalopods and mussels, cadmium is accumulated to amounts that exceed any legal limits by far. When not eviscerated immediately after catch, cadmium from hepatopancreas penetrates into the edible part (mantle) during storage, leading to elevated cadmium concentrations also in this body compartment. In products that have not undergone thermal treatment and that are offered to the consumer as ready to eat (e.g., cold smoked products, gravad products, sushi, and sashimi), there is an inherent microbial risk. Fish and other aquatic animals from areas that are polluted (rivers, inshore waters, estuaries, seas with no or limited water exchange with world oceans such as Baltic Sea, Mediterranean Sea, Caspian Sea, or Black Sea) can carry a high burden of environmental pollutants, especially in their organs responsible for detoxification such as liver and kidney. Aquatic animals from some areas of the world can carry viruses and microorganisms (e.g., Vibrio sp.) that are harmful to human health and must be destroyed or removed before marketing of the products.

1.4 Benefits and Risks Seafood is a rich source for a great number of nutritive and important components. The high amount of long-chain polyunsaturated fatty acids of the n-3 series such as eicosapentanoic acid (20:5) and docosahexanoic acid (22:6); the vitamins A, D, E, and B12; the well-balanced content of essential amino acids; the high amount of taurine; the presence of antioxidants such as tocopherols; the exceptional concentrations of essential elements such as selenium and iodine; and the good digestibility of fish protein due to low amounts of connective tissue are some examples of the many benefits seafood offers when consumed. On the other hand, we have the risk of viruses and microorganisms; we are confronted with toxins in mussels and fish; we have sometimes a parasitical problem; we may find high amounts of inorganic toxic elements and organic pollutants (POP, persistent organic pollutants), and residues of pharmaceuticals and hormones used in aquaculture can be detected and more. All of these parameters and substances have to be carefully analyzed and quantified to allow a risk benefit analysis, which can give reliable advice and guidance for wise and responsible seafood consumption. Unfortunately, only few quantitative analytical data have entered these assessments, with the consequence that recommendations are mostly restricted to a few factors being appropriately analyzed but not based on all factors. Considering the great variability of seafood described here, a tremendous amount of analytic work in seafood has to be done.

1.5

Sampling

Sampling, which means here the selection of an appropriate number and part of aquatic animals under well-defined conditions, is very often underestimated. Most errors and most erroneous results arising from analytical methods are based on poor or even wrong sampling plans and practices. Before starting the sampling procedure, a sampling plan has to be developed describing the numbers of samples to be taken, the body compartments to be dissected, and the measures to be taken to avoid any contamination as well as the storage and transport conditions of the samples after sample preparation.

Introduction ◾ 7

The number of individuals should be big when a small specimen has to be analyzed, smaller when medium-sized animals are the target, and only a few samples are taken from big individuals. In small specimens that are consumed totally, the whole body may be sampled and analyzed (mussels, sprat, snails); in medium-sized specimen, always the whole edible part (fillet, tail muscle) must be taken due to intrinsic variations in fillet parts and after homogenization subsamples can be taken; and in big fish (tuna, shark), it is advisable to concentrate on a muscle part that is simple to identify and can be dissected without destroying the fish completely (examples are muscle below gill cover, head end, or tail end of fillet). While sampling is done, precaution must be taken not to contaminate the sample by instruments used during manipulation (scissors, knives) or by protective clothes or gloves. When sampling is done onboard a vessel, a careful selection of individuals that have not been mechanically damaged by the catching technique, other species or mud, sand, and so forth, is necessary. When sampling for later microbiological analyses, it has to be made under strict hygienic conditions to avoid any microbial contamination. After sampling is completed successfully, it is recommended to store all samples (also solutions) in deep frozen conditions (0.3. The connective tissue proteins are often called the insoluble proteins and can be extracted using alkali or acid. The methods for extraction are not standardized so the amount of proteins extracted will vary with the method used. However, changes in solubility can be used to measure changes in protein structure caused by denaturation during storage and processing. Fish muscle proteins are more sensitive and less stable than proteins from mammals. A few examples of methods to extract proteins from fish muscle are given here. Hultmann and Rustad [11] used a modification of the method by Anderson and Ravesi [12] and Licciardello and coworkers [13]. Four grams of muscle was homogenized for 20 s in 80 mL 50 mM phosphate buffer, pH 7. After centrifugation, the supernatant was decanted and the volume made up to 100 mL—this was the water-soluble fraction. The precipitate was homogenized in 80 mL phosphate buffer with 0.5 M KCl and centrifuged as above. The volume of the supernatant was made up to 100 mL. This was the salt-soluble fraction. Martinez-Alvarez and Gomez-Guillen [14] used a modification on the method of Stefansson and Hultin [15]. The soluble protein was extracted in distilled water (low ionic strength), and in 0.86 M NaCl solution (high ionic strength). Two grams of minced muscle was homogenized at low temperature for 1 min in 50 mL of distilled water. The homogenates of these solutions were stirred constantly for 30 min at 2°C, then centrifuged (6000 g) for 30 min at 3°C. Kelleher and Hultin compared the use of NaCl, KCl, and LiCl for extraction of protein from fish muscle and concluded that LiCl was a better extractant of fish muscle proteins over a wider range of conditions than NaCl or KCl [16].

14

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Solubility of collagen can be determined by extraction in alkali or acid as described by Eckhoff and coworkers [17], which is a modification of the method described by Sato et al. [18]. Samples were homogenized in 0.1 M NaOH and centrifuged. The extraction in NaOH was repeated five times and the supernatants were pooled for the analysis of alkali-soluble collagen content. The precipitate was stirred with 0.5 M acetic acid for 2 days at room temperature and centrifuged as above. This was the acid-soluble collagen. After extraction, the concentration of the soluble proteins can be analyzed with a wide variety of methods. Since all proteins absorb UV/visible light to varying degrees, one of the simplest methods is to determine absorbance in the far UV range. The protein concentration can then be calculated from the Lambert–Beer law: A = ε cl where A is the absorption at a given wavelength c is the molar protein concentration l is the path length for the light (cm) e is the molar absorption or extinction coefficient (M−1 cm−1) The molar absorptivity can be determined by dry weight estimation of a purified protein, by absorbance at 205 nm or from knowledge of amino acid composition [19]. Measurement of UV absorption at 280 nm is a simple and popular method to determine protein concentration. However mg quantities of protein are generally required. Absorption at 280 nm is mainly due to tryptophan and tyrosine residues with smaller contributions from phenylalanine and the sulfur-containing amino acids, the method therefore has protein-to-protein variations. In addition, the presence of nonprotein UV-absorbing groups such as nucleic acids and nucleotides which absorb strongly at 260 nm further complicate matters. Light scattering because of large particles or aggregates can also lead to errors. Methods exist to correct for the influence of light scattering and nucleic acids/ nucleotides [19].

2.4 Analysis of Soluble Proteins There are many indirect colorimetric methods to determine protein content, and a few of them will be treated here. The Biuret method is based on the formation of complexes between copper salts and peptide bonds under alkaline conditions. The purple complex is relatively stable and has an absorption maximum at 540–560 nm. A standard curve is needed, but the method is simple and inexpensive. The method is not very sensitive, measuring concentrations between 1 and 10 mg/mL. The sensitivity can be increased by measuring absorbance at 310 nm or by increasing the time for the Biuret reaction. However, some of these methods reduce the speed and simplicity of the method [19]. The Lowry method [20] is based on a Biuret-type reaction between protein and copper(II) ions under alkaline conditions, the complexes react with the Folin-phenol reagent a mixture of phosphotungstic acid and phosphomolybdic acid in phenol. The product becomes reduced to molybdenum/tungsten blue and can be measured at 750 nm. The reactions are highly pH dependent. Peterson have reviewed the Lowry method [21] and listed interfering substances, giving upper tolerable limits for a long range of these as well as some methods for coping with the effect of these substances. Reducing agents and sucrose as well as several common buffers interfere with the Lowry method. The review also discusses many of the modifications that

Peptides and Proteins ◾

15

have been suggested for the Lowry method. Finally he compares the Lowry method with other methods to determine protein concentration and concludes that the advantages of the Lowry method are simplicity, sensitivity, and precision, the disadvantages are interfering substances and time—compared to some of the dye-binding methods such as the Coomassie Blue methods. Use of bicinchonic acid (BCA) was introduced as an easier way to determine protein; it uses only one reagent instead of two as in the Lowry procedure [1]. Sensitivity is similar to the Lowry procedure, but detergents, buffer salts, and denaturing agents such as urea and guanidine hydrochloride cause less interference. However, for lipids, reducing agents, chelators such as EDTA, and acids and alkali cause interference. There are different dye-binding methods, and one of the most widely used is the Biorad method based on binding of Coomassie Brilliant Blue G (CBBG) [22]. This method is based on the color change taking place when CBBG binds to proteins under acidic conditions. Th is method is faster to perform than the Lowry procedure (5 min development compared to 30–45 min), and stable reagents and kits are available. The method is compatible with a wide range of buffers/substances. The Coomassie Brilliant Blue method is also used for visualizing proteins in electrophoretic gels. The ability of proteins to bind silver has also been used as a very sensitive method to visualize proteins in gel electrophoresis. The silver staining methods are 100 times more sensitive than the CBBG staining. Silver binding is also being used as a method to analyze concentration of soluble proteins [19]. All the methods discussed above are highly protein dependent and this should be kept in mind when applying these methods for analysis of the protein content. It would be best if the protein being analyzed could be used as the standard protein; however, this is often not possible or practical. The Lowry method determines both proteins, small peptides and free amino acids, while methods such as Biuret and Biorad only determine peptide chains above a certain length. However, as different amino acids and peptides give different colors in the Lowry method, the method is highly protein dependent (Table 2.1). The amount of collagen can be determined by analysis of the hydroxylysine content by the Neuman and Logan method as modified by Leach [23]. Hydroxylysine is an amino acid that is almost exclusively found in collagen. However, an accurate determination requires that the amount of hydroxylysine residues per 100 residues in the collagen is known. Th is figure varies for different collagen types such as collagen from fish skins from different fish species [24]. Table 2.1 Comparison of Useful Range for Methods to Determine Protein Concentration Method

Range (μg)

Kjeldahl

500–30,000

Biuret

1,000–10,000

Lowry

10–300

Biorad (Coomassie Brilliant Blue)

20–140

Biorad (Coomassie Brilliant Blue)—micro

1–20

Bicinchonic acid

1–50

Absorption at 280 nm

100–300

16 ◾ Handbook of Seafood and Seafood Products Analysis

2.5

Immunoassays

The amount of a specific protein in a mixture can be determined by enzyme-linked immunosorbent assays (ELISA). It is then necessary to have the antibody of the protein that one seeks to quantify. A polyclonal or monoclonal antibody against the protein of interest is then bound to a film through the Fc region of the antibody. Bovine serum albumin (BSA) is then added to block nonspecific binding sites. After washing, a second antibody is bound to the protein bound to the primary antibody. The amount of secondary antibody bound is proportional to the amount of the specific protein in the sample. The secondary antibody is usually linked to peroxidase or alkaline phosphatase. These enzymes can convert a colorless substrate to a colored product which can then be detected. The method is very sensitive but requires available antibodies.

2.6 Electrophoresis-Based Methods The molecular weight of proteins and peptides is often of interest and this can be determined by several different methods. In native gel filtration chromatography, the proteins are separated based on their size and shape (Stokes’ radii). For low- and medium-pressure chromatography, beads are made of open, cross-linked three-dimensional polymer networks such as agarose, dextrans, cellulose, polyacrylamide, and combinations of these. For high-pressure systems, macroporous silica, porous glass, or inorganic–organic composites are used as support media [1]. Small proteins can enter all the pores in the beads, while larger proteins can only enter the largest pores. As the protein solution moves down the column, smaller proteins will, on the average, spend more time inside the beads and the larger proteins will emerge from the column first. How a certain protein behaves in a gel filtration column can be described by the coefficient Kav which defines the proportion of pores that are accessible to that molecule. Kav = (Ve − V0)/(Vt − V0), where Ve is the elution volume of the molecule, V0 is the void volume of the column, and Vt is the total volume of the column. By using standard proteins of known molecular weight, a standard curve can be made allowing determination of the molecular weight distribution in a protein mixture. Molecular weight can also be determined by electrophoresis. One of the most commonly used methods is SDS-PAGE, using gels of polyacrylamide and denaturing the samples by boiling in a solution of sodium dodecyl sulfate (SDS). SDS binds to proteins in a weight ratio of 1:1.4, which gives one SDS molecule for every two amino acids. Since SDS is charged, this results in a charged complex where the charge is proportional to the molecular weight of the protein. Dithiothreitol (DTT) or mercaptoethanol is often added to reduce disulfide bonds. The most commonly used system is that of Laemmli [25]. The denatured proteins are applied to the gel and an electric current is applied, causing the negatively charged proteins to migrate across the gel toward the anode. The proteins will migrate based on their size; smaller proteins will travel farther down the gel, while larger ones travel a shorter distance. By using markers of known molecular weight, a standard curve can be made in the same way as for gel chromatography and the weight of the unknown proteins determined.

2.7 Peptide Characterization Studying the composition and properties of peptides in seafood is often of interest, for instance after enzymatic hydrolysis of proteins or during processing and storage of seafood. Many peptides are bioactive and have physiological properties, such as immunostimulating or antihypertensive

Peptides and Proteins ◾

17

properties. For characterization of mixtures of peptides, especially after enzymatic degradation/ hydrolysis, the term degree of hydrolysis describes the extent to which peptide bonds are broken by the enzymatic hydrolysis reaction. The measurement shows the number of specific peptide bonds broken in hydrolysis as a percent of the total number of peptide bonds present in the intact protein. Several methods to determine this value exist. One of these is the determination of free amino groups after reaction with trinitrobenzene-sulfonic acid (TNBS) [26]; this is spectrophotometric method determining the amount of the chromophore formed when TNBS reacts with primary amines. The reaction takes place under slightly alkaline conditions and is stopped by lowering the pH in the solution. Another widely used method is the determination of free amino groups after titration with formaldehyde [27]. Formaldehyde reacts with unprotonated primary amine groups resulting in loss of protons. The amount of liberated protons can be determined by titration. Studying the peptide fraction can give a lot of useful information as peptides may have several functions in the food. The peptides may also give valuable information about the quality of the food, such as provide information about the enzymes that are active during storage. For determination of the amount of peptides below a certain chain length, selective precipitation using ethanol, methanol, or trichloroacetic acid can be used [28]. The amount of peptides soluble in different concentrations of ethanol was found to be dependent on the chain length as well as on the hydrophobicity of the peptides. Precipitation of the proteins makes it possible to study peptides which are found in lower concentrations using different chromatographic methods such as LC–MS or electrophoretic methods. Mass spectroscopy can be used to determine the molecular mass of the peptides, and by using tandem mass spectroscopy detailed information of the structure of the peptides can be found. Bauchart and coworkers [29] studied the peptides in rainbow trout using precipitation with perchloric acid followed by electrophoresis and MS-analysis in order to study proteolytic degradation.

2.8

Protein Modifications

During storage and processing of marine raw materials, changes take place in the proteins and it is often of interest to quantify these changes. In addition to lipids and pigments, muscle proteins are also vulnerable to oxidative attack during processing and storage of muscle foods [30]. Oxidation can occur at both the protein backbone and on the amino acid side chains, and can result in major physical changes in protein structure ranging from fragmentation of the backbone to oxidation of the side chains. Oxidation of protein side chains can give rise to unfolding and conformational changes in protein and also to dimerization or aggregation [31]. Oxidative modification often leads to alterations in the functional, nutritional, and sensory properties of the muscle proteins, including gelation, emulsification, viscosity, solubility, and water-holding capacity. Several methods are used to determine protein oxidation, the most used are determination of formation of carbonyl groups [32,33] and reduction in SH-groups. The content of sulfhydryl groups can be determined using DTNB by the method of [34] with the modification of [35]. Formation of dityrosine is also used to determine the degree of protein oxidation. In addition oxidation can be measured as loss of functional properties such as loss of solubility, loss of water-holding capacity, gelling and emulsification properties, and formation of aggregates. However, these properties are not only dependent on the oxidation state of the proteins, and changes in these properties may be due to other factors. Changes in proteins during storage and processing will often result in changes in the functional properties of the proteins. One much used definition of functional properties is this: Those physical and chemical properties that influence the behavior of proteins in food systems during

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processing, storage, cooking, and consumption [36]. A description of the properties of the proteins important for functional properties was given by Damodaran [37]: The physicochemical properties that influence functional behavior of proteins in food include their size, shape, amino acid composition and sequence, net charge, distribution, hydrophobicity, hydrophilicity, structures (secondary, tertiary, and quaternary), molecular flexibility/rigidity in response to external environment (pH, temperature, salt concentration), or interaction with other food constituents. Nutritional, sensory, and biological values are sometimes included in the functional properties. Functional properties can be divided in several groups. It is usual to classify them according to mechanism of action into three main groups: (1) properties related with hydration (absorption of water/oil, solubility, thickening, wettability), (2) properties related with the protein structure and rheological characteristics (viscosity, elasticity, adhesiveness, aggregation, and gelation), and (3) properties related with the protein surface (emulsifying and foaming activities, formation of protein–lipid films, whippability). Methods to determine functional properties are often developed for a particular use in a specific food system resulting in a vast number of different methods. It is therefore difficult to compare results from different laboratories. The book edited by Hall [38] gives a good overview of methods to determine protein functionality.

References 1. Owusu-Apenten, R.K., Food Protein Analysis: Quantitative Eff ects on Processing. New York: Marcel Dekker. 2002, p. 463. 2. Venugopal, V., Methods for processing and utilization of low cost fishes: A critical appraisal. Journal of Food Science & Technology, 1995. 32: 1–12. 3. Venugopal, V. and F. Shahidi, Value added products from underutilised fish species. Journal of Food Science & Nutrition, 1995. 35: 431–435. 4. Kirsten, W.J., Automatic methods for the simultaneous determination of carbon, hydrogen, sulphur and sulphur alone in organic and inorganic materials. Analytical chemistry, 1979. 51: 1173–1179. 5. Mariotti, F., D. Tome, and P.P. Mirand, Converting nitrogen into protein—Beyond 6.25 and Jones’ factors. Critical Reviews in Food Science and Nutrition, 2008. 48: 177–184. 6. Isaksson, T. et al., Non-destructive determination of fat, moisture and protein in salmon fillets by use of near-infrared diff use spectroscopy. Journal of the Science of Food and Agriculture, 1995. 69: 95–100. 7. Uddin, M. et al., Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. Food Chemistry, 2006. 96: 491–495. 8. Bock, J.E. and R.K. Connelly, Innovative uses of near-infrared spectroscopy in food processing. Journal of Food Science, 2008. 73: R91–R98. 9. Foegeding, E.A., T.C. Lanier, and H. Hultin, Characteristics of edible muscle tissue, in Food Chemistry, O.R. Fennema, Ed., Marcel Dekker: New York. 1996, pp. 879–942. 10. Haard, N.F., Control of chemical composition and food quality attributes of cultured fish. Food Research International, 1992. 25: 289–307. 11. Hultmann, L. and T. Rustad, Iced storage of Atlantic salmon (Salmo salar)—Effects on endogenous enzymes and their impact on muscle proteins and texture. Food Chemistry, 2004. 87: 31–41. 12. Anderson, M.L. and E.M. Ravesi, Relation between protein extractability and free fatty acid production in cod muscle aged in ice. Journal of Fisheries Research Board Of Canada, 1968. 25: 2025–2069. 13. Licciardello, J.J. et al., Time–temperature tolerance and physical-chemical quality tests for frozen Red Hake. Journal of Food Quality, 1982. 5: 215–234.

Peptides and Proteins ◾ 19 14. Martinez-Alvarez, O. and M.C. Gomez-Guillen, Effect of brine salting at different pHs on the functional properties of cod muscle proteins after subsequent dry salting. Food Chemistry, 2006. 94: 123–129. 15. Stefansson, G. and H.O. Hultin, On the solubility of cod muscle proteins in water. Journal of Agricultural and Food Chemistry, 1994. 42: 2656–2664. 16. Kelleher, S.D. and H.O. Hultin, Lithium chloride as a preferred extractant of fish muscle proteins. Journal of Food Science, 1991. 56: 315–317. 17. Eckhoff, K.M. et al., Collagen content in farmed Atlantic salmon (Salmo salar L.) and subsequent changes in solubility during storage on ice. Food Chemistry, 1998. 62: 197–200. 18. Sato, K. et al., Isolation of types I and V collagen from carp muscle. Comparative Biochemistry & Physiology, 1988. 90B: 155–158. 19. Yada, R.Y. et al., Analysis: Quantitation and physical characterization, in Food Proteins: Properties and Characterization, S. Nakai and H.W. Modler, Eds., VCH: New York, 1996. 20. Lowry, O.H. et al., Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 1951. 193: 265–275. 21. Peterson, G.L., Review of the Folin Phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Analytical Biochemitry, 1979. 100: 201–220. 22. Bradford, M.M., A rapid and sensitive method for the determination of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976. 72: 248–254. 23. Leach, A.A., Notes on a modification of the Neuman & Logan method for the determination of the hydroxyproline. Biochemistry Journal, 1960. 74: 70–71. 24. Almås, K.A., Muskelcellehylsteret hos torsk: Ultrastruktur og biokjemi, in Dep. Technicla Biochemistry. Norges Tekniske høgskole: Trondheim. 1981, p. 175. 25. Laemmli, U.K., Cleavage and structural proteins during assembly of the head of bacteriophage T4. Nature, 1970. 227: 680–685. 26. Adler-Nissen, J., Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry, 1979. 27(6): 1256–1262. 27. Taylor, W.H., Formol titration: An evaluation of its various modifications. Analyst, 1957. 82: 488–498. 28. Rohm, H. et al., Comparison of ethanol and trichloracetic acid fractionation for measurement of proteolysis in Emmental cheese. International Dairy Journal, 1996. 6: 1069–1077. 29. Bauchart, C. et al., Peptides in rainbow trout (Oncorhynchus mykiss) muscle subjected to ice storage and cooking. Food Chemistry, 2007. 100: 1566–1572. 30. Choe, E. and D.B. Min, Mechanisms and factors for edible oil oxidation. Comprehensive Reviews in Food Science and Food Safety, 2006. 5: 169–186. 31. Davies, M.J., The oxidative environment and protein damage. Biochimica Biophysica Acta, 2005. 1703: 93–109. 32. Baron, C.P. and H.J. Andersen, Myoglobin-induced lipid oxidation. A review. Journal of Agricultural and Food Chemistry, 2002. 50: 3887–3897. 33. Baron, C.P. et al., Protein and lipid oxidation during frozen storage of rainbow trout (Oncorhynchus mykiss). Journal of Agricultural and Food Chemistry, 2007. 55: 8118–8125. 34. Ellman, G.L., Tissue sulfhydryl groups. Archives in Biochemistry & Biophysics, 1959. 82: 70–78. 35. Sompongse, W., Y. Itoh, and A. Obtake, Effect of Cryoprotectants and a reducing reagent on the stability of actomyosin during ice storage. Fisheries Science, 1996. 62: 73–79. 36. Kinsella, J.E., Functional properties of food proteins: A review. CRC Critical Reviews Food Science & Nutrition, 1976, 7: 219–280. 37. Damodaran, S., Food Proteins: An Overview, in Food Proteins and their applications, S. Damodaran and A. Paraf, Eds., Marcel Dekker, Inc.: New York. 1997, pp. 1–24. 38. Hall, G.M., Ed., Methods for Testing Protein Functionality. Blackie Academic and Professional: London, U.K., 1996, p. 265.

Chapter 3

Proteomics Hólmfríður Sveinsdóttir, Ágústa Guðmundsdóttir, and Oddur Vilhelmsson Contents 3.1 Introduction ..................................................................................................................... 22 3.2 Proteome Analysis by 2DE ............................................................................................... 22 3.2.1 Sample Matrix Considerations .............................................................................. 22 3.2.1.1 Whole Larval Proteomes......................................................................... 22 3.2.1.2 Muscle Proteomes ................................................................................... 24 3.2.1.3 The Degradome ...................................................................................... 24 3.2.2 Basic 2DE Methods Overview ...............................................................................25 3.2.2.1 Sample Extraction and Cleanup ..............................................................25 3.2.2.2 First-Dimension Electrophoresis ..............................................................25 3.2.2.3 Equilibration .......................................................................................... 27 3.2.2.4 Second-Dimension Electrophoresis ......................................................... 27 3.2.2.5 Staining .................................................................................................. 28 3.2.2.6 Analysis .................................................................................................. 28 3.2.3 Protein Identification by Peptide Mass Fingerprinting .......................................... 28 3.3 Applications of 2DE in Seafood Analysis ..........................................................................31 3.3.1 Development .........................................................................................................31 3.3.2 Quality Involution ................................................................................................ 32 3.3.2.1 Protein Autolysis and Oxidation during Storage and Processing ............. 32 3.3.2.2 Aquaculture and Antemortem Effects on Quality and Processability ........33 3.3.3 Species Authentication .......................................................................................... 34 3.3.4 Allergen Identification .......................................................................................... 34 Acknowledgments ......................................................................................................................35 References ..................................................................................................................................35 21

22 ◾


3.1 Introduction As with all living matter, foodstuffs are in large part made up of proteins. This is especially true of fish and meat, where the bulk of the food matrix is constructed from proteins. Furthermore, the construction of the food matrix, both on the cellular and tissue-wide levels, is regulated and brought about by proteins. It stands to reason, then, that proteome analysis, also known as proteomics, is a tool that can be of great value to the food scientist, giving valuable insight into the composition of the raw materials, quality involution within the product before, during, and after processing or storage, the interactions of proteins with one another or with other food components, or with the human immune system after consumption. Proteomics, succinctly defined as “the study of the entire proteome or a subset thereof”1 is currently a highly active field possessing a wide spectrum of analytical methods that continue to be developed at a brisk pace. While high-throughput, gel-free methods, for example, based on liquid chromatography tandem mass spectrometry (LC–MS/MS),2 surface-enhanced laser desorption/ionization3 or protein arrays,4 hold great promise and are deserving of discussion in their own right, the “classic” process of two-dimensional (2D) gel polyacrylamide electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Figure 3.1) remains the workhorse of most proteomics work, largely because of its high resolution, simplicity, and mass accuracy. This chapter will therefore focus on 2DE.

3.2 Proteome Analysis by 2DE 2DE, the cornerstone of most proteomics research, is the simultaneous separation of hundreds, or even thousands, of proteins on a 2D polyacrylamide slab gel. The method most commonly used was originally developed by Patrick O’Farrell and is described in his seminal and thorough 1975 paper5 and briefly outlined, along with some of the main improvements that have developed since, in the following sections.

3.2.1

Sample Matrix Considerations

Unlike the genome, the proteome varies from tissue to tissue, as well as with time and in response to environmental stimuli. Selection of tissues for protein extraction is therefore an important issue that needs to be considered before a seafood proteomic study is embarked upon. Like other vertebrates, fish possess a number of tissues amenable to 2DE-based proteome analysis. Studies on whole larvae,6–8 liver,9–11 heart,12 kidney,12,13 skeletal muscle,14–18 gill,12 brain,12,19 intestine,12 and rectal gland12 have been reported. In the following sections, we present some issues and challenges related to sample matrices of particular interest to the seafood scientist.

3.2.1.1 Whole Larval Proteomes The production of good quality larvae is still a challenge in marine fish hatcheries. Several environmental factors can interfere with the protein expression of larvae leading to poor larval quality like malformations, growth depression, and low survival rate. Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression,

Proteomics ◾

23

2D PAGE

Trypsin digestion

MS fingerprinting

MS/MS sequencing

Figure 3.1 An overview over the “classic approach” in proteomics. First, a protein extract (crude or fractionated) from the tissue of choice is subjected to 2D PAGE. Once a protein of interest has been identified, it is excised from the gel, subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry, yielding a peptide mass fingerprint. In many cases this is sufficient for identification purposes, but if needed, peptides can be dissociated into smaller fragment and small partial sequences obtained by MS/MS. See text for further details.

posttranslational modifications and redistribution of specific proteins within cells,20 all important information for controlling factors influencing the aptitude to continue a normal development until adult stages. Only a few proteome analysis studies on fish larvae have been published.6,7,21,22 Three of these publications have focused on the whole larval proteomes in Atlantic cod (Gadus morhua)6,22 and zebrafish (Danio rerio).7 These studies provided protocols for the production of high-resolution 2D gels. Peptide mass mapping using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed only on the cod larval proteins, allowing identification of ca. 85% of the of the selected protein spots.6,22 The advantage of working with whole larvae versus distinct tissues is the ease of keeping the sample handling to a minimum in order to avoid loss or modification of the proteins. Nevertheless, there are several drawbacks when working with the whole larval proteome, like the overwhelming presence of muscle and skin proteins. These proteins may mask subtle changes in proteins expressed in other tissues or systems, such as the gastrointestinal tract or the central nervous system. The axial musculature is the largest tissue in larval fishes as it constitutes approximately 40% of their body mass.23 This is reflected in our studies on whole cod larval proteome, where the majority of the highly abundant proteins were identified as muscle proteins.6,22 Also, cytoskeletal

24

◾


proteins were prominent among the identified proteins. Removal of those proteins may increase detection of other proteins present at low concentrations. However, it may also result in a loss of other proteins, preventing identification of holistic alterations in the analyzed proteomes. Various strategies have been presented for the removal of highly abundant proteins24 or enrichment of lowabundant proteins.25,26

3.2.1.2 Muscle Proteomes In most seafood products, fish skeletal muscle is the main component. The fish muscle proteome is therefore likely to be of comparatively high interest to the seafood scientist. Structural proteins, such as actin and tubulin, are particularly abundant in the skeletal muscle proteome. An unfractionated 2DE map of the muscle proteome therefore tends to be dominated by comparatively few high-abundance protein spots, rendering analysis of low-abundance proteins difficult or impossible. Swamping of low-abundance spots by highly abundant ones may not be a problem for applications relating specifically to structural proteins, but for other applications low-abundance proteins, which include most regulatory proteins and many important metabolic enzymes, are of keen interest. No amplification method analogous to PCR exists for proteins, and simply increasing the amount of sample is usually not an option, as it will give rise to overloading artifacts in the gels.5 The remaining option, then, is fractionation of the protein sample in order to weed out the high-abundance proteins, allowing a larger sample of the remaining proteins to be analyzed. A myriad of methods suitable for subsequent 2DE exist for fractionating the proteome into defined subproteomes, such as those associated with individual organelles or cell compartments28 or by protein biochemical methods such as affi nity chromatography,29,30 preparative isoelectrofocusing31 or solubility in the presence of various detergents32 or chaotropes33 have been described. Fractionation methods for a variety of sample matrices have been reviewed recently.34–41

3.2.1.3 The Degradome The degradome may be a subproteome of particular interest to the food scientist, as many textural and other quality factors of muscle foods are related to proteolytic activity in the muscle tissue before, during and after processing. In addition to having a hand in controlling autolysis determinants, protein turnover is a major regulatory engine of cellular structure, function, and biochemistry. Cellular protein turnover involves at least two major systems: the lysosomal system and the ubiquitin–proteasome system.42,43 The 20S proteasome has been found to have a role in regulating the efficiency with which rainbow trout (Oncorhynchus mykiss) deposit protein.44 It seems likely that the manner, in which protein deposition is regulated, particularly in muscle tissue, has profound implications for quality and processability of the fish flesh. Protein turnover systems, such as the ubiquitin–proteasome or the lysosome systems, are suitable for rigorous investigation using proteomic methods. For example, lysosomes can be isolated and the lysosome subproteome queried to answer the question whether and to what extent lysosome composition varies among fish expected to yield flesh of different quality characteristics. Proteomic analysis on lysosomes has been successfully performed in mammalian (human) systems.45,46 An exploitable property of proteasome-mediated protein degradation is the phenomenon of polyubiquitination, whereby proteins are targeted for destruction by the proteasome by covalent

Proteomics ◾

25

binding to multiple copies of ubiquitin.43,47 By targeting these ubiquitin-labeled proteins, it is possible to observe the ubiquitin–proteasome “degradome,” i.e., which proteins are being degraded by the proteasome at a given time or under given conditions. Gygi and coworkers have developed methods to study the ubiquitin–proteasome degradome in the yeast Saccharomyces cerevisiae using multidimensional LC–MS/MS.2 Some proteolysis systems, such as that of the matrix metalloproteases, may be less directly amenable to proteomic study. Activity of matrix metalloproteases is regulated via a complex network of specific proteases.48–50 Monitoring of the expression levels of these regulatory enzymes, and how they vary with environmental or dietary variables, may be more conveniently carried out using transcriptomic methods.

3.2.2

Basic 2DE Methods Overview

O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF), where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes have been deposited. This separates the proteins according to their molecular charge. The tube gel is then transferred onto a polyacrylamide slab gel and the isoelectrically focused proteins are further separated according to their molecular mass by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), yielding a two-dimensional map (Figure 3.2) rather than the familiar banding pattern observed in one-dimensional (1D) SDS-PAGE. The map can be visualized and individual proteins quantified by radiolabeling or by using any of a host of protein dyes and stains, such as Coomassie blue, silver stains, or fluorescent dyes. Although a number of refinements have been made to 2DE since O’Farrell’s paper, most notably the introduction of immobilized pH gradients (IPGs) for IEF,51 the procedure remains essentially as outlined earlier. In the following sections, a general protocol is outlined briefly with some notes of special relevance to the seafood scientist. For more detailed, up-to-date protocols, the reader is referred to any of a number of excellent reviews and laboratory manuals.52–57

3.2.2.1

Sample Extraction and Cleanup

For most applications, sample treatment prior to electrophoresis should be minimal in order to minimize in-sample proteolysis and other sources of experimental artifacts. We have found direct extraction into the gel reswelling buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-1-propanesulfonate], 0.3% (w/v) DTT [dithiothreitol], 0.5% Pharmalyte ampholytes for the appropriate pH range) supplemented with a protease inhibitor cocktail to give good results for proteome extraction from whole Atlantic cod larvae6,22 and Arctic charr (Salvelinus alpinus) liver.58 Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome. Cleanup of samples using commercial 2D sample cleanup kits may be beneficial for some sample types.

3.2.2.2 First-Dimension Electrophoresis The extracted proteins are first separated by IEF, which is most conveniently performed using commercial dry IPG gel strips. These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing. Ready-made IPG strips are currently available in a variety of linear and

26 ◾

Handbook of Seafood and Seafood Products Analysis MW (kDa) 60

42

30

22

17

4

5

pl

6

7

Figure 3.2 A 2DE protein map of whole Atlantic cod (G. morhua) larval proteins with pI between 4 and 7 and molecular mass about 10–100 kDa. The proteins are separated according to their pI in the horizontal dimension and according to their mass in the vertical dimension. Isoelectrofocusing was by pH 4–7 IPG strip and the second dimension was in a 12% polyacrylamide slab gel.

sigmoidal pH ranges. This method is thus suitable for most 2DE applications and has all but completely replaced the older and less reproducible method of IEF by carrier ampholytes in tube gels. Broad-range linear strips (e.g., pH 3–10) are commonly used for whole-proteome analysis of tissue samples, but for many applications narrow-range and/or sigmoidal IPG strips may be more appropriate as these will give a better resolution of proteins in the fairly crowded pI 4–7 range. Narrow-range strips also allow for higher sample loads (since part of the sample will run off the gel) and thus may yield improved detection of low-abundance proteins. Before electrophoresis, the dried gel needs to be reswelled to its original volume. A recipe for a typical reswelling buffer is presented in Section 3.2.2.1. Reswelling is normally performed overnight at 4°C. Application of a low voltage current may speed up the reswelling process. Optimal conditions for reswelling are normally provided by the IPG strip manufacturer. If the protein sample is to be applied during the reswelling process, extraction directly into the reswelling buffer is recommended. IEF is normally performed for several hours at high voltage and low current. Typically, the starting voltage is about 150 V, which is then increased stepwise to about 3,500 V, usually totaling about 10,000–30,000 Vh, although this will depend on the IPG gradient and the length of the strip. The appropriate IEF protocol will depend not only on the sample and IPG strip, but also on

Proteomics ◾ 27

the equipment used. The manufacturer’s instructions should be followed. Görg et al.56 reviewed IEF for 2DE applications.

3.2.2.3 Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel, it needs to be equilibrated for 30–45 min in a buffer-containing SDS and a reducing agent such as DTT. During the equilibration step, the SDS–polypeptide complex that affords protein-size-based separation will form and the reducing agent will preserve the reduced state of the proteins. A tracking dye for the second electrophoresis step is also normally added at this point. A typical equilibrationbuffer recipe is as follows: 50 mM Tris–HCl at pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 1% DTT, trace amount of bromophenol blue. A second equilibration step in the presence of 2.5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applications. This will alkylate thiol groups and prevent their reoxidation during electrophoresis, thus reducing vertical streaking.59

3.2.2.4 Second-Dimension Electrophoresis Once the gel strip has been equilibrated, it is applied to the top edge of an SDS-PAGE slab gel (Figure 3.3) and cemented in place using a molten agarose solution. Optimal pore size depends on the size of the target proteins, but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate. Ready-made gels suitable for analytical 2DE are available commercially. While some reviewers recommend alternative buffer systems,60 the Laemmli method,61 using glycine as the trailing ion and the same buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at both electrodes, remains the most popular one. The gel is run at a constant current of 25 mA until the bromophenol blue dye front has reached the bottom of the gel.

Figure 3.3 Orientation and placement of an isoelectrofocused IPG strip onto the top of the second-dimension gel. Care must be taken that the (+) end of the strip is on the same side of all slab gels, that the gel side of the IPG strip faces the notched side of the glass plate, and that the strip is pressed gently onto the SDS gel, avoiding trapping air bubbles. This is best performed using a dentist’s tool or other appropriate implement, taking care to put the pressure on the IPG strip’s plastic backing rather than the gel itself.

28 ◾


3.2.2.5 Staining Visualization of proteins spots is commonly achieved through staining with colloidal Coomassie Blue G-250 due to its low cost and ease of use. A typical staining procedure includes fi xing the gel for several hours in 50% ethanol/2% ortho-phosphoric acid, followed by several 30 min washing steps in water, followed by incubation for 1 h in 17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid, followed by staining for several days in 0.1% Coomassie Blue G-250/17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid, and followed by destaining for several hours in water. There are, however, commercially available colloidal Coomassie staining kits that do not require fi xation or destaining. A great many alternative visualization methods are available, many of which are more sensitive than colloidal Coomassie and thus may be more suitable for applications where the visualization of low-abundance proteins is important. These include radiolabeling, such as with [35S] methionine, and staining with fluorescent dyes, such as the SYPRO or Cy series of dyes. Multiple staining with dyes fluorescing at different wavelengths offers the possibility of differential display allowing more than one proteome to be compared on the same gel, such as in difference gel electrophoresis (DIGE). Patton published a detailed review of visualization techniques for proteomics.62

3.2.2.6 Analysis Although commercial 2DE image analysis software, such as ImageMaster (Amersham), PDQuest (BioRad), or Progenesis (Nonlinear Dynamics), has improved by leaps and bounds in recent years, analysis of the 2DE gel image, including protein spot definition, matching, and individual protein quantification, remains the bottleneck of 2DE-based proteome analysis and still requires a substantial amount of subjective input by the investigator.63 In particular, spot matching between gels tends to be time-consuming and has proved difficult to automate.64 These difficulties arise from several sources of variation among individual gels, such as protein load variability due to varying IPG strip reswelling or protein transfer from strip to slab gel. Also, gene expression in several tissues varies considerably among the individuals of the same species, and therefore individual variation is a major concern and needs to be accounted for in any statistical treatment of the data. Pooling samples may also be an option and this depends on the type of experiment. These multiple sources of variation has led some investigators63–65 to cast doubt on the suitability of univariate tests, such as Student’s t-test, commonly used to assess the significance of observed protein expression differences. Multivariate analysis has been successfully used by several investigators in recent years.65–67

3.2.3

Protein Identification by Peptide Mass Fingerprinting

Identification of proteins on 2DE gels is most commonly achieved via mass spectrometry of trypsin digests. Briefly, the spot of interest is excised from the gel, digested with trypsin (or another suitable protease), and the resulting peptide mixture is analyzed by mass spectrometry. The most popular mass spectrometry method is MALDI-TOF mass spectrometry,68 where peptides are suspended in a matrix of small, organic, UV-absorbing molecules (such as 2,5-dihydroxybenzoic acid) followed by ionization by a laser at the excitation wavelength of the matrix molecules and acceleration of the ionized peptides in an electrostatic field into a flight tube where the time of flight of each peptide is measured and this gives its expected mass.

Peptides identified as those derived from Atlantic cod β-tubulin Trypsin autolysis peaks

0 741.0

1652.6

2108.4

2798.43

2506.36

2212.25

1974.00

1659.96 1697.90

1229.61 1272.70

1196.8

1822.98 1886.07

10

1131.56

20

856.50

30

1028.50

40

1575.35 1621.801616.86

50

1287.69

60

1061.54

 Intensity

70

870.54

80

1960.06

1258.71

90

29

1159.63

1040.60

100

842.51

Proteomics ◾

2564.2

Mass (m/z)

Figure 3.4 A trypsin digest mass spectrometry fingerprint of an Atlantic cod larval protein spot, identified as b-2 tubulin. The open markers indicate mass peaks corresponding to trypsin self-digestion products and were, therefore, excluded from the analysis. The solid markers indicate the peaks that were found to correspond to expected b-2 tubulin peptides.

The resulting spectrum of peptide masses (Figure 3.4) is then used for protein identification by searching against expected peptide masses calculated from data in protein sequence databases, such as the National Centre for Biotechnology Information (NCBI) nonredundant protein sequences database, using the appropriate software. Several programs are available, many with a web-based open-access interface. The ExPASy Tools web site (http://www.expasy.org/tools) contains links to most of the available software for protein identification and several other tools. Attaining a high identification rate is problematic in fish and seafood proteomics due to the relative paucity of available protein sequence data for these animals. As can be seen in Table 3.1, this problem is surprisingly acute for species of commercial importance. To circumvent this problem, it is possible to take advantage of the available nucleotide sequences, which in many cases is more extensive than the protein sequences available, to obtain a tentative identity. How useful this method is will depend on the length and quality of the available nucleotide sequences. It is important to realize, however, that an identity obtained in this manner is less reliable than that obtained through protein sequences and should be regarded only as tentative in the absence of corroborating evidence (such as 2D immunoblots, correlated activity measurements, or transcript abundance). In their work on the rainbow trout liver proteome, Martin et al.10 and Vilhelmsson et al.9 were able to attain an identification rate of about 80% using a combination of search algorithms that included the open-access Mascot program69 and a licensed version of Protein Prospector MS-Fit70 by searching against both protein databases and a database containing all salmonid nucleotide sequences. In those cases where both the protein and nucleotide databases yielded results, 100% agreement was observed between the two methods.

30

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Table 3.1 Families of Some Commercially Important Seafood Species and the Availability of Protein and Nucleotide Sequence Data as of March 27, 2008 Protein Sequences

Nucleotide Sequences

185,533

5,782,086

Anguilliformes (eels and morays)

1,680

2,208

Clupeiformes (herrings)

1,407

2,284

Cypriniformes (carps)

84,896

2,046,798

Siluriformes (catfishes)

3,845

81,845

10,063

999,489

3,122

130,353

268

287

26,424

170,381

2,442

45,626

237

138

2,344

16,210

3,592

768,007

Carcharhiniformes (ground sharks and dogfishes)

735

911

Lamniformes (mackrel sharks)

179

303

Rajiformes (skates and rays)

585

18,008

32,287

898,006

8,158

121,762

20,245

726,864

2,380

47,751

21,656

467,933

Caridea (shrimps, etc.)

2,585

3,871

Astacidea (lobsters and crayfishes)

1,237

32,138

Brachyura (short-tailed crabs)

2,203

36,557

Actinopterygii (Ray-Finned Fishes)

Salmoniformes (salmons and trout) Gadiformes (cod-likes, incl. cod, haddock, saithe, and pollock) Lophiiformes (anglerfishes, incl. monkfish) Perciformes (perch-likes, incl. sea bream, sea bass, mackrel, tuna, and wolffish) Pleuronectiformes (flatfishes, incl. halibut, turbot, sole, and plaice) Zeiformes (dories) Scorpaeniformes (scorpionfishes, incl. redfish and lumpfishes)

Chondrichthyes (Cartilagenous Fishes)

Mollusca (Mollusks) Bivalvia (mussels, scallops, etc.) Gastropoda (incl. whelks and abalone) Cephalopoda (squid and octopi)

Crustacea (Crustaceans)

Proteomics ◾ 31

A more direct, if rather more time-consuming, way of obtaining protein identities is by direct sequence comparison. Until recently, this was accomplished by N-terminal or internal (after proteolysis) sequencing by the Edman degradation of eluted or electroblotted protein spots.71,72 Today, the method of choice is tandem mass spectrometry (MS/MS). In the peptide mass fingerprinting discussed earlier, each peptide mass can potentially represent any of a large number of possible amino acid sequence combinations. The larger the mass (and longer the sequence), the higher the number of possible combinations. In MS/MS one or several peptides are separated from the mixture and dissociated into fragments that are then subjected to a second round of mass spectrometry, yielding a second layer of information. Correlating this spectrum with the candidate peptides identified in the first round narrows down the number of candidates. Furthermore, several short stretches of amino acid sequence will be obtained for each peptide, which, when combined with the peptide and fragment masses obtained, enhances the specificity of the method even further.73–75 Mass spectrometry methods in proteomics have been reviewed, for example, by Yates,76 Nyman,77 Damodaran et al.,78 Thiede et al.,79 Rappsilber et al.,80 Mo and Karger,81 Gygi and Aebersold,82 Lin et al.,83 and Delahunty and Yates.84

3.3 Applications of 2DE in Seafood Analysis The two-dimensional electrophoresis has been in use within food science for at least two decades. Early studies focused on relatively small, clearly defined subproteomes and included such applications as the characterization of bovine caseins,85 wheat flour baking quality factors,86 and soybean protein bodies.87 With the lower cost, improved reproducibility and resolving power of electrophoretic separation techniques, and vastly superior protein spot identification techniques, proteomic investigations on fish and seafood products, as well as in aquaculture, fish physiology, and development, have gained considerable momentum.88,89 A brief discussion of a few emerging areas within fish and seafood proteomics is given as follows.

3.3.1 Development Fishes go through different developmental stages (embryo, larva, and adult) during their life span that coincide with changes in the morphology, physiology, and behavior of the fish.23,90–92 The morphological and physiological changes that occur during these developmental stages are characterized by differential cellular and organelle functions.93 This is reflected in the variations of global protein expression and posttranslational modifications of the proteins that may cause alterations in protein function.94 Proteome analysis provides valuable information on the variations that occur within the proteome of organisms. These variations may, for example, reflect a response to biological perturbations or external stimuli9–11,95 resulting in different expression of proteins, posttranslational modifications, or redistribution of specific proteins within cells.20 To date few studies on fish development exist in which proteome analysis techniques have been applied. Recent studies on global protein expression during early developmental stages of zebrafish7 and Atlantic cod6 revealed that distinctive protein profiles characterize the developmental stages of these fishes even though abundant proteins are largely conserved during the experimental period. In both these studies, the identified proteins consisted mainly of proteins located in the cytosol, cytoskeleton, and nucleus. Proteome analyses in developing organisms have shown that many

32 ◾


of the identified proteins have multiple isoforms96 that reflect either different gene products97 or posttranslationally modified forms of these proteins.98 Different isoforms generated by posttranslational modifications are largely overlooked by studies based on RNA expression. This fact further indicates the importance of the proteome approach to understand cellular mechanisms that underlie fish development. Studies on various proteins have shown that during fish development sequential synthesis of different isoforms appear successively.8,21,94,99–107 In this context, developmental stage specific muscle protein isoforms have gained a special attention.8,21,99–107 The developmental changes in the composition of muscle protein isoforms have been tracked by proteome analysis in African catfish (Heterobranchus longifilis),102 common sole (Solea solea),21 and dorada (Brycon moorei).101 These studies demonstrated that the muscle shows the usual sequential synthesis of protein isoforms in the course of development. For example, in the common sole 2DE revealed two isoforms (larval and adult) of myosin light chain 2 and likewise in dorada larval and adult isoforms of troponin I were sequentially expressed during development. Proteomic techniques have thus been shown to be applicable for investigating cellular and molecular mechanisms involved in the morphological and physiological changes that occur during fish development. The major obstacle on the use of proteomics in embryonic fish has been the high proportion of yolk proteins. These interfere with any proteomic application that intends to target the cells of the embryo proper. In a recent study on the proteome of embryonic zebrafish, the embryos were deyolked to enrich the pool of embryonic proteins and to minimize ions and lipids found in the yolk prior to 2D gel analysis.7 Despite this undertaking, a large number of yolk proteins remained prominently present in the embryonic protein profiles. Link et al.27 published a method to efficiently remove the yolk from large batches of embryos without losing cellular proteins. The success in the removal of yolk proteins by Link et al.27 is probably due to dechorionation prior to the deyolking of the embryos. By dechorionation, the embryos fall out of their chorions facilitating the removal of the yolk.99,108

3.3.2 Quality Involution Degradation of proteins during chilled storage, and their oxidation during frozen storage, are among persistent quality problems in the seafood industry and have deleterious effects on fish flesh texture. Furthermore, several commercially important fish muscle processing techniques, such as curing, fermentation, and production of surimi and conserves occur under conditions conducive to endogenous proteolysis.109,110 Problems of this kind, where differences are expected to occur in the number, molecular mass, and pI of the protein present in a tissue, are well suited for investigation using 2DE-based proteomics. It is also worth noting that protein isoforms other than proteolytic ones, whether they be encoded in structural genes or brought about by posttranslational modification, usually have different molecular weight or pI and can, therefore, be distinguished on 2DE gels. Thus, specific isoforms of myofibrillar proteins, many of which are correlated with specific textural properties in seafood products, can be observed using 2DE or other proteomic methods.88,111

3.3.2.1 Protein Autolysis and Oxidation during Storage and Processing The specifics of fish muscle protein autolysis during storage and processing still remain in large part to be elucidated, although degradation of myofibrillar proteins by calpains and cathepsins112,113

Proteomics ◾ 33

and degradation of the extracellular matrix by the matrix metalloproteases and matrix serine proteases114,115 are thought to be among the main culprits. Whatever may be the mechanism, it is clear, that these quality changes are species dependent116,117 and, furthermore, appear to display seasonal variations.112,118 Several 2DE studies have been performed on postmortem changes in seafood flesh14–17, 67,111,117,119,120 and have demonstrated the importance and complexity of proteolysis and oxidative changes in seafood proteins during storage and processing. For example, Martinez et al.121 used a 2DE approach to demonstrate different protein composition of surimi made from prerigor versus postrigor cod and found that 2DE could distinguish between the two. Kjærsgård et al.17 used 2DE, 2D-immunoblots and LC–MS/MS to study changes in protein oxidation during frozen storage of rainbow trout. They found fish muscle proteins to be differentially carbonylated during frozen storage and were able to identify several carbonylated proteins using LC–MS/MS.

3.3.2.2

Aquaculture and Antemortem Effects on Quality and Processability

It is well known that an organism’s phenotype, including quality characteristics, is determined by environmental as well as genetic factors. Indeed, Huss noted in his review122 that product quality differences within the same fish species can depend on feeding and rearing conditions, differences that can affect postmortem biochemical processes in the product which, in turn, affect the involution of quality characteristics in the fish product. The practice of rearing fish in aquaculture, as opposed to wild fish catching, therefore raises the tantalizing prospect of managing quality characteristics of the fish flesh antemortem, where individual physiological characteristics, such as those governing gaping tendency, flesh softening during storage, etc., are optimized. To achieve that goal, the interplay between these physiological parameters and environmental and dietary variables needs to be understood in detail. With the ever increasing resolving power of molecular techniques, such as proteomics, this is fast becoming feasible. We are aware of two recent studies where Atlantic cod muscle proteomes have been compared between farmed and wild fish.15,123 Both studies indicated that several proteins are differentially expressed in farmed versus wild cod. Olsson et al.123 found these to comprise several members of the glycolytic and Krebs cycle pathways. In a recent study on the feasibility of substituting fish meal in rainbow trout diets with protein from plant sources, various quality characteristics of fillet and body were measured124,125 and the liver proteome was analyzed9,10,126 in fish fed with the experimental diets. The diet was found to have a marked effect on product texture, and the amount and composition of free amino acids in the fish flesh. Furthermore, the proteome analysis identified a number of metabolic pathways sensitive to plant protein substitution in rainbow trout feed, such as pathways involved in cellular protein degradation, fatty acid breakdown, and NADPH metabolism. In the context of this chapter, the effects on the proteasome are particularly noteworthy. The proteasome is a multisubunit enzyme complex that catalyzes proteolysis via the ATP-dependent ubiquitin–proteasome pathway which, in mammals, is thought to be responsible for a large fraction of cellular proteolysis.127,128 In rainbow trout, the ubiquitin–proteasome pathway has been shown to be downregulated in response to starvation129 and have a role in regulating protein deposition efficiency.44 The results led the authors to speculate that the difference in texture and postmortem amino acid-free pool development are affected by antemortem proteasome activity.1

34 ◾

3.3.3


Species Authentication

Processed fish products are increasingly common in the market and, as different fish species have different market values, this makes the issue of species authentication an area of increasing economic importance, as well as being relevant from a public health standpoint. While DNA-based species identification130–132 and isotope distribution techniques for determining geographical origin133 are powerful tools in this area and likely to remain the methods of choice in the near term, proteomics-based species identification methods are likely to develop rapidly and find commercial uses within this field, particularly for addressing questions on the health status of the fish in question, the presence of stress-factors or contamination levels at the place of breeding, and postmortem treatment.14 Unlike the genome, the proteome varies from tissue to tissue and with environmental conditions. Proteome analysis can therefore potentially yield more information than genomic methods, possibly indicating freshness and tissue information in addition to species. Martinez et al.134 recently reviewed proteomic and other methods for species authentication in foodstuffs. From early on, proteomic methods have been recognized as a potential way of fish species identification. During the 1960s, 1D electrophoretic techniques were developed to identify the raw flesh of various species,135–137 which was soon followed by methods to identify species in processed or cooked products.138,139 These early efforts were reviewed in 1980.140,141 More recently, 2DE-based methods have been developed to distinguish various closely related species, such as the gadoids or several flat fishes.18,142,143 Indeed, the proteomes of even closely related fish species are be easily distinguishable by eye from one another on 2D gels1 indicating that diagnostic protein spots may be used to distinguish closely related species. Piñeiro and coworkers have found that Cape hake (Merluccius capensis) and European hake (Merluccius merluccius) can be distinguished on 2D gels from other closely related species by the presence of a particular protein spot identified as corresponding to nucleoside diphosphate kinase.143 Lopez and coworkers, studying three species of European mussels: Mytilus edulis, Mytilus galloprovincialis, and Mytilus trossulus, found that M. trossulus could be distinguished from the other two species on foot extract 2D gels by a difference in a tropomyosin spot. They found the difference to be due to a single T to D amino acid substitution.144 Martinez and Jakobsen Friis concluded that the identification of not only the species present, but also their relative ratios in mixtures of several fish species and muscle types14 would become viable once a suitable number of markers have been identified.

3.3.4 Allergen Identification Allergenic potential is food safety issue of particular concern to the seafood producer. Allergic reactions to seafood affect a significant part of the population. For example, about 0.5% of young adults are allergic to shrimp.145 Seafood allergies are caused by an immunoglobulin E-mediated response to particular proteins, including structural proteins such as tropomyosin.146 Proteome analysis can be a valuable tool for the identification and the characterization of allergens as exemplified by the study of Yu et al.147 at National Taiwan University. These authors, studying the cause of shrimp allergy in humans, performed a 2DE on crude protein extracts from the tiger prawn, Penaeus monodon, blotted the 2D gel onto a PVDF membrane, and probed the membranes with serum from confirmed shrimp allergic patients. The allergens were then identified by MALDITOF MS of tryptic digests. The allergen was identified as a protein with close similarity to arginine kinase. The identity was further corroborated by cloning and sequencing the relevant cDNA. A final proof was obtained by purifying the protein, demonstrating that it had arginine kinase

Proteomics ◾

35

activity and reacted to serum IgE from shrimp allergic patients and, furthermore, induced skin reactions in sensitized shrimp allergic patients.

Acknowledgments This work was supported by grants from the Icelandic Graduate Research Fund, the University of Iceland Research Fund, the University Research Fund of Eimskipafélag Íslands, and the AVS Research Fund.

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Proteomics ◾ 41 120. Martinez, I., Jakobsen Friis, T., and Careche, M., Post mortem muscle protein degradation during ice-storage of Arctic (Pandalus borealis) and tropical (Penaeus japonicus and Penaeus monodon) shrimps: A comparative electrophoretic and immunological study. Journal of the Science of Food and Agriculture 81, 1199–1208, 2001. 121. Martinez, I., Solberg, C., Lauritzen, C., and Ofstad, R., Two-dimensional electrophoretic analyses of cod (Gadus morhua L.) whole muscle proteins, water soluble fraction and surimi. Effect of the addition of CaCl 2 and MgCl2 during the washing procedure. Applied and Theoretical Electrophoresis 2, 201–206, 1992. 122. Huss, H. H., Quality and Quality Changes in Fresh Fish, FAO, Rome, 1995. 123. Olsson, G. B., Friis, T. J., Jensen, E., and Cooper, M., Metabolic disorders in muscle of farmed Atlantic cod (Gadus morhua). Aquaculture Research 38, 1223–1227, 2007. 124. De Francesco, M., Parisi, G., Médale, F., Lupi, P., Kaushik, S. J., and Poli, B. M., Effect of long-term feeding with a plant protein mixture based diet on growth and body/fillet quality traits of large rainbow trout (Oncorhynchus mykiss). Aquaculture 236, 413–429, 2004. 125. Parisi, G., De Francesco, M., Médale, F., Scappini, F., Mecatti, M., Kaushik, S. J., and Poli, B. M., Effect of total replacement of dietary fish meal by plant protein sources on early post mortem changes in the biochemical and physical parameters of rainbow trout. Veterinary Research Communications 28, 237–240, 2004. 126. Martin, S. A. M., Vilhelmsson, O., and Houlihan, D. F., Rainbow trout liver proteome—Dietary manipulation and protein metabolism, in Progress in Research on Energy and Protein Metabolism, Souffrant, W. B. and Metges, C. C., Eds., Wageningen Academic Publishers, Wageningen, The Netherlands, 2003, pp. 57–60. 127. Craiu, A., Akopian, T., Goldberg, A., and Rock, K. L., Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide. Proceedings of the National Academy of Science USA 94, 10850–10855, 1997. 128. Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., and Goldberg, A. L., Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78, 761–771, 1994. 129. Martin, S. A., Blaney, S., Bowman, A. S., and Houlihan, D. F., Ubiquitin-proteasome-dependent proteolysis in rainbow trout (Oncorhynchus mykiss): Effect of food deprivation, Pflügers Archives European Journal of Physiology 445 (2), 257–66, 2002. 130. Sotelo, C. G., Piñeiro, C., Gallardo, J. M., and Pérez-Martín, R. I., Fish species identification in seafood products. Trends in Food Science and Technology 4, 395–401, 1993. 131. Mackie, I. M., Pryde, S. E., Gonzales-Sotelo, C., Medina, I., Pérez-Martín, R., Quinteiro, J., Rey-Mendez, M., and Rehbein, H., Challenges in the identification of species of canned fish. Trends in Food Science and Technology 10, 9–14, 1999. 132. Martinez, I., Jakobsen Friis, T., and Seppola, M., Requirements for the application of protein sodium dedecyl sulfate-polyacrylamide gel electrophoresis and randomly amplified polymorphic DNA analyses to product speciation. Electrophoresis 22, 1526–1533, 2001. 133. Campana, S. E. and Thorrold, S. R., Otoliths, increments, an elements: Keys to a comprehensive understanding of fish populations? Canadian Journal of Fisheries and Aquatic Sciences 58, 30–38, 2001. 134. Martinez, I., Aursand, M., Erikson, U., Singstad, T. E., Veliyulin, E., and van den Zwaag, C., Destructive and non-destructive analytical techniques for authentication and composition analyses of foodstuffs. Trends in Food Science and Technology 14, 489–498, 2003. 135. Cowie, W. P., Identification of fish species by thin slab polyacrylamide gel electrophoresis. Journal of the Science of Food and Agriculture 19, 226–229, 1968. 136. Mackie, I. M., Identification of fish species by a modified polyacrylamide disc electrophoresis technique. Journal of the Association of Public Analysts 5, 83–87, 1969. 137. Tsuyuki, H., Uthe, J. F., Roberts, E., and Clarke, L. W., Comparative electropherograms of Coregonis clupeoformis, Salvelinus namaycush, S. alpinus, S. malma and S. fontinalis from the family Salmonidae. Journal of the Fisheries Research Board of Canada 23, 1599–1606, 1966.

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138. Mackie, I. M., Some improvements in the polyacrylamide disc electrophoretic method of identifying species of cooked fish. Journal of the Association of Public Analysts 8, 18–20, 1972. 139. Mackie, I. M. and Taylor, T., Identification of species of heat-sterilized canned fish by polyacrylamide disc electrophoresis. Analyst 97, 609–611, 1972. 140. Mackie, I., A review of some recent applications of electrophoresis and isoelectric focusing in the identification of species of fish in fish and fish products, in Advances in Fish Science and Technology, Connell, J. J., Ed., Fishing News Books Ltd., Aberdeen, U.K., 1980. 141. Hume, A. and Mackie, I., The use of electrophoresis of the water-soluble muscle proteins in the quantitative analysis of the species components of a fish mince mixture, in Advances in Fish Science and Technology, Connell, J. J., Ed., Fishing News Books Ltd., Aberdeen, U.K., 1980. 142. Piñeiro, C., Barros-Velázquez, J., Sotelo, C. G., and Gallardo, J. M., The use of two-dimensional electrophoresis for the identification of commercial flat fish species. Zeitschrift für Lebensmitteluntersuchung und -forschung 208, 342–348, 1999. 143. Piñeiro, C., Vázquez, J., Marina, A. I., Barros-Velázquez, J., and Gallardo, J. M., Characterization and partial sequencing of species-specific sarcoplasmic polypeptides from commercial hake species by mass spectrometry following two-dimensional electrophoresis. Electrophoresis 22, 1545–1552, 2001. 144. Lopez, J. L., Marina, A., Alvarez, G., and Vazquez, J., Application of proteomics for fast identification of species-specific peptides from marine species. Proteomics 2, 1658–1665, 2002. 145. Woods, R. K., Thien, F., Raven, J., Walters, E. H., and Abramson, M., Prevalence of food allergies in young adults and their relationship to asthma, nasal allergies, and eczema. Annals of Allergy Asthma and Immunology 88, 183–189, 2002. 146. Lehrer, S. B., Ayuso, R., and Reese, G., Seafood allergy and allergens: A review. Marine Biotechnology 5, 339–348, 2003. 147. Yu, C.-J., Lin, Y.-F., Chiang, B.-L., and Chow, L.-P., Proteomics and immunological analysis of a novel shrimp allergen, pen m 2, The Journal of Immunology 170, 445–453, 2003.

Chapter 4

Seafood Genomics Astrid Böhne,* Delphine Galiana-Arnoux,* Christina Schultheis,* Frédéric Brunet, and Jean-Nicolas Volff Contents 4.1 Introduction ..................................................................................................................... 43 4.2 Genetics and Genomics.................................................................................................... 44 4.3 Genomic Resources and Genome Projects for Aquatic Species ..........................................45 4.4 Genomics, Fisheries, and the Management of Biodiversity ................................................47 4.5 Genomics and Aquaculture .............................................................................................. 49 4.6 Concluding Remarks ........................................................................................................51 Acknowledgments ......................................................................................................................52 References ..................................................................................................................................52 There the nets brought up beautiful specimens of fish: Some with azure fins and tails like gold, the flesh of which is unrivalled; some nearly destitute of scales, but of exquisite flavour; others, with bony jaws, and yellow-tinged gills, as good as bonitos; all fish that would be of use to us. Jules Verne, Twenty Thousand Leagues under the Sea

4.1 Introduction The development of high-throughput DNA sequencing methods has opened the era of genomics, which has revolutionized biology, medicine, and biotechnology over the last decade. The rise of * Equal contributors.

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genomics has generated an impressive wave of novel information concerning genome structure, function, and evolution. Massive analysis of functional gene variability in many organisms has allowed to better understand the molecular basis of biodiversity and disease. In the field of biotechnology, genomics is principally used to identify molecular markers, genes and alleles of zootechnical interest for the genetic improvement of economically important species, and to contribute to the management of biodiversity. Genomics has important applications for fisheries and aquaculture [1], which are reviewed in this chapter.

4.2 Genetics and Genomics Genetics can be defined as the science of heredity and variation in organisms. Heredity is based on genes, which are carried by chromosomes, which themselves constitute the genome. Most genes are located in the nucleus, but organelles (mitochondria and chloroplasts) have their own genome too. The science dealing with the analysis of genomes as a whole is called genomics. There are different but complementary ways to analyze genomes. One of them, called genetic mapping, consists in delineating intervals on the genome with genetic markers. This generates a genetic linkage map, with the distance between markers being directly proportional to the frequency of recombination between them. Genetic loci and genes of interest can then be mapped relative to these markers, providing an estimation of their localization in the genome. In addition, comparative mapping provides important information on the structure and evolution of genomes in different species. Genetic markers must be polymorphic to allow the analysis of their segregation, and therefore of their linkage. Different types of DNA markers are used for mapping, such as restriction fragment length polymorphisms (RFLPs, caused by sequence polymorphisms at restriction sites) [2]. DNA markers with a polymorphic number of tandem repeats are called minisatellites (repeat units up to 25 bp in length) and microsatellites (shorter repeat units, usually dinucleotides or tetranucleotides), the latter being of wide use in genotyping and mapping experiments. Other important markers are single nucleotide polymorphisms (SNPs), that is, one nucleotide differences within otherwise identical, generally orthologous sequences [3]. Since SNPs can occur not only in noncoding but also in coding sequences, they are likely to be less neutral than other markers from the functional point of view. SNP analysis can therefore uncover genes and residues that are targeted by evolution and lead to the identification of disease-associated genes. Random amplified polymorphic DNA (RAPD) markers are amplified enzymatically by polymerase chain reaction (PCR) using short arbitrary oligonucleotide primers. Amplified fragment length polymorphism (AFLP) markers combine the principle of RFLP with PCR: fragments cut with restriction enzymes are ligated with adaptors; DNA fragments are amplified enzymatically using primers matching both adaptor and restriction site. Finally, polymorphic insertions of retrotransposable elements, increasingly used for phylogenetic reconstructions [4], can also be used for mapping purposes. Such markers might be further developed in fish, which have genomes with very diverse transposable elements [5]. Molecular markers are not only useful for genome mapping but also represent important tools in other domains, for example, in population genetics. In order to investigate gene content, arrangement, and structure, nuclear and organelle genomes can be sequenced to (almost) completion, as done for the human genome [6,7]. Traditionally, genomes are sequenced using the “shotgun” strategy, with randomly sheared pieces of DNA massively cloned, sequenced, and subsequently assembled in “contigs” in silico. Gene regulatory and coding sequences are then predicted through bioinformatic analysis involving sequence prediction and database comparisons. The development of efficient methods in bioinformatics is a condition sine qua non for progresses in the field of genomics.

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A clone-by-clone approach can be used as an alternative to, or even better in combination with, shotgun sequencing. Parts of the genome, cloned in a bacterial vector and constituting a so-called genomic library, can be sequenced either to completion or from their ends. Such an approach is, for example, useful in the case of regions rich in repetitive sequences posing problems to assembly after whole genome shotgun sequencing. Bacterial artificial chromosomes (BACs) accepting inserts from several hundreds of kilobases are frequently used as vectors. The overlapping between these clones and their relative arrangement in the genome can be determined through fingerprint analysis (e.g., through the identification of common restriction fragments). This provides a physical map respecting the “real” base pair distance between genes and markers, which can be very useful to precisely determine the relative position of sequence contigs assembled “in silico” from whole genome shotgun sequencing data. The relative position of two contigs can also be estimated cytogenetically using double fluorescent in situ hybridization [8]. Probes specific to each contig marked with different fluorochromes are cohybridized on chromosome preparations to test if they are located on the same or on different chromosomes. Physical maps can also be constructed by analyzing the segregation of genomics markers (also called STSs for sequence-tagged sites) in randomly fragmented parts of the genome. These fragments are either integrated in the genome of a host cell line from a different organism in radiation hybrid (RH) mapping [9] or diluted to give aliquots containing approximately one haploid genome equivalent (HAPPY mapping [10]). Importantly, a new revolution of large-scale sequencing is ushering in a second era of genomics, with novel methods allowing very rapid and much cheaper sequencing of large amounts of DNA [11–13]. Sequence data can be used among others to identify similarities and differences between species and study genome evolution (comparative genomics [14]) or to infer reliable phylogenetic relationships between organisms (molecular phylogenetics and phylogenomics [15]). A method called “DNA barcoding” should help to identify species and phylogenetic units, hereby contributing to species conservation and management of global fish biodiversity (http://www.fishbol.org/). Barcoding is based on a sequence of short standard parts of the genome. Generally, a 650 bp fragment of the 5′ end of the mitochondrial gene cytochrome c oxidase I is used as a global standard in fish and other animals (for review, see Ref. [16]). Additional approaches are required to study gene expression (transcriptomics, proteomics) and function (functional genomics) as well as interactions with the environment (environmental genomics). Large-scale expression studies at the transcriptional level are generally performed using microarrays or other methods of high-throughput expression profiling. Of particular interest are expressed sequence tags (ESTs), obtained through sequencing of complementary DNA (cDNA) libraries. EST analysis not only provides important data on genes expressed in particular tissues/ organs or at specific stages of development but also allows the characterization of gene structure through comparison with genomic sequences. ESTs can also be used, for instance, for SNP detection and phylogenetic reconstructions.

4.3

Genomic Resources and Genome Projects for Aquatic Species

Genetic and genomic resources have been generated for many aquatic species of economical interest. In addition, aquatic model organisms of insignificant importance such as seafood have been developed for other scientific purposes and have been targeted for whole genome-sequencing projects [17]. For example, zebrafish and medaka are two complementary fish models to study

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vertebrate development [18]. These models are nevertheless useful to decipher gene content in species targeted by fisheries and aquaculture through comparative genomics [19,20]. Compared with agricultural plants and terrestrial livestock, genomic studies on aquatic species are relatively recent. SNPs and other polymorphic markers as well as linkage maps have now been generated for many aquaculture species, including fish (sea bream, sea bass, Atlantic salmon, rainbow trout, and other salmonids, tilapia, carp, catfish, Japanese flounder, and others) and invertebrates (oyster, abalone, mussel, scallop, sea urchin, shrimp, and others) (for review, see Refs. [1,21]). Expressed sequence tags are also available for many fish species, providing useful information on gene sequence and expression in different tissues and organs or at different stages of development (http://www.ncbi.nlm.nih.gov/dbEST/). Aquatic invertebrate species with well-developed EST resources include scallop and oyster (mollusks) as well as blue/green crabs, shrimp, and lobster (crustaceans). A variety of genomic libraries, particularly BAC libraries, as well as RH panels and cDNA microarrays have been constructed for aquatic organisms; physical maps are available for species such as Nile tilapia, Atlantic salmon, and channel catfish [22–28]. For some species like the rainbow trout, assignment of linkage groups to specific chromosomes has been performed through fluorescent in situ hybridization [29]. Most genome drafts available so far are for aquatic model species without any real economic importance (for review, see Ref. [17]). However, these sequencing projects have provided valuable general information on the structure, evolution, and gene content of fish genomes. Particularly, they have revealed some evolutionary peculiarities possibly linked to biodiversity, such as the high diversity of transposable elements and presence of numerous duplicated genes that are remnants of an ancestral whole genome duplication [30–33]. Fishes with sequenced genomes include the pufferfish species Takifugu rubripes ([34]; http:// www.fugu-sg.org/) and Tetraodon nigroviridis [35]. Both species have an extremely compact genome with low repeat content and short intronic and intergenic sequences and have been useful to identify conserved genes and noncoding sequences in the human genome [36]. Other species with advanced or completed genome projects include the medaka Oryzias latipes [37,38], the three-spined stickleback Gasterosteus aculeatus (http://www.ensembl.org/Gasterosteus_ aculeatus/), and the zebrafish Danio rerio (http://www.ensembl.org/Danio_rerio/). A genomesequencing project is underway for the tilapia Oreochromis niloticus, an aquaculture species of high economical value, in association with low-coverage sequencing projects for three additional cichlids (http://www.genome.gov/10002154). For Atlantic salmon and other salmonids, no draft genome is available now, but many other genomic resources have been developed, particularly by the Genomics Research on All Salmon Project consortium (cGRASP) (http://web.uvic.ca/grasp/). Atlantic salmon genome should be sequenced soon, possibly followed by the genome of the rainbow trout. Other projects aim to enhance genomic resources for economically important species, for example, for the Atlantic cod (Cod Genomics and Broodstock Development Project, http:// codgene.ca/index.php). For cartilaginous fish, the genome of the elephant shark Callorhinchus milii, which is relatively compact, has been sequenced at low coverage [39,40]; http://esharkgenome.imcb.a-star.edu.sg/). A genome project is in the pipeline for another cartilaginous fish, the little skate Leucoraja erinacea. (http://www.mdibl.org/research/skategenome.shtml). Further projects aim to sequence the genome of coelacanth, gar, skate, lamprey, and hagfish, which occupy strategic taxonomic positions within and relative to vertebrates (http://www.genome.gov/10002154). The genome of an echinoderm, the purple sea urchin Strongylocentrotus purpuratus, has been sequenced [41]. Beside the genome of the zooplankton Daphnia pulex (water flea; http://wfleabase. org/), the sequencing of the genome of other crustaceans is planned, including the amphipod

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crustacean Jassa slatteryi (http://www.genome.gov/10002154) as well as the genome of the Atlantic horseshoe crab (chelicerate) (http://www.jgi.doe.gov/sequencing). Genome projects are performed for the cnidarian species Hydra magnipapillata (green hydra) and Nematostella vectensis (sea anemone) (http://hydrazome.metazome.net/; http://genome.jgi-psf.org/Nemve1/Nemve1.home.html). Genome sequencing should follow for many other aquatic animal species of economical interest, for example, the Pacific oyster [42]. Seaweed, constituted by several groups of multicellular algae (red algae, green algae, and brown algae), is used as food by coastal populations, particularly in East Asia. Organelle genome sequences and EST resources are available for many algal species, for example, for the red alga Porphyra yezoensis (http://est.kazusa.or.jp/en/plant/porphyra/EST/). Genome drafts have been generated for the red alga Cyanidioschyzon merolae, the green algae or chlorophytes Chlamydomonas reinhardtii and Volvox carteri, the marine picoeukaryote Ostreococcus tauri, the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum, and the haptophyte Emiliania huxleyi (for review, see Ref. [43]).

4.4 Genomics, Fisheries, and the Management of Biodiversity Many aquatic populations have been overexploited through overfishing or collapsed and even become extinct through other factors such as pollution, habitat degradation and loss, introduction of exogenous species, climate change, and perturbations of ocean biogeochemistry [44–47]. About 30% of seafood stocks available in 1950 have already collapsed; it has been predicted that all commercial fish and seafood species will have done so by 2048 [48]. Harvesting and other forms of stress can cause strong alterations in population structure as well as a reduction in biodiversity. In addition, exploitation can act as a selective pressure and induce phenotypical shifts as evolutionary responses. For example, fisheries targeting large individuals will select for early maturation at smaller sizes, leading to a reduction of fisheries’ yield [49,50]. Biodiversity decline is associated with a collapse of seafood resource and a reduction in species stability and recovery potential, as well as with a decrease in water quality. Restoration of biodiversity increases fisheries productivity. Hence, the loss of marine biodiversity impairs the ability of ocean to provide food, to maintain water quality, and to recover from perturbations [48]. Consequently, description, monitoring, and conservation of biodiversity of aquatic organisms are now high priorities, with a major role for genomics, particularly in the assessment and follow-up of biodiversity in wild stocks, the estimation of fisheries-induced evolution, and the definition of conservation units and priorities for sustainable fishery management. Genetic monitoring, that is, the quantification of temporal changes in populations using molecular markers, provides information relevant to both the ecological and evolutionary time frame [51]. Important demographic and evolutionary parameters to be considered include organism abundance and vital rates, population structure and interactions, site occupancy, reproductive structure and behavior, pedigrees and social structure, gene flow, and hybridization, and invasion of disease and invasive species [51,52]. Nuclear and mitochondrial molecular markers can be used to identify units of management for fisheries and priorities for the conservation of biodiversity. Populations and ecosystems, with their particular adaptations and contributions to biodiversity, can be considered as conservation units [52]. Characterization of minimum viable population size is required to assess if they are facing a risk of extinction [45]. Population genetics is determined using various polymorphic genetic markers, including mitochondrial DNA polymorphisms, micro/minisatellites, AFLP and

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RAPD markers, SNPs, and others (for review, see Ref. [1]). For example, multiple SNPs have been generated for Atlantic cod. Populations of North-East Arctic cod and Norwegian coastal cod have been analyzed, thereby identifying loci potentially influenced by natural selection [53]. Different types of markers have been used for the estimation of natural population and the determination of conservation genetic parameters in salmonids [54] and to estimate quantitative genetic parameters under wild conditions [55]. For several species, including Atlantic herring, Atlantic salmon, brown trout, European eel, turbot, and pike, sufficient genetic data might be available to provide at least basic information on genetic structure and genetic units for biologically sustainable use [56]. In contrast, the available population genetic information is insufficient for most other species. Genetic monitoring of diversity using polymorphic markers allows monitoring population size and diversity over time. For example, microsatellite data indicated marked genetic changes in declining North Sea cod [57]. Population genomics is a form of population genetics extending the analysis of genetic variation in natural populations to the scale of the genome itself, heralding a new era in the analysis of adaptive evolution and functional variation [58,59]. With the development of much faster and cheaper high-throughput sequencing methods, this field will certainly be of major importance in the future of fisheries management and biodiversity conservation. Genome-wide gene expression profiling can also be used to detect variations in gene expression within and among natural populations [60]. This approach has already been used to identify adaptive differences between natural populations in several species, including the European flounder and the brown trout [61,62]. Beside populations, taxa can also be considered as conservation units, with poorly represented phylogenetic groups receiving high conservation priorities [52]. Accordingly, phylogenetics and phylogenomics are of major importance for the recognition of endangered taxa from the systematic point of view, with the discovery of new groupings and the determination of divergence times and molecular clocks [63]. Finally, conservation efforts could focus on the preservation of genetic diversity allowing biota to adapt to new conditions. For example, species-rich groups such as the East African cichlids [64] might be preserved with priority since their evolution potential might predispose them to serve as progenitors of future biodiversity [52]. Quantitative genetics as well as evolutionary genetics and genomics can help to identify such groups of high evolvability and to study the mechanisms driving their adaptability and speciation, with possible detection of DNA sequences promoting evolution in their genomes [17]. Evolutionary genetics and genomics might also help to understand the interplay between fishing and natural selection on population and species targeted by fisheries [65]. DNA barcoding and other methods have applications not only for species identification and molecular phylogenies but also in the field of population genetics to describe genetic diversity within species [16]. Molecular markers can be used to monitor the efficiency of programs aiming to supplement declining wild populations through individuals reared in captivity. Through pedigree reconstruction with microsatellite markers, it has been, for example, observed that reintroduced steelhead trout presented reduced reproductive capabilities caused by genetic effects of domestication [66]. Genomics and transcriptomics can allow assessing the genetic and functional consequences of interbreeding between farmed and wild fish. This type of study has been performed on Atlantic salmon, for which large annual escapees of farmed Atlantic salmon enhance the risk of extinction of wild populations. Gene transcription profiling suggested that interbreeding of fugitive farmed salmon and wild individuals can substantially modify gene transcription in natural populations exposed to high migration from fish farms, resulting in potentially detrimental effects on survival of these populations [67]. The effects of stress factors contributing to species collapse and

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extinction, for example, pollution (ecotoxicogenomics), as well as the development of resistance mechanisms by the targeted species can be studied using transcriptomics [25,68].

4.5 Genomics and Aquaculture Fish consumption has doubled over the past 50 years and would need to double again over the next 25 years ([69] and references therein). In order to reduce the ecological disaster of overfishing and contribute to solve the problem of global feeding, especially in developing countries, aquaculture including marine aquaculture (mariculture) has increased its production by a 20-fold factor over the last 30 years. Aquaculture needs to be further developed in the future; diversification and genetic improvement of cultivated species should lead to both a reduction in production costs and an increase in fish production. The genetic basis of important zootechnical traits, such as resistance to viral and bacterial diseases, fillet quality (color, texture, and fat deposition), growth and feed efficiency, sexual development, and others must be analyzed to allow efficient breeding and management programs. Significant improvements have been obtained through efficient breeding programs for several species such as farmed salmon and trout. Molecular methods have contributed to the significant increase in aquaculture production worldwide, but genetics and genomics remain poorly developed for aquaculture species compared with crops and livestocks [70]. Genomic sequences, particularly polymorphic DNA markers such as microsatellites, can be used for parental assignment and construction of DNA pedigrees to analyze the heritability of zootechnical parameters and reproductive success or to avoid inbreeding and estimate genetic diversity [71]. These methods are particularly useful when classical individual tagging is difficult or when individual tanks are not available to separate families. Linkage maps are used to map onto genomes genetic loci such as quantitative trait loci (QTLs) influencing traits of economical interest in aquaculture fish species. Linkage analysis allows determining the segregation of a trait of interest relative to polymorphic molecular markers. Examples include the mapping of QTLs involved in development rate, body weight and size, disease resistance and thermal tolerance in salmonids [72–78], cold tolerance, innate immunity, response to stress, biochemical parameters of blood and fish size in tilapia [79–81] and growth-related traits in sea bass [82], as well as in growth-related traits in the Pacific abalone [83], disease resistance in oyster [84], body weight and length in the Kuruma prawn [85], and virus resistance in shrimp [86]. DNA markers linked to a locus of zootechnical interest can subsequently be used to perform marker-assisted selection (MAS). Marker-assisted selection is an indirect process based on the selection of a DNA marker linked to a trait of interest to choose animals for selective breeding programs instead of selecting on the trait itself. Selection against an allele, conferring for example a disease, is also feasible with this method (for review, see Ref. [2]). MAS can be performed at early stages of development and is particularly appropriate for traits that are difficult to measure, exhibit low heritability, and/or are expressed late in development. This method also allows monitoring the transfer of genes that control desired phenotypes between breeds, for example, a gene conferring disease resistance into a strain selected for production. In this case, individuals backcrossed with the “production” parent will be selected for the presence of a molecular marker linked to the resistance locus. The efficiency of the method depends on the predictability provided by the marker, that is, on its linkage with the locus of interest. Accordingly, the most effective markers to perform this method of selection are the functional mutations within the trait genes (“direct” markers). A variation of MAS using markers covering the whole genome to assess the status of multiple QTLs is called genomic selection

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[87]. For the great majority of aquaculture species, MAS has not been used so far, particularly due to the lack of high-resolution genetic maps [1]. A trait of particular interest for aquaculture is sex determination. In order to avoid overcrowding and stress induced by sexual maturation and exploit advantageous sex-linked traits (growth rate, flesh quality, behavior, etc.), monosex cultures (either all-male or all-female populations, depending on the species) are frequently used in fish farming. Such monosex populations can be obtained with parents sex-reversed through hormone treatment or produced by androgenesis or gynogenesis. Molecular sexing of individuals at early stages of their development using sex-specific markers would allow the early selection of breeders of a chosen genotype for the production of monosex populations and the rapid analysis of breeding, androgenesis, and gynogenesis products. A better knowledge of sex determination is also required for environment-friendly manipulation of phenotypic sex, for example through temperature, as an alternative to exogenous hormone treatment. Interestingly, in contrast to the situation observed for example in birds and mammals, sex determination is hypervariable in fish [88]. Several hundreds of fish species are sequential hermaphrodites and develop either first as a male and subsequently as a female (protandrous) or vice versa (protogynous). Synchronous hermaphrodites also exist in fish. In gonochoristic (with distinct sexes) species, all possible forms of genetic sex determination have been observed, from male and female heterogamety with or without influence of autosomal loci to more complicated systems involving several loci but without sex chromosomes (polyfactorial sex determination) or more than two sex chromosomes and even several pairs of sex chromosomes. In numerous species, sex determination can be influenced by temperature and other environmental factors such as the pH of water and even social parameters [89]. Phenotypic sex can frequently be fully reversed by hormone treatment, a method largely used in aquaculture to control fish reproduction. Interestingly, even closely related fish species can have very different mechanisms of sex determination, thus reflecting a frequent switching between sex determination systems during evolution. Due to this variability, sex-linked markers for molecular sexing at early stages of development are generally restricted to a single species or are even population-specific within a same species. Sex-specific molecular markers linked to the master sex-determining gene on the sex chromosomes have been identified in many aquaculture fish species, including salmonids, tilapia, and African catfish [90–97]. The only master sex-determining gene identified so far in fish, dmrt1bY from the medaka fish Oryzias latipes [98,99], is not present in any fish species of economical interest. Once DNA markers linked to a locus controlling a trait of economical interest have been identified, the gene itself and the sequence polymorphism involved in phenotypic variation can be identified through positional cloning. When a genomic library is available, for example, a BAC library, genomic clones containing markers linked to the locus can be isolated from the library and sequenced to determine their gene content. When a physical map is available, sequencing can be performed on the tilling path, the minimal set of overlapping clones covering the region of interest. Genes identified through sequencing can be chosen for further analysis according to their described function or their pattern of expression. Sequencing of genomic clones covering a region of interest can also provide new DNA markers that can be used to refine the mapping of the locus, thereby reducing the number of genes to be tested. Alternatively, gene candidates with described functions related to the trait of interest can be directly mapped on the linkage map, with the hope of revealing a colocalization with the locus itself. Sequencing and sequence comparison of the different versions of the gene in individuals polymorphic for the phenotypes studied can allow the identification of the sequence variation at the origin of phenotype differences. Further characterization can be performed at the functional level in vitro or in vivo. Gene candidates with potentially interesting functions can be also directly sequenced in different families without

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mapping in order to test for associations between sequence and phenotype variation. One example is the identification of associations between SNPs in candidate genes and the growth rate in Arctic charr [100]. The detection of genes of zootechnical interest can also be performed through large-scale transcriptional analysis (transcriptomics). EST- and microarray-based transcription profiling for specific tissues, organs and stages of development has been performed in a variety of aquaculture species (for review, see Wenne et al. [1]). Transcriptomics is useful to detect genes differentially expressed in different genetic backgrounds or conditions. For example, genes differentially expressed in progenies exhibiting opposed susceptibility to summer mortality have been identified by suppression subtractive hybridization in oyster [101]. The effect of artificial selection on gene expression has been monitored through transcriptome analysis in Atlantic salmon [102]. The effect of dietary fish oil and fishmeal replacement by vegetable oils and plant proteins on farmed fish metabolism has been investigated in juvenile rainbow trout through hepatic gene expression profiling (nutrigenomics [103]). Phosphorus-responsive genes have been identified through transcriptomics in rainbow trout [104]. The effects of hormone treatments can be also monitored using microarrays [105–107]. Transcriptomics is frequently used to analyze disease and other stress response gene expression and identify resistance gene candidates. Immune response genes downregulated in the gills of amoebic gill disease-affected Atlantic salmons have been found through transcriptome analysis [108], and stress response genes have been investigated in the gilthead sea bream [109]. Microarray analysis of gene expression changes in catfish liver after infection with the gram-negative bacterium Edwardsiella ictaluri indicated a strong upregulation of several pathways involved in the inflammatory immune response and potentially in innate disease resistance [110]. Genes expressed in response to infection with white spot syndrome virus have been identified in shrimp [111].

4.6 Concluding Remarks In the future, seafood genetics and genomics might revolutionize fisheries management and aquaculture development. From systematic, ecological, and evolutionary perspectives, genomics has important applications in biodiversity analysis, exploitation, and conservation, with strong consequences on fisheries productivity. In this domain, much work is still to be done, since information on resource status and extinction risk is available for only a minority of marine fish species [45]. In aquaculture, a better knowledge of genes involved in the control of economically important traits will contribute to improve the production and reduce the costs for current aquaculture species and to identify and develop new potential target species for aquaculture. Such new species might include halibut, cod, wolf fish, flounder, bream, jack, dolphin fish, cobia, and grouper for marine species, and Arctic char, hybrid striped bass, and Australian Murray cod for fresh water species [69]. Genomics will also help to improve and control transgenesis and other methods of modification of gene expression, with the potential of increasing growth, environmental tolerance, and disease resistance ([69]; but see Ref. [112]). Comparative genomics will need to be further developed to increase the transfer of knowledge from models to aquaculture. Importantly, selection methods based on molecular makers remain extremely underdeveloped for aquatic species and will require further exploration based on denser genetic maps. Finally, genomics will boost the discovery of new bioactive molecules in aquatic organisms [113,114] and will be further developed for the identification/authentication of the composition of sea food products put on the market [115].

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Accordingly, many collaborative projects dealing with marine and aquaculture genomics have been or are currently funded by various agencies. For example, the European Union supports different projects. “Aquafirst” aims to combine genetic and functional genomic approaches for stress and disease resistance MAS in fish and shellfish (http://aquafirst.vitamib.com/). “Marine Genomics” is a network of excellence devoted to the development, utilization, and spreading of high-throughput approaches for the investigation of the biology of marine organisms (http:// www.marine-genomics-europe.org/). “Bridgemap” (http://www.bridgemap.tuc.gr/) develops an integrated genomic approach toward the improvement of aquacultured fish species. “AquaFunc” wants to generate an integrated knowledge on functional genomics in sustainable aquaculture (http://genomics.aquaculture-europe.org/index.php?id = 3). Finally, “AquaGenome” aims to coordinate the ongoing and future national and international research projects in the field of genomics in fish and shellfish European aquaculture and support diff usion of genomic approaches within research laboratories. Importantly, recent impressive progresses in large-scale DNA sequencing technology are currently re-revolutionizing the field of genomics (next generation rapid sequencing technology; for review, see Refs. [11–13]). New sequencing platforms allow rapid and much cheaper sequencing of large amounts of DNA, with major applications in genome sequencing, SNP analysis, and most other aspects of genomics. The first full human genome to be sequenced using next generation rapid-sequencing technology has been already published [116]. Genomics is a fast evolving discipline, with a strong potential impact of such new technologies on seafood production for the future.

Acknowledgments Our work is supported by grants from the Association pour la Recherche contre le Cancer (ARC), the Fondation de la Recherche Médicale (FRM), the Centre National de la Recherche Scientifique (CNRS), and the Institut National de la Recherche Agronomique (INRA).

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Chapter 5

Nucleotides and Nucleosides M. Concepción Aristoy, Leticia Mora, Aleida S. Hernández-Cázares, and Fidel Toldrá Contents 5.1 Introduction ......................................................................................................................57 5.2 Chemical Structure of Main Seafood Nucleosides and Nucleotides ..................................59 5.3 Analysis of ATP-Related Compounds ...............................................................................59 5.3.1 Extraction of Nucleotides and Nucleosides ........................................................... 60 5.3.2 Nucleotides and Nucleosides Determination .........................................................61 5.3.2.1 31Phosphorous-Nuclear Magnetic Resonance Spectroscopy .....................61 5.3.2.2 Capillary Electrophoresis .........................................................................61 5.3.2.3 Chromatography......................................................................................61 5.3.2.4 Enzymatic Analysis................................................................................. 64 References ..................................................................................................................................65

5.1

Introduction

Bacterial growth is the main factor limiting fish commercial life by producing its alteration and unpleasant flavor. Nevertheless, the autolytic process derived from tissue enzymatic activity and lipid oxidations also contributes to fish maturation and subsequent spoilage. Sensory methods to evaluate fish quality are subjective and difficult to use in the evaluation of processed (fillets, beheaded, or eviscerated fish) or canned fish. Thus, objective methods for freshness determination are required and the determination of the biochemical changes occurring in early postmortem in fish constitute a helpful tool. The first autolytic process taking place in fish affects carbohydrates and nucleotides. After death, the adenosine triphosphate (ATP) regeneration that occurs in vivo stops and ATP is degraded until 57

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rigor mortis is reached. This process involves a series of reactions commonly represented according to the sequence shown in Figure 5.1. As a result of endogenous enzymes action, ATP molecule is rapidly degraded to adenosine monophosphate (AMP) and afterward to inosine monophosphate (IMP), which is accumulated in postharvest fish.1 The following IMP dephosphorylation to obtain inosine is mainly autolytic and occurs at a slower rate during the first stage of cold storage, although it might be accelerated by the action of different bacteria. IMP degradation to inosine (Ino) and its disappearance have been correlated with lack of freshness in some fish species. Inosine is transformed to hypoxanthine (Hx) by the action of the enzyme nucleoside phosphorylase (NP), which is oxidized to xanthine (Xa) and uric acid in the presence of xanthine oxidase (XO) enzyme. This enzyme is mainly generated in muscle from biochemical processes of microorganisms. The speed of each step in this reaction chain and especially in the Ino to Hx and Hx to Xa conversion depends on the fish species. IMP is the main nucleotide present in fish species, whereas AMP remains major in crustaceans.2 Howgate et al. (2006) published a review of the concentration of IMP, Ino, and Hx in the flesh of some species of fish during chilled storage.3 In all cases, Ino and Hx concentrations increased during storage, and either of the two may be used as freshness indicators.4 However, the use of a single compound as freshness indicator is not always advisable, because many factors can affect

O HO

N H 2N

N N

P O

N

OH O P

O

O

N

O

P

N

N

O P

O O

Pi

OH HO

HO

OH ADP Myokinase

Pi

N

N Pi

N N

N OH

HO

N N

HO

O

N

OH O

OH

OH ATP

HO

O P

ATP ase

OH O

HO

N

H2N

NH3

OH O P OH AMP deaminase

Nucleotidase HO

OH Ino

N

O

O

N

H2N N

O

O

N

O

OH

HO

IMP

OH AMP

Pi Nucleosidase phosphorilase Ribose 1-phosphate O N

HN

N H

N Hx

Figure 5.1

O

O2

O H2O2

N

HN

Xanthine oxidase

O

O2

N H

N H Xa

Degradation of ATP in postmortem fish muscle.

O H2O2

H N

HN

Xanthine oxidase

O N H UA

N H

P

OH

OH

Nucleotides and Nucleosides ◾

59

nucleotide degradation such as the type of spoilage bacteria and mechanical handling of fish,5 and, also, the disappearance of the degradation products differs from one species to another3 as mentioned here. This is the main reason for the use of indexes with more than one compound from the ATP-degradation chain. Some of them are briefly described here. K value is defined as the ratio of Ino and Hx to the sum of ATP and related compounds expressed as a percentage.6 This value has been used as one of the freshness indexes to evaluate the quality change of postharvest fish.2,7–9 Nevertheless, ATP, adenosine diphosphate (ADP), and AMP disappear early postmortem, generally within 1 day of storage in ice after death in all fish species,10,11 and, consequently, a revised K value, often designed K ′ value or Ki index, is more often considered as monitoring the loss of IMP and is defined as the ratio of Ino and Hx to the sum of IMP, Ino, and Hx expressed as percentage.12 However, for several species, a high accumulation of Ino occurs during ATP degradation, making K value inadequate as a freshness indicator. For this reason, a hypoxanthine ratio or H value (Hx/(IMP + Ino + Hx) × 100) was considered as a better indicator of fish freshness in this type of species. The ratio Hx/AMP was considered an adequate alternative to characterize fish freshness due to its constant increment with time.13,14 Measurement of ATP-related compounds is also useful for the quality control of retorted fishes, as shown when comparing high-temperature short-time process at 125°C for 9 min with a common retort process at 115°C for 90 min.15,16 Another suggestion to use nucleotide compounds as a measurement of seafood quality is their relation with sensory attributes. In this way, a high content of Hx is related with the bitter off taste of spoiled fish, whereas IMP evokes a fresh meaty taste sensation.16,17

5.2

Chemical Structure of Main Seafood Nucleosides and Nucleotides

To a better understanding of the methods of analysis of these compounds, the knowledge of their molecular structure is important. Nucleosides are glycosylamines that are formed when a nucleobase (purine or pyrimidine base) attaches to a ribose or deoxyribose ring. Nucleosides currently analyzed in seafoods are those in which a purine ring, adenine, or hypoxanthine is attached to a ribose, forming the adenosine or inosine, respectively. Nucleotides are o-phosphoric acid esters of the nucleosides, and thus, AMP or adenylic acid is derived from the adenosine in which a phosphate group is attached at the 5-ribose carbon. ADP and ATP are derived from the AMP, to which one or two additional phosphate groups are attached through pyrophosphate bonds (∼P) (Figure 5.2). On the other hand, IMP is derived from the inosine in which a phosphate group is attached to the 5-ribose carbon.

5.3 Analysis of ATP-Related Compounds The correct analysis of ATP-related compounds must take into account that early postmortem fish muscle is very sensitive to temperature. ATP-chain degradation occurs very fast, even at refrigeration temperatures, and it is important to stop this reaction drastically at the sampling time. This is achieved by immediately freezing the excised muscle under liquid nitrogen to stop all enzymatic reactions. In order to achieve this rapid freezing, it is advisable to collect small tissue samples and immerse them into liquid nitrogen. These cold conditions must be held along the sample preparation.18 After this, nucleotides and nucleosides should be extracted and analyzed.

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Handbook of Seafood and Seafood Products Analysis Adenosine nucleoside

N NH2

N

OH

OH HO

P O

O

P O

O

O O

P O

N

N

OH OH

HO

Ribose

Adenine purine base

AMP ADP ATP Adenosine nucleotide

Figure 5.2

5.3.1

Structure of adenosine-derived nucleotides.

Extraction of Nucleotides and Nucleosides

A typical extraction procedure for the analysis of fish samples by reversed-phase chromatography, with or without employing an ion-pairing agent, is the following: 5 g or less of muscle tissue are excised and quickly frozen with liquid nitrogen. The frozen tissue is minced, avoiding any thawing, 3–5 vol. cold 0.6 M perchloric acid is added, and the tissue is homogenized with a stomacher-type homogenizer for a few minutes under cold conditions. Once the extract is centrifuged (15,000 g for 20 min), the supernatant is filtered through glass wool and neutralized to pH 6.5–6.8 by adding solid potassium carbonate or 1 M potassium hydroxide. This neutralized extract is kept in an ice bath for 15 min and centrifuged again (15,000 g for 10 min). The supernatant is filtered through a 0.2 μm membrane filter and stored under frozen storage at temperatures below −20°C until analysis, although storage at −18°C has been demonstrated to be enough to preserve fish samples and fish extracts for the analysis of IMP, Ino, and Hx.17 Other extraction methods consist in the homogenization of 2.5 g of fish sample with 10% trichloroacetic acid and, after centrifugation (27,000 g for 15 min), they are neutralized with 2 M sodium hydroxide. The neutralized extract must be made up to 5 mL with 20 mM phosphate buffer pH 7.8 and then filtered with a 0.45 mm membrane. These fish extracts are used in enzymatic assays with biosensors19,20 and/or spectrophotometers as well as in capillary electrophoresis (CE)21 or ion chromatography (IC).22


61

In the development of biosensor analysis, some authors have described extraction methods that consisted of heated fish sample. In this way, both a microwave oven at 500 V for 5 s and heating at 100°C for 60 min have been used.23,24

5.3.2

Nucleotides and Nucleosides Determination

Several methods have been used to measure nucleotides and nucleosides, including nuclear magnetic resonance spectroscopy (NMR), high-performance capillary electrophoresis (HPCE), radioimmunoassay,25 thin-layer chromatography (TLC),26 reversed-phase high-performance liquid chromatography (RP-HPLC) with and without ion pair, ion-exchange HPLC,27 IC,22 and enzymatic assays.

5.3.2.1

31

Phosphorous-Nuclear Magnetic Resonance Spectroscopy

31

The phosphorous nuclear magnetic resonance spectroscopy (31P-NMR) technique makes it possible to perform multiple determinations of high-energy phosphates in vivo in the same muscle sample. Thus, in vivo 31P-NMR spectroscopy has been used as a powerful technique to characterize the biochemical changes that occur in live, intact fishes after being submitted to physical and chemical stressors such as hypoxia.28 Also in vitro 31P-NMR spectroscopy has been applied to both excised tissue and perchloric acid extracts of fish muscle.

5.3.2.2 Capillary Electrophoresis CE is a powerful separation technique that can provide high separation efficiency and high sample throughput with minimal sample volume and buffer consumption. Capillary electrophoresis has been used in many nucleotide analysis applications as in the study of nucleotide degradation in fish tissues. In the analysis of complex biological samples, including fish extract, this technique can present problems in reproducibility, because these samples usually contain significant amounts of ions, which may be adsorbed on capillary walls. However, the reconditioning of the capillary surface is ensured by washing 1 min with 1M NaOH, followed by 2 min of the running buffer used.21 Typical conditions to get a good separation of IMP, inosine, and hypoxanthine would be a potential of 416 V/cm of capillary using 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer, pH 11.19

5.3.2.3

Chromatography

At present, among other chromatographic techniques, HPLC has been shown to be the most widely used technique to analyze nucleotides and nucleosides. In particular, RP-HPLC and ion-paired reverse-phase are the methods of choice for this analysis. The mode of separation will depend on the analyte of interest. Thus, to analyze nucleotides, the addition of an ion-pair to the mobile phase greatly improves the separation by increasing the retention time of charged molecules (ATP, ADP, AMP). Nevertheless, nucleotides will disappear at the rigor mortis state (normally 1 day after catch), and the K′ or K i index will be usually enough to characterize fish

62

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freshness or quality. Then, a simple RP-HPLC with a phosphate buffer as mobile phase will be adequate.

5.3.2.3.1 Reversed-Phase HPLC The chromatographic analysis should be performed in a liquid chromatograph equipped with an UV detector (254 nm). The column used is an analytical reversed-phase RP-18 column. There are many approaches to analyze nucleotides and nucleosides by this technique, which differ mainly in the pH of the mobile phase. All of them use a phosphate buffer as the mobile phase and a gradient with methanol or acetonitrile should be accomplished to improve the Ino resolution and reduce the chromatogram time.17,29 With buffer pH 7,29 phosphorylated metabolites are also well separated in the chromatogram.15 The identification of the chromatographic peaks can be performed by comparing the peak retention times and spectral characteristics (if a diode array detector is available) with those of standards. Quantitative analysis can be performed by external or internal standard method. In Figure 5.3 both chromatograms of standards and hake nucleosides and nucleotides are shown. The separation was achieved with an RP C-18 column at 35°C and a gradient between phosphate buffer at pH 7 and acetonitrile. 1000

(a) 6

800

600

Absorbance at 254 nm (mAU)

400

2 1

200

4 5

3

0 (b)

1200

1 1000 6

800 600 400

5

200

2 3

4

0 0

2

4

6

8

10

Retention time (min)

Figure 5.3 RP-HPLC chromatograms of standards (a) and hake (b) ATP-derived compounds. (1) IMP, (2) ATP, (3) ADP, (4) AMP, (5) hypoxanthine, and (6) inosine.


63

5.3.2.3.2 Ion-Pair RP-HPLC The most common technique used for the separation of nucleotides is ion-pair RP-HPLC, which is especially useful in separating mixtures of charged and uncharged molecules. The separation is achieved in a reversed-phase column, and the key is to add an ion pair (an ion of charge opposite to that of the analyte molecule). Due to the negative charge of the phosphorylated groups of nucleotides, the ion pair should be a positive ion with a hydrophobic rest to improve the affinity with the stationary phase. Thus, either tetrabutylammonium hydrogen sulfate or phosphate is the ion pair most used.17,30 This ion-paired technique is especially useful when di- and tri-nucleotides have to be analyzed, because the ion pair enhances the retention time and separation, making it less dependant on the type of column, as well as the resolution, due to the ionic nature of the phosphate esters that facilitates strong interactions with the ion-pair reagent at the appropriate pH. Nevertheless, this method is more expensive than the more simple technique previously described. Figure 5.4 shows an ion-paired chromatogram of a 48 h postmortem sardine extract.

1400

(a)

1

1200 5

1000 800


600 400 200

6

2

3

4

0 1200

(b)

6 1000

1

800 600 400 5 200 2

4

3

0 0

5

10

15

20


Figure 5.4 Ion-paired HPLC chromatograms of salmon (a) and sardine (b). ATP-derived compounds. (1) IMP, (2) ATP, (3) ADP, (4) AMP, (5) hypoxanthine, and (6) inosine.

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5.3.2.4 Enzymatic Analysis The use of enzymatic methods to analyze nucleotides in seafood is widespread due to their high specificity. These assays may be carried out with the enzymes in solution31,32 or immobilized, constituting what is known as enzyme sensors o biosensors.20,33,34 A biosensor is a system composed of a biological recognition element and a biochemical or physical transducer in intimate contact or in close proximity with each other in order to relate the concentration of an analyte to a measurable signal.35 Some details about the use of different biomaterials in order to select the best recognition elements and the most adequate methods for the enzyme immobilization have been described.36–38 The most used biosensors for the nucleotide-related compound analysis are electrochemical sensors, in which an enzyme or a group of enzymes are immobilized in a membrane or other supports, which is further coupled to a chemical transducer. These enzymes act by oxidizing the substrates (analyte) while consuming oxygen or producing hydrogen peroxide or uric acid. The depletion of oxygen or the formation of hydrogen peroxide or uric acid may be detected amperometrically. This option offers some advantages in relation to the free enzyme, due to its specificity, simplicity, and rapid response.36 Nevertheless, although biosensors have shown its utility in some applications such as clinical, environmental, agricultural, and biotechnology, the application in the food industry is still restricted36 mainly due to critical stages such as enzyme immobilization or sample preparation for analysis. Indeed, all the approaches to date need the sample preparation described earlier. In addition, the use of commercial kits or disposals presents some problems, because the denaturalization of the enzymes with time, and thus test kits, enzyme-coated strips, electrodes, or sensors have a limited shelf life.

5.3.2.4.1 Enzymatic Methods with the Enzyme in Solution The concentration of Hx, Ino, and IMP may be determined spectrophotometrically by a sequential addition of XO, NP, and 5′-nucleotidase (NT) into a reaction phosphate buffer containing the fish extract sample at pH 7.6–7.8 and 30°C–37°C. In these conditions, IMP, inosine, and hypoxanthine will be oxidized to uric acid and H2O2, which will be further quantified by measuring the absorbance at 290 nm and by polarimetry, respectively.20,39 This procedure was also used to analyze ATP and related compounds in fish sauces with very good results, because no interference of salt in the medium was observed here as was in the case using the HPLC method.31 Another possibility consisted in monitoring the oxygen consumption after these enzymatic reactions with an amperometric-type sensor (oxymeter), although these applications used to be achieved with at least one of these enzymes immobilized as described earlier.

5.3.2.4.2 Enzymatic Methods with Immobilized Enzymes In this case, the analysis may be performed with one or more enzymes, but they remain immobilized in different supports. Prodomidis and Karayannis85 reported a review on enzyme-based amperometric sensors applied to food analysis in which the principles and materials commonly used for the construction of the electrodes are described.35 The most used is the biosensor based on the measure of hypoxanthine. This sensor has been developed mainly for assessing the freshness of fish meat40,41 or for the evaluation of chicken32 and beef meat33 aging. In this sensor, XO enzyme, which is immobilized in a membrane fi xed in the sensing area of the electrode, oxidizes the hypoxanthine to xanthine and uric acid, while the depletion of oxygen is measured by a Clark-type elec-


65

trode at a platinum cathode (−0.6 to −0.9 V) vs. an Ag/AgCl reference electrode. The consumed oxygen produces a current decrease that can be correlated to the concentration of Hx. Both Hx and X are substrates for the XO action and will be oxidized either simultaneously or sequentially. The proposed relation 1 Hx for ½ X for each oxygen molecule formed must be taken into account to quantify the Hx,33 although this relation should be confirmed in each particular system. In the measurement of hypoxanthine, different supports have been used for the immobilization of the XO enzyme. Most of these supports have been developed with the aim of eliminating interferences due to ascorbic acid, uric acid, or H2O2, which can be present in the sample or formed during the enzymatic reaction. In this way, cellulose triacetate,41 preactivated nylon,18 and a silk fibron membrane in combination with a cellulose acetate membrane42 or a nylon net43 have been used. Most recent approaches to determine Hx are based on the incorporation of the XO enzyme in a graphite/Teflon matrix,44 a polyaniline film by electropolimerization,45 a nafion-coated platinum disc electrode,46 or even in a carbon paste electrode modified with electrodeposited gold nanoparticles.47 On the other hand, specific biosensors to determine AMP,48 IMP,49 and Ino50 and a multienzymatic sensor to analyze simultaneously AMP, IMP, Ino, and Hx using a cellulose triacetate membrane have been described.51 The use of multienzymatic biosensors to measure fish freshness has been very helpful for the simultaneous determination of AMP, IMP, Ino, and Hx amounts, necessary to obtain K, K i, and H values. Thus, Luong and Male20 used a multienzymatic biosensor system to determine the H value as a fish freshness indicator. An immobilized NT was used for the previous conversion of IMP to Ino, and then after adding a soluble NP, Ino was converted to Hx. Formed Hx was measured with an amperometric sensor that detected uric acid + hydrogen peroxide in an additive matter, and, thus, 1 mol of Hx would be converted to 1 mol of uric acid and 2 mol of hydrogen peroxide. This method was patented by Luong, Male, and Nguyen52 and afterward it has been commercialized as a Freshness Meter KV-101 (Oriental Electric Co. Ltd., Japan). Comparable results to that of HPLC were reported. A similar application was proposed12,34 to obtain the Ki parameter as a freshness indicator. Flow injection analysis (FIA) has been widely used in the development of these multienzymatic biosensors constituting different types of reactors in which different enzyme combinations can be immobilized as well as introduced as soluble enzyme.53,54 In fact, some authors have described this type of biosensor coupled with an oxygen electrode.23,53,55,56

References 1. Massa, A.E.; Palacios, D.L.; Paredi, M.E. et al. Postmortem changes in quality indices of ice-stored flounder (Paralichthys patagonicus). J. Food Biochem. 29: 570–590, 2005. 2. Mendes, R.; Quinta, R.; Nunes, M.L. Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast. Eur. Food Res. Technol. 212: 141–146, 2001. 3. Howgate, P. A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage. Int. J. Food Sci. Technol. 41: 341–353, 2006. 4. Özogul, F.; Özogul, Y.; Kuley, E. Nucleotide degradation in sardine (Sardina pilchardus) stored in different storage condition at 4°C. J. Fish. Sci. 1: 13–19, 2007. 5. Surette, M.E.; Gill, T.A.; LeBlanc, P.J. Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage. J. Agric. Food Chem., 36: 19–22, 1988. 6. Saito, T., Arai, K., Matsuyoshi, M. A new method for estimating the freshness of fish. Bull. Jpn. Soc. Sci. Fish., 24: 749–750, 1959. 7. Pacheco-Aguilar, R.; Lugo-Sánchez, M.E.; Robles-Burgueno, M.R. Postmortem biochemical and functional characteristic of Monterey sardine muscle stored at 0°C. J. Food Sci. 65: 40–47, 2000.

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8. Aubourg, S.P.; Quitral, V.; Larrain, M.A. et al. Autolytic degradation and microbiological activity in farmed Coho salmon (Oncorhynchus kisutch) during chilled storage. Food Chem. 104: 369–375, 2007. 9. Castillo-Yanez, F.J.; Pacheco-Aguilar, R.; Marquez-Rios, E. et al. Freshness loss in sierra fish (Scomberomorus sierra) muscle stored in ice as affected by postcapture handling practices. J. Food Biochem. 31: 56–67, 2007. 10. Jones, N.R. Meat and fish flavors—Significance of ribomononucleotides and their metabolites. J. Agric. Food Chem. 17: 712, 1969. 11. Jones, N.R.; Murray, J. Degradation of adenine- and hypoxanthine nucleotide in the muscle of chillstored, trawled cod (Gadus callarius). J. Sci. Food Agric. 13: 475–480, 1962. 12. Karube, I.; Matsuoka, H.; Suzuki, S. et al. Determination of fish freshness with an enzyme sensor system. J. Agric. Food Chem. 32: 314–319, 1984. 13. Massa, A.E.; Paredi, M.E.; Crupkin, M. Nucleotide catabolism in cold stored adductor muscle of scallop (Zygochlamys patagonica). J. Food Biochem. 26: 295–305, 2002. 14. Márquez-Rios, E.; Moran-Palacio, E.F.; Lugo-Sanchez, M.E. et al. Postmortem biochemical behavior of giant squid (Dosidicus gigas) mantle muscle stored in ice and its relation with quality parameters. J. Food Sci. 72: C356–C362, 2007. 15. Kuda, T.; Fujita, M.; Goto, H. et al. Effects of freshness on ATP-related compounds in retorted chub mackerel Scomber japonicus. LWT-Food Sci. Technol. 40: 1186–1190, 2007. 16. Kuda, T.; Fujita, M.; Goto, H. et al. Effects of retort conditions on ATP-related compounds in pouched fish muscle. LWT-Food Sci. Technol. 41: 469–473, 2008. 17. Veciana-Nogués, M.T.; Izquierdo-Pulido, M.; Vidal-Carou, M.C. Determination of ATP related compounds in fresh and canned tuna fish by HPLC. Food Chem. 59: 467–472, 1997. 18. Aristoy, M.C.; Toldra´, F. Nucleotides and its derived compounds. In Nollet L.M.L.; Toldra´, F. (Eds.), Handbook of Muscle Foods Analysis, CRC Press, Boca Raton, FL, pp. 279–288, 2009. 19. Luong, J.H.T.; Male, K.B.; Masson, C. et al. Hypoxanthine ratio determination in fish extract using capillary electrophoresis and immobilized enzymes. J. Food Sci. 57–1: 77–81, 1992a. 20. Luong, J.H.T.; Male, K.B. Development of a new biosensor system for the determination of the hypoxanthine ratio, an indicator of fish freshness. Enzyme Microb. Technol. 14: 125–130, 1992b. 21. Nguyen, A.L.; Luong, J.H.T.; Masson, C. Determination of nucleosides in fish tissues using capillary electrophoresis. Anal. Chem. 62: 2490–2493, 1990a. 22. Gao, R.; Xue, C.; Yuan, L. et al. Determination of the big head carp myofibrillar (Aristichthys nobilis) adenosine triphosphatase activity by ion chromatography. J. Chromatogr. A 1118: 278–280, 2006. 23. Okuma, H.; Watanabe, E. Flow system for freshness determination based on double multi-enzyme reactor electrodes. Biosens. Bioelectron. 17: 367–372, 2002. 24. Watanabe, E.; Tamada, Y.; Hamada-Sato, N. Development of quality evaluation sensor for fish freshness control based on K1 value. Biosens. Bioelectron. 21: 534–538, 2005. 25. Roberts, B.; Morris, B.A.; Clifford, M.N. Comparison of radioimmunoassay and spectrophotometric analysis for the quantitation of hypoxanthine in fish muscle. Food Chem. 42: 1–17, 1991. 26. Dingle, J.R.; Hines, J.A.; Fraser, D.I. Post-mortem degradation of adenine nucleotides in muscle of the lobster, Homarus americanus. J. Food Sci. 33: 100–103, 1968. 27. Borgese, T.A.; Nagel, R.L.; Roth, E. et al. Guanosine triphosphate (GTP): The major organic phosphate in the erythrocytes of the elasmobranch Mustelus canis (smooth dogfish). Comp. Biochem. Physiol. 60: 317–321, 1977. 28. Van der Thillart, G.; Van Waarde, A.; Muller, H.J. et al. Determination of high-energy phosphate compounds in fish muscle: 31P-NMR spectroscopy and enzymatic methods. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol. 95(4): 789–795, 1990. 29. Özogul, F.; Taylor, K.D.A.; Quantick, P.C. et al. A rapid HPLC-determination of ATP-related compounds and its application to herring stored under modified atmosphere. Int. J. Food Sci. Technol. 35: 549–554, 2000.


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30. Meynial, I.; Paquet, V.; Combes, D. Simultaneous separation of nucleotides and nucleotide sugars using an ion-pair reversed-phase HPLC: Application for assaying glycosyltransferase activity. Anal. Chem. 67: 1627–1631, 1995. 31. Cho, Y.J.; Im, Y.S.; Kim, S.M. et al. Enzymatic method for measuring ATP related compounds in fish sauces. J. Korean Fish. Soc. 32: 385–390, 1999. 32. Fujita, T., Hori, Y., Otani, T. et al. Applicability of the K0 value as an index of freshness for porcine and chicken muscles. Agric. Biol. Chem. 52: 107–112, 1988. 33. Yano, Y.; Kataho, N.; Watanabe, M. et al. Evaluation of beef ageing by determination of hypoxanthine contents: Application of a xanthine sensor. Food Chem. 52: 439–445, 1995. 34. Volpe, G.; Mascini, M. Enzyme sensors for determination of fish freshness. Talanta 43: 283–289, 1996. 35. Prodomidis, M.I.; Karayannis, M.I. Enzyme based amperometric biosensors for food analysis. Electroanalysis 14: 214–261, 2002. 36. Mello, L.D.; Kubota, L.T. Review of the use of biosensors as analytical tools in the food and drink industries. Food Chem. 77: 237–256, 2002. 37. Venugopal, V. Biosensor in fish production and quality control. Biosens. Bioelectron. 17: 147–157, 2002. 38. Sharma, S.K.; Sehgal, N.; Kumar, A. Biomolecules for development of biosensors and their applications. Curr. Appl. Phys. 3: 307–316, 2003. 39. Luong, J.H.T.; Male, K.B.; Nguyen, A.L. Application of polarography for monitoring the fish post-mortem metabolite transformation. Enzyme Microb. Technol. 11: 277–282, 1989. 40. Nguyen, A.L.; Luong, H.T.; Yacynych, A.M. Retention of enzyme by electropolymerized film: A new approach in developing a hypoxanthine biosensors. Biotech. Bioeng. 37: 729–735, 1990b. 41. Watanabe, E.; Ando, K.; Karube, I. et al. Determination of hypoxanthine in fish meat with an enzyme sensor. J. Food Sci. 48: 496–500, 1983. 42. Qiong, Q.; Tuzhi, P.; Liju, Y. Silk fibroin/cellulose acetate membrane electrodes incorporating xanthine oxidase for the determination of fish freshness. Anal. Chim. Acta 369: 245–251, 1998. 43. Shen, L.; Yang, L.; Peng, T. Amperometric determination of fish freshness by hypoxanthine biosensor. J. Sci Food Agric. 70: 298–302, 1996. 44. Cayuela, G.; Peña, N.; Reviejo, A.J.; Pingaron, J.M. Development of a bioenzymatic graphite-Teflon composite electrode for the determination of hypoxanthine in fish. Analyst 123: 371–377, 1998. 45. Hu, S.; Xu, C.; Luo, J. et al. Biosensor for detection of hypoxanthine based on xanthine oxidase immobilized on chemically modified carbon paste electrode. Anal. Chim. Acta 412: 55–61, 2000. 46. Nakatani, H.S.; Viera dos Santos, L.; Pelegrine, C.P. et al. Biosensor based on xanthine oxidase for monitoring hypoxanthine in fish meat. Am. J. Biochem. Biotechnol. 1(2): 85–892, 2005. 47. Agüí, L.; Manso, J.; Yañez-Sedeño, P. et al. Amperometric biosensor for hypoxanthine based on immobilized xanthine oxidase on nanocrystal gold-carbon paste electrodes. Sens. Actuators B 113: 272–280, 2006. 48. Watanabe, E.; Ogura, T.; Toyama, K. Determination of adenosine 5′-monophosphate in fish and shellfish using an enzyme sensor. Enzyme Microb. Technol. 6: 207–211, 1984a. 49. Watanabe, E.; Toyama, T.; Karube, I. et al. Determination of inosine-5-monophosphate in fish tissue with an enzyme sensor. J. Food Sci. 48: 114–116, 1984b. 50. Watanabe, E.; Toyama, T.; Karube, I. et al. Enzyme sensor for hypoxanthine and inosine determination in edible fish. Appl. Microbiol. Biotechnol. 19(1): 18, 1984c. 51. Watanabe, E.; Tokimatsu, S.; Toyama, K. Simultaneous determination of hypoxanthine, inosine-5′ phosphate and adenosine 5′-phosphate with a multielectrode enzyme sensor. Anal. Chim. Acta 164: 139–146, 1984d. 52. Luong, H.T.; Male, K.B.; Nguyen, A.L. 1994. http://www.freepatentsonline.com/5288613.html. 53. Okuma, H.; Takahashi, H.; Yazawa, S. et al. Determination of a system with double enzyme reactors for the determination of fish freshness. Anal. Chim. Acta 260: 93–98, 1992.

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54. Carsol, M.-A.; Volpe, G.; Mascini, M. Amperometric detection of uric acid and hypoxanthine with xanthine oxidase immobilized and carbon based screen-printed electrode. Application for fish freshness determination. Talanta 44: 2151–2159, 1997. 55. Park, I.-S.; Kim, N. Simultaneous determination of hypoxanthine, inosine and inosine 5′-monophosphate with serially connected three enzyme reactors. Anal. Chim. Acta 394: 201–221, 1999. 56. Park, I.-S.; Cho, Y.-J.; Kim, N. Characterization of meat freshness application of a serial three-enzyme reactor system measuring ATP-degradative compounds. Anal. Chim. Acta 404: 75–81, 2000.

Chapter 6

Lipid Compounds Santiago P. Aubourg Contents 6.1 Introduction ..................................................................................................................... 70 6.1.1 General Aspects of Lipid Compounds .................................................................. 70 6.1.2 Marine Lipid Characteristics ................................................................................ 70 6.1.3 Lipid Analysis in Marine Products ........................................................................ 71 6.2 First Steps in Marine Lipid Analysis ................................................................................. 72 6.2.1 Isolation of Lipids from Tissues ............................................................................ 72 6.2.2 Removal of Nonlipid Contaminants ..................................................................... 72 6.2.3 Lipid Manipulation and Storage ........................................................................... 73 6.2.4 Lipid Quantification ............................................................................................. 73 6.3 Marine Fatty Acid Analysis .............................................................................................. 73 6.3.1 Fatty Acid Methyl Esters Preparation ................................................................... 73 6.3.1.1 Acid-Catalyzed Esterification and Transesterification ............................. 73 6.3.1.2 Base-Catalyzed Transesterification ......................................................... 75 6.3.2 GLC Analysis of FAME........................................................................................ 75 6.3.2.1 Qualitative Analysis of Fatty Acid Composition ..................................... 75 6.3.2.2 Quantitative Estimation of Fatty Acid Composition ...............................76 6.4 Analysis of Marine Nonsaponifiable Matter ......................................................................76 6.4.1 Lipid Saponification...............................................................................................76 6.4.2 Analysis of Sterols ..................................................................................................76 6.4.3 Analysis of Ether Lipids ........................................................................................ 77 6.5 Qualitative and Quantitative Analyses of Marine Lipid Classes ....................................... 77 6.5.1 Spectrophotometric Assessments of Total Lipid Extract ........................................ 78 6.5.2 Stereospecific Analysis of Lipid Classes ................................................................. 78 6.5.3 Column Chromatography..................................................................................... 79 69

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6.5.4 Thin-Layer Chromatography ................................................................................ 79 6.5.5 High-Performance Liquid Chromatography ......................................................... 79 6.5.6 Silver Ion Chromatography ................................................................................... 80 6.5.7 Nuclear Magnetic Resonance (NMR) Spectrometry ............................................ 80 6.5.8 Mass Spectrometry ................................................................................................81 6.5.9 Supercritical Fluid Chromatography ..................................................................... 82 References ................................................................................................................................. 82

6.1 Introduction 6.1.1 General Aspects of Lipid Compounds Lipids are found in all living organisms and have been shown to play two critical roles: (1) maintaining the integrity of plants and animals as structural compounds by forming a barrier separating the living cell from the outside world and (2) being a major source of cellular energy and function in living organisms where they can be stored. Different attempts have been carried out to define what is meant by the term lipid, although no satisfactory or widely accepted definition exists. Most textbooks describe lipids as a group of naturally occurring compounds, which have in common a ready solubility in organic solvents, such as hexane, toluene, chloroform, ethers, and alcohols. Such diverse compounds as hydrocarbons, carotenoids, steroids, soaps, triacylglycerols (TG), phospholipids (PL), gangliosides, and lipopolysaccharides would be included. Because of their structural and functional variety, a widely accepted division has been difficult. A simple physicochemical classification that empirically groups lipid molecules according to the hydrophilic–lipophilic balance has been proposed [1]. An alternative division into two broad classes has been shown to be convenient for lipid analysts [2]; in it, “simple lipids” (fatty acid and alcohol components) would be those that yield on hydrolysis at most two types of different products per mol, whereas “complex lipids” (glycerophospholipids, glycoglycerolipids, and sphingolipids) would yield three or more types of products per mol.

6.1.2 Marine Lipid Characteristics In many marine organisms, lipid is usually the second largest biochemical constituent after protein. Seafood lipids are known to provide high contents of important components for the human diet, such as nutritional lipid-soluble vitamins (namely A and D) and essential and ω3 polyunsaturated fatty acids (PUFA) that have shown a positive role in preventing certain human diseases, including cardiovascular ones [3]. Most animal and plant lipids from terrestrial and marine sources are similar in that they contain mainly even-numbered saturated and unsaturated fatty acids combined with glycerol (glycerides and glyceryl ethers), fatty alcohols (wax esters), sterols (sterol esters), phosphoric acid, and amines (PL). Marine lipids, however, differ from the other sources in that they contain a wider range of fatty acids, longer-chain fatty acids, and a larger proportion of highly unsaturated fatty acids, particularly C20:5ω3 (eicosapentaenoic acid, EPA) and C22:6ω3 (docosahexaenoic acid, DHA) [4]. Marine species have shown large variations in lipid content and composition as a result of endogenous and exogenous effects [5–7]. Related to exogenous effects, the catching season has been shown to play a key role regarding temperature and feeding availability; indeed, an inverse

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relationship between unsaturated fatty acid content and environmental temperature has been confirmed for many marine fish. With respect to endogenous effects, lipid matter has been described to exhibit a heterogeneous distribution throughout the body of marine species, probably affected by physiological and anatomical factors. Thus, differences according to the type of muscle and its location, age, sex, and sexual maturation have been pointed out. In all cases, content variations have specially been observed in fish locations to be employed as lipid depots.

6.1.3

Lipid Analysis in Marine Products

Researchers are required to analyze the lipid composition and its changes that arose during processing of food material from marine sources. The approach to the analysis of lipids in a given marine sample will depend on the amount of material in the sample, the equipment, and instrumentation available, but mainly the amount of information required. The present chapter is focused on describing the available traditional and advanced analytical methodology to assess the lipid composition of marine species on the basis of a food technologist and nutritionist requirements. A basic protocol procedure is exposed in Figure 6.1.

Marine products

Lipid isolation from tissues

Removal of nonlipid contaminants

Frozen storage

Fatty acid analysis

Lipid classes analysis

Traditional and advanced analytical methodology

Figure 6.1

Basic steps to be carried out for the lipid analysis of marine products.

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6.2 First Steps in Marine Lipid Analysis 6.2.1 Isolation of Lipids from Tissues Ideally, marine tissues should be extracted from the living organism as soon as possible after catching or slaughtering [2]. When this is not feasible, the tissue should be kept frozen (about −60°C or less) as rapidly as possible. Two main problems can arise with lipid fraction when employing inconvenient storage conditions. First, PUFA can autoxidize as a result of endogenous oxidant enzymes, although endogenous tissue antioxidants can provide some protection. The second problem is endogenous lipolytic enzymes that can lead to large amounts of unesterified fatty acids, diacylglycerides, phosphatidic acid, or lyso-phosphatidylglycerols in lipid extracts. Pure single lipid classes are soluble in a wide variety of organic solvents, but many of these are not suitable for extracting lipids from tissues as they are not sufficiently polar to overcome the strong forces of association between tissue lipids and the other cellular constituents, such as proteins. However, polar complex lipids, which do not normally dissolve readily in nonpolar solvents, may on occasion be extracted by these when they are in the presence of large amounts of simple lipids such as TG. The ideal solvent or solvent mixture for extracting lipids from tissues should be sufficiently polar to remove all lipids from their association with cell membranes or with lipoproteins but should not react chemically with those lipids. At the same time, it should not be so polar that TG and other nonpolar simple lipids do not dissolve and are left adhered to the tissues. Although there are limitations to its use and alternatives are frequently suggested, most workers in the field appear to accept two basic routines currently in general use, which yield essentially quantitative extractions of the major lipid classes when applied to homogenates of whole marine tissue extractions. The most popular is the method of Folch et al. [8], which employs chloroform–methanol (2:1) in a solvent–tissue ratio of 20:1. Where large amounts of tissue have to be extracted, the procedure of Bligh and Dyer [9] offers some advantages as it does not use large volumes of solvent; this method applies a single-phase solubilization of the lipids using chloroform–methanol (1:1) in a solvent–tissue ratio of 4:1. For all extraction methods, it is advisable to include an additional antioxidant at a level of 50–100 mg/L to the solvents. As an advanced alternative, supercritical fluid extraction shows an increasing demand, a major driving force being the environmental concern regarding the use of organic solvents. Its employment has recently been reviewed [10].

6.2.2

Removal of Nonlipid Contaminants

Most polar organic solvents used to extract lipids from tissues also extract significant amounts of nonlipid contaminants such as sugars, urea, amino acids, peptides, and salts. In addition, all solvents can contain contaminants, and as large volumes of solvents may be used to obtain small amounts of lipids, any such impurities can be troublesome. Most of the contaminating compounds can be removed from the lipid extract mixtures simply by shaking the combined solvents with one-quarter their total volume of a dilute salt solution (e.g., 0.88% KCl) [8]. A more elegant and complete, though more time-consuming, method of removing nonlipid contaminants is to carry out the washing procedure by liquid–liquid partition chromatography on a column of a dextran gel such as Sephadex G-25. This type of washing procedure was first developed by Wells and Dittmer [11] and simplified later by Wuthier [12] for large numbers of samples.

Lipid Compounds

6.2.3

◾ 73

Lipid Manipulation and Storage

Wherever possible, lipids should be handled in an atmosphere of nitrogen, since PUFA will oxidize rapidly in air [2]. Autoxidation of double bonds in marine lipid fatty acids is particularly troublesome, and care must be taken at all steps in the analysis of lipids. When it is necessary to concentrate lipid extracts, large volumes of solvents are best removed by means of a rotatory film evaporator at a temperature that, in general, should not exceed about 40°C. Small volumes of solvent can be evaporated by carefully directing a stream of nitrogen onto the surface of the solvent. Lipids should not be left for any time in the dry state and should be stored in an inert nonalcoholic solvent such as chloroform from which air is excluded by flushing with a stream of nitrogen. Storage temperature should be −30°C as the highest temperature. Natural tissue antioxidants, such as tocopherols, afford some protection to lipid extracts, but it is usually advisable to add further synthetic antioxidants to storage solvents at the level of 50–100 mg/L [2]. As storage containers, glass is the best choice. Plastic ware of all kinds (other than that made from TeflonTM) can be specially troublesome and is best avoided, since plasticizers are very easily leached out. Conversely, it has been shown that lipids can themselves dissolve in some plastics, leading to selective losses of a proportion of the less polar constituents.

6.2.4

Lipid Quantification

For most common purposes, a known aliquot of the purified lipid extract is softly heated and the resulting dry lipid matter weighted, provided water absorption onto the dry extract lipid is avoided. For fast purposes, the Soxhlet method of extraction has been developed [13]. In it, a large diethyl ether volume is employed, and the resulting lipid extract can no more be employed for further analysis. This method proved to be accurate in the case of a high lipid content (low complex lipid content); if not, relatively important errors are obtained. According to the special relevance recently acquired by noninvasive technologies, application of nuclear magnetic resonance (NMR), near-infrared (NIR) spectrometry, and Fatmeter measurements is proving to be of increasing interest [14].

6.3 Marine Fatty Acid Analysis 6.3.1 Fatty Acid Methyl Esters Preparation Fatty acids are essential components of lipids. Their measurement by gas–liquid chromatography (GLC) is the most commonly used method for lipid analysis. Owing to the wide variety of fatty acid compounds in marine lipids (Table 6.1), this analysis is more complicated than that with other kinds of living organisms. Lipid extracts have to be converted into methyl ester derivatives. Two basic strategies can be applied [15,16]: acid catalysis and base catalysis. Then, fatty acid methyl esters (FAME) obtained are usually introduced in the GLC system without previous removal of contaminants.

6.3.1.1

Acid-Catalyzed Esterification and Transesterification

Free fatty acids (FFA) are methylated and O-acyl lipids transmethylated by heating them with a large excess of anhydrous methanol in the presence of an acidic catalyst. In addition, fatty acids

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Handbook of Seafood and Seafood Products Analysis Table 6.1

Fatty Acids Commonly Present in Marine Speciesa

Abbreviated Name

Systematic Name

Trivial Name

Saturated Fatty Acids 14:0

Tetradecanoic

Myristic

15:0

Pentadecanoic

16:0

Hexadecanoic

Palmitic

17:0

Heptadecanoic

Margaric

18:0

Octadecanoic

Stearic

20:0

Eicosanoic

Arachidic

22:0

Docosanoic

Behenic

24:0

Tetracosanoic

Lignoceric

—

Monounsaturated Fatty Acids 16:1 ω7

9-Hexadecenoic

Palmitoleic

18:1 ω9

9-Octadecenoic

Oleic

18:1 ω7

11-Octadecenoic

Vaccenic

20:1 ω11

9-Eicosenoic

Gadoleic

20:1 ω9

11-Eicosenoic

Gondoic

22:1 ω11

11-Docosenoic

Cetoleic

22:1 ω9

13-Docosenoic

Erucic

24:1 ω9

15-Tetracosenoic

Nervonic

Polyunsaturated Fatty Acids

a

18:2 ω6

9,12-Octadecadienoic

Linoleic

18:3 ω3

9,12,15-Octadecatrienoic

Linolenic

18:4 ω3

6,9,12,15-Octadecatetraenoic

Stearidonic

20:4 ω6

5,8,11,14-Eicosatetraenoic

Araquidonic

20:5 ω3

5,8,11,14,17-Eicosapentaenoic

EPA

22:5 ω3

7,10,13,16,19-Docosapentaenoic

DPA or clupanodonic

22:6 ω3

4,7,10,13,16,19-Docosahexaenoic

DHA or cervonic

In all cases, the double-bond configuration is “cis.”

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from amide-bound lipids (sphingolipids) are also transesterified, whereas aldehydes are liberated from plasmalogens under acidic conditions. The commonest and mildest reagent used for the purpose is anhydrous hydrogen chloride in methanol. This is simply prepared by adding acetyl chloride slowly to cooled dry methanol. FAME are obtained by heating the reaction mixture in a stoppered tube at 50°C overnight. In order to guarantee complete solution of nonpolar lipid classes, a further solvent such as toluene should be employed. This reagent has been applied directly to fish muscle to obtain FAME without previous lipid extraction [17]. A different possibility consists of employing a solution of 1%–2% (v/v) concentrated sulfuric acid in methanol. Transesterification is carried out in the same manner and at much the same rate as with methanolic hydrogen chloride. Boron trifluoride in methanol is also used as a transmethylation catalyst and in particular as a rapid esterifying reagent for FFA. The reagent has a limited shelf life unless refrigerated, and the use of old or too concentrated solutions may result in the production of methoxy-substituted acids from unsaturated fatty acids and, accordingly, a PUFA loss.

6.3.1.2 Base-Catalyzed Transesterification O-acyl lipids are transesterified very rapidly in anhydrous methanol in the presence of a basic catalyst. However, FFA are not esterified. As with acid-catalyzed procedures, an additional solvent is necessary to solubilize nonpolar lipids such as cholesterol esters or TG. However, under base catalysis, aldehydes are not liberated from plasmalogens and amide-bound fatty acids are not affected. The commonest reagent used for this purpose has been sodium methoxide in anhydrous methanol, prepared simply by dissolving fresh clean sodium in dry methanol, although potassium methoxide or hydroxide have also been used as catalysts.

6.3.2

GLC Analysis of FAME

The advent of GLC revolutionized the analysis of the fatty acid components of lipids, so most performances have been carried out for qualitative and quantitative analysis [16]. Initially, glasspacked columns were widely employed [18]. Later on, the application of wall-coated open tubular (WCOT) columns to the analysis of fatty acids has provided a better knowledge of the complexity of marine fatty acids [19].

6.3.2.1

Qualitative Analysis of Fatty Acid Composition

During the previous decades, parameters known as equivalent chain lengths (ECLs) or carbon numbers have considerably been employed. ECL values can be calculated from an equation similar to that for Kovats’ indices or found by reference to the straight line obtained by plotting the logarithms of the retention times of a homologous series of straight-chain saturated FAME against the number of carbon atoms in the aliphatic chain of each acid. The retention times of the unknown acids should be measured under identical operating conditions, and the ECL values are read directly from the graph. Parallel to ECL value employment, known commercial FAME have been employed for the provisional identification of fatty acids by direct comparison of their retention times and those of the unknown esters on the same columns under identical conditions.

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In recent years, GLC/mass spectrometry (MS) has been widely accepted as one of the most valuable techniques for the identification of fatty acids and their derivatives [20]. A high proportion of the available data has been obtained for the methyl ester derivatives of fatty acids, as these are easily prepared and are widely used in chromatographic analysis. However, pyridinecontaining derivatives, such as picolinyl esters, have been shown to be suitable for direct mass spectrometric structural analysis of acids containing straight, branched, unsaturated, cyclic, or oxygenated chains. Finally, 4,4-dimethyloxazoline derivatives of fatty acids have been found to show several advantages and have been applied successfully to the structural determination of PUFA and cyclopropenoid fatty acids [21].

6.3.2.2

Quantitative Estimation of Fatty Acid Composition

With reliable modern gas chromatographs equipped with flame ionization detectors (FID), the areas under the peaks on the GLC traces are, within limits, linearly proportional to the amount (by weight) of material eluting from the columns [15,16]. Problems of measuring this area arise mainly when components are not completely separated, and there is no way of overcoming this difficulty entirely. A known quantity of an internal standard should be added to the lipid sample. In most cases, nonadecanoic acid is employed and added before the methylation step; quantitative results would first be calculated on its basis. On the other hand, commercially available standard mixtures containing accurately known amounts of methyl esters of saturated, monoenoic, and polyenoic fatty acids should be analyzed under the same GLC conditions for checking the quantification results. If necessary, calibration factors may have to be calculated for each fatty acid component to correct the areas of the relevant peaks in the mixtures analyzed; this is specially relevant for PUFA. Results can be expressed as weight percentages of the fatty acids present or as molar amounts of each fatty acid.

6.4 Analysis of Marine Nonsaponifiable Matter 6.4.1

Lipid Saponification

Lipids may be hydrolyzed (saponified) by heating them under reflux with an excess of dilute aqueous ethanolic alkali; the fatty acids on one side and diethyl-ether-soluble nonsaponifiable materials on the other side are separately recovered for further analysis [2]. According to the previous section, the resulting FFA have to be transformed into their corresponding FAME for further analysis by an acid-catalyzed method. On the other hand, the nonsaponifiable layer will contain any long-chain alcohols and sterols originally present in the lipid sample in the esterified form, as well as the deacylated residues of ether lipid compound. Such compounds can be divided into sterols and “ether lipids.”

6.4.2

Analysis of Sterols

Sterols are biological compounds, the basic structure of which includes the cyclopentanophenanthrene ring. Total sterol content can be determined directly and spectrophotometrically from the lipid extract by using the method of Huang et al. [22], based on the Liebermann–Buchardt reaction. For a complete analysis, sterols can be fractionated and analyzed by means of different

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chromatographic techniques [23,24]. For GLC analysis, marine sterols have to be converted into more volatile compounds such as acetate [25] and trimethylsilyl (TMS) [26] derivatives. Cholesterol has been shown to be the most abundant sterol in all marine living beings, although invertebrates have shown a significant presence of other sterols [27]. Chromatographic methods for cholesterol analysis [28] are of relevant importance in foods in relation to human health concerns. Although the GLC is normally carried out with cholestane as internal standard, high-performance liquid chromatography (HPLC) methods can offer a nondestructive alternative, but they suffer from the limitation of the lack of a distinctive chromophore in the analyte. Great attention has been accorded to the assessment of cholesterol oxide formation in marine products [29].

6.4.3 Analysis of Ether Lipids Marine lipids may contain fatty acid residues as the only radicals, or they may include alkyl and alkenyl radicals. The first type is the major one in marine lipids, and its analysis has already been discussed in Section 6.3. The others are often united into a group called “ether lipids.” Such compounds are basically found as PL classes (specially in phosphatidylethanolamine), being normally placed as the radical in position 1 and specially abundant in marine invertebrates [5,27]. Unlike fatty acids, information on ether lipid composition in marine PL is less abundant, although a great interest has been accorded to their isolation because of their medical and cosmetic applications [30]. Methods for separating and quantifying ether-linked glycerides have been reviewed [31,32]. The alkyl groups of 1-alkyl-2,3-diacyl-sn-glycerols are generally saturated or cis-monoenoic even– numbered components (16:0, 18:0, and 18:1, mostly). The alkyl moieties are usually analyzed in the form of 1-alkylglycerol or as volatile nonpolar derivatives of this compound, such as acetate, trifluoroacetate, TMS, or isopropylidene derivatives by GLC. The determination of double-bond positions in long alkyl chains has been carried out by means of picolinyl and nicotinylidene derivatives by GLC-MS [33]. Further, supercritical fluid chromatography (SFC) has been employed for the glycerol ether analysis of liver oils of shark species [34]; thus, chimyl, batyl, and selachyl alcohols were found to be the most abundant. Concerning alkenylglycerols, such compounds tend to be decomposed during GLC analysis and are best reduced by catalytic hydrogenation to alkylglycerols. Although the vinyl ether linkage is unaffected by basic hydrolysis conditions, it can be cleaved by acid-catalyzed transmethylation, which generates dimethyl acetals from the liberated fatty aldehydes. Accordingly, alkenyl compounds have been directly identified and quantified by GLC together with FAME [35]. Adsorption thin-layer chromatography (TLC) on silica gel layers can be used to separate simple alkyl and alkenyl lipids; neutral plasmalogens tend to migrate ahead of alkyldiacylglycerols, which in turn migrate just in front of TG. They can be separated by a double development in a single direction with hexane–diethyl ether (95:5, v/v) as a solvent system. Neutral plasmalogens may be detected by spraying the TLC plates with 2,4-dinitrophenyl-hydrazine (0.4%) in 2M HCl, whereas no simple spot test is available for the identification of alkyldiacylglycerols.

6.5

Qualitative and Quantitative Analyses of Marine Lipid Classes

Lipid samples obtained from extraction of biological material are complex mixtures of individual lipid classes [16]. Often, no single procedure will achieve the desired analysis, and combinations of techniques must be used until the required purposes are served. In this section, different analytical

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approaches will be discussed, focused on the qualitative and quantitative analyses of marine lipid classes. The fatty acid composition of each lipid class can be determined by GLC of the methyl ether derivatives, prepared by esterification or transesterification of the purified lipid class, according to details explained in Section 6.3.

6.5.1 Spectrophotometric Assessments of Total Lipid Extract Some classical methods are available for the analysis of lipid classes or lipid class groups when applied directly onto the lipid extract without prior separation. For FFA assessment, titrimetric methods were used for many years, although some interference of polar lipids was found. Procedures that involve spectrophotometric measurement of highly colored copper complexes are now favored. A very popular one is that proposed by Lowry and Tinsley [36], where an FFA-cupric acetate-pyridine complex is involved. More recently, a rapid NIR spectrometry has been applied for the direct FFA determination in fish oil [37]. Traditional determination of PL content in lipid extracts has involved the digestion of PL with the release of inorganic phosphate; then, this is made to react with ammonium molybdate to form phosphomolybdic acid, which is reduced and determined spectrophotometrically [38]. An alternative and successful method has been proposed by Raheja et al. [39] without previous digestion; in it, PL present in the lipid extract are made to react with ammonium molybdate in an organic phase and then measured spectrophotometrically. Ester linkages can be quantified by the method of Vioque and Holman [40]. In it, such functional groups are made to react with hydroxamic acid and further complexed with Fe (III). This method can be applied to total lipid extract or to any lipid class after previous isolation [7,41]. Finally, a method for the quantification of esterified and unesterified total sterols is mentioned in Section 6.4.2 [22].

6.5.2 Stereospecific Analysis of Lipid Classes The determination of fatty acid composition at each location in lipid classes has ever since attracted great attention. Compared with the data compiled for plant oils and for fats from land animals, the results so far reported for aquatic animals are few. This is due to the great complexity of fatty acids present in these oils and fats, giving rise to a tremendous number of species. The advent of new NMR, HPLC, and GLC technologies combined with MS in the last decades has provided quick and useful procedures for the stereospecific lipid analysis, according to the information provided in following sections. However, traditional methodologies are still employed in cases where such advanced technologies are not available and are reviewed in this section. Most living organisms have developed lipolytic enzyme systems that are able to distinguish between bonds to the various positions of glycerol or between certain types of bonds in specific lipids. In many cases, these enzymes can be isolated and used in simple incubations in vitro as an aid in structural analyses of lipids, this including chromatographic separation and further analysis of fatty acids after previous methylation and transmethylation. A wide use was found for lipase hydrolysis, although it turned out not to be accurate enough for marine lipids, since the presence of double bonds in the proximity of a carbonyl group of fish PUFA reduces the rate of deacylation of glycerides. Accordingly, the Grignard reagent has widely been employed in the case of marine substrates [42]. Additionally, preparative TLC on silicic acid impregnated with 5% (w/w) boric acid has been applied to prevent acyl migration during chromatographic separation.

Lipid Compounds

◾

79

Traditional research accounts for consecutive series of methods combining chemical reactions and enzymatic releases of fatty acids in different positions for resolution of the molecular species. Such stereospecific studies have widely been focused onto TG [43,44] and PL [45,46] classes.

6.5.3 Column Chromatography Normal-pressure or low-pressure column chromatography (CC) was widely employed in the past and is now mostly used as a way of preliminary fractionation of lipid classes. Separation can be carried out on silicic acid, acid-washed florisil, or florisil as adsorbents, being simple lipids eluted in a stepwise sequence with hexane containing increasing proportions of diethyl ether, whereas complex lipids are recovered by elution with methanol [41,47]. The principal advantages of the method are the ease of preparation of a column and the comparatively large amount of lipid that can be separated. Column chromatography on diethylaminoethyl (DEAE)-cellulose has shown to be a valuable method for the isolation of particular groups of complex lipids in comparatively large amounts, although lengthy conditioning may be necessary before columns can be employed [2,47]. Aminopropyl-bonded phase cartridges have been much used for the isolation of simple and complex lipid fractions, although particular care is required to recover the acidic lipids quantitatively.

6.5.4 Thin-Layer Chromatography Many text books and reviews report TLC application on lipids for routine separations, identification, and quantification [48,49]. A variety of solvent systems have been used to separate simple lipids on an analytical or semipreparative scale by single or two-dimensional TLC. Those used most frequently contain hexane, diethyl ether, and acetic (or formic) acid in various proportions. For preparative purposes, 20–50 mg of marine lipid may be applied with ease as a band on a 20×20 cm plate coated with a layer of silica gel of 0.5 mm thickness [7,41]. However, precoated plates are much more convenient than laboratory-made plates, in spite of the relatively higher costs [50]. In all cases, lipid classes can be detected by any of the nonspecific available reagents and identified by their migration characteristics relative to authentic standards chromatographed simultaneously alongside the samples under investigation. The improvement and versatility of TLC enable it to be used for several modern applications, which include highly automated techniques right from sample application and development to detection and quantification. Such techniques would include high-pressure TLC (HPTLC), overpressure TLC (OPTLC), and tubular TLC (TTLC). In addition, coupling of TLC with other techniques such as HPLC, infrared (IR) spectrometry, MS, and NMR has increased its analytical power in several applications. The perceived weakness of TLC has been recognized as the quantification aspect, and this has led to the evolution of the TLC/FID Iatroscan system, which has been used routinely for lipid analysis in the last decades. It combines the separation capabilities of conventional TLC with the quantification power of the FID and has application in the quantitative analysis of all substances separable by conventional TLC. The system has been successfully used for marine lipid class analysis [51].

6.5.5 High-Performance Liquid Chromatography In recent years, HPLC has undoubtedly been the most widely applied separation technique in the analysis of most simple and complex lipid classes [48,52]. HPLC is much more expensive than

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TLC in terms of both equipment and running costs, but it can be automated to a considerable degree and gives much cleaner fractions in micropreparative applications. It can give better and more consistent separations of minor components, while no oxidation of the unsaturated fatty acid constituents needs to occur during fractionation on an HPLC column. An isocratic and gradient elution procedure with ultraviolet (UV) detection has been employed for marine PL analysis. In the detection, some of the more impressive separations have made use of FID systems, but others have obtained satisfactory results, with UV detection at 206 nm both on an analytical and on a preparative scale. Finally, evaporative light-scattering detection has successfully been applied [16]. HPLC has specially been applied to the most abundant lipid classes; therefore, quantification and stereospecific analyses have been carried out. Thus, TG separation according to the carbon number or partition number has been achieved [53]. For PL classes, HPLC analysis has been accepted as the most accurate one, by employing both gradients of polar solvents and microparticulate silicic acid [6,54].

6.5.6 Silver Ion Chromatography Silver ions, like the ions of other transition metals, interact specifically with the olefinic double bonds of unsaturated compounds to form weak charge transfer complexes. The complexes are usually unstable and exist in equilibrium with the free form of the olefin. However, such complexation is favorable for use in chromatography and enables the performance of the various Ag+-chromatographic techniques developed so far. Thus, Ag+-chromatography has been performed in conjunction with CC, TLC, and HPLC, being successfully applied to all lipid classes in marine species by separating molecules according to unsaturation degree [55]. Ag+-TLC is used mostly in the preparative mode, as a complementary separation method to GLC or GLC-MS. Both homemade and precoated glass plates are used in Ag+-TLC. The usual supporting materials are silica gel G for FAME and TG and silica gel H for complex lipids. Thus, the complementary employment of GLC or GLC-MS together with Ag+-TLC is considered one of the most powerful tools for elucidation of fatty acid composition in complex lipid samples [56]. On the other hand, Ag+-HPLC and reverse phase (RP)-HPLC applied in complementary ways were effective in the analysis of TG in fish oils [57]. Perona and Ruiz-Gutiérrez [53] were able to resolve a large number of sardine TG molecular species by RP-HPLC; further identification of most peaks was carried out by using preparative Ag+-TLC followed by fatty acid analysis by GLC.

6.5.7 Nuclear Magnetic Resonance (NMR) Spectrometry In recent years, high-resolution NMR spectrometry (1H-NMR, 13C-NMR, 31P-NMR) has increasingly been applied to the identification of lipid structures to determine patterns of branching, or substitution, and in particular to the detection, and often the location, of the double-bond systems in fatty acid chains. In the past 20 years, some important articles and reviews have been published [58], so it has become an extremely powerful technique for obtaining qualitative and quantitative information of the lipid class profile of a marine tissue extract. The procedure is rapid and nondegradative.

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Based on 1H-NMR spectrometry [59], a rapid and structure-specific method for the determination of ω3-PUFA in fish lipids was presented. The different chemical shifts observed for the methyl resonance of ω3-PUFA (δ = 0.95 ppm) with respect to the methyl resonance of all other fatty acids (δ = 0.86 ppm) provided the possibility of proposing this new analytical tool. In a first attempt for 13C-NMR application [60], the high-resolution NMR spectra of four fish oils were recorded. Signals in the spectra were assigned, and attention was focused on the identification of specific signals for ω3 fatty acid group and also individually for DHA, EPA, and stearidonic acid. Later on [61], 13C-NMR spectrometry was successfully used to determine the proportions of saturated, mono- and diene-, ω6, ω3, and highly unsaturated fatty acids of lipid extract of Atlantic salmon muscle. A good agreement could be observed between NMR values and those from the GLC analysis. Quantitative analysis of fatty acid composition and alpha-beta distribution in TG tuna fish was achieved [62]; results obtained using high-resolution 13C-NMR were in good agreement with those obtained by GLC. FFA carbonyl resonances were detected at the lower field of the carbonyl region, thus providing a suitable tool for lipolysis analysis. The positional distribution (1, 2, and 3 locations) of ω3 fatty acids in depot fat of several fish species was examined by 13C-NMR [63]. It could be observed that DHA was concentrated in the 2-location of TG in depot fats. Finally, 13C-NMR was employed for the plasmalogen analysis in fish lipid samples showing a good agreement with the data obtained by GLC [64]. Application of 31P-NMR has shown to be far shorter than with 1H and 13C, so little application is specially available for marine lipids [58]. This NMR technique can provide a single signal for each PL class, according to each corresponding resonance; its intensity should be proportional to the quantity. The 31P-NMR application has also shown the possibility of analyzing the ether structures within the glycerol backbone of phosphatidylethanolamine and phosphatidylcholine.

6.5.8 Mass Spectrometry MS has long been used as a powerful tool for the analysis of the molecular weight, empirical formula, and complete structure of an unknown compound, although an increasing importance has been obtained lately for quantitative analysis [20,65,66]. The first step for any MS method is ionization of the sample molecules in the gas phase. Following ionization to a negatively or positively charged species (most commonly the later), the molecules or their fragments can be separated and identified on the basis of their mass-to-charge ratio (m/z). Over the years, many of the advances in MS have involved new ionization techniques. The information-rich nature of MS makes it the most desirable detector for many explanations, but, although GLC is conveniently coupled to electron impact ionization (EI) and chemical ionization (CI) sources, the condensed mobile phase used for liquid separations is not readily compatible with high vacuum ionization sources. After different approaches, Arpino [67] likened the HPLC-MS union. This development paralleled the development of atmospheric pressure chemical ionization (APCI). Recent developments in MS have been very interesting for complex lipid molecules. Thus, soft ionization MS techniques such as fast atom bombardment, thermospray, and electrospray have the ability to ionize lipid molecules without causing extensive fragmentation. Thus, fragmentation of the molecular ion species produced by soft ionization processes can further be achieved in a second mass spectrometer (MS/MS) by collision-induced dissociation. Among the different food lipids, marine lipids have received lesser attention, probably due to their more complicated structure. Some applications concerning the marine lipids’ study will now be mentioned.

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The oxidative decomposition of cholesterol in different fish products was investigated by means of MS analysis of cholesterol oxide TMS derivatives with a quadrupole mass spectrometer fitted with an EI source [68]. A wide range of cholesterol oxides were identified and quantified. The qualitative and quantitative compositions of 1-O-alk-1-enylglycerolipids of albacore tuna (Thunnus alalunga) were studied along the canning process [35]; analysis was carried out in conjunction with FAME by means of their dimethyl acetal derivatives resulting from the acid transmethylation of lipid extracts. Minor fatty acids from mussels (Mytilus galloprovincialis) were enriched by Ag+-TLC and then analyzed by GLC-MS as 2-alkenyl-4,4,-dimethyloxazoline derivatives [69]. The mass spectrometer was operated in the EI mode (70 eV), and several nonmethylene interrupted fatty acids were singled out. Lately, Rezanka [70] described a method for the enrichment of long-chain fatty acids from fatty acids of a green freshwater alga and their identification as picolinyl esters by means of HPLCMS with APCI; the method was based on the use of preparative reversed-phase HPLC followed by subsequent identification by APCI HPLC-MS.

6.5.9 Supercritical Fluid Chromatography In this advanced technique [10,71], analytes are eluted from a capillary chromatographic column, which uses a highly compressed gas above its critical temperature and critical pressure. Carbon dioxide is by far the most commonly used SFC mobile phase because of its low critical temperature, whereas its critical pressure and critical density are high enough for good solvation of many potential analytes. An important advantage is that it is compatible with FID, which has great sensitivity and linearity. In addition, the use of SFC can substantially reduce the dependence on organic solvents in solvent extraction or HPLC analysis. Analytical SFC has been shown to be particularly applicable to the analysis of higher molecular weight lipid moieties, such as mixed glyceride compositions ranging from 200 to 900 in molecular weight. Concerning marine species analysis, in a first attempt Baltic herring flesh TG were separated in eight fractions by Ag+-TLC, and the four most unsaturated fractions were analyzed by capillary SFC according to their acyl carbon numbers [72]. Later on, simple classes from marine oils of different species were separated and quantified by capillary SFC [73]; carbon dioxide as the mobile phase, a nonpolar capillary column, and a FID were employed in it. The liver oils of several shark species were analyzed by SFC [34]; thus, the method was capable of direct quantification of squalene and cholesterol, whereas quantification of TG, cholesterol esters, and diacylglycerol ethers required TLC fractionation before SFC analysis. Purification of PUFA (DHA and EPA) ethyl esters from tuna oil was carried out by SFC [74]; an optimization of process parameters was achieved to obtain a maximal production rate.

References 1. Small, D., Handbook of Lipid Research, The Physical Chemistry of Lipids, Plenum Press, New York, 1986, Vol. 4, p. 89. 2. Christie, W., Lipid Analysis, 2nd edn., Pergamon Press, Oxford, U.K., 1982, p. 17. 3. Simopoulos, A., Nutritional aspects of fish, in Seafood From Producer to Consumer, Integrated Approach to Quality, Luten, J., Börrensen, T., and Oehlenschläger, J., eds., Elsevier Science, London, U.K., 1997, p. 589.

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4. Ackman, R., Fatty Acids, in Marine Biogenic Lipids, Fats, and Oils, Ackman, R., ed., CRC Press, Boca Raton, FL, 1989, Vol. 1, p. 103. 5. Pearson, A., Love, J., and Shorland, F., “Warmed-over” flavor in meat, poultry and fish. Adv. Food Res., 23, 2, 1977. 6. Vaskowski, V., Phospholipids, in Marine Biogenic Lipids, Fats, and Oils, Ackman, R., ed., CRC Press, Boca Raton, FL, 1989, p. 199. 7. Sieiro, P., Aubourg, S. and Rocha, F., Seasonal study of the lipid composition in different tissues of the common octopus (Octopus vulgaris). Eur. J. Lipid Sci. Technol., 108, 479, 2006. 8. Folch, J., Lees, M., and Stanley, G., A simple method for the isolation and purification of total lipids from animal tissue, J. Biol. Chem., 226, 497, 1957. 9. Bligh, E. and Dyer, W., A rapid method of total extraction and purification. Can. J. Biochem. Physiol., 37, 911, 1959. 10. King, J., Supercritical fluid chromatography (SFC)-Global perspective and applications in lipid technology, in Advances in Lipid Methodology—Five, Adlof, R., ed., The Oily Press, Bridgwater, England, U.K., 2003, p. 301. 11. Wells, M. and Dittmer, J., The use of sephadex for the removal of nonlipid contaminants from lipid extracts. Biochemistry, 2, 1259, 1963. 12. Wuthier, R., Purification of lipids from nonlipid contaminants on sephadex bead columns, J. Lipid Res., 7, 558, 1966. 13. Prost, E. and Wrebiakowski, H., Evaluation of soxhlet’s and Bligh and Dyer’s methods in the determination of fat in meat. Z. Lebensm. Unters. Forsch., 149, 193, 1972. 14. Nielsen, D., Hyldig, G., Nielsen, J., and Nielsen H., Lipid content in herring (Clupea harengus L.). Influence of biological factors and comparison of different methods of analyses: Solvent extraction, Fatmeter, NIR and NMR. Food Sci. Technol., 38, 537, 2005. 15. Kuksis, A., Separation and determination of structure of fatty acids, in Handbook of Lipid Research, Fatty Acids and Glycerides, Kuksis, A., ed., Plenum Press, New York, 1978, Vol. 1, p. 1. 16. Christie, W., Lipid Analysis, 3rd edn., The Oily Press, Bridgwater, U.K., 2003, p. 37. 17. Lepage, G. and Roy, C., Direct transesterification of all classes of lipids in a one step reaction. J. Lipid Res., 27, 114, 1986. 18. Hammond, E., Packed-column gas chromatography, in Analysis of Oils and Fats, Hamilton, R. and Rossell, J., eds., Elsevier Applied Science, London, U.K. and New York, 1986, p. 113. 19. Ackman, R., WCOT (capillary) Gas–liquid chromatography, in Analysis of Oils and Fats, Hamilton, R. and Rossell, J., eds., Elsevier Applied Science, London, U.K. and New York, 1986, p. 137. 20. Le Quéré, J., Gas chromatography-mass spectrometry and tandem mass spectrometry in the analysis of fatty acids, in New Trends in Lipid and Lipoprotein Analyses, Sebedio, J. and Perkins, E., eds., AOCS Press, Champaign, IL, 1995, p. 191. 21. Medina, I. and Garrido, J., One-step conversion of fatty acids into their 2-alkenyl-4,4,-dimethyloxazoline derivatives directly from total lipids. J. Chrom. A, 673, 101, 1994. 22. Huang, T., Chen, C., Wefler, V., and Raftery, A., A stable reagent for the Liebermann-Buchardt reaction. Anal. Chem., 33, 1405, 1961. 23. Teshima, S., Sterols and crustaceans, mollusks and fish, in Physiology and Biochemistry of Sterols, Patterson, G. and Nes, W., eds., American Oil Chemists’ Society Press, Champaign, IL, 1990, p. 229. 24. Hoving, E., Chromatographic methods in the analysis of cholesterol and related lipids. J. Chrom. B, 671, 341, 1995. 25. Aubourg, S., Pérez-Martín, R., and Gallardo, J., Stability of lipids of frozen albacore (Thunnus alalunga) during steam cooking. Int. J. Food Sci. Technol., 24, 341, 1989. 26. Krzynowek, J. and Panunzio, L., Cholesterol and fatty acids in several species of shrimp. J. Food Sci., 54, 237, 1989. 27. Joseph, J., Distribution and composition of lipids in marine invertebrates, in Marine Biogenic Lipids, Fats and Oils, Ackman, R., ed., CRC Press Inc., Boca Raton, FL, 1989, Vol. 2, p. 49. 28. Fenton, M., Chromatographic separation of cholesterol in foods. J. Chrom., 624, 369, 1992.

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29. Ohshima, T., Formation and content of cholesterol oxidation products in seafood and seafood products, in Cholesterol and Phytosterol Oxidation Products, Guardiola, F., Dutta, P., Codony, R. and Savage, G., eds., American Oil Chemists’ Society Press, Champaign, IL, 2002, p. 186. 30. Hayashi, K., Occurrence of diacyl glycerol ethers in liver lipids of gonatid squid Gonatopsis borealis. Nippon Suisan Gakkaishi, 55, 1383, 1989. 31. Sargent, J., Ether-linked glycerides in marine animals, in Marine Biogenic Lipids, Fats and Oils, Ackman, R., ed., CRC Press Inc., Boca Raton, FL, 1989, Vol. 1, p. 175. 32. Urata, K. and Takaishi, N., Ether lipids based on the glyceryl ether skeleton: Present state, future potential, J. Am. Oil Chem. Soc., 73, 819, 1996. 33. Harvey, D., Nicotinylidene derivatives for the structural elucidation of glycerol mono-ethers and mono-esters by gas chromatography/mass spectrometry. Biol. Mass Spectrom., 20, 87, 1991. 34. Borch-Jensen, C., Magnussen, M., and Mollerup, J., Capillary supercritical fluid chromatographic analysis of shark liver oils. J. Am. Oil Chem. Soc., 74, 497, 1997. 35. Medina, I., Aubourg, S., and Pérez-Martín, R., Analysis of 1-O-alk-1-enyl glycerophospholipids of albacore tuna (Thunnus alalunga) and their alterations during thermal processing. J. Agric. Food Chem., 41, 2395, 1993. 36. Lowry, R. and Tinsley, I., Rapid colorimetric determination of free fatty acids. J. Am. Oil Chem. Soc., 53, 470, 1976. 37. Zhang, H. and Lee, T., Rapid near-infrared spectroscopic method for the determination of free fatty acid in fish and its application in fish quality assessment. J. Agric. Food Chem., 45, 3515, 1997. 38. Barlett, G., Phosphorus assay in column chromatography. J. Biol. Chem., 234, 466, 1959. 39. Raheja, R., Kaur, C., Singh, A., and Bhatia, I., New colorimetric method for the quantitative determination of phospholipids without digestion. J. Lipid Res., 14, 695, 1973. 40. Vioque, E. and Holman, R., Quantitative estimation of esters by thin-layer chromatography. J. Am. Oil Chem. Soc., 39, 63, 1962. 41. Gallardo, J., Aubourg, S., and Pérez-Martín, R., Lipid classes and their fatty acids at different loci of albacore (Thunnus alalunga): Effects of the pre-cooking, J. Agric. Food Chem., 37, 1060, 1989. 42. Aubourg, S., Sotelo, C., and Gallardo, J., Zonal distribution of fatty acids in albacore (Thunnus alalunga) triglycerides and their changes during cooking. J. Agric. Food Chem., 38, 255, 1990. 43. Christie, W., Stereospecific analysis of triacyl-sn-glycerols, in New Trends in Lipid and Lipoprotein Analyses, Sebedio, J. and Perkins, E., eds., AOAC Press, Champaign, IL, 1995, p. 93. 44. Myher, J., Kuksis, A., Geher, K., Park, P., and Diersen-Schade, D., Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids. Lipids, 31, 207, 1996. 45. Brockerhoff, H., Determination of the positional distribution of fatty acids in glycerolipids. Methods Enzymol., 35, 315, 1975. 46. Aubourg, S., Medina, I., and Pérez-Martín, R., Polyunsaturated fatty acids in tuna phospholipids: Distribution in the sn-2 location and changes during cooking. J. Agric. Food Chem., 44, 585, 1996. 47. Hemming, F. and Hawthorne, J., Lipid Analysis. Editorial Acribia, S. A., Zaragoza (Spain), 2001, p. 5. 48. Hamilton, R., Thin layer chromatography and high-performance liquid chromatography, in Analysis of Oils and Fats, Hamilton, R. and Rossell, J., eds., Elsevier Applied Science, London, U.K., 1986, p. 243. 49. Shukla, V., Thin-layer chromatography of lipids, in New Trends in Lipid and Lipoprotein Analyses, Sebedio, J. and Ackman, R., eds., AOCS Press, Champaign, IL, 1995, p. 17. 50. Nakamura, T., Fukuda, M., and Tanaka, R., Estimation of polyunsaturated fatty acid content in lipids of aquatic organisms using thin-layer chromatography on a plain silica gel plate. Lipids, 31, 427, 1996. 51. Shantha, N. and Napolitano, G., Lipid analysis using thin-layer chromatography and the Iatroscan, in Lipid Analysis in Oils and Fats, Hamilton, R., ed., Blackie Academic and Professional, London, U.K., 1998, p. 1.

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52. Shukla, V., High-performance liquid chromatography: Normal-phase, reverse-phase detection methodology, in New Trends in Lipid and Lipoprotein Analyses, Sebedio, J. and Ackman, R., eds., AOCS Press, Champaign, IL, 1995, p. 38. 53. Perona, J. and Ruiz-Gutiérrez, V., Characterization of the triacylglycerol molecular species of fish oil by reversed-phase high performance liquid chromatography. J. Liq. Chrom. Rel. Technol., 22, 1699, 1999. 54. Medina, I., Aubourg, S., and Pérez-Martín, R., Composition of phospholipids of white muscle of six tuna species. Lipids, 30, 1127, 1995. 55. Nikolova-Damyanova, B., Lipid analysis by silver ion chromatography, in Advances in Lipid methodology—Five, Adlof, R., ed., The Oily Press, Bridgwater, England, U.K., 2003, p. 43. 56. Joh, Y., Elenkov, I., Stefanov, K., Popov, S., Dobson, G., and Christie, W., Novel di-, tri- and tetraenoic fatty acids with bis-methylene-interrupted double-bond systems from the sponge Haliclona cinerea. Lipids, 32, 13, 1997. 57. Laakso, P. and Christie, W., Combination of silver ion and reversed-phase high-performance liquid chromatography in the fractionation of herring oil triacylglycerols. J. Am. Oil Chem. Soc., 68, 213, 1991. 58. Diehl, B., Multinuclear high-resolution nuclear magnetic resonance, in Lipid Analysis in Oils and Fats, Hamilton, R., ed., Blackie Academic and Professional, London, U.K., 1998, p. 87. 59. Sacchi, R., Medina, I., Aubourg, S., Addeo, R., and Paolillo, L., Proton nuclear magnetic resonance rapid and structure-specific determination of ω-3 polyunsaturated fatty acids in fish lipids. J. Am. Oil Chem. Soc., 70, 225, 1993. 60. Gunstone, F., High resolution NMR studies of fish oils. Chem. Phys. Lipids, 59, 83, 1991. 61. Aursand, M., Rainuzzo, J., and Grasdalen, H., Studies of fatty acids in Atlantic salmon (Salmo salar) by 13C and 1H nuclear magnetic resonance (NMR) spectroscopy, in Quality Assurance in the Fish Industry, Huss, H., ed., Elsevier Science Publishers B. V., Amsterdam (Holland), 1992, p. 407. 62. Sacchi, R., Medina, I., Aubourg, S., Giudicianni, I., Addeo, F., and Paolillo, L., Quantitative high resolution 13C-NMR analysis of lipids extracted from the white muscle of Atlantic tuna. J. Agric. Food Chem., 41, 1247, 1993. 63. Aursand, M., Jørgensen, L., and Grasdalen, H., Positional distribution of ω3 fatty acids in marine lipid triacylglycerols by high-resolution 13C nuclear magnetic resonance spectroscopy. J. Am. Oil Chem. Soc., 72, 293, 1995. 64. Sacchi, R., Medina, I., and Paolillo, L., One and two-dimensional NMR study of plasmalogens (alk-1-enyl-phosphatidylethanolamine). Chem. Phys. Lipids, 76, 201, 1995. 65. Kuksis, A., Mass spectrometry of complex lipids, in Lipid Analysis in Oils and Fats, Hamilton, R., ed., Blackie Academic and Professional, London, England, U.K., 1998, p. 181. 66. Byrdwell, W., APCI-MS in lipid analysis, in Advances in Lipid Methodology—Five, Adlof, R., ed., The Oily Press, Bridgwater, England, U.K., 2003, p. 171. 67. Arpino, P., On-line liquid chromatography/mass spectrometry? An odd couple! Trends Anal. Chem., 1, 154, 1982. 68. Oshima, T., Li, N., and Koizumi, C., Oxidative decomposition of cholesterol in fish products. J. Am. Oil Chem. Soc., 70, 595, 1993. 69. Garrido, J. and Medina, I., Identification of minor fatty acids in mussels (Mytillus galloprovincialis) by GC-MS of their 2-alkenyl-4,4-dimethyloxazoline derivatives. Anal. Chim. Acta, 465, 409, 2002. 70. Rezanka, T., Identification of very long chain fatty acids by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry from green alga Chlorella kesslerri. J. Sep. Sci., 25, 1332, 2002. 71. Blomberg, L., Demirbuker, M., and Andersson, M., Characterization of lipids by supercritical fluid chromatography and supercritical fluid extraction, in Lipid Analysis in Oils and Fats, Hamilton, R., ed., Blackie Academic and Professional, London, U.K., 1998, p. 34.

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72. Kallio, H., Vauhkonen, T., and Linko, R., Thin-layer silver ion chromatography and supercritical fluid chromatography of Baltic herring (Clupea harengus membras) triacylglycerols. J. Agric. Food Chem., 39, 1573, 1991. 73. Staby, A., Borch-Jensen, C., Balchen, S., and Mollerup, J., Quantitative analysis of marine oils by capillary supercritical fluid chromatography. Chromatographia, 39, 697, 1994. 74. Alkio, M., Gonzales, C., Jäntti, K., and Aaltonen, O., Purification of polyunsaturated fatty acid esters from tuna oil with supercritical fluid chromatography. J. Am. Oil Chem. Soc., 77, 315, 2000.

Chapter 7

Lipid Oxidation Turid Rustad Contents 7.1 Introduction ..................................................................................................................... 87 7.2 Analysis of Lipid Oxidation ............................................................................................. 88 7.2.1 Primary Oxidation Products ................................................................................. 88 7.2.2 Secondary Oxidation Products ............................................................................. 89 7.2.3 Stability Methods ................................................................................................. 92 7.2.4 Instrumental Methods .......................................................................................... 92 7.2.5 Sensory Analysis of Rancidity ............................................................................... 93 7.3 Summary ......................................................................................................................... 93 References ................................................................................................................................. 93

7.1

Introduction

Marine lipids are good and natural sources of polyunsaturated n-3 fatty acids (PUFA) such as docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3). These fatty acids have beneficial health effects and are reported to prevent coronary heart diseases and have a positive effect on the brain and nervous system as well as stimulating the immune system [1,2]. However, due to the high content of long-chain PUFAs, marine lipids are highly susceptible to oxidation. Lipid oxidation is the most important factor limiting the shelf life of marine oils and is also an important factor determining the shelf life of seafood products, except when microbial processes limit the shelf life. Reaction products from lipid oxidation have a negative effect on the sensory properties of fish products. The volatile, secondary oxidation products, especially those that originate from n-3 PUFAs are components that have a low threshold and therefore have a negative impact on the sensory quality of the food even in low concentrations [3]. This can lead to 87

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loss of products, complaints from the consumers, and reduced sales. Some of the reaction products from lipid oxidation may also have negative health effects. Lipid oxidation can be divided into three types, autoxidation, photooxidation, and enzymatic oxidation. Autooxidation of lipids takes place when the unsaturated fatty acids are exposed to oxygen and proceeds through an autocatalytic chain reaction [3]. Free radicals are formed when hydrogen ions are extracted from the fatty acids. The radicals react with oxygen forming peroxy radicals and hydroperoxides. The peroxides are easily broken down to alkoxy radicals, leading to a wide variety of reaction products. These include nonradical species such as aldehydes, ketones, acids, and alcohols, and also more complex reaction products such as epoxy and polymeric compounds are formed during the propagation and termination steps [4]. The secondary oxidation products include both low molecular weight, volatile compounds and nonvolatile components with a relatively high molecular weight. The secondary oxidation products can also react further, resulting in a wide variety of degradation products, which makes it difficult to find where the components originated. This also makes the determination of the degree of oxidation a challenging task. When the decomposition of a hydroperoxide has resulted in the formation of a low-molecular weight volatile compound, the parent triglyceride is left with a shorter fatty acid. If this contains a terminal carbonyl group, the molecule is called a core aldehyde. However, the influence of these compounds has been little studied [3]. The fatty acids and the lipid oxidation products in foods can also react with other components in the food such as proteins, carbohydrates, and water, making it even more difficult to determine the degree of rancidity.

7.2 Analysis of Lipid Oxidation Many different methods have been implemented both by the industry and in research to determine the degree of lipid oxidation both in marine oils and in seafood. Methods to determine the degree of lipid oxidation can be divided into two main groups, methods that determine the primary oxidation products and methods that measure the secondary oxidation products.

7.2.1

Primary Oxidation Products

The most common methods to determine primary oxidation products are peroxide value (PV) and conjugated dienes. PV is one of the classical methods for determination of oxidative status; it is both one of the oldest and one of the most used methods. For determination of PVs in foods, the lipid can be extracted using the methods of Ref. [5] or [6] before analysis. Several analytical procedures are available, but it is important to keep in mind that the results for PV measurements will vary both according to the method used and how the procedure is performed [3]. A simple titration method where the sample is dissolved in chloroform–acetic acid (or isooctane–acetic acid) is often used for fats and oils. Potassium iodide is added, this is oxidized by the hydroperoxides or other components present in the sample, and the liberated iodine is titrated with sodium thiosulfate with starch as an indicator. The PV is expressed in milliequivalent of iodine per kilogram of lipid or as millimolar of peroxide per kilogram of lipid [7]. This method requires a sample of 5 g if the PV is below 10 and about 1 g if the PV is higher [3]. The sensitivity is about 0.5 meq/kg, but this can be improved by determining the endpoint colorimetrically or by

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89

determining the liberated iodine electrometrically using a platinum electrode. Oxygen in the air, light, and absorption of iodine by the unsaturated fatty acids in the oil may interfere and cause variations in the results. Care should therefore be taken in standardizing how the procedure is performed. Several colorimetric methods for determination of PV values are used. One of these is the colorimetric ferric thiocyanate method. In this procedure ferrous ions are oxidized to ferric ions, which react with ammonium thiocyanate forming ferric thiocyanate, which is a red complex with an absorption maximum of 500 nm [3]. This method is more sensitive and requires smaller samples. The method of The International Dairy Federation—often called the IDF method [8] as modified by Ueda et al. [9] and Undeland et al. [10]—requires a low amount of sample (less than 10 mg). Nielsen et al. compared five different methods for determination of PVs [11]—the titration method, the colorimetric ferro method, the micromethod determining oxidation of iodide to free iodine, the FOX2 method determining oxidation of ferrous salts to ferric ions and reaction with xylenol orange, and the modified IDF method. Even if new instrumental methods now have been developed for determination of PVs, it is often desirable to use a method that either does not require instruments or requires only a spectrophotometer. The different methods gave different PVs for the same sample, and there was no consistency in the levels of PV determined by the different methods. Based on the fact that the methods chosen should have a large linear range, a high reproducibility, and use a low amount of solvent, the IDF method was chosen as the best of these methods. However, also for this method care should be taken in standardizing the procedure, with regard to chemicals used, how these are stored, and how the procedure is performed. Small changes in quality of ethanol can give widely different standard curves and thereby influence the results. In order to determine individual peroxides, high-performance liquid chromatography (HPLC) methods can be used [3]. After the initiation phase, the level of primary oxidation products increases and passes through a maximum. Using PV as a sole determination of oxidation level can therefore be misleading, and it is important to know the history of the oil or the seafood to interpret the measurement of PV. Due to rapid polymerization of EPA and DHA compared with the formation of stable peroxides of these fatty acids, PV is reported to be an unreliable indicator of lipid peroxidation in fish [4]. Conjugated diene hydroperoxides are formed when polyunsaturated fatty acids oxidize. The fatty acid chain then contains a structure with alternating simple and double bonds. Conjugated dienes have a strong absorption maximum at 230–235 nm [12]. Frankel [3] suggests measuring the absorbance of conjugated dienes at 243 nm. A known amount of sample is diluted in methanol (esters), isooctane, or hexane [13]. Conjugated dienes are useful for bulk lipids, and the AOCS method requires a sample size of around 10 mg. For use on tissue extracts, extraction and separation techniques are necessary. The sensitivity and specificity can be increased by using second derivative spectra [12].

7.2.2

Secondary Oxidation Products

Development of peroxides and conjugated dienes follows the same process and can be reduced after a certain oxidation level. These methods are therefore most useful as a measure of lipid oxidation for lipids with a low level of oxidation. Peroxides are unstable and are rapidly transformed into secondary [14] oxidation products, and determinations of PV have to be combined with the determination of secondary products such as thiobarbituric acid-reactive substances (TBARS) and

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anisidine value (AnV). These methods determine the presence of aldehydes, which are secondary oxidation products. For determination of secondary oxidation products, the AnV is a common method. This determines the amount of aldehydes (mainly 2-alkenals and 2,4-dienals). The sample is dissolved in isooctane, p-anisidine dissolved in acetic acid is added, and the absorbance at 350 nm is determined after 10 min [15]. The AnV of freshly deodorized oils is caused by core aldehydes. AnV can also be determined using Fourier transform infrared (FT-IR) [16]. The Totox value is still one of the most commonly used oxidation parameters used in commercial laboratories and laboratories in the edible oil industry. This value is a combination of the PV and the AV. The Totox value is given as 2*PV + AnV. The determination of TBARS (or TBA) is a common method to determine secondary oxidation products. There are many published methods to determine TBARS, but as for the determination of PV, different methods give different results. All the methods are based on the pink color absorbance formed by reaction between TBA and oxidation products of polyunsaturated lipids. Originally, the colored complex was ascribed to the condensation of two moles of TBA and one mole of malonaldehyde (MDA), which is formed as a decomposition product from lipid hydroperoxides under the acidic test conditions [3]. However, the reaction is not specific, and the color is formed by many different secondary oxidation products, hence the name TBARS. Many factors influence the color in the TBA test—temperature, time of heating, pH, metal ions, and antioxidants. Some of the MDA detected in this test is formed during the peroxidation of the lipids, but most of it is formed during the decomposition of the lipid peroxides during the acid heating stage. This process is accelerated by metal ions [12]; in addition H2O2, antioxidants, and chelating agents may also influence the peroxide decomposition during the assay. Many variations of this test are being used. In the AOCS method [13], the lipids are dissolved in a solution of thiobarbituric acid in butanol, the sample is incubated at 95°C for 2 h, and the absorbance of the solution is read at 530 nm. In another method, the oxidation products are extracted in trichloroacetic acid (TCA) before the reaction with TBA. In the micromethod of Ke and Woyewoda [17], the lipids are boiled for 45 min with a mixture of TBA, sulfite, and chloroform before adding TCA, and the optical density of the water phase is determined at 538 nm. In other variations, the TBARS are separated by steam distillation or HPLC to increase selectivity. The TBA test can be standardized using MDA, which is generated by acid hydrolysis of 1,1,3,3-tetraethoxypropane [3]. However, alkanals, alkenals, and 2,4-dienals also react with TBA, forming a yellow pigment absorbing light at 450 nm. Dienals also give a red pigment absorbing at 530 nm. In addition, many other components in foods can react with TBA or interfere with the measurements. Protein, amino acids, nucleic acids, nitrite, sucrose and other sugars, reaction products from browning reactions, antioxidants, and trace metals can influence the result [3,13]. The TBARS values for different foods with the same level of oxidation (based on flavor scores) can vary significantly [3,13,18]. However, TBARS values have been found to correlate with sensory scores within the same materials [19]. The volatile compounds formed as a result of lipid oxidation can be analyzed using electronic noses/gas-sensor array systems [20]. Different types of headspace analyses can be used, where the headspace volatiles over the samples are sampled, separated, and identified using different gas sensors. Of these methods, static headspace and solid-phase microextraction (SPME) are the least sensitive. Purge and trap techniques, where the samples are flushed or purged with nitrogen and the volatiles in the gas flow are trapped on a solid absorber, are highly sensitive. After sampling, the volatiles can be thermally desorbed into a gas chromatograph for separation. The mass spectra of the compounds can also be compared with spectra of pure standard compounds and

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identified [21]. The advantages of this method are that it is flexible, and the amount of sample and sampling conditions can be varied according to the needs. However, quantification of headspace data, especially from solid matrixes, is complicated, and the results are dependant on the sample material. Small variations in sampling procedures can give large variations in the data; the data handling is also difficult. Analysis of volatiles is discussed by Ólafsdóttir and Jónsdóttir in Chapter 8. Lipid oxidation products can interact with other components in food, such as amino acids, peptides, proteins, nucleic acids, deoxyribonucleic acid (DNA), phospholipids, and so on, and form fluorescent products. Reactions between lipid oxidation products and other components in seafood or seafood products may lead to underestimation of the degree of lipid oxidation, as measured by methods such as PV and TBARS. Hydroperoxides (primary lipid oxidation products) and aldehydes (secondary oxidation products) can react with amino groups in proteins, forming Schiff bases. This reaction can lead to formation of brown-colored compounds [22,23]. The fluorescent compounds formed from lipids are the result of oxidation of phospholipids or are formed from oxidized fatty acids in the presence of phospholipids. Fluorescence techniques are highly sensitive and 10–100 times more sensitive for detection of MDA than TBARS [3]. The different chromophores formed as a result of oxidized lipids, reactions between oxidized lipids and proteins/ peptides, or reactions between oxidized lipids and DNA have different excitation and emission maxima as shown in Table 7.1. Fluorescence has traditionally been applied to samples in solution, for example, for assessment of lipid oxidation during fish processing [24–26]. Aubourg and Medina [26] extracted fish muscle with a 2/2/1.8 chloroform/methanol/water mixture and measured fluorescence both in the water and in the organic phase. They measured the fluorescence intensity both at 393/463 nm and 327/415 nm. The fluorescence intensities were divided by the fluorescence intensity of quinine sulfate and the fluorescence shift calculated. The fluorescence shift was found to be a more effective index of changes in fish quality than other commonly used methods. When fluorescence measurements are done on samples in solution, and the concentration is below a certain level, the measured intensity follows the Beer–Lambert law. When the samples are turbid or solid or the concentration is high, scatter, quenching, and so on, destroy this relationship. Instead, front-face fluorescence

Table 7.1 Excitation and Emission Maxima for Chromophores Formed as a Result of Oxidized Lipids, Reactions between Oxidized Lipids and Proteins/Peptides or Reactions between Oxidized Lipids and DNA Chromophore

Excitation Maxima (nm)

Emission Maxima (nm)

Oxidized phospholipids/oxidized fatty acids + phospholipids

365

435–440

MDA + phospholipids

400

475

Oxidized arachidonic acid + dipalmityl phosphatidylethanolamine

360–390

430–460

Oxidized arachidonic acid + DNA

315

325

Peroxides/secondary oxidation products + DNA in the presence of metal ions or reducing agents

320

420

92

◾


spectroscopy can be used. Using solid-phase fluorescence is a relatively new approach, but studies on the use of this technique in dried fish were published in 1992 [27]. Fluorescence spectroscopy on intact samples has been shown to be a sensitive technique, comparable to sensory analysis and gas chromatography, for measuring lipid oxidation [28]. One challenge is that fluorescence spectra can be very complex and that not only the oxidation products but also connective tissue, adipose tissue, porphyrins, and additives may contribute to the spectra. So far, little has been done to study the fluorescence spectra of the different oxidation products that are formed in foods. In a study of different model systems including fish and meat, Veberg et al. [28] concluded that fluorescence spectroscopy may be able distinguish between different oxidation products formed but that this would require using the whole spectrum and not only the intensity at the maximum wavelength. Fluorescence spectroscopy has a great potential for on-line or at-line applications.

7.2.3

Stability Methods

Several techniques based on accelerated oxidation are used for evaluation of oxidation; these include the oil stability index method [29], the Rancimat test [30], and oxidative stability measurement by Oxidograph [31] and they are all suitable for analyzing oil systems. The Rancimat, oil stability index (OSI), active oxygen method (AOM), and Oxidograph are techniques for measuring the stability of oils toward oxidation. In Rancimat and OSI instruments, the oil can be heated to 80°C or more while air is bubbled through it. This results in the formation of low molecular weight acids that are flushed out with the air and collected in vessels containing distilled water. The change in conductivity is measured, and the point where it changes most is called the induction time. The AOM method is performed in a somewhat similar way, but it measures the time taken to reach a certain PV. The Oxidograph instrument finds the induction time based on measurement of the decline in pressure caused by the absorption of oxygen in a closed vessel.

7.2.4

Instrumental Methods

Many instrumental methods have been developed for the determination of oxidation parameters in oils and foods, including near-infrared spectroscopy (NIR), Fourier-transform near-infrared (FT-NIR), and FT-IR spectroscopy methods [16,32,33]. Lipid oxidation products can produce very weak chemiluminescence (CL). It has been shown that sodium hypochlorite-induced decomposition of hydroperoxides gives strong CL [34,35]. The level of hydroperoxides in fish oil can be determined using a rapid CL method [14]. In recent years, new methods have been developed, and these include assessment of free radicals using electron spin resonance (ESR) spectroscopy and use of different chromatographic methods to determine both primary and secondary oxidation products. The gas chromatography–mass spectrometry (GC–MS) techniques can be used to determine a wide range of volatile secondary lipid oxidation products [36]. The liquid chromatography–mass spectrometry (LC–MS) techniques can also determine nonvolatile products—of special interest are the core aldehydes [3,37]. Free radical assessments by the ESR spin-trapping technique detected the very early stages of lipid oxidation, and a few minutes of oxidation of docosahexaenoate (DHA) resulted in significant changes in the ESR spectra. The levels of free radicals trapped in cod liver oil and salmon oil during the first hours of oxidation were in accordance with the oxidative stability measured by conventional methods [4]. 1H NMR spectra can be used to study specific lipid oxidation products, such as different hydroperoxides, aldehydes, and also cyclic compounds, obtaining information

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that cannot usually be obtained by single conventional analytical methods [4]. Multivariate data analysis is a valuable tool in elucidating changes in spectra during storage and showed the resonances that came from n-3 fatty acids during oxidation. However, the sensitivity was low (detection levels ∼0.01 nM). The sensitivity could be improved by the use of CryoProbe technology.

7.2.5

Sensory Analysis of Rancidity

The ultimate measurement of rancid odor and taste is sensory analysis by a trained panel. A trained panel can be a very valuable tool for detection of early lipid oxidation of foods containing n-3 fatty acids. Some of the degradation products from long-chain n-3 PUFAS have a profound effect on odor and flavor in concentrations as low as in the parts per billion range [3]. In general, the oxidation products from n-3 fatty acids have a lower sensory threshold than those of oxidation products from other fatty acids. The detection of these low levels is not straightforward with classical lipid oxidation measurement methods. Odor threshold values vary both with the chemical structure of the carbonyl compounds and with the food matrix and based on how the sensory detection is performed, through the nose (nasal) or through the mouth (retronasal). Even if sensory methods can give sufficient information, their use is limited by the cost of employing a trained panel. It can also be difficult to compare data from different panels using different vocabularies or data from the same panel analyzed at different times. In addition, sensory analysis requires relatively large amounts of samples, and the use of chemical and instrumental analyses is recommended to support and complement the sensory analysis [3].

7.3 Summary Many different methods for the analysis of lipid oxidation exist. However, for many of these methods the results obtained vary not only with the method used but also with the analytical procedure that is performed, so care should be taken in standardizing the procedures. The ultimate wish from the food industry would be a rapid nondestructive method that can be applied on-line to analyze the oxidative or sensory quality in raw materials, intermediary goods, and finished products during seafood processing. However, even if there are many different methods that are used to determine lipid oxidation, today it is not possible to use only one method to determine lipid oxidation. There is, however, a rapid development in analytical methods to determine lipid oxidation, but for many of these methods calibration and verification are needed before they can be used for routine analysis.

References 1. Boissonneault, G.A., Dietary fat, immunity, and inflammatory disease, in Fatty Acids in Foods and Their Health Implications, C.K. Chow, Ed., Marcel Dekker: New York, 2000, pp. 777–795. 2. Narayan, B., K. Miayshita, and M. Hosakawa, Physiological effects of eicosapentanoic acid (EPA) and docosahexanoic acid (DHA)—A review. Food Rev. Int., 2006. 22: 291–306. 3. Frankel, E.N., Lipid Oxidation, 2nd ed., The Oily Press: Bridgewater, U.K., 2005. 4. Falch, E., Lipids from residual fish raw material, in Department of Biotechnology. 2005, Norwegian University of Science and Technology: Trondheim, 2005, 206. 5. Folch, J., M. Lees, and G.H. Sloan Stanley, A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 1957. 226: 497–509.

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6. Bligh, E.G. and W.J. Dyer, A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 1959. 37: 911–917. 7. AOCS, Method Cd 8-53, in Official Methods and Recommended Practices of the American Oil Chemists’ Society, D. Firestone, Ed., AOCS: Champaign, IL, 1995. 8. Sato, K. et al., Type V collagen in trout (Salmo gairdneri) muscle and its solubility change during chilled storage of muscle. J. Agric. Food Chem., 1991. 39: 1222–1225. 9. Ueda, S., M. Hayahashi, and M. Namiki, Effect of ascorbic acid in a model food system. Agric. Biol. Chem., 1986. 50: 1–7. 10. Undeland, I., M. Stading, and H. Lignert, Influence of skinning on lipid oxidation in different horizontal layers of herring (Clupea harengus) during frozen storage. J. Sci. Food Agric., 1998. 78: 441–450. 11. Nielsen, N.S., M. Timm-Heinrich, and C. Jacobsen, Comparison of wet-chemical methods for determination of lipid hydroperoxides. J. Food Lipids, 2003. 10: 35–50. 12. Halliwell, B. and J.M.C. Gutteridge, The measurement and mechanism of lipid peroxidation in biological systems. Trends Biochem. Sci., 1990. 15: 129–135. 13. AOCS, AOCS Official Method Ti 1a-64, D. Firestone, Ed., AOCS: Champaign, IL, 1995. 14. Pettersen, J., Chemiluminescence of fish oils and its flavour quality. J. Sci. Food Agric., 1994. 65: 307–313. 15. AOCS, Method Cd 18–90, in Official Methods and Recommended Practices of the American Oil Chemists’ Society, D. Firestone, Ed., AOCS: Champaign, IL, 1995. 16. Guillen, M.D. and N. Cabo, Fourier transform infrared spectra data versus peroxide and anisidine values to determine oxidative stability of edible oils. Food Chem., 2002. 77: 503–510. 17. Ke, P.J. and A.D. Woyewoda, Microdetermination of thiobarbituric acid values in marine lipids by a direct spectrophotometric method with a monophasic reaction system. Anal. Chim. Acta, 1979. 106: 279–284. 18. Nawar, W.W., Lipids, in Food Chemistry, O.R. Fennema, Ed., Marcel Dekker Inc.: New York, 1996, pp. 225–319. 19. Wold, J.P. et al., Rapid assessment of rancidity in complex meat products by front face fluorescence spectroscopy. J. Food Sci., 2002. 67: 2397–2404. 20. Olsen, E., Analysis of early lipid oxidation in foods with n-3 fatty acids, in Dept. of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences: Ås., Norway, 2005, 160. 21. Hübschmann, H.-J., Basics, in Handbook of GC/MS-Fundamentals and Applications. Wiley-VCH Verlag GmbH: Weinheim, 2001, pp. 7–212. 22. Tironi, V.A., M.C. Tomas, and M.C. Anon, Structural and functional changes in myofibrillar proteins of sea salmon (Pseudopercis semifascata) by interaction with malonaldehyde (RI). J. Food Sci., 2002. 67: 930–935. 23. Pokorny, J., Interaction of oxidised lipids with protein. La Rivista Italiana Delle Sostanze Grasse, 1977. 27: 389–393. 24. Aubourg, S., I. Medina, and J.M. Gallardo, Quality assessment of blue whiting (Micrometistius poutassou) during chilled storage by monitoring lipid damages. J. Agric. Food Chem., 1998. 46: 3662–3666. 25. Aubourg, S., Lipid damage detection during the frozen storage of an underutilized fish species. Food Res. Int., 1999. 32: 497–502. 26. Aubourg, S.P. and I. Medina, Influence of storage time and temperature on lipid deterioration during cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) frozen storage. J. Sci. Food Agric., 1999. 79: 1943–1948. 27. Hasegawa, K., Y. Endo, and K. Fujimoto, Oxidative deterioration in dried fi sh model systems assessed by solid sample fluorescence spectrophotometry. J. Food Sci., 1992. 57: 1123–1126. 28. Veberg, A., G. Vogt, and J.P. Wold, Fluorescence in aldehyde model systems related to lipid oxidation. LWT-Food Sci. Technol., 2006. 39: 562–570.

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29. Jebe, T.A., M.-G. Matlock, and R.T. Sleeter, Collaborative study of the oil stability index analysis. J. Am. Oil Chem. Soc., 1993. 70: 1055–1061. 30. Mendez, E. et al., Comparison of Rancimat evaluation modes to assess oxidative stability in fi sh oils. J. Am. Oil Chem. Soc., 1997. 74: 331–332. 31. Vinter, H. The Oxidograph. A development within accelerated measurement of stability, in Scandinavian Symposium of Lipids (Lipidforum) 16th, 1991. pp. 160–162. 32. Moh, M.H. et al., Determination of peroxide value in thermally oxidized crude palm oil by near infrared spectroscopy. J. Am. Oil Chem. Soc., 1999. 76: 19–23. 33. Li, H. et al., Determination of peroxide value by Fourier transform near-infrared spectroscopy. J. Am. Oil Chem. Soc., 2000. 77: 137–142. 34. Yamamoto, Y. et al., Study of oxidation by chemiluminescence. IV. Detection of low levels of lipid hydroperoxides by chemiluminescence. J. Am. Oil Chem. Soc., 1985. 62: 1248–1250. 35. Matthäus, B., C. Wiezorek, and K. Eichner, Fast chemiluminescence method for detection of oxidized lipids. Fat Sci. Technol., 1994. 96: 95–99. 36. Jonsdottir, R., M. Bragadottir, and G. Olafsdottir, The role of volatile compounds in odor development during hemoglobin-mediated oxidation of cod muscle membrane lipids. J. Aquat. Food Prod. Technol., 2007. 16: 67–86. 37. Kuksis, A., H. Kamido, and A. Ravandi, Glycerophospholipid core aldehydes: Mechanism of formation, methods of detection, natural occurrence, and biological significance, in Lipid Oxidation Pathways, A. Kamal-Eldin, Ed., AOCS Press: Champaign, IL, 2003, pp. 138–189.

Chapter 8

Volatile Aroma Compounds in Fish Guðrún Ólafsdóttir and Rósa Jónsdóttir Contents 8.1 8.2 8.3 8.4

Introduction ..................................................................................................................... 98 Development of Fish Aroma............................................................................................. 98 Fresh Fish Odors .............................................................................................................. 99 Identification of Quality Indicators ................................................................................ 100 8.4.1 Microbial Spoilage Odors ....................................................................................103 8.4.1.1 Sweet, Sour, and Malty Odors ...............................................................103 8.4.1.2 Dried Fish, Ammonia-Like, and Stale Odors .........................................105 8.4.1.3 Putrid, Onion, and Cabbage-Like Odors ...............................................105 8.4.1.4 Miscellaneous ........................................................................................105 8.4.2 Oxidatively Derived Odors ..................................................................................106 8.4.2.1 Cooked Odor—Boiled Potato and Rancid Odors .................................106 8.4.2.2 Washed Cod Muscle System ..................................................................108 8.4.3 Processing Odors ................................................................................................. 111 8.4.3.1 Smoked Fish Odors ............................................................................... 111 8.4.3.2 Ripening Odor—Salted and Dried Fish Odor.......................................112 8.5 Conclusions .....................................................................................................................113 References ................................................................................................................................113

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98

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8.1 Introduction Health and wellness are the main drivers in new product development. Fish being a valuable source of polyunsaturated fatty acids (PUFA) and other nutrients is a prominent candidate as the healthy choice for consumers. Research has aimed at strengthening the marine-based food industry in the development of fish products of acceptable quality to meet new trends in lifestyles. A prerequisite for increased consumption of fish products is their availability on the market as fresh and high-quality products of delicate flavor. Volatile compounds play an important role in the odor quality characteristics and consumer acceptance of fish. The understanding of odor development by chemical, biochemical, and microbiological processes in fish postharvest is of importance to be able to control the various extrinsic factors that influence the formation of volatile degradation products and consequently the quality of fish products. Research over the years has led to improved chilling and packaging technologies aimed at reducing microbial growth. As a result, extension in shelf life of fresh chilled fish has been achieved. However, oxidative processes causing odors and texture changes become noticeable during extended storage and limit the shelf life. Enhanced oxidation during cooking resulting in off odor development is of concern and an obstacle for application of fish in convenience food. Improved understanding of the role of oxidation of polyunsaturated fatty acids in the development of off odors in fish products has directed research efforts to search for effective means to control oxidative processes. Consequently, studies on application of natural antioxidants are of prime interest to underpin further utilization of fish in innovative product development as fresh, cooked, processed, or hydrolyzed products and as ingredients in functional foods.

8.2 Development of Fish Aroma An overview of changes during handling and processing influencing the development of aroma in fish is generalized in Figure 8.1. Initially, the changes are dominated by autolytic activity, including degradation of nucleotides, formation of taste, active inosine, and accumulation of hypoxanthine (Hx), lowering of pH and endogenous enzyme activity, followed by oxidation processes. Finally, the proliferation of the specific spoilage organisms (SSO) results in the development of volatile compounds, contributing to spoilage changes and thus influencing the freshness and quality of the end product of chilled fish [1–3]. It is well established that enzyme lipoxygenase (LOX)-mediated conversions of polyunsaturated fatty acids (PUFA) to volatile aroma compounds initiates the development of plant-like aroma of fresh fish [4–6]. Other prooxidants like hemeproteins (hemoglobin and myoglobin) are also involved in the initiation of the oxidative processes in fish muscle [7], leading to the formation of secondary oxidation products and off flavors [8]. Degradation of soluble muscle constituents such as sarcoplasmic proteins and microbial metabolism contributes to changes in the aroma profile of fish during storage. The pool of components that are degraded and cause off flavors because of microbial growth are mainly soluble substances in the muscle. They are composed of the various nonprotein nitrogenous components (NPN), including small peptides such as carnosine and anserine, amino acids, guanidine compounds like creatine, TMAO, and nucleotides. Some of these compounds influence the taste of fish-like peptides (i.e., anserine), and the individual amino acids glycine, valine, alanine, and glutamic acid are known to contribute to taste together with the degradation components of the nucleotides such as inosine. Proteolysis plays a critical role in postmortem changes, resulting in undesirable texture changes in fish. Endogenous enzyme

Volatile Aroma Compounds in Fish

Handling chilling, freezing, and cooking

99

Processing smoking, salting, drying, and hydrolysis

Endogenous enzymes

Microbial metabolism

i.e., LOX proteases, hydrolases, phospholipases TMAOase

Specific spoilage organisms (SSO)

Fresh fish aroma seaweedy, cucumber, metallic, neutral

◾

Spoilage aroma sweet, malty, sour, putrid, dried fish, ammonia-like

Oxidation Prooxidants: metals (Fe,Cu) Hb, Mb Antioxidants: α-tocopherol, ascorbic acid, polyphenols

Lipids phosholipids/PUFA Proteins sarcoplasmic, peptides Soluble substances, NPN, nucleotides, amino acids

Oxidized aroma Processed aroma green-like, boiled potato, popcorn, caramel, malty, stockfish, stale, rancid potato, mushroom, cucumber

Figure 8.1 Overview of changes in fish influencing the development of characteristic aroma of fresh, oxidized, spoiled, and processed fish. LOX, lipoxygenase; SSO, specific spoilage organisms; Hb, hemoglobin; Mb, myoglobin; PUFA, polyunsaturated fatty acids; NPN, nonprotein nitrogen-containing compounds; TMAO, trimethylamineoxide.

activity influences the deterioration of fish muscle, including calpains (neutral calcium-dependent proteases) and cathepsins (lysosomal proteases), but the mechanism of this activity is not fully elucidated [9].

8.3 Fresh Fish Odors The delicate flavor of fish is mostly contributed by volatile compounds and taste active substances in the aqueous phase, whereas volatiles generated from fat result in variation in the specific flavor character of different fish species. Newly caught marine fish contains low levels of volatile compounds and is nearly odorless. Soon after harvest, LOX activity on the skin and gills of both freshwater and marine species (rainbow trout, river trout, and sardines) plays a role in the formation of odorous volatiles, contributing to green, pleasant aromas of fish [6,10–13]. The compounds that contribute to the characteristic plant-, cucumber-, melon-, and mushroom-like odors are unsaturated carbonyl compounds and alcohols with six, eight, or nine carbon atoms [4,5,14,15]. Josephson et al. [5] summarized the occurrences of volatile compounds in freshwater and saltwater species and concluded that the four common compounds found in saltwater species, hexanal, 1-octen-3-ol, 1,5-octadien-3-ol, and 2,5-ocatadien-1-ol, were responsible for the moderate, faint odor of saltwater species. On the other hand, the unsaturated C9 carbonyl compounds such as 2,6-nonadienal, which have potent green, plant-, cucumber-, and melon-like odors, were characteristic for freshwater and euryhaline fish. The overall perceived odor is dependent on the level of influential compounds and their odor thresholds along with possible synergistic effects. Some components are desirable at low levels, but


if their concentration increases, they may contribute to off odors. An example is the enzymically derived long-chain alcohols and carbonyls that exhibit characteristic fresh, plant-like notes in fresh fish, but when accumulated in higher levels because of autooxidation, they contribute to oxidized and fishy odors in stale fish [16]. Another example is iodine-like off flavor in prawns associated with bromophenols originating from the feed chain [17]. However, in nominal levels the bromophenols appear to contribute to natural sea-, iodine-, and marine-like flavors of seafood [18]. Environmental conditions and seasonal effects like spawning can influence the odor quality of fish. The volatile pattern changes in mature salmon when migrating from the sea for spawning. C9 LOX-derived compounds have been found in higher levels in spawning euryhaline and freshwater fish [5]. Seasonal effects have also been reported for capelin, a saltwater species, which has a very characteristic cucumber odor during spawning. 2,6-Nonadienal was identified to be the most characteristic compound for the cucumber-like capelin odor [19]. Studies performed in Japan, on accumulation of hydroperoxides in fish tissues, indicate their involvement in the development of fresh fish aroma associated with seasonal variation. Accumulation of certain hydroperoxide isomers coincided with the period of enhancement of characteristic aroma in sweet smelt. They were suggested as the possible precursors of nine-carbon volatile compounds, including (E)-2-nonenal, (E,Z)-2,6-nonadienal, and 3,6-nonadien-1-ol in sweet smelt tissues [20].

8.4 Identification of Quality Indicators Different characteristic odors develop in various fish species during storage. Fatty species develop rancid odors and taste; lean species typically develop sweet, boiled potato-, and amine-like odors; and species of the salmonidae family develop earthy, muddy, and sweet odors. Volatile compounds formed by microbial metabolism and oxidation contributing to these odors have been identified by gas chromatography methods and suggested as indicators of quality. Some of the influential odor compounds that have very low odor thresholds are often present in low levels, and these are difficult to detect by analytical techniques. Therefore, it is useful to monitor the overall pattern of volatile compounds and select indicator compounds, which are present in higher levels and can be quantified. Rapid methods can then be applied to detect indicators or alternatively classes of compounds if the pattern of the volatile compounds is known and a connection has been verified between the indicator compounds and the compounds that are responsible for the odors and quality changes. This has been the approach in our studies, where it has been demonstrated by monitoring key volatiles to study changes in different fish products during storage, odor, and quality changes can be explained in, for example, cod during storage [21,22] and in smoked salmon [23]. Both single compounds such as TMA and ethanol and multicompound indices based on combination of alcohols, amines, and sulfur compounds representing the different changes occurring during storage have been suggested by numerous researchers as indicators for freshness and spoilage [22,24–29]. Volatile degradation compounds as quality indicators can be detected by rapid techniques such as electronic nose to monitor and predict quality changes in various fish species and in smoked salmon [19,21–22,30–36]. Table 8.1 summarizes the occurrence of volatile compounds detected in our studies on cod [22] and haddock fillets [31] and smoked salmon [23]. Purge and trap on Tenax and SPME methods were applied for sampling, and identification was based on GC–FID, GC–MS, and GC–O. The main classes of compounds detected during storage are alcohols, aldehydes, ketones, amines, acids, esters, and sulfur compounds. The aldehydes contribute most to the spoilage odors because of their low flavor thresholds, as seen by the detected odors listed in Table 8.1.


◾

101

Table 8.1 Volatile Compounds Detected in Cod [22], Haddock Fillets [31], and Smoked Salmon [23] during Chilled Storagea Raw Cod

Boiled Cod

Raw Haddock

Smoked Salmon

Odor Description (GC–O)

Ethanol

×

×

×

×

—

2-Methyl-1-propanol/pentane

×

×

×

1-Penten-3-ol

×

×

×

×

—

3-Methyl-1-butanol

×

×

×

×

—

2-Methyl-1-butanol

×

×

—

2,3-Butandiol

×

×

—

Compound

Alcohols

1-Octen-3-ol 2-Ethyl-1-hexanol

× ×

1-Octanol

—

×

Mushroom

×

—

×

—

Aldehydes Acetaldehyde

×

×

×

—

2-Methyl-propanal

×

—

2-Methyl-butanal

×

Sweet, caramel, fish fillet

3-Methyl-butanal

×

×

×

×

Hexanal

×

×

×

×

cis-4-Heptenal Heptanal

×

2,4-Heptadienal, (E,E)-

×

×

×

Sweet, caramel, flowery —

×

Rancid

×

Boiled potato, earthy

×

Sweet, fatty

Nonanal

×

×

×

×

—

Decanal

×

×

×

×

Fresh, floral

Undecanal

×

×

×

Sweet, candy

×

×

×

Ketones 2-Butanone 2,3-Butandione

×

— N/A (continued)

102 ◾


Table 8.1 (continued) Volatile Compounds Detected in Cod [22], Haddock Fillets [31], and Smoked Salmon [23] during Chilled Storagea Compound

Raw Cod

Boiled Cod

Raw Haddock

2-Pentanone 3-Pentanone

×

×

Smoked Salmon


×

—

×

Sweet, caramel

2,3-Pentanedione

×

—

3-Hexanone

×

—

3-Methyl-2-butanone

×

3-Hydroxy-2-butanone

×

6-Methyl-5-hepten-2-one

×

×

— ×

×

Sweet, sour Flowery, sweet, heavy, spicy

Amine Trimethylamine

×

×

×

×

×

×

×

TMA-like, dried fish

Acid Acetic acid

×

—

×

—

Esters Ethyl acetate Ethanthiocacid, S-methylester

× ×

Propanoic acid, ethyl ester

×

N/A —

Propanoicacid-2-methyl, ethylester

×

N/A

Acetic acid, 2-methylpropyl ester

×

N/A

Butanoic acid, ethyl ester

×

×

×

Sickenly sweet, vomit

2-Butenoic acid, ethyl ester

×

N/A

Butanoic acid, 2-methyl, ethylester

×

N/A

Butanoic acid, 3-methyl, ethylester

×

N/A

Hexanoic acid, ethyl ester

×

N/A

Sulfur Compounds Methanethiol Dimethyl sulfide

× ×

×

— ×

—


◾

103

Table 8.1 (continued) Volatile Compounds Detected in Cod [22], Haddock Fillets [31], and Smoked Salmon [23] during Chilled Storagea Raw Cod

Boiled Cod

Raw Haddock

Dimethyl disulfide

×

×

×

Onion like

Dimethyl trisulfide

×

×

×

Rotten, sulfur, cabbage

Compound

Smoked Salmon


a

Volatiles in boiled cod were analyzed in samples of raw chilled cod fillets [22] by heating corresponding samples at 80°C for 60 min. —, not detected by GC–O; N/A, data not available for haddock.

Seaweedy and marine-like odors, as well as green plant-, cucumber-, mushroom-, or geraniumlike odors are characteristic sensory odor descriptors for fresh whole fish. In general when fish is cooked, the aroma of the fillet is described as sweet and reminiscent of shellfish, meat-like, and sometimes metallic. After several days of storage, the freshness notes disappear and the odor of the uncooked fish becomes neutral. Sweet-milky and vanilla/caramel-like odors are typical in cooked fish. During prolonged storage boiled potato odor develops, and when combined with frozen storage odor, dried fish/stockfish, and TMA-like smell, and finally sour and dirty tablecloth odor, the fish is no longer fit for consumption. The odor descriptors in Table 8.1 based on GC–O analysis of cod and smoked salmon represent most of these overall changes.

8.4.1 Microbial Spoilage Odors The spoilage odors in chilled fish vary depending on the dominant microflora in the products, which is mostly affected by handling, cooling, packaging, and temperature conditions during storage [33,34]. An example of the spoilage pattern of volatile compounds in chilled fish is illustrated in Figure 8.2, showing results from a storage study of cod fillets packed in styrofoam boxes during chilled storage (0.5°C) [22]. The aim was to screen for potential quality indicators and determine which compounds and classes of compounds were most abundant in the headspace and also to identify the most influential spoilage odors contributing to sensory rejection. Identification of volatile compounds was based on GC–MS analysis (see Table 8.1), and quantification of the main classes of compounds was based on the sum of the PAR for respective compounds in each class. The loss of freshness of cod fillets and early spoilage changes were related to the formation of ketones, alcohols, and aldehydes, contributing to sweet, sour, and malty odors. Late spoilage changes, development of spoilage odors, and the end of shelf life of cod fillets on day 12 of storage are explained by the presence of TMA, esters, acids, and sulfur compounds produced by microbial degradation of fish components, mainly amino acids.

8.4.1.1

Sweet, Sour, and Malty Odors

Ketones, alcohols, and aldehydes detected on day 4 of storage and their increasing levels on days 7 and 10 (Figure 8.2) were associated with the development of sweet, sour, caramel-like, and malty spoilage odors. The microbially derived alcohols 2-methyl-1-propanol, 3-methyl-1-butanol, and 2,3-butandiol were found in the highest levels on day 12 at sensory rejection. The flavor thresholds

104 ◾

Handbook of Seafood and Seafood Products Analysis 120

Peak area ratio (PAR)

100 80

Alcohols Aldehydes

60

Ketones TMA

40

Aceticacid Esters

20 0 0

2

4 6 8 10 Days of storage

12

14

Figure 8.2 GC–MS analysis of volatile compounds showing changes in the levels (PAR, peak area ratio) of the main classes of compounds contributing to spoilage in cod fillets packed in styrofoam boxes during storage at 0.5°C until sensory rejection on day 12. (Modified from Ólafsdóttir, G., Volatile compounds as quality indicators in fish during chilled storage: Evaluation of microbial metabolites by an electronic nose, PhD thesis, University of Iceland, Reykjavík, 2005.)

of alcohols are higher than those of carbonyls, and they did not contribute to the odor of the fillets as evaluated by GC–O (Table 8.1) [22]. Ethanol was detected in high levels initially (on days 4 and 7) and then declined. The initial high levels of ethanol in spoilage of fish has been related to the utilization of carbohydrate sources, whereas the formation of branched-chain alcohols and aldehydes such as 2-methyl-1-propanol, 3-methyl-1-butanol, and 3-methyl-butanal probably originate from degradation of valine and leucine, respectively. The branched chain aldehyde, 3-methyl-butanal, was characterized by sweet, caramel, and fish-fillet-like odors by GC–O in our study. Lindsay [8] suggested using short-chain alcohols such as ethanol, butanol, and 3-methyl-1-butanol as potential indices of refrigerated fish spoilage based on studies of freshwater whitefish. Propanol was suggested as a potential indicator when using modified atmosphere packaging techniques. In chilled haddock fillets stored in styrofoam boxes, TMA, 2-methyl-1-propanol, 3-methyl-1-butanol, 3-hydroxy-butanone, ethyl acetate, and butanoic acid ethyl ester were found in the highest amounts and increased with storage. Dimethyl disulfide and dimethyl trisulfide were detected at the end of storage time when samples were spoiled, whereas dimethyl sulfide was detected initially and throughout storage [31]. In cultured and wild sea bream stored in ice for 23 days, TMA, 3-methyl-1-butanol, 1-penten-3-ol, piperidine, methanethiol, dimethyl disulfide, dimethyl trisulfide, and acetic acid were identified as spoilage indicators [29]. The formation of acetoin (3-hydroxy-2-butanone) was characteristic for the spoilage of chilled cod fillets packed in styrofoam boxes and was attributed to the growth of Photobacterium phosphoreum [22]. Levels of acetoin increased earlier than those of TMA, and, therefore, it is more useful to monitor the loss of freshness as an early indicator of spoilage. The concentration of acetoin was much higher than the lipid derived ketones detected, such as 2-butanone, 3-pentanone, and the carotenoid-derived 6-methyl-5-heptene-2-one, that were present in cod fillets throughout


◾

105

storage, but no obvious increase occurred until at the end of shelf-life and during continued storage. Ketones can influence the overall odor because of their low odor thresholds. The lipid-derived saturated aldehydes detected on day 12 at sensory rejection also contributed to the overall sweet aroma. The odor of ethyl butanoate, described as sickeningly sweet and nauseous, contributed to the sensory rejection of chilled cod fillets on day 12 and suggested the role of Pseudomonas fragi in the development of sweet, fruity off odors [37,38]. Pseudomonas species have also been found responsible for the formation of volatile sulfides, alcohols (3-methyl-1-butanol, 1-penten-3-ol), and ketones (2-butanone), contributing to the stale and putrid off odors in fish because of amino acid and lipid degradation [39].

8.4.1.2 Dried Fish, Ammonia-Like, and Stale Odors The development of dried fish, ammonia-like, and stale odors by amines during fish spoilage is well known, and measurements of volatile amines such as TMA or total volatile bases (TVB-N) have been used in the fish industry as indicators of quality for fish and fish products. Enzymically produced DMA (dimethylamine), which forms very early after harvest of fish, has been suggested as a freshness indicator along with its precursor TMAO (trimethylamine oxide) [27]. TMA is a potent odorant with a characteristic fishy, dried fish, ammonia-like odor. Figure 8.2 shows that TMA was detected in high levels on day 12. At this point there was an increase in the pH value, which may have influenced the overall odor perception leading to the sensory rejection of the fillets. Additionally, TMA has been noted for intensifying fishiness by a synergistic action with certain volatile unsaturated aldehydes derived from autoxidation of polyunsaturated fatty acids [40]. TMA is characteristic for the spoilage odors of fish, whereas DMA may influence the overall fresh flavor of fish in combination with oxidatively formed aldehydes from long-chain fatty acids in fish. The onset of stale odors can be explained by cis-4-heptenal and heptanal, which contributed to boiled potato-like odors (Table 8.1).

8.4.1.3

Putrid, Onion, and Cabbage-Like Odors

Low levels of sulfur compounds (Figure 8.2) indicated that they were not important in the spoilage of chilled cod fillets stored in styrofoam boxes. In whole fish stored in ice, volatile sulfur compounds such as hydrogen sulfide, methyl mercaptan, methyl sulfide, and dimethyl disulfide have been suggested as the main cause of putrid spoilage aromas [41]. Dimethyl trisulfide has also been associated with spoilage in fish and associated with the growth of Shewanella putrecfaciens [25,38,39]. Milo and Grosch [42] evaluated the headspace of boiled cod by gas chromatography olfactometry (GC–O) and found that dimethyl trisulfide was the most potent odorant contributing to off odors in cod formed when the raw material was inappropriately stored. The origin of the sulfur compounds is microbial degradation of cysteine and methionine to form hydrogen sulfide and methyl mercaptan, respectively [41]. Oxidative processes are involved in the formation of dimethyl sulfide from methyl mercaptan and further oxidation of dimethyl disulfide, and the incorporation of hydrogen sulfide yields dimethyl trisulfide [38].

8.4.1.4 Miscellaneous The concentration of the straight chain alkanes (nonane, decane, and undecane) appeared to be similar throughout storage in chilled cod fillets [22]. Additionally, numerous branched chain

106

◾


alkanes were detected, and overall the alkanes showed an increasing trend with storage time. However, they are not considered of interest as quality indicators, since they are not aroma active. A characteristic earthy odor in many species residing in ponds has been associated with piperidine and its reaction products, but the knowledge of the formation of these compounds is obscure. Piperidine levels have been reported to increase in spawning salmon and contribute to off odors [43]. Piperidine was tentatively identified in chilled cod fillets [22] and has also been suggested as a quality indicator in sea bream [29]. Several odor active terpene derivatives have been identified in fish. Limonene has low odor threshold and a fresh lemon odor was detected by GC–O analysis of cod, suggesting that it may have an impact on the overall odor of fish fillets [22]. Limonene has also been detected in sea bream during storage [29]. The origin of limonene in fish is most likely related to the diet derived from algae or plant source. Similarly, the feed may have influenced higher levels of aldehydes, ketones, aromatics, and terpenes found in wild sea bream compared with those of its cultured counterpart [29].

8.4.2 Oxidatively Derived Odors Initiation of lipid oxidation in fish is generally associated with the polyunsaturated fatty acids in phospholipids of muscle cell membranes [44], which are known to be more susceptible to oxidation than triacylglycerols in fat deposits [45]. Various pro and antioxidants influence the stability of the muscle and have been studied in relation to the oxidative stability of phospholipids [46]. Phospholipids are the main membrane-bound lipids, and because of their high unsaturation, they are in particular sensitive to oxidation, which is further enhanced by preprocessing and storage of fish. Oxidative processes occurring during storage of fish result in the accumulation of aldehydes, such as hexanal, cis-4-heptenal, 2,4-heptadienal, and 2,4,7-decadienal, that contribute to the development of rancid cold store flavors [47]. Our studies on the development of volatile compounds in chilled cod fillets packed in styrofoam boxes during storage at 0°C showed that oxidatively formed, lipid-derived saturated aldehydes, such as hexanal, heptanal, and decanal, were detected in the fillets throughout the storage time, in similar or slightly increasing levels. These oxidation products contributed to the overall characteristic sweet, fish-like odors of chilled cod fillets in combination with other carbonyls (3-hydroxy-2-butanone, 3-methyl-butanal, 2-butanone, 3-pentanone, and 6-methyl-5-heptene-2one). Aldehydes generally have low odor thresholds, and, therefore, their impact was greater than alcohols and ketones, although their overall levels were lower. 6-Methyl-5-heptene-2-one derived from carotenoids was described as spicy and flowery by GC–O and suggested to contribute along with other ketones and aldehydes to the characteristic sweet odor of cod fillets [22]. The influence of other aroma active compounds present in lower levels such as the unsaturated autoxidatively derived aldehydes (2,4-heptadienal and 2,4,7-decatrienal) should not be overlooked. These compounds have been associated with rancid and dried fish odors, but the sampling techniques used were not sensitive enough to allow quantification of these compounds.

8.4.2.1

Cooked Odor—Boiled Potato and Rancid Odors

Characteristic odors and key volatile compounds in boiled cod stored in closed plastic bags for 22 days compared with fresh boiled cod are shown in Figure 8.3 to demonstrate which odors are most dominating in the aroma profile [48]. Boiled potato- and potato-like odors contributed by

Volatile Aroma Compounds in Fish DMS Sulfur 5

Cucumber, sweet, melon Cucumber

2-Nonenal

Fishy

3-Pentanone 1-Penten-3-ol Flowery 2-Penten-1-ol

3

Flowery Fatty, green-like odors Grass Hexanal

2 1

2,4-Heptadienal Rancid

Heavy

Geranium-like 1-Octen-3-ol

107

Fishy odors

4

Fatty Fatty, green-like, rancid odors Flowery

◾

Mushroom, earthy

Mushroom

Potato-like

Earthy, pop-like

Boiled potato

cis-4-Heptenal Heptanal

Earthy-like odors

Figure 8.3 Odor profile (GC–O analysis) of boiled cod stored in plastic bags (-♦-) after 22 days of refrigerated storage (3°C) compared with freshly boiled cod (---▲---). (From Jónsdóttir, R. and Ólafsdóttir, G., Unpublished data, 2004.)

heptanal and cis-4-heptenal were the most potent odors. Overall earthy, sweet, sour, and fish oil notes were characteristic for fresh cooked salmon, and the most pronounced attribute was a boiled potato odor [49]. The fresh raw salmon odor was characterized as cucumber-like with weak sweet, sourish, and fish oil notes in the same study. The occurrence of cis-4-heptenal has been associated with the “cold storage flavor” of cod [47]; however, some confusion exists about the role of cis-4heptenal as the “cold-storage compound” [8]. In fact, this aldehyde does not exhibit a fishy-type aroma by itself, but it rather participates in the expression of the overall fishy odor. Its odor has been described both as cardboardy, paint-like [50], as well as boiled potato-like [51,52]. Other pronounced odors detected in boiled cod (Figure 8.3) were fatty, green-like, and rancid odors contributed by 2-nonenal and 2,4-heptadienal. Fatty, sweet, flowery, and green-like odors were associated with oxidatively derived 3-pentanone, 1-penten-3-ol and hexanal. Baltic herring has been reported to have a similar development of volatiles, although the level of the compounds may vary and explain the differences in the characteristic odor of these species. In fresh baked herring (200°C; 20 min) 3-methylbutanal, 2-methylbutanal, and hexanal were abundant in headspace, and after storage for 3 days the proportions of 4-heptenal, 2-heptanone, and octatriene increased significantly. Hexanal, heptanal, 1-penten-3-ol, and octadienes also increased many-fold during further storage, and after 8 days of storage at 6°C, microbial metabolites such as 3-methyl1-butanol and cresol were identified [53]. Ideally, quality indicators should demonstrate clear increasing or decreasing levels with storage time. However, this is not always the trend for dynamic microbial and oxidative changes and the formation of volatiles in fish during storage [22]. Taking into account the complexity of the spoilage processes, multivariate data analysis is useful to explore the overall trend of the main quality indicators. Principal component analysis (PCA) was performed (Figure 8.4) on data from our studies on volatiles in cod [22] during prolonged storage for 17 days and compared with corresponding

108

◾


1.0

PC2

B-D17

Bi-plot

1-Penten-3-ol

3-me-butanal Acetic acid 0.5

Undecanal Ethanol 3-me-1-butanol Ethylbutanoate B-D4 Heptanal Nonanal Acetaldehyde Ethylacetate 2-Butanone

B-D10 0

R-D4 R-D12 R-D7 R-D10

3-HO-2-Butanone 2-me-1-propanol TMA Decanal R-D14 6-me-5-h-2-one Hexanal

–0.5 –0.4 –0.2 Raw and boild c…, X-expl: 53%, 19%

0

0.2

0.4

0.6

R-D17

0.8

PC1 1.0

Figure 8.4 Principal component analysis of raw and boiled cod. Samples are labeled with R, raw and B, boiled and storage days, D (4, 7, 10, 12, 14, and 17 days).

samples after heating (see Table 8.1). The PCA demonstrates how volatile compounds can explain the variation in quality of samples according to storage time and handling (raw and boiled), in particular the role of volatile compounds derived from oxidation in heated/boiled samples. The characteristic pattern or trend in volatiles in raw and boiled fish is clearly different, as indicated by the arrows (Figure 8.4). It is in particular interesting to demonstrate that the influence of heating gives a very different volatile profile compared with that of the raw samples that are all clustered on the left of the PCA plot. Only the spoiled raw samples (R-D14 and R-D17) can be correlated with the freshly boiled (B-D4) sample. The effect of oxidation induced by cooking and formation of oxidation products such as heptenal and nonanal characterizes the (B-D4) sample. Other oxidatively formed compounds like 2-butanone and aldehydes were in higher levels in the B-D4 sample compared with the corresponding raw sample (R-D4). Sulfur compounds dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide were detected in higher levels in the boiled samples (data not shown). The oxidatively formed compounds, that is, hexanal, decanal, and 6-methyl-5-hepten-2-one, increased with time and were pronounced in the spoiled raw samples (R-D14 and R-D17). Autoxidatively produced unsaturated carbonyl compounds were the most abundant components in boiled and canned fish, especially in trout [15]. In boiled trout, methional with a characteristic boiled potato-like odor dominated the odor of the aldehyde fraction of the headspace volatiles. The malty flavor of 3-methyl butanal was suggested earlier to be mainly responsible for the malty off flavor defect of boiled cod [54]. Interestingly, 3-methyl-butanal was correlated to the boiled stored cod (B-D17) (Figure 8.4), in agreement with earlier studies [54]. On the basis of odor evaluation, 3-methyl-butanal in combination with acetaldehyde, methional, and oxidatively derived (Z)-1,5-octadien-3-one, (E,Z)-2,6-nonadienal, and (E,E)-2,4-decadienal from PUFA were determined as character impact odorants of boiled cod [54].

8.4.2.2

Washed Cod Muscle System

Rancid odor development during chilled storage of fish has commonly been associated with fatty species. However, oxidation of membrane-bound phospholipids in lean species can cause fishy,


◾

109

rancid, dried fish-like off odors as discussed before. Studies on the development of the odorous degradation compounds of phospholipid oxidation can lead to a better understanding of the kinetics and reaction pathways of oxidation in lean fish. Consequently, this may facilitate the selection of preventive measures to limit oxidation and guide new technological developments with the aim to ensure the delicate taste and nutritional value of lean fish products. To accurately evaluate the potential of antioxidants in foods, it is necessary to apply models that take into account the chemical, physical, and environmental conditions expected in food products. This is because the activity of antioxidants in food systems depends not only on the chemical reactivity of the antioxidant (e.g., free radical scavenging and chelation) but also on factors such as physical location, interaction with other food components, and environmental conditions (e.g., pH) [55,56]. The role of antioxidants (a-tocopherol, ascorbic acid, and glutathione peroxidase) and aqueous prooxidants in fish muscle, including blood components like inorganic metals iron (Fe) and copper (Cu), has been studied to understand better the mechanisms of oxidation in the muscle [57,58]. In lean fish such as cod, lipid oxidation of muscle phospholipids may be induced by several catalysts, including hemoglobin from blood [7,59]. Sohn et al. [60] studied lipid oxidation and rancid odor during the early stage of ice storage of ordinary and dark muscle of yellowtail and concluded that myoglobin was the main cause in the development of the unpleasant color and undesirable odor during ice storage of fish muscle. Washed cod muscle system has been widely used to study oxidation and the influence of prooxidative and antioxidative factors [59,61]. Odor development in lean fish studied by hemoglobininduced oxidation in washed cod muscle system showed that sweet, green, earthy, cucumber-like, floral, and rancid odors dominated the aroma profile [62]. The added hemoglobin was very effective as a prooxidant, and the overall odor was an intense dried fish, painty, rancid fish oil like. To monitor the development of rancidity, the concentration and composition of volatile oxidation products analyzed by GC were compared with TBARS measurements, sensory assessments, and color. The most potent odors detected in the model system were malty, sweet, and caramel-like odors contributed by 3-methylbutanal, 2,3-pentandione, and 1-penten 3-ol; grass odor contributed by hexanal; rancid, potato-like odor caused by cis-4-heptenal and heptanal; mushroom odor caused by 1-octen-3-ol; and spicy and flowery notes exhibited by 6-methyl-5-hepten-2-one. Furthermore, rancid, fatty, soapy, and lemon-like odors were explained by 2,4-heptadienal [62]. These odors were also detected in cod fillets during chilled storage (Table 8.1), but the compounds were detected in much lower levels [22]. Preconcentration techniques are necessary for the analysis of unsaturated aldehydes, which is not practical for rapid determination of oxidation. On the other hand, it is possible to detect the most volatile oxidation products like propanal and hexanal by rapid, static headspace sampling methods. These compounds can be used as indicator compounds for oxidation, as demonstrated by Boyd et al. [63]. They showed that direct analysis of propanal can provide a quick and economical method for the determination of oxidation of n-3 fatty acids and pentane and hexanal analysis can give an indication of the oxidation of linoleic acid. Similarly, we found in our studies on the washed cod muscle system that hexanal could be used as indicator for rancid odor development. The prooxidative effect of hemoglobin was evident by the formation of hexanal in high levels, and a similar trend was observed in the development of cis-4-heptenal (Figure 8.5) as well as 2,4heptadienal that contributed to rancid odor caused by oxidation, in agreement with TBARS and changes in color [62]. The effect of thermal treatment on hemoglobin-mediated oxidation in the phospholipid model system from cod muscle was studied by monitoring oxidative changes during chilled storage on ice by sensory analysis, TBARS (thiobarbituric reactive substances), and instrumental color changes.

110 ◾


1000

Hexanal

25

800

20

600

ng/g

ng/g

cis-4-Heptenal

30

400

15 10

200

5 0

0 0

1 Blank-II

2 Hb-Char-II

3

0

4

1

3

Hb-Char-II

Blank-II

Hb-Cod-II

2

4

Hb-Cod-II

Figure 8.5 Gas chromatography analysis (FID) of characteristic volatile compounds contributing to rancid odor (hexanal and cis-4-heptenal) in hemoglobin (from Arctic char and cod) mediated oxidation in washed cod model stored at 0°C for 4 days (-♦-, Blank-II; -▲-, Hb-Char-II; -■-, HbCod-II). ( Adapted from Jónsdóttir, R. and Ólafsdóttir, G., J. Aquat. Food Prod., 16, 67, 2007.)

100 90 80 70 60 50 40 30 20 10 0

40 35

TBARS (μmol/kg)

Odor score (rancidity)

Thermal treatment of the cod model system significantly enhanced the oxidation of the model on day 1, as measured by rapid increase in rancid odor, described as rancid, painty, and dried fish odors, and in TBARS (Figure 8.6) as well as more rapid loss of red color (not shown) already on the first day of storage. The studies on the washed cod muscle system verify the importance of oxidation in off odor development in fish muscle and consequently the benefit of being able to control oxidation to prevent the formation of the aldehydes. Active research is ongoing on the application of various natural antioxidants based on polyphenols like flavonoids (i.e., catechins from tea) and cinnamic acid derivatives (i.e., caffeic acid) [65] as well as application of tocopherol, citric acid, and EDTA [66]. Studies on LOX inhibitors are of interest in preventing the initiation of oxidation in fish. Some promising results have been reported, where commercially available green tea polyphenols were shown to effectively inhibit the LOX activity of mackerel muscle [67].

30 25 20 15 10 5 0

0

1 Blank

2 Raw

3 Cooked

4

0

1

2

Blank

Raw

3 Cooked

Figure 8.6 Sensory analysis of rancid odor (odor score) and TBARS measurements in raw and cooked washed cod model stored at 0°C for 4 days, with added hemoglobin (raw and cooked, respectively) and raw without hemoglobin (blank). (From Jónsdóttir, R. et al., Matis Report 08, 73, 2008. With permission.)


◾ 111

8.4.3 Processing Odors Flavor development in processed seafood is a result of complex proteolytic and lipolytic reactions induced by different processing parameters like enzymes and temperature. Maillard reaction, including Strecker degradation, thermal degradation, and lipid oxidation, plays important roles in the formation of complicated processing flavors. Thermally generated aroma-active compounds via the Maillard reaction such as pyrazines are characteristic for enzymatically hydrolyzed seafood products like crayfish processing by-products [68]. Volatile compounds like alkyl-pyrazines and sulfur-containing compounds have been found in cooked crustaceans, and furans have been found in spray-dried shrimp powder and shrimp hydrolysate [69]. Key volatile compounds identified in enzymatically produced seafood flavorants are formed via Maillard reaction and Strecker degradation of amino acids. These are compounds like methional, the Strecker aldehyde produced from methionine, which has a characteristic potato-like odor, and 2-acetyl-1-pyrroline, giving a popcorn-like odor that can be thermally generated. Lipid-derived components, like cis-4-heptenal, 1-octen-3-ol, 2,6-nonadienal, hexanal, 2,4-heptanal, and 2,4-decadienal, also contribute to the aroma of seafood flavorants [70]. Lipid-derived aldehydes play an important role in flavor formation and have been reported to contribute to the characteristic fish-like, sweet odors of processed seafood like those in smoked salmon [23,71,72].

8.4.3.1

Smoked Fish Odors

Degradation compounds from Maillard reactions and lipid oxidation are the main compounds contributing to the aroma of smoked salmon [72]. The typical smoked salmon aroma results from a number of chemicals found in the smoke, but it is mostly attributed to the phenols. Figure 8.7 illustrates the main odors that were present in smoked fish samples after 14 days of chilled storage, which is typical for products on the market [23]. Phenolic derivatives like guaiacol (2-methoxyphenol) and syringol (2,6-dimethoxyphenol) have been identified as the most characteristic smoke-related compounds in smoked fish-like herring (Clupea harengus) [73] and in smoked salmon (Salmo salar) [23,72]. Guillén et al. [74] analyzed headspace components of cod and swordfish, where groups of phenol pyrolysis were most noticeable in the smoke flavor volatiles. In addition to phenolic compounds, furan-like compounds have been reported to be responsible for the smoked odor in smoked salmon, whereas carbonyl compounds, such as heptanal and (E,Z)-2,6-nonadienal, were characteristic in unsmoked fish, giving the flesh its typical fishy odor [71,72]. The oxidatively derived compounds cis-4-heptenal and heptanal, giving rancid, potato-like odors, and 1-octen-3-ol, contributing to mushroom-like odor, gave the most intense odors of smoked salmon and contributed to the fish-like earthy odors and fatty and rancid odors (Figure 8.7) [23]. Other oxidatively derived compounds like 1-penten-3-ol, hexanal, nonanal, and decanal were among key volatiles, and although they contributed less to the odors, it is clear that their presence contributes to the characteristic fish odor of smoked salmon products. Microbially produced ketones, aldehydes, and alcohols were abundant in the headspace of cold smoked salmon products during storage, associated with spoilage off flavors, like 3-methyl butanal, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-penten-3-ol, and 1-propanol [28,75]. Some of these compounds were selected as key spoilage indicators for smoked salmon based on their high levels and contribution to sweet and fruity spoilage off odors in our study on smoked salmon (Figure 8.7) (e.g., ethanol, 2-butanone, 2-pentanone, 3-methyl-butanal, 3-hydroxy-2-butanone, and 3-methyl-1-butanol) [23]. Additionally, it was verified that selected key volatile compounds performed better as predictors to explain variation in sensory attributes (smoked, sweet/sour rancid, and off odor and flavor) than traditional chemical and microbial variables.

112 ◾


Sweet and fruity-like odors

Smoked salmon odors Characteristic smoke odor Smoke-like

Sweet, fruity Flowery, sweet

2- and 3-Methyl phenol Guaiacol 4-Methyl-guaiacol

Wood, burnt, smoke

3-Methyl butanal Sweet, caramel

Smoke-house, sweet

Wood, smoke, sweet

Flowery, sweet

Burnt, smoke

Flowery, earthy, mushroom 2, 4-Heptadienal Sweet, fatty

Mushroom, geranium

Boiled potato-like Fatty, and rancid-like odors

Rancid cis-4-Heptenal Heptanal Earthy-like odors

1-Octen-3-ol

Figure 8.7 GC–O evaluation of volatile compounds detected in cold smoked salmon after 14 days of storage at 5°C. (Modified from Jónsdóttir, R. et al., Food Chem., 109, 184, 2008.)

8.4.3.2

Ripening Odor—Salted and Dried Fish Odor

Numerous volatile compounds have been detected in ripened products like dry cured ham, most of them generated from chemical or enzymatic oxidation of unsaturated fatty acids and further interactions with proteins, peptides, and free amino acids. [76–78]. Similar processes have been reported in ripened seafood products, where methional derived from methionine and 2,6-nonadienal from fatty acid oxidation were the main odorants in sugar salted, ripened roe products [79] Similarly, Triqui and Reineccius [80] found that 2,4-heptadienal and 3,5-octadien-2-one were associated with the development of the typical flavor obtained after anchovy ripening. Thus, they suggested that lipid autoxidation during ripening was primarily responsible for aroma development. However, manufacturers of ripened products have observed that some degree of proteolysis is necessary before flavor can develop. Methional and (Z)-1,5octadien-3-one were also identified as potent odorants in ripened anchovy [81], and aldehydes such as acetaldehyde, 2-methylpropanal and 3-methylbutanal were the key, highly volatile components of ripened anchovy, probably originating from amino acids. Salted cod are traditional products from the North-Atlantic fisheries and are highly regarded as ripened fish products in many countries, especially those in the Mediterranean. During ripening of salted cod, the desired flavor and texture develop as a consequence of protein and fat degradation. In our study, where the ripening of salted cod (Gadus morhua) produced by different salting methods was studied, the highest odor scores were given for boiled potato and rancid, potato-like odors together with cucumber-like odor [82]. The rancid, potato-like odor was identified as cis4-heptenal and the boiled potato-like odor, as heptanal, both oxidatively derived compounds.


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Methional, derived from methionine and eluting at a similar time as cis-4-heptenal and heptanal, could also be responsible for the boiled potato-like odor. The cucumber-like odor detected is possibly 2,6-nonadienal, according to retention index (RI) of standard and odor evaluation, although the compound could not be identified by GC–MS. Other key volatile compounds in salted cod are derived form lipid oxidation, for example, 1-penten-3-ol, hexanal, and 2-butanone. A certain degree of lipid oxidation is both necessary and desirable for sufficient ripening of the products but the process should be controlled to obtain a desirable degree of ripening based on consumer preferences [82,83].

8.5 Conclusions Although aldehydes, such as heptanal and (E,Z)-2,6-nonadienal, cause off odors in fish during storage, their presence at nominal levels gives the characteristic and desirable fishy odor in fresh and processed fish. Lipid oxidation during ripening appears to be primarily responsible for desirable aroma development in processed fish. The oxidatively derived compounds cis-4-heptenal and heptanal, exhibiting rancid, potato-like odors, and 1-octen-3-ol, contributing to mushroom-like odor, were the most intense character impact compounds of salted cod and smoked salmon. Studies on hemoglobin-induced oxidation in the washed cod model system and enhanced oxidation after heating verified the role of the oxidatively derived compounds contributing to off odors in chilled stored and boiled cod. Proper handling and application of natural antioxidants to control oxidative processes caused by lipoxygenase, hemoglobin, and myoglobin, and other prooxidants in combination with mild heating treatment are important factors to maintain the delicate flavor and odor of fish products. In addition, microbial growth can be limited by effective cooling techniques, temperature control, proper handling, and new packaging technologies. Therefore, careful control of handling and processing conditions should open up possibilities for fish to become a favored choice in new product development of convenience food and in functional food because of its health beneficial properties. However, careful evaluation of the quality of product is needed to ensure acceptable flavor. Volatile compounds as indicators of freshness quality and spoilage can be monitored to determine the quality of fish products. Development of smart sensor technologies like the electronic nose to detect microbial metabolites and oxidation products is of interest to verify the quality of products to facilitate process management, to increase trust between buyers and sellers in trade, and in retail for consumers as smart sensors imprinted on packaging. Detection of microbial metabolites originating mainly from soluble aqueous fractions of the muscle can be directly related to the quality of products. Knowledge of the spoilage pattern of volatile compounds is the basis for the development of rapid techniques like smart sensor technologies. A similar set of sensors with selectivity and sensitivity toward the main quality-indicating classes of compounds, such as ketones, amines, alcohols, aldehydes, acids, esters, and sulfur compounds, can be used for a variety of fish species that are stored and processed by different techniques.

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41. Herbert, R.A., Ellis, J.R., and Shewan, J.M. Isolation and identification of the volatile sulphides produced during chill-storage of North sea cod (Gadus morhua). J. Sci. Food Agric., 26, 1195, 1975. 42. Milo, C. and Grosch, W. Detection of defects in boiled cod and trout by gas chromatographyolfactometry of headspace samples. J. Agric. Food Chem., 43, 459, 1995. 43. Yamanaka, H. Offensive odour of fish and shellfish, in Odour of Marine Products, Koizumi, C., Ed., Koseisha-Koseikaku, Tokyo, Japan, 1989, p. 53. 44. Decker, E.A. and Xu, Z. Minimizing rancidity in muscle foods. Trends Food Sci. Technol., 9, 241, 1998. 45. Frankel, E. M. Lipid Oxidation. The Oily Press, Dundee, Scotland, 1998, 303. 46. Hultin, H.O. Oxidation of lipids in seafoods, in Seafoods: Chemistry, Processing Technology and Quality, Shahidi, F., and Botta, J.F., eds., Blackie Academic and Professional, Glasgow, U.K., 1994, 49. 47. McGill, A.S., Hardy, T., Burt, R.J., and Gunstone, F.D. Hept-cis-4-enal and its contribution to the off-flavour of cold-stored cod. J. Sci. Food Agric., 25, 1477, 1974. 48. Jónsdóttir, R. and Ólafsdóttir, G. Unpublished data, 2004. 49. Refsgaard, H.H.F., Brockhoff, P.B., and Jensen, B. Sensory and chemical changes in farmed Atlantic salmon (Salmo salar) during frozen storage. J. Agric. Food Chem., 46, 3473, 1998. 50. Hardy, R., McGill, A.S., and Gunstone, F.D. Lipid and autoxidative changes in cold stored cod (Gadus morhua). J. Sci. Food Agric., 28, 999, 1979. 51. Josephson, D.B. and Lindsay, R.C. Retro-aldol degradations of unsaturated aldehydes: Role in the formation of c4-heptenal from t2,c6-nonadienal in fish, oyster, and other flavours. J. Food Sci., 64, 1186, 1987. 52. Josephson, D.B. and Lindsay, R.C. c4-Heptenal: An influential volatile compound in boiled potato flavour. J. Food Sci., 52, 328, 1987. 53. Aro, T., Tahvonen, R., Koskinen, L., and Kallio H. Volatile compounds of Baltic herring analysed by dynamic headspace sampling-gas chromatography-mass spectrometry. Eur. Food Res. Technol., 216, 483, 2003. 54. Milo, C. and Grosch, W. Changes in the odourants of boiled salmon and cod as affected by the storage of the raw material. J. Agric. Food Chem., 44, 2366, 1996. 55. Decker, E.A., Warner, K., Richards, M.P. and Shahidi, F. Measuring antioxidant effectiveness in food. J. Agric. Food Chem., 53, 4303, 2005. 56. Jacobsen, C., Let, M.B., Nielsen, N.S., and Meyer, A.S. Antioxidant strategies for preventing oxidative flavour deterioration of foods enriched with n-3 polyunsaturated lipids: A comparative evaluation. Trends Food Sci. Technol., 19, 76, 2008. 57. Hultin, H.O. and Kelleher, S.D. Surimi processing from dark muscle fish, in Surimi and Surimi Seafood, Park, J.W., Ed., Marcel Dekker Inc., New York, 2000, p. 59. 58. Undeland, I., Hall, G., and Lingnert, H. Lipid oxidation in fillets of herring (Clupea harengus) during ice storage. J. Agric. Food Chem., 47, 524, 1999. 59. Richards, M.P. and Hultin, H.O. Rancidity development in a fish model system as affected by phospholipids. J. Food Lipids, 8, 215, 2001. 60. Sohn, J.H., Taki, Y., Ushio, T., Kohata, T., Shioya, I., and Ohshima, T. Lipid oxidations in ordinary and dark muscles of fish: Influences on rancid off-odor development and colour darkening of yellowtail flesh during ice storage. J. Food Sci., 70, 7, 490, 2005. 61. Undeland, I., Kristinsson, H.G., and Hultin, H.O. Hemoglobin-mediated oxidation of washed minced cod muscle phospholipids: Effect of pH and hemoglobin source. J. Agric. Food Chem., 52, 4444, 2004. 62. Jónsdóttir, R., Bragadóttir, M., and Ólafsdóttir, G. The role of volatile compounds in odor development during hemoglobin-mediated oxidation of cod muscle membrane lipids. J. Aquat. Food Prod., 14, 67, 2007. 63. Boyd, L.C., King, M.F., and Sheldon, B. A rapid method for determining the oxidation of n-3 fatty acids. J. Am. Oil Chem. Soc., 69, 325, 1992.


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64. Jónsdóttir, R., Bragadóttir, M., and Ólafsdóttir, G. Oxidation in fish muscle: The role of phosholipids, proteins, pro-oxidants, antioxidants and the effect of heating. Matis report 08, Reykjavík, Iceland, 2008, 73. 65. Medina, I., Gallardo, J.M., Gonzalez, M.J., Lois, S., and Hedges, N. Effect of molecular structure of phenolic families as hydroxycinnamic acids and catechins on their antioxidant effectiveness in minced fish muscle. J. Agric. Food Chem., 55, 3889, 2007. 66. Jittrepotch, N., Ushio, H., and Ohshima, T. Effects of EDTA and a combined use of nitrite and ascorbate on lipid oxidation in cooked Japanese sardine (Sardinops melanostictus) during refrigerated storage. Food Chem., 99, 70, 2006. 67. Banerjee, S. Inhibition of mackerel (Scomber scombrus) muscle lipoxygenase by green tea polyphenols. Food Research International, 39, 486, 2006. 68. Baek, H.H. and Cadwallader, K.R. Volatile compounds in flavour concentrates produced from crayfish-processing byproducts with and without protease treatment. J. Agric. Food Chem., 44, 3262, 1996. 69. Pan, B.S. and Kuo, J.M. Flavour of shellfish and kamaboko flavourants, in Seafoods: Chemistry, Processing, Technology and Quality, Shahidi, F., and Botta, J.R., Eds., Blackie Academic and Professional, Glasgow, U.K., 1994, 85. 70. Jónsdóttir, R., Ólafsdóttir, G., Hauksson, S., and Einarsson, J.E. Flavorants from seafood byproducts, in Handbook of Food Products Manufacturing: Health, Meat, Milk, Poultry, Seafood, and Vegetables, Vol 2, Hui, Y.H., Ed., John Wiley & Sons, Hoboken, NJ, 2007, 931. 71. Varlet, V., Knockaert, C., Prost, C., and Serot, T. Comparison of odour-active volatile compounds of fresh and smoked salmon. J. Sci. Food Agr., 54, 3391, 2006. 72. Varlet, V., Prost, C., and Serot, T. Volatile aldehydes in smoked fish: Analysis methods, occurrence and mechanisms of formation. Food Chem., 105, 1536, 2007. 73. Sérot, T., Baron, R., Knockaert, C., and Vallet, J.L. Effect of smoke processes on the content of 10 major phenolic compounds in smoked fillets of herring (Cuplea harengus). Food Chem., 85, 111, 2004. 74. Guillén, M.D., Errecalde, M.C., Salmerón, J., and Casas, C. Headspace volatile components of smoked swordfish (Xiphias gladius) and cod (Gadus morhua) detected by means of solid phase microextraction and gas chromatography–mass spectrometry. Food Chem., 94, 151, 2006. 75. Joffraud, J.J., Leroi, F., Roy, C., and Berdagué, J.L. Characterisation of volatile compounds produced by bacteria isolated from the spoilage flora of cold-smoked salmon. Int. J. Food Microbiol., 66, 175, 2001. 76. Toldrá, F. Proteolysis and lipolysis in flavour development of dry-cured meat products. Meat Science, 49, 101, 1998. 77. Toldrá, F. and Flores, M. The role of muscle proteases and lipases in flavour development during the processing of dry-cured ham. Crit. Rev. Food Sci., 38, 331, 1999. 78. Toldrá, F., Aristoy, M.C., and Flores, M. Contribution of muscle aminopeptidases to flavour development in dry-cured ham. Food Res. Int., 33, 181, 2000. 79. Jónsdóttir, R., Ólafsdóttir, G., Martinsdóttir, E., and Stefánsson, G. Flavour characterization of ripened cod roe by gas chromatography, sensory analysis and electronic nose. J. Agric. Food Chem., 52, 6250, 2004. 80. Triqui, R. and Reineccius, G.A. Changes in flavour profiles with ripening of anchovy (Engraulis encrasicholus). J. Agric. Food Chem., 43, 1883, 1995. 81. Triqui, R. and Guth, H. Determination of potent odourants in ripened anchovy (Engraulis encrasicholus L.) by aroma extract dilution analysis and by gas chromatography-olfactometry of headspace samples, in Flavour and Lipid Chemistry of Seafoods, Shahidi, F., and Cadwallader, K.R., Eds., ACS Symposium Series 674 American Chemical Society, Washington, DC, 1997, 31. 82. Jónsdóttir, R., Lauritzesen, K., and Thórarinsdóttir, K. Unpublished data, 2007. 83. Lauritzesen, K. and Olsen, R.L. Effects of antioxidants on copper induced lipid oxidation during salting of cod (Gadus morhua). J. Food Lipids, 1, 105, 2004.

PROCESSING CONTROL

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Chapter 9

Basic Composition: Rapid Methodologies Heidi Nilsen, Karsten Heia, and Margrethe Esaiassen Contents 9.1 Near-Infrared Spectroscopy............................................................................................ 122 9.1.1 Determination of Basic Composition.................................................................. 122 9.1.2 Theory, Measurement Principles, and Data Analysis ........................................... 122 9.1.3 Analysis of Basic Constituents ............................................................................ 124 9.2 Imaging Spectroscopy .................................................................................................... 128 9.2.1 Theory, Measurement Principles, and Analysis ................................................... 128 9.2.2 Analysis of Basic Constituents ............................................................................ 128 9.3 NMR Spectroscopy ........................................................................................................ 130 9.3.1 Determination of Basic Composition.................................................................. 130 9.3.2 Theory and Measurement Principles ................................................................... 130 9.3.3 Analysis of Basic Constituents .............................................................................131 9.4 X-Ray Imaging ................................................................................................................132 9.4.1 Theory and Measurement Principles ....................................................................132 9.4.2 Analysis ...............................................................................................................132 9.5 Summary ........................................................................................................................133 Acknowledgment .................................................................................................................... 134 References ............................................................................................................................... 134

Fish and seafood consumption has gained increased attention during the last years as a consequence of increased focus on nutritional quality as well as aspects related to healthy living. 121

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Compared with the production and distribution of meat from the agricultural sector, seafood is considered highly fragile and perishable with a short shelf life and delicate texture, and hence these issues must be considered during the processing and characterization of the material. Another aspect to be considered is the increased consumer awareness regarding the quality of their food; frequently consumers want readily accessible information about nutritional parameters and food quality. The documentation of basic nutritional composition of foods is a legal requirement in many countries, and so the need for measurement and documentation of such parameters is both a consumer requirement and also issued by law. In this perspective there is an obvious need for objective methods for evaluating and documenting the basic composition of fish and seafood. Regarding industrialized food production, requirements for such a method would preferably be that it is rapid and nondestructive. In this context seafood is particularly challenging as it comprises a vast number of different species with their own characteristics and qualities. In this chapter, we review some of the most relevant methods for assessing the basic composition of fish and seafood as presented in scientific literature. These methods are near-infrared (NIR) spectroscopy, magnetic resonance, imaging techniques, as well as x-rays. The basic principles of the techniques are described as well as a presentation of the use and applicability of quality measures of fish. The four methods presented fit with the requirements of speed and nonobtrusiveness; hence, these techniques may be applied in or at a production line.

9.1 9.1.1

Near-Infrared Spectroscopy Determination of Basic Composition

The development and usage of near-infrared spectroscopy (NIR) as an analytical tool has proven useful in areas varying from food quality, pharmaceutical applications, to analysis related to the environment and the petrochemical sector [1]. During the last 30 years the use of NIR spectroscopy has gained increased importance in the evaluation of a number of different food quality parameters [2–7]. The work in food analysis tends to have a focus within the agricultural sector [1]. However, throughout the years the method has also proven useful for the analysis of seafood and seafood products [8]. There are several reasons why NIR as a food analytical tool has caught attention and approval during the last decennia. The measurements are based on light interaction with material, facilitating a rapid response, which is a prerequisite for a methodology to be applied along a production line. Another benefit is the potential of simultaneous measurements of more parameters, and additionally the method may be applied with little or no obtrusion to the material sample. In the following section, we give a short introduction to the principles of NIR spectroscopy, followed by a presentation of the usage of NIR measurements for the rapid determination of basic constituents in fish and seafood products.

9.1.2 Theory, Measurement Principles, and Data Analysis The electromagnetic range applied in NIR spectroscopy spans from 700 to 2500 nm, comprising the frequencies just below those of visible light. A food sample exposed to emission in this wavelength range will absorb certain parts of the energy depending on the chemical composition of the sample. The absorption of light is due to the response of the molecular bonds O−H, C−H, C−O,

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and N−H [9] and corresponds mainly to overtones and combinations of fundamental vibrations. A thorough theoretical description of the NIR theory as well as the designation of numerous bands of absorptions may be found in Osborne and Fearn [2] and reviews on the subject [1,9,10]. Over the years there has been a steadily ongoing development of instrumentation for NIR spectroscopy, both with respect to the detectors and the capture of the spectral information [10,11]. In the context of rapid methodologies, we view this in terms of the measurement setup enabled by technology, developed toward the facilitation of nondestructive, nondisruptive, and noncontact methods. Different measurement modes for NIR spectroscopy are illustrated in Figure 9.1. A setup as shown in (a), where the light passes through the sample from one side to another, enables “transmission” measurements. The amount of light entering the detector unit depends on the scattering and absorption features of the sample as well as the sample thickness and lamp characteristics. If the light source and the detector are placed on the same side of the sample as shown in (b), the system is operated in “reflection” mode. For both (a) and (b), the transmission and reflection may be either direct or diff use, depending on the scattering properties of the medium under investigation. Finally, (c) illustrates how measurements are performed in “transflection” mode, placing the light source and the detector at the same side of the sample, but focusing the two devices so as to ensure that the light has traversed some region of the sample before detection. NIR spectroscopy is an indirect measurement technique. The broad spectral bands may be an indication of the material constituents, but an immediate look at an NIR spectrum is not sufficient to quantify the different substances. Hence, the spectral readings must be correlated to a relevant reference method such as, for example, traditional chemical determination of the constituents. NIR spectroscopy would not have had such an impact as an analytical tool had it not been for the development of mathematical tools for spectral analysis. A common methodology is chemometrics or

Shelter

Detector

Sample

Light source (a)

(b)

(c)

Figure 9.1 Different measurement setups for NIR spectroscopy. In (a) the transmission setup is shown; light from the source penetrates the sample and enters the detector. The setup in (b) displays the reflection setup where light reflected from the sample surface enters the detector. In (c) the light source and detector are located to register light that has traversed the sample before detection. In order to prevent direct reflection from the surface, a screen is placed between the directly emitted area and the area of inspection.

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multivariate data analysis. If there is good correlation between the spectral measurements and the method of reference, the reference method may be replaced by the spectral reading and the analytical model. Typically, a model based on several wavelengths is required to extract useful information from the spectroscopic data. Among the most used multivariate techniques are principal component analysis (PCA), partial least square (PLS) regression, and soft independent modeling of class analogies (SIMCA) [12].

9.1.3 Analysis of Basic Constituents As found in NIR analysis of foods in general, a substantial part of the work related to NIR analysis of fish and food from fish concerns the quantification of the chemical constituents, fat, protein, and water. Being the basic nutritional components of any food, an easy, reliable, and rapid method for the assessment and quantification of these constituents is considered a valuable tool in the quality evaluation of any foodstuff. In the following paragraphs, we give several examples of the use of NIR spectroscopy for the determination of basic food constituents in fish and seafood and how the method has been applied and developed over the last 20 years. The earliest reports of NIR spectroscopy to measure chemical components in fish appeared more than two decades ago. In 1987 Gjerde and Martens [13] demonstrated the applicability of NIR to predict water, fat, and protein content in rainbow trout. The same year Mathias et al. [14] reported the use of NIR spectroscopy to determine lipid and protein content in freshwater fish, namely, fingerling Arctic charr and rainbow trout. In both studies reflectance measurements were performed, and the sample preparation included mincing and freeze drying of the material to be evaluated. Both of these early reports concluded that the method was promising in terms of speed and efficiency when measuring a large number of samples. Darwish and others [15] used the technique in 1989 to measure fat, water, and protein in cod, mackerel, and tuna. For the measurement of fat and protein, the samples were minced and dissolved in a milk-like emulsion, whereas water determination was made on the water extracted from the fish mince. In spite of the rather cumbersome sampling procedure, the study concluded that the method could be a useful tool for rapid quality control. Consecutive research articles proved the feasibility of the tool in developing the method to apply with simpler procedures of sample preparation. Sollid and Solberg [16] measured the fat content in salmon by transmission spectroscopy on raw minced muscle. As early as in 1992 Lee and others [17] showed how NIR spectroscopy could be used noninvasively to estimate the lipid content of small-sized, intact rainbow trout. The measurements were performed by use of fiber optic bundles conveying the light to and from the sample site. Based on measurements through scales and skin, it was possible to estimate the lipid content of the intact muscle. This measurement setup clearly displayed how NIR spectroscopy could be used in a nondestructive way. Downey [18] applied a similar spectroscopic setup to measure fat and water content of intact farmed salmon; and, as in the work of Lee et al. [17], the measurement locations for obtaining the best calibration results were also addressed. In addition, by use of NIR in connection with fiber optics Solberg et al. [19] performed a study on live anesthetized farmed salmon, demonstrating the possibility to determine fat content in live fish. The prospect of measuring the chemical composition of intact fish could facilitate the use of the method in connection with selection in breeding programs [17] as well as for quality grading in terms of nutritional quality [19]. Farmed salmon is of high commercial value and a worldwide favorable product. This could account for the many studies relating to the rapid analysis of the basic chemical composition of


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salmon. Isaksson et al. [20] conducted a study in which they compared NIR measurements on intact salmon fillet, as well as on minced salmon muscle. In this work they applied a fiber optic measurement setup. Spectroscopic readings obtained on the minced samples correlated better with the reference measurements on fat, water, and protein than those made on intact muscles. The study concluded that NIR is well suited for nondestructive quality evaluation of salmon fillets. Wold et al. [21,22] also conducted studies documenting the efficiency of applying NIR spectroscopy in different measurement modes to assess fat and water content in salmon. An example illustrating the use of NIR spectroscopy for assessing fat content in farmed salmon is given in Figure 9.2. NIR spectroscopy has been used for evaluating the chemical composition of several other fish species as well. Nortvedt, Torrissen, and Tuene [24] made use of NIR transmission spectroscopy to assess protein, fat, and dry matter in halibut fillet. Transmission spectroscopy was also employed for the analysis of fat and dry matter in capelin [25], applying minced samples for the spectral readings. This work also emphasized the impact of the conditional state of the fish when making calibration models, whether pre-, in, or postspawning. In a research article published in 2004, Xiccato et al. [26] showed that NIR spectroscopy could be used to estimate lipid, water, and protein content of European sea bass, and additionally the spectroscopic measurements could be used for origin identification or authentication of the samples. In a recent work by Khodabux et al. [27], NIR spectroscopy was proven to be a useful tool for the evaluation of basic constituents of different types of tuna. The fat content of herring has also been assessed by the use of NIR spectroscopy. In both the works of Vogt et al. [28] and Nielsen et al. [29] one question of interest was the comparison of different methods for measuring fat content, Torry fatmeter, microwave, and NIR spectroscopy,

Measured Y Elements: Slope: Offset: Correlation: RMSEP: SEP: 21 Bias: 24

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Figure 9.2 The plot shows the predicted versus measured fat content in farmed salmon based on multivariate analysis of 78 spectra from salmon fillets and the respective chemical analyses of the fillets. (From Nilsen, H. and Sørensen, N.K., Unpublished data, 1998.) Spectral measurements were performed on intact fillets by transflection measurements by use of the fiber optic probe of the instrument NIRS6500 (Perstorp Analytical Inc., Silver Spring).

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and Distell fatmeter, NIR, and NMR, respectively. In both studies NIR spectroscopy resulted in favorable outcomes with respect to speed and accuracy. Vogt et al. [28] however, also commented on the cost aspect of the different methods as part of the feasibility of the methods. The use and results described above were all on raw fish samples, either intact fish/muscle or minced muscle. NIR spectroscopy has proven applicable also for the analysis of frozen products as well as processed and refined products. Shimamoto et al. [30] used NIR spectroscopy in connection with an interactance probe as a means of determining the fat content in frozen horse mackerel nonintrusively. A few years later the same group used NIR to assess the fat content in frozen skipjack [31]. Smoked and cured fish have also been subject to investigation by the use of NIR spectroscopy. Huang et al. [32] performed a study to show that moisture and salt content in cold smoked salmon could be evaluated using NIR measurements. Similar findings were made on hot smoked portions of salmon fillets by Lin et al. [33]. The salting, smoking, and exposure to elevated temperatures, about 63°C for the hot smoking process, alter the physical and chemical properties as well as the textural properties of the fish muscle. NIR spectroscopy, however, still proved viable for assessing the chemical constituents of the samples. Moisture and sodium chloride in cured Atlantic salmon were measured nondestructively by NIR diff use reflectance spectroscopy [34], although the assessment of salt did not prove as effective as that of water content. Of the most recent studies in the field is work by Wold et al. [35] applying the NIR technique to determine water content in salted dried cod—clipfish. They addressed the sampling/measurement location and the method of performing measurements in a representative way. They did, however, combine the NIR technique with imaging—further described later in this chapter—which facilitates a novel way of measuring and analyzing fish quality. In addition to the analysis on raw fish and processed fish material, NIR spectroscopy has also been applied for the analysis of basic chemical constituents in other types of fish products. In 2001 Huang et al. [36] presented a study where NIR spectroscopy was used for the investigation of salt content in cured salmon roe. It was argued that the sensitivity of the method could have been better; however, the nonintrusive method would still be an interesting alternative for rapid testing of high-value food products. The spectroscopic method has been used to assess moisture, fat, and protein content in another roe-based product, namely, the Greek dish taramosalata. A work by Adamopoulos and Goula [37] showed that the chemical composition could be assessed with a high degree of accuracy in addition to the obvious benefit of the ease and simplicity of the measurement method. For surimi products, refined fish-based products made by washing mechanically deboned fish to remove constituents such as blood, lipids, enzymes, and certain proteins, NIR spectroscopy was applied to determine water and protein content [38]. A further use of NIR measurements for the evaluation of basic food constituents was suggested by Svensson et al. [39]. In this work it was demonstrated how NIR spectroscopy could be used to assess the protein content in brine from salted herring and thus indirectly be a measure of the maturity and ripening of the salted herring. In addition to the many studies assessing the basic chemical constituents in fish and seafood, the spectroscopic method has confirmed its applicability for the evaluation of several other quality issues in fish. Examples of these are nondestructive texture analysis of farmed salmon [40], differentiation between fresh and frozen-thawed fish [7], storage time of frozen fish [41], evaluation of freshness or storage time of fresh fish [41,42], and the detection of bruises in the fish muscle [33]. The versatility of the method is one reason for its relevance and growing popularity during the recent years. The broadbanded spectra contain information about several parameters, and


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the possibility of simultaneously monitoring a number of different issues, with one reading, is considered intriguing. As illustrated by the above, the ease of use of the methodology has increased through instruments facilitating little or no sample preparation as well as measurement setups for rapid and nonintrusive registration. Instrument development has come from the grand-size laboratory desktop versions to portable or handheld instruments as illustrated in Figure 9.3. These developments have enabled the use of at-line or online methodology. The technique has, however, not yet become an everyday instrumental tool for food-quality control nor, say, fish-quality inspection. There may be several reasons for this. The high price of the instrumentation, on one side, has been a reason for the method not gaining a broader range of applicability. High-cost instrumentation designed for versatile use and flexibility has probably better met the requirements of laboratory use than those of industrial application. Another issue is the need for modeling the correlation between the spectroscopic reading and the quality parameter in question. Th is is a challenging task in view of the variety and the heterogeneity of the material and so may have contributed to the reluctance in investing in and developing this technology to a commercial tool for assessment of fish quality. The development in recent years in instrumentation, combining imaging techniques with the spectral information, may promote the future applicability and usefulness of the information in

Figure 9.3 Prototype version of handheld spectroscopic instrument for quality assessment of fish. This instrument was used for the determination of freshness of cod as well as the assessment of frozen storage time of hake.

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the near-infrared spectra. A novel example of this is the development of the QMonitor (QVision AS, Oslo, Norge), an analytical tool for industrial quality control of clipfish and salmon fillets. The realization of a commercial processing analytical tool for the simultaneous analysis of several parameters makes the technology interesting for a broad range of fish and seafood processing industries.

9.2 Imaging Spectroscopy 9.2.1

Theory, Measurement Principles, and Analysis

Imaging spectroscopy, also known as multispectral imaging or hyperspectral imaging, is a new technique that has been developed during the last decade [43,44]. It has become a widely used technique within fields spanning microscopy to satellite remote sensing. In addition to what traditional spectroscopy can facilitate, this technique also provides spatial information. Th is means that for each spatial location it is possible to access the full spectral information. To simplify the concept, this can be illustrated as simultaneously recording information about shape and color. This implies that this technique is a powerful tool for segmentation and classification and that it may also map the chemical composition into the spatial domain [45]. Imaging spectroscopy can be implemented for transmission, reflection, as well as transflection measurements. Depending on the applied sensor technology, the spectra may be recorded in the visible and near-infrared region. Typically, an imaging spectrograph operates in the following way; it uses a two-dimensional sensor, and each frame captured provides full spectral information for one line across the object to be imaged. Between each captured frame, the spectrograph and the object must move relative to each other. In this way an image of the object is built line by line. Typically, the relative motion is accomplished by mounting the imaging spectrograph above a conveyer belt where each captured frame images a line perpendicular to the direction of motion. As described in Section 9.1 on NIR spectroscopy, this method is an indirect measurement technique. The analytical techniques described in that section are also applied to imaging spectroscopy data. As these techniques only use the spectral information, improved results can be obtained by combining these techniques with more traditional image processing techniques. For instance, the hyperspectral data can be preprocessed based on spatial features before applying analytical spectral techniques, or the result from these techniques can be postprocessed to utilize the spatial information [46].

9.2.2

Analysis of Basic Constituents

During the last decade several applications within food-quality inspection have been developed based on imaging spectroscopy. Most of them are on foods such as fruits, vegetables, and meat. There are still relatively few reports on imaging spectroscopy applied for the analysis of fish and seafood. However, the feasibility of the method for the analysis of basic composition of foods, in general, demonstrates the potential of the method in the seafood sector as well. In order to illustrate the potential parameters to be assessed by imaging spectroscopy, some examples related to the agricultural sector are referred. For fruits and vegetables more articles report on determination of chemical constituents such as moisture content, total soluble solids, and acidity (expressed as pH) [47–49]. Several solutions have also been developed for detection of defects and contaminations on fruits. It has been shown that NIR hyperspectral imaging techniques are


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useful for automatic online detection of surface defects and contaminations on apples [50–52]. A thorough review of imaging spectroscopy applications within fruits and vegetables is presented by Nicolai et al. [53]. Inspection systems based on hyperspectral imaging have been tested for poultry carcass inspection focusing on classification of carcasses into normal, septicemic, and cadaver [54,55]. Further on, imaging spectroscopy solutions for detection of contaminants such as fecal and ingesta on poultry carcasses have been studied [56–58]. A recent work on quality assessment of pork has been reported by Qiao et al. [59,60] where several quality parameters were evaluated by imaging spectroscopy. The parameters included were drip loss, pH, color, and different texture features. The first article addressing analysis of fish or seafood by imaging spectroscopy was published in 2000 by Sigernes et al. [61]. Peeling of shrimps and detection of nematodes were mentioned as possible applications for the future. Since 2000, the main activities within imaging spectroscopy and fish analysis have been focused on online solutions for assessing chemical composition and detection of quality defects in fish products. Regarding the determination of basic chemical composition of fish and seafood, there is one recent publication on assessing water content in salted dried cod by Wold et al. [35]. In this publication the importance of including spatial information is illustrated. When drying fish, the moisture content of the fish varies from the thinner parts to the thicker parts of the fish. Hence, measuring the water content in one spot is not necessarily representative for the whole fish. QVision (Oslo, Norway) has also developed an industrial solution based on multispectral imaging for measuring the fat content in salmon fi llets (see Figure 9.4).

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Figure 9.4 Fat distribution in salmon fillet measured by the multispectral imaging system QMonitor fabricated by QVision (Oslo, Norway). The color bar to the right indicates the correspondence between color and fat content in percentage. The mean fat content for this fillet is 18.3%, whereas the local fat content varies from approximately 6% up to 43%.

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In addition to the measurement and documentation of basic composition, imaging spectroscopy of fish has been applied to address other quality issues. For detection of flaws or defects in fish, a lot of effort has been invested in the detection of nematodes, blood spots, black lining, and skin remnants in whitefish fillets [62–64]. Still the number of imaging spectroscopy applications with fish and seafood is low, but looking at reported applications within other areas the potential for new applications is high. For NIR spectroscopy several applications within fish and seafood are reported, and the methods that are feasible by spot measurements may also be implemented using imaging spectroscopy. Even more important is that for some applications imaging spectroscopy can provide better results, since it is possible to use spectra from dedicated relevant areas on the sample. For instance, if blood oxidation should be quantified spectra from blood-infested area of a fillet can easily be extracted for analysis based on imaging spectroscopy data. Furthermore, experience with NIR spectroscopy shows that more than one attribute can be estimated based on one recording, but this requires that the same spot be used. With imaging spectroscopy this is not a problem since spectra are available for all spatial locations. Imaging spectroscopy is well suited for application in the fish processing industry as an online technique. Using the interaction between light and the sample object, measurements may be performed at high speed as well as in noncontact mode. With respect to commercial implementation of imaging spectroscopy, this is a relatively new field, and currently there are a limited number of equipment suppliers. A low-resolution (spectral and spatial) instrument is available for industrial assessment of chemical composition such as fat and water content (QMonitor, QVision, Oslo, Norway) in fish. In addition to this a high-resolution prototype imaging spectrograph has been developed for detection of defects as well as determination of chemical constituents in fish fillets as reported by Heia et al. [63].

9.3 NMR Spectroscopy 9.3.1

Determination of Basic Composition

Nuclear magnetic resonance (NMR) has evolved from being an expensive and academic analytical technique into being a technique applicable for the food industry in both size and price of the equipment as well as speed of analyses. The main technique used is NMR spectroscopy, but during the last few years magnetic resonance imaging (MRI) has also been explored for its usefulness in food analyses.

9.3.2 Theory and Measurement Principles NMR provides a large amount of information regarding composition and structure of components in food. NMR techniques use electromagnetic radiation and magnetic fields to obtain chemical information, and they are based on the magnetic properties of atomic nuclei. All nuclei that contain odd numbers of protons or neutrons have an intrinsic magnetic moment and angular momentum. The most commonly measured nuclei are 1H and 13C. Additionally, 31P-NMR and 23Na-NMR have also been used for food analyses. When an external magnetic field is applied, NMR active nuclei absorb at a frequency characteristic of the isotope. The energy absorptions of the atomic nuclei are also affected by the nuclei of neighboring atoms within the same molecule as well as nuclei in surrounding molecules. Hence, NMR spectroscopy may provide detailed


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information regarding the molecular structure of a food sample. Numerous applications of NMR in food analyses have been reported in the literature, and some examples of analyses of seafood are given here. Today, different NMR equipments are available, and they may provide different information regarding the food properties. For analyses of seafood products, low-resolution NMR (LR-NMR) and high-resolution NMR (HR-NMR) spectroscopy as well as MRI and NMR-mobile universal surface explores (NMR-MOUSE) have been used. HR-NMR has been used in many studies and has the advantage over LR-NMR that it is possible to obtain detailed information regarding the molecular structure.

9.3.3 Analysis of Basic Constituents For several years 1H- and 13C-NMR have been applied to measure the lipid or water content of many different foods including fish, and there are numerous reports available. Low-field (LF) NMR spectroscopy requires little or no sample preparation, and it has mainly been used for analyses of water in food samples, but the technique has been applied in the recent years for determination of both fat and water content in different food products and also seafood. For example, LF-1H-NMR has been used for studying water distribution in smoked salmon [65], cod [66], water distribution, and mobility in herring [67] and oil and water content of salmon and cod [68]. Studies of large objects like whole fish are impossible using most traditional LF-NMR instruments. A new type of LF-NMR instrument, the Bruker Professional MOUSE ® (Bruker Optik GmbH, Rheinstetten, Germany) has been developed to handle such samples. Aursand et al. [69] demonstrated that this equipment could be applied to determine fat in homogenates from salmon, whereas Veliyulin et al. [70] demonstrated that NMR-MOUSE could also be used for in vivo determination of fat content in Atlantic salmon. Additionally, in a study focusing on both 23Na-NMR and low-field 1H-NMR spectroscopy, it was shown that 23Na-NMR has proven useful for quantitative salt determinations in salted cod, whereas LF- 1H-NMR seems to correlate to fillet pH and water-holding capacity [71]. High-resolution NMR can be used to provide information on lipid classes, degree of saturated/ unsaturated fatty acids, fatty acid composition, and studies of lipid degradation processes in lipid mixtures such as fish oils. As recent examples, Tyl et al. [72] used HR-NMR to measure the content of n-3 polyunsaturated fatty acids in four types of unoxidized fish oils, whereas Siddiqui et al. [73] used HR-1H- and HR-13C-NMR for multicomponent analyses of encapsulated marine oil supplements. Falch et al. [74] reported the use of HR-NMR to determine oxidation products in marine lipids. Due to the provision of very detailed information regarding the molecular structure of a food sample, high-resolution NMR has been applied in many food authenticity studies. Extensive reviews on different techniques, including NMR, used for seafood authenticity have been provided by Martinez et al. [75] and Arvanitoyannis et al. [76]. Among more recent work, Standal et al. [77] used NMR to discriminate cod liver oil according to whether the origin was wild/ farmed as well as geographic origin. Rezzi et al. [78] demonstrated the use of NMR lipid profiling for classification of gilthead sea bream according to geographic origin, whereas Thomas et al. [79] and Masoum et al. [80] used this technique to determine the origin of Atlantic salmon. Additionally, 1H NMR spectroscopy has been explored to identify the fate of some bioactive compounds during processing of seafood. Martinez et al. [81] showed that it was possible to identify taurine, betaine, anserine, creatine, trimethylamine oxide, and dimethylamine in extracts

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from processed cod. Gribbestad et al. [82] showed that it was possible to identify single chemical compounds such as hypoxanthine, amino acids, anserine, lactate, and some fatty acids in extracts and muscle from salmon using high-resolution 1H NMR spectroscopy. As illustrated here, NMR is a versatile tool for the identification and quantification of numerous compounds in fish related to nutritional quality. An objection to the method, however, has been that conventional NMR is an expensive technique. However, the high spectral resolution is not always required, and lately many low-field, low-resolution NMR spectrometers have been developed and commercialized. Such equipment is cheaper, smaller, and less sensitive to fluctuations in the environment and thus more applicable in industry as well as in many research fields.

9.4

X-Ray Imaging

9.4.1 Theory and Measurement Principles X-ray imaging is a technique based on the emission of x-rays through a sample and recording the amount of attenuation. For online applications this can be implemented as a line-by-line imaging or a frame-by-frame imaging. The decrease in x-ray intensity inside a sample will be due to absorption by different materials. A more dense material will absorb more x-ray energy, and the attenuation will also be influenced by the sample thickness. It is not possible to accurately characterize the observed sample by applying only one x-ray energy level. There are two interactions, the photoelectric effect and the Compton scattering that causes the x-ray attenuation, and their relative contributions are energy dependent [83]. By using two x-ray energy levels, more specific information about the sample can be revealed. This technique is referred to as dual-energy x-ray absorptiometry (DXA, previously DEXA) and may be implemented using a two-layer detector, one layer for each energy level. Within the field of medicine, computed tomographic (CT) scanning is widely used. This is also an x-ray imaging system, but it provides a three-dimensional image of the sample. This is achieved by rotating the x-ray/detector unit around the sample. Making profiles from different angles and then combining them by software, a two-dimensional cross section of the sample can be made. Then the third dimension is accomplished by the sample movement. Th is is a powerful imaging technique that can be used both as a single-energy and a dual-energy module.

9.4.2 Analysis X-ray imaging provides spatial information in two dimensions (2D) or three dimensions (3D) (CT). Typical applications within fish and fish products are related to the detection of bones and bone fragments as well as chemical composition and localization. A study has been conducted on the applicability of CT scanning as a nondestructive and rapid way of measuring muscle dry matter content and liquid leakage in cod fillets [84]. The results obtained showed that CT scanning could be used as a rapid method for the assessment of these attributes and would add valuable information to be used in genetic studies and breeding programs. Further on the CT scans gave significant information about dry matter distribution from head to tail of the cod. In another study Kolstad et al. [85] tested CT scanning as a tool for estimating the relative size of fat deposits and lean tissue and fat content in Atlantic halibut. Based on the results obtained the


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Figure 9.5 Detection of pin bones in fish fillets by x-ray imaging using the SensorX instrumentation (Marel, Iceland). To the left is the original x-ray image of one cod fillet and to the right is the processed image where only the bones identified in the fillet are shown.

authors recommended CT scanning as an online technique for carcass evaluation. A similar work has been carried out by Hancz et al. [86] showing good results predicting fat content of common carp based on CT scanning. With respect to bone detection in fish fillets there are commercial solutions available today (Marel Hf, Iceland). Marel developed an X-ray-based bone detection unit (SensorX) that was commercially available on the market in 2003 [87]. This instrument can detect bones and bone fragments down to a diameter of 0.3 mm when operating at industrial speed (see Figure 9.5 for an example).

9.5 Summary The methods and applications presented in the above clearly illustrate that there are more tools and techniques that could serve as an easy and useful way of rapid quality determination of fish and seafood; instrumental means capable of objective and rapid determination of basic composition are also available. Throughout development all presented techniques have met the requirements

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of simplicity in sample preparation, and progress in data processing and analytical tools has facilitated usability and ease of interpretation of measurement results. In addition, NIR, NMR, imaging, and x-rays are operated at a speed that makes it possible to perform measurements at or in a processing line, hence allowing for measurements to be performed on large-scale quantities. This chapter, however, also makes it clear that although proven useful and promising in laboratory-scale trials, these techniques have—with a few commercial exceptions—still not been shown to be commercially valid for quality determination in the fish and seafood processing industry. Part of the explanation for this could be the cost level of the equipment in question. The price of measurement equipment for NIR, NMR, and x-rays is considerable and, therefore, not easily applicable for small-scale industries as is often the case in the fish processing industry. Another issue is the substantial variety and heterogeneity of the material to be analyzed. Due to the spread and diversity in fish species and sizes as well as the seasonal difference in bodily composition, the finding of a universal measurement tool to meet with this variety is a challenging task. However, the technological development exemplified by SensorX (Marel hf, Reykjavik, Iceland) and QMonitor (QVision AS, Oslo, Norway) confirms that these techniques may be applied in commercial and industrial high-speed fish processing applications. Introducing and applying these methods to industrial applications and enabling production of well-documented, quality seafood products will contribute to retaining the good reputation of fish and seafood in the years to come.

Acknowledgment The authors would like to thank Dr Jens Petter Wold, Nofima Food, Norway, for providing the example picture used in Figure 9.4.

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Chapter 10

Microstructure Isabel Hernando, Empar Llorca, Ana Puig, and María-Angeles Lluch Contents 10.1 Main Microscopy Techniques for Studying Seafood .....................................................139 10.2 Fish Muscle Microstructure...........................................................................................140 10.2.1 Herring ............................................................................................................145 10.2.2 Hake ................................................................................................................146 10.3 Processed Fish Microstructure .......................................................................................148 10.3.1 Smoked Salmon............................................................................................... 148 10.3.2 Salted Cod....................................................................................................... 149 10.3.3 Surimi ..............................................................................................................150 10.4 Squid Microstructure .................................................................................................... 151 References ................................................................................................................................ 151

10.1 Main Microscopy Techniques for Studying Seafood The microstructure of foods forms a link between the molecular and macroscopic levels and constitutes a key factor for studying the properties of foods and for improving and optimizing food processes. The organization of the chemical components of foods (proteins, carbohydrates, fats, etc.) is responsible for their microstructure, so any chemical or enzymatic change that takes place in the chemical components has an effect on the microstructural organization of the food matrices and their functionality. Several strategies can be used to study food microstructure. Pérez-Munuera et al. (2008) gave an overview of the most important techniques for studying muscle food structure. This chapter 139

140 ◾


provides a detailed description of the protocols often followed to obtain information about seafood microstructure. The light microscope (LM) is a very versatile tool that works in different applications such as bright field, phase contrast or differential interference contrast (Nomarski), polarizing microscopy, or fluorescence microscopy. The most useful application for studying seafood structure is bright field microscopy. For this, the sample has to be prepared in semithin sections of about 0.1–2 mm (Figure 10.1). The sections are obtained using a microtome after embedding the food in paraffin or resin or using a cryotome after freezing the sample with CO2 or liquid N2. Once the semithin sections are obtained, they are mounted in glass slides and stained with different dyes (toluidine blue, sudan, red oil, iodine, light green, etc.) before examination in the LM. Electron microscopy (EM) allows food structures to be studied at higher magnifications than those used in LM. Two types of microscopes use electron beams as their source of illumination: transmission electron microscopes (TEM) and scanning electron microscopes (SEM). In both methods, the samples need to be prepared first. The steps in preparing samples for TEM observation (Figure 10.2) are primary fi xation with aldehydes such as glutaraldehyde, secondary fi xation with osmium tetroxide, dehydration in a series of ethanol dilutions of increasing concentration, infiltration and embedding in resin, cutting ultrathin sections (5–100 nm) in an ultramicrotome, and staining the ultrathin sections with heavy metal solutions such as lead citrate or uranyl acetate. The SEM method observes the surface of the sample, so there is no need to section it. There are two ways of preparing samples for SEM: chemical fi xation and physical fi xation (Figure 10.3). In the former, the sample preparation steps are chemical fi xation (with aldehydes and osmium tetroxide, as for TEM), dehydration in a series of ethanol dilutions of increasing concentration, critical point drying, and coating with a conducting metal for SEM imaging or with carbon for x-ray. When physical fixation is used, the sample is frozen in liquid nitrogen and then freeze-dried before being coated and observed. In recent years considerable progress has been made in the field of SEM through vitrification techniques. In Cryo-SEM, the sample is frozen in slush nitrogen (Figure 10.4) and quickly transferred under vacuum to a cold stage fit on a microscope where the frozen sample is fractured, etched, coated, and observed; in this way, the sample can be observed with all its constituent water. Besides the secondary electrons, other emanations or signals such as x-rays, backscattered electrons, and so on, may be generated as a result of the electron beam striking the specimen (Pérez-Munuera et al., 2008); these different signals can be captured by the appropriate detector in each case. In this way, ions or molecules can be identified and quantified in situ using specific detectors coupled to the electron microscope, so microanalysis can be carried out by means of x-ray. Finally, image analysis relies heavily on computer technology to obtain quantitative results from microscopy observation.

10.2

Fish Muscle Microstructure

Fish muscle consists of myotomes. They are arranged in concentric circles forming subdivisions of striated muscle (Figure 10.5). At each subdivision there are macroscopic collagenous dividing lines (myocommata). The muscle cells are short and 0.02–1.0 mm in diameter. They are each surrounded by the sarcolemma membrane and by a thin layer of connective tissue (endomysium). Many of the endomysia are connected to the perymisium, which is contiguous to the myocommata (Ofstad et al., 2006). The fibers are essentially the same as those of terrestrial animals in terms of the arrangement of the thick and thin filaments, showing alternate arrangements of

Microstructure ◾

Food

Specimen portions

Embedding in paraffin or resin

Freezing (CO2, liquid N2)

Cold knife

Preparation for slicing

Slice

CO2

Semithin sections (0,11–2 μm)

Knife Cold stub Microtome

Cryotome

Mounting in glass slides

Staining specimen

1% Toluidine blue 1% Lugol, 1% red oil, ...

LM observation

Figure 10.1 Preparation of samples for LM observation.

141

142 ◾


Food

Specimen portions

Fixation 2,5% Glutaraldehyde

2% Os O4

Dehydration Ethanol (30%, 50%, 70%, 90%, 100%)

Infiltration and embedding in resine Epoxi resin, Araldite Spurr’s, LR white

Cured resin

Glass or diamond knife

Ultrathin sectioning (5–100 nm)

Ultramicrotome Specimen block

Trimmed block

Tweezers Section collection Ultrathin section staining

Specimen block face Grid Knife

4% Lead citrate 2% Uranyl acetate

TEM observation

Figure 10.2 Preparation of samples for TEM observation.

Microstructure ◾ 143

Food

Specimen portions

Fixation Physical fixation

Quick freezing in liquid N2

Chemical fixation

2,5% Glutaraldehyde

2% Os O4

Ethanol (30, 50, 70, 90, 100%) Sublimated H2O

P

T Dehydration

(To vaccum) Freeze dryer

CO2

Critical point dryer

(To transformer) (To pumps) Sputter metal coater (or evaporation coater)

Coating (Au, Pd, for SEM imaging) (C, for X-ray)

SEM observation

Figure 10.3

Preparation of samples for SEM observation.

144

◾


Food

Specimen portions

Quick freezing in slush N2 (T < –210°C)

Freezing

Transfer to Cryo-SEM

Specimen transfer

Knife Specimen fracturing (into Cryo-SEM) Specimen fracturing (–180°C, vaccuum)

Etched H2O

Specimen fracturing (–90°C, vaccuum)

Au deposition

Etching (into Cryo-SEM)

Coating (Au, C, ...) (into Cryo-SEM)

(–130°C, vaccuum)

Cryo-SEM observation

Figure 10.4

Preparation of samples for Cryo-SEM observation.


a

b

Figure 10.5 Schematic representation of fish muscle with myotomes. a: myotome, b: myocommata.

A and I bands (Pérez-Munuera et al., 2008), but the total collagen content is lower, since the water in which the fish live lends support for the body (Lampila, 1990). Examples of different fresh fish tissues observed by several techniques are described here.

10.2.1 Herring Figure 10.6A shows a cross section of herring tissue fi xed with glutaraldehyde and observed by SEM. The typical fish muscle fibers can be seen, surrounded by the sarcolemma and by the endomysial connective tissue, which is mainly composed of collagen. The separation that can be observed between the muscle cells is usually attributed to the effect produced by chemical fixation and dehydration during preparation for SEM. The myofibrils are shown in longitudinal section in this sample (Figure 10.6B), where the Z disks can be distinguished. Fixing in osmium tetroxide shows the distribution of fat in the herring tissue. At low magnification, the fat can be observed covering the fibers in a longitudinal section of herring muscle (Figure 10.6C). Figure 10.6D shows the microstructure of herring tissue at a higher magnification, where the fat is viewed as globules on the surface of the fiber. The fiber is composed of myofibrils in which Z disks are distinguished in the areas where the sarcolemma is broken. In a cross section of the sample fi xed in osmium tetroxide (Figure 10.6E), fat globules of different sizes are observed occupying the interfibrillar spaces and myofibrils are distinguished inside the cells. When the muscle fibers are observed using the Cryo-SEM technique, the aggregation of solutes produced during the etching of the sample generates the typical eutectic artifact observed in Figure 10.6F. A micrograph cross section of the fibers shows them surrounded by the sarcolemma, with the endomysial connective tissue keeping the muscle fibers firmly attached to one another. Figure 10.7A shows a herring sample stained with toluidine blue and observed by LM; the perymisial connective tissue that surrounds the muscle bundles can be seen. Figure 10.7B, obtained by the same technique but observed at a higher magnification, reveals the myofibrils inside each cell; the myofibrils at the cell edges have a less rounded section than the central myofibrils and are arranged like a palisade. When ultrathin sections of herring muscle tissue are studied by TEM, it is possible to observe ultrastructural details. The longitudinal section in Figure 10.7C shows the inside of a muscle fiber with the myofibrils perfectly bundled. The layouts of the Z disks that mark the length of the sarcomere are visible. The myofibrils are

146

◾


Z

300 μm

(A)

6 μm

(B)

I 40 μm

(C)

a

f

f

f (E)

10 μm

(D)

c 60 μm

(F)

30 μm

Figure 10.6 Herring tissue. (A–E) SEM, (F) Cryo-SEM. Z, Z disk; f, fat globule; a, eutectic artifact; c, connective tissue.

connected to each other at the Z disk level by the costameres, which are the components of the cytoskeletal network that links the myofibrils to one another and to the sarcolemma. The structural elements that constitute the sarcomere, the A and I bands, can be seen, along with the M and Z lines. The same structure has also been observed in different meat products, for example, pork meat (raw ham) (Larrea et al., 2007). The TEM technique allows images to be obtained at higher magnification and with better resolution than other microscopy techniques (Figure 10.7D).

10.2.2

Hake

The observation of hake muscle by SEM after fi xing with glutaraldehyde allows distinguishing that the fibers of hake muscle tissue are very similar to those of herrings. The main difference is their size: hake fibers are thicker than herring ones. Hake fibers surrounded by connective tissue can be observed in Figure 10.8. The cytoskeletal ultrastructure of hake was studied by Pagano et al. (2005) after depleting the thick and thin filaments with a potassium iodide treatment. TEM and SEM studies demonstrated an extensive network of filaments connecting Z structures that were regularly spaced and connected by sets of longitudinal, continuous, and roughly parallel filaments (Figure 10.9).

Microstructure ◾

147

p m m

30 μm

10 μm

(A)

(B) m

c M

m A Z (C)

(D)

Figure 10.7 Herring tissue. (A and B) LM, (C and D) TEM. P, perymisial connective tissue; m, myofibrils in a “palisade” ringing the edge; Z, Z disks; c, costameres; A, A band; I, I band; M, M line.

100 μm

Figure 10.8 Hake tissue observed by SEM.

148 ◾


LZ

Z

IZ DZ

8.000 X

Figure 10.9 Cytoskeletal structure of hake observed by TEM. Z, Z-disk; LZ, longitudinal filament connecting Z-Z. (Reprinted from Pagano, M.R. et al., Com. Biochem. Physiol. B, 141, 13, 2005. With permission.)

10.3 Processed Fish Microstructure 10.3.1

Smoked Salmon

A cross section of a smoked salmon sample obtained using the Cryo-SEM technique is seen in Figure 10.10A. The micrograph shows geometrically shaped fibers surrounded by a connective tissue. Figure 10.10B shows a detail of an intercellular space created by the conjunction of three fibers or cells. Sigurgisladottir et al. (2000) used LM to observe the changes that occurred in the salmon during the smoking process and quantified them by image analysis. The data of the average cross-sectional area of muscle fibers showed that the smoking process produces shrinkage of the fibers; the higher the smoking temperature, the greater the shrinkage. The fiber shrinkage and the space between the fibers both increased to a greater extent in the muscle from the salmon that were frozen before smoking than in muscle smoked from fresh salmon. Gudmundsson and

(A)

Figure 10.10

100 μm

(B)

Smoked salmon. (A and B) Cryo-SEM.

10 μm


Hafsteinsson (2001) studied the effect of pulsed electric fields (PEF) and a combination of PEF and high pressure on smoked salmon microstructure; these treatments decreased the cell size compared with fresh salmon, and gaps were formed in the tissue structure. A combination of PEF and high pressure had a more detrimental effect on smoked salmon microstructure than PEF treatment alone.

10.3.2 Salted Cod The presence of salt deposits in the cod tissue can be observed by SEM (Figure 10.11A) in samples that have been obtained using physical fi xing (freeze-drying) instead of chemical fi xing. Figure 10.11A shows a longitudinal section of salted cod, where two fibers can be observed completely covered by salt deposits. Figure 10.11B shows a cross section of salted cod tissue

(A)

Figure 10.11

100 μm

(B)

300 μm

Salted cod. (A) SEM, (B) Cryo-SEM.

(A)

(B)

(C)

Figure 10.12 Seafood stick (surimi) observed by SEM. (A) longitudinal section, (B) cross section, and (C) protein network.

150

◾


observed by Cryo-SEM, where the presence of salt makes the etching of the sample for observation difficult and masks the underlying structures.

10.3.3 Surimi One of the most common surimi products on the market is artificial crab muscle. Such a product is often sold as “crab sticks” or “seafood sticks.” Lean fish meat is minced to a paste; after adding different additives, the paste is shaped and an “artificial fish muscle” is obtained. Figure 10.12A, obtained by SEM, shows a longitudinal section of a crab stick where the “artificial fibers” can be observed. The cross section (Figure 10.12B) shows the typical concentric layers of this type of surimi product. The formation of a new network with the myofibrillar protein (Figure 10.12C) is responsible for the water-holding capacity and functional properOuter lining Outer tunic Muscle tunic Inner tunic Visceral lining Radial fibers

Circumferential fibers

(A) Radial fibers

Circumferential fibers (B)

Figure 10.13 Schematic representation of (A) squid mantle and (B) arrangement of muscle cells. (From Lluch, M.A. et al., The Chemical and Functional Properties of Food Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 2001. With permission.)

Microstructure ◾ 151 –200 μm

(A) 10 μ

–10 μm

(B) 3.75 μ

m

l

s (C)

(D)

Figure 10.14 Squid. (A and B) SEM, (C) LM, and (D) TEM. s, central sarcoplasm; m, myofibril; l, sarcolemma. (From Llorca, E. et al., Eur. Food Res. Technol., 225, 807, 2007. With permission.)

ties of surimi. Th is gel network structure gives surimi its characteristic elasticity and texture (Sikorski, 1990).

10.4

Squid Microstructure

The squid mantle is composed of muscle tissue sandwiched between two tunics of connective tissue (Figure 10.13). The inner and outer tunics are covered by a visceral lining and outer lining, respectively. Muscle fibers are grouped in bands that are arranged orthogonally. Circumferential muscle bands (100–200 mm) comprise fibers running about the entire circumference of the mantle cone. Radial bands (10–15 mm thick) comprise fibers that connect two tunics of connective tissue. Regardless of their orientation, all the muscle fibers are thin, approximately 3.5 mm in diameter (Lluch et al., 2001). The fibers arranged in circumferential and radial bands were observed by SEM in samples fi xed with glutaraldehyde (Figure 10.14A and B) (Llorca et al., 2001). This fiber distribution can also be observed by LM in samples stained with toluidine blue (Figure 10.14C). LM makes it possible to distinguish a peripheral area in blue and a central core in white inside each cell. When TEM is used to study the ultrastructure of fresh squid (Figure 10.14D), a central sarcoplasm is shown to be surrounded by myofibrils; the intermyofibrillar spaces between these can be observed. Each fiber is surrounded by a sarcolemma (Llorca et al., 2007).

References Gudmundsson, M. and Hafsteinsson, H., Effect of electric field pulses on microstructure of muscle foods and roes, Trends Food Sci. Tech., 12, 122–128, 2001. Lampila, L.E., Comparative microstructure of red meat, poultry and fish muscle, J. Muscle Foods, 1, 247–267, 1990.

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Larrea, V., Pérez-Munuera, I., Hernando, I., Quiles, A., Llorca, E., and Lluch, M.A., Microstructural changes in Teruel dry-cured ham during processing, Meat Sci., 76, 574–582, 2007. Llorca, E., Hernando, I., Pérez-Munuera, I., Fiszman, S.M., and Lluch, M.A., Effect of frying on the microstructure of frozen battered squid rings, Eur. Food Res. Technol., 213(6), 448–455, 2001. Llorca, E., Hernando, I., Pérez-Munuera, I., Quiles, A., Larrea, V., and Lluch, M.A., Protein breakdown during the preparation of frozen batter-coated squid rings, Eur. Food Res. Technol., 225(5–6), 807–813, 2007. Lluch, M.A., Pérez-Munuera, I., and Hernando, I., Proteins in food structures, in The Chemical and Functional Properties of Food Proteins, Sikorski, Z.E. (Ed.), Technomic Publishing Co., Inc., Lancaster, PA, 2001, chap. 2. Ofstad, R., Olsen, R.L., Taylor, R., and Hanneson, K.O., Breakdown of intramuscular connective tissue in cod (Gadus morhua L.) and spotted wolfish (Anarhichas minor O.) related to gaping, Lebens. Wiss. Tech., 39, 1143–1154, 2006. Pérez-Munuera, I., Larrea, V., Quiles, A., and Lluch, M.A., Microstructure, in Handbook of Muscle Foods Analysis, Nollet, L. and Toldrá, F. (Eds.), CRC Press, Boca Raton, FL, 2008. Pagano, M.R., Paredi, M.E., and Crupkin, M., Cytoskeletal ultrastructure and lipid composition of I-Z-I fraction in muscle from pre- and post spawned female hake (Merluccius hubbsi), Com. Biochem. Physiol. Part B 141, 13–21, 2005. Sigurgisladottir, S., Ingvarsdottir, H., Torrisen, O.J., Cardinal, M., and Hafsteinsson, H., Effects of freezing/ thawing on the microstructure and the texture of smoked Atlantic salmon (Salmo salar), Food Res. Int., 33, 857–865, 2000. Sikorsi, Z.E., Seafood: Resources, Nutritional Composition and Preservation. CRC Press, Boca Raton, FL, 1990, chap. 1.

Chapter 11

Chemical Sensors Corrado Di Natale Contents 11.1 Introduction ..................................................................................................................153 11.2 Sensor Parameters..........................................................................................................154 11.3 Chemical Sensor Technologies ......................................................................................157 11.3.1 Sensors Based on Conductance Changes ..........................................................157 11.3.1.1 Metal-Oxide Semiconductors ..........................................................157 11.3.1.2 Conducting Polymers and Molecular Aggregates .............................158 11.3.2 Amperometric Gas Sensors ..............................................................................158 11.3.3 Mass Transducers ............................................................................................158 11.3.4 Field-Effect Transistors ....................................................................................159 11.3.5 Color Indicators ...............................................................................................159 11.4 Electronic Noses ............................................................................................................160 11.5 The Application of Electronic Noses for Fish Freshness and Quality Measurement ...................................................................................................160 11.6 Conclusions ...................................................................................................................164 References ................................................................................................................................165

11.1

Introduction

Among the thousands of molecules composing food complex mixtures, some are of great importance to define overall properties such as freshness or quality [1]. The relationship between chemistry and food properties is particularly interesting in the case of fish and seafood in general, for which the human perception of airborne chemicals, called odor, is one of the most used method to assess freshness by both consumers and industries [2]. For these reasons the knowledge of the chemical 153

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profile of food is considered of great value, and the development of rapid and reliable chemical analyzers has been pursued since decades. Chemical analysis of foodstuff is a large part of the analytical chemistry discipline, and a number of methods and protocols for different food are available. Analytical chemistry is naturally based on “separation” approaches: namely, it develops methods to decompose complex mixtures (foods contains thousands of different molecules) in order to target either a single molecular species or a molecular family. These methods require in some cases complex sample treatments and instrumentation such as gas chromatography or spectrophotometers. On the other hand, it is known that Nature provides living beings chemical senses that, in order to be reliable, do not require any sample treatment. Differently from analytical instrumentations, natural senses are not analytical, in the sense that the interaction of human senses with complex mixtures provides a global perception rather than a list of compounds. Global perceptions may be enough in many cases to detect freshness or edibility, and ultimately they are of paramount importance to determine the acceptance of foodstuff [3]. A sort of combination of natural and analytical approaches has been pursued since decades, and it resulted in a class of chemical analyzers that have the advantage of interacting directly with samples and of providing signals bearing the notion of the chemical composition of a sample being a liquid or gas. These analyzers are chemical sensors. In the rest of this chapter an overview of the technologies related to these devices is provided together with examples of their use for fish freshness and quality determination.

11.2

Sensor Parameters

A sensor is an electronic device whose parameters depend on some external quantity of whatever nature [4]. As an example, according to this definition there are resistors whose resistance is a function of external temperature (thermistors) or diodes whose current–voltage relationship is strongly altered once they are illuminated by light (photodiodes). In the same way there are devices that from the electronic point of view are resistors, capacitors, or even field effect transistors, whose electrical parameters may depend on the chemical composition of the environment in which they are in contact. Electronic properties of materials may hardly be directly influenced by the ambiental chemistry; in order to achieve chemical sensors, a complex structure is necessary. Figure 11.1 shows what can be considered as the general structure of a chemical sensor. The device is composed of two parts. The first is a chemically interactive material, namely a solid-state layer of molecules that can interact with the molecules in the environment. These interactions can be of different nature, and the more utilized are adsorption and reaction phenomena. The interaction with a target molecule (hereafter called analyte) and a solid-state layer is a chemical event that, as a consequence, can modify the physical properties of the sensing layer. Properties such as conductivity, work function, mass, or optical absorbance are among those that can be transduced into an electric signal by suitable transducers. These transducers are the second component of a chemical sensor, and they are sometimes called “basic devices.” The matching between sensitive material and transducer is not univocal: a single sensitive material can be coupled with many different transducers and vice versa. In practice, there are many possibilities of assembling a chemical sensor. The optimal matching between a sensitive layer and transducer is fundamental to achieving a well-performing sensor.

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ΔT

Δm

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Δn

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Δv

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Figure 11.1 Schematic representation of a generic chemical sensor. Targeted molecules interact with a chemically interactive material. As a consequence of the interaction, one or more physical properties of the interactive material change. These quantities can be the temperature (DT), mass (Dm), electric conductance (Ds), refraction index (Dn), or work function (DF). For each, and many others, of these quantities, there are a number of devices that, once properly connected in an electric circuit, provide an electrical signal that is a function of the quantity of interactions occurring at the interface between the sensor and the environment.

Before illustrating the technological basis of chemical sensors, it is important to introduce the fundamental parameters that allow a correct interpretation of the performance of any sensor. These parameters are sensitivity, resolution, and selectivity. The fundamental action of a chemical sensor is the conversion of the information about the concentration of a chemical species into an electric signal. The relationship between the signal and the chemical concentration can be represented by an analytical function that embodies the sensor operation. V = f (C ) where V is a generic signal C is the analyte concentration The knowledge of the response function is necessary to estimate from the sensor signal the amount of concentration. This estimation is straightforward if the response function is linear, and in more general cases, the estimation may require the solution of a nonlinear equation. Besides response function, other important quantities are necessary to be known to appreciate sensor performances [5]. One of these quantities is sensitivity. The sensitivity expresses the capability of a sensor to modify its signal as a consequence of a change in concentration. Analytically, it is the first derivative of the response function S=

dV df (C ) = dC dC

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Only in case of a linear response function, the sensitivity is a constant quantity. In all the other cases, it is a function of the concentration. Let us consider the generic case of a chemical sensor based on a sensitive material characterized by a limited number of adsorption sites. The amount of adsorbed molecules as a function of the concentration is ruled by the Langmuir isotherm [6]. The response curve is almost linear at low concentration and tends to saturation corresponding to the complete occupation of available adsorption sites. A sensor containing such a sensing material and a basic transducer simply providing a signal proportional to the number of adsorbed molecules is represented by the response curve shown in Figure 11.2a. In Figure 11.2b the corresponding sensitivity is shown. The sensitivity is larger at low concentrations, and it tends gradually to zero as the sensor response reaches saturation. In order to fully appreciate the importance of sensitivity, it is necessary to evaluate the influence of measurement errors. The knowledge of the signal V is affected by an error and this error is propagated in an error on the estimation of the concentration. Simple mathematical considerations lead to the conclusion that given an error ΔVerr affecting the signal V, the error ΔC on the estimated concentration is given by the following relationship: ΔC =

ΔVerr S

The error in concentration is then inversely proportional to the sensitivity. It is worth mentioning that in case of electrical signals, the error ΔV is limited by the electronic noise that determines the ultimate uncertainty of any electric signal. The previously mentioned quantities are totally general, and their importance holds for any kind of sensor. For chemical sensors, an additional parameter of great importance is selectivity. Selectivity defines the capability of a sensor to be sensitive only to one quantity rejecting all the others. In the case of physical sensors, the number of quantities is limited to a dozen, and the selectivity can be achieved in many practical applications. For chemical sensors, it is important to consider that the number of chemical compounds is in millions and that the structural differences among them may be extremely subtle. With these conditions, the selectivity of a chemical sensor can be obtained only in very limited conditions. Lack of selectivity means that the sensor responds with comparable intensity to different species, and with such a sensor, it is not possible to deduce

Saturation

Sensitivity

Signal

Nonlinear region

Linear region Concentration

Concentration

Figure 11.2 Typical response curve (left) and sensitivity (right) of a generic chemical sensor based on adsorption of target molecules in a sensing layer characterized by a limited amount of adsorption sites.

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any reliable information about the chemical composition of the measured sample. Selectivity will be reconsidered in Section 11.4.

11.3

Chemical Sensor Technologies

In this section the basic principles of the most popular categories of chemical sensors are illustrated. Sensors are here classified according to the physical intermediate quantity.

11.3.1 Sensors Based on Conductance Changes 11.3.1.1 Metal-Oxide Semiconductors Changes in conductance become appreciable in materials characterized by a limited number of mobile charge carriers. In practical, semiconductors are subjected to large changes of conductance also in the presence of a modest variation in the number of conductance electrons or holes. The most popular materials undergoing a conductance change on interaction with gases are metaloxide semiconductors. These are oxides of transition metals, the most known and studied of which is SnO2 [7]: a wide band gap n-type semiconductor. The main sensitivity mechanism is related to the role played by oxygen. At sufficiently high temperature (above 200°C), dissociative adsorption sites of molecular oxygen are active on the oxide surface. A charge transfer occurs between the material and the adsorbed oxygen atom with the consequence that the conductance band in proximity of the surface becomes depleted, and a surface potential barrier is formed. The amount of depletion and the barrier height are proportional to the number of adsorbed molecules. Since the material is a semiconductor, the number of conductance electrons is limited, and then the amount of oxygen molecules that can be adsorbed at the surface is also limited. The consequence of the exposure to oxygen is a reduction of the surface conductance. The exposure to any molecule interacting on the sensor surface with adsorbed oxygen atoms may result in a release of electrons back to the conductance band, a reduction of the surface conductance band depletion, and a lowering of the potential barrier. Paradigmatic, in this regard, is the case of carbon monoxide, which reacts with the bounded oxygen to form carbon dioxide, releasing an electron back to the conductance band. This is only one of the many interactions taking place on the surface of metal oxides, and the sensitivity of these devices is extended to many different kinds of volatile compounds [8]. The sensitivity can be further modified adding ultrathin amounts of noble catalytic metal atoms on the surface. It is important to remark that this kind of sensors needs to be operated at high temperature, and as a consequence, an electrically actuated heater is integrated in the device. Metal-oxide semiconductor sensors can be prepared in many different ways; in any case, the general advice is to produce a nanocrystalline material in such a way that the modulation of the surface conductance band population becomes dominant in the whole sensor, providing the maximum sensitivity. Recently, metal oxide growth in regular shapes such as nanosized belts [9] has shown peculiar properties. The characteristics of these structures, although interesting, have not yet resulted in practical improvements of performances. Metal oxide semiconductor chemoresistors have been used several times in fish freshness applications. For instance, the sensitivity to trimethylamine and dimethylamine of aluminum-doped ZnO films was demonstrated [10] as well as the sensitivity to trimethylamine of SnO2 and CuO [11,12].

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11.3.1.2 Conducting Polymers and Molecular Aggregates The conductance properties of organic materials based either on polymers or on molecular aggregates have been studied since several years, with broader scopes related to the possibility of developing a novel sort of electronics based on carbon chemistry [13]. Chemical sensors based on conducting polymers may be considered as a lateral result of these studies. Indeed, aggregates of polypyrrole or polythiophene have a semiconducting character, and their conductance can change after exposure to volatile compounds. With respect to metal oxides these sensors have two important advantages: they are operated at room temperature and, most important, their chemical sensitivity can be changed at synthesis level modifying the chemical structure of the monomer [14]. Thanks to this versatility, conducting polymers sensors can be prepared for different applications, and food freshness is among them [15]. One of the drawbacks of these sensors is the instability mainly due to the degradation of doping radicals that are added to increase the conductance.

11.3.2 Amperometric Gas Sensors Electrolytic cells based on either solid-state or liquid-ionic conductors are used to detect several kinds of gases. The main mechanism is the catalytic reaction occurring on the surface of a noble metal electrode. Although designed for polluting gases, these sensors demonstrated a good sensitivity for compounds relevant for fish freshness. For instance, sensors designed for CO are found to be sensitive toward alcohols, aldehydes, and esters; a sensor for ammonia can detect amines; and a sensor for SO2 can detect volatile sulfides. Due these cross-selectivities, these sensors were properly used to detect fish freshness [16].

11.3.3 Mass Transducers The adsorption of molecules into a sorbent layer produces a change of mass; the measurement of these mass shifts can allow the evaluation of the amount of adsorbed molecules. The measurement of small mass changes is made possible by piezoelectric resonators. A piezoelectric resonator is a piece of piezoelectric crystal properly cut along a well-specified crystalline axis. Due to the piezoelectric effect, the mechanical resonance of the crystal is coupled with an electric resonance. Since crystal resonance is extremely efficient, the electric resonance is characterized by a very large quality factor (Q). This property is largely exploited in electronics to build stable oscillators as clock references. The same effect is exploited for chemical sensing adopting particularly shaped crystals such as in quartz microbalances (QMB). These are thin slabs of AT cut quartz oscillating at a frequency between 5 and 50 MHz approximately [17]. The frequency of the mechanical oscillation decreases almost linearly with the mass gravitating onto the quartz surface. If the quartz is connected to an oscillator circuit, the electric frequency decreases linearly with the mass. A typical QMB has a limit of detection around 1 ng, an amount that is sufficient in many practical applications. QMB coated by sensitive layers was used for many applications. As an example, the possibility of using these sensors to measure fish freshness was demonstrated with metalloporphyrin coating [18]. Piezoelectric effect can also be exploited in other configurations such as those based on surface acoustic waves. More sophisticated mass transducers were proposed by using resonant cantilevers similar to those adopted in atomic force microscopy [19]. In spite of the claimed properties, these sensors were never demonstrated in practical applications.

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Field-Effect Transistors

Most of the properties of field-effect transistors (FET) depend on the difference between the work function of electrons in the metal gate and in the semiconductor. This difference can be modulated by a layer of electric dipoles that can reach the metal–oxide interface. The principle was adequately exploited with a palladium gate FET exposed to hydrogen gas [20]. H2 molecules dissociate into atomic hydrogen at the palladium surface, and hydrogen atoms can diffuse through the palladium film until they reach the oxide surface, where they form an ordered dipoles layer. As a result, although under constant bias, the current flowing in the FET changes revealing the chemical interaction. This basic structure was successively modified changing the gate metal and thickness to extend the range of measured gases. In this way sensitivity to ammonia, an important gas for fish freshness and quality, was also obtained [21]. FET structures were also modified to accommodate, as a sensing part, organic molecular layers, such as metalloporphyrins [22], whose sensitivity toward amine was also recently measured [23].

11.3.5 Color Indicators Although known for several years [24], the colorimetric detection of fish freshness recently received a novel interest. In particular, the importance of amines as spoilage markers leads to consider their reducing role and then the possibility to detect them with functional layers sensitive to pH changes. The feasibility of this approach has been demonstrated using as sensitive layer a film of a sodium salt (bromocresol green) [25]. This salt exhibits a rather large change in color, also appreciable by eye. Nonetheless, the use of pH indicators is limited by the fact that mainly amines are considered (limiting the detection not to freshness but rather to spoilage), and, furthermore, the visual determination limits the performance and may greatly vary between individuals. Chemical sensing based on optical sensitive layers is a captivating strategy due to the strong influence of target chemicals on the absorption and fluorescence spectra of chosen indicators [26]. Nonetheless, the chemical practice of this approach is badly balanced by the transducer counterpart. Indeed, standard optical instrumentations are usually expensive. On the other hand, in the last decade we have seen rapid growth in performance in fields such as consumer electronics, giving rise to a number of low-cost advanced optical equipments such as digital scanners, cameras, and screens, whose characteristics largely fit the requirements necessary to capture change in optical properties of sensitive layers in many practical applications. The first demonstration in this direction was given by Suslick and colleagues when they showed that a digital scanner has enough sensitivity to detect the color changes in chemical dyes due to the adsorption of volatile compounds [27]. The method demonstrated also the possibility to identify a number of different amines [28]. Furthermore, Lundström and Filippini proved that it is possible to assemble a sort of spectrophotometer using the computer screen monitor as a programmable source and a web camera as detector [29]. This last technique, known as computer screen photo assisted technique (CSPT), is based on the fact that a computer screen can be easily programmed to display millions of colors, combining wavelengths in the optical range. Compared with the use of digital scanners, to probe the sample with a variable combination of wavelengths instead of using the white light of scanners gives the possibility of performing an optical fingerprint measurement, allowing a simultaneous evaluation of absorbance and fluorescence of samples. Due to the large diffusion of portable computers, PDAs, and cellular phones all endowed with color screen,

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camera, and an even more extended computation capabilities, the application of the CSPT concept may be foreseen as greatly extending the analytical capacity worldwide. CSPT has demonstrated its utility in particular to classify airborne chemicals reading absorbance and fluorescence changes in chemical dyes such as metalloporphyrins [30]. Standard optochemical sensors are based either on absorbance or on fluorescence, whereas CSPT arrangement gives the possibility of evaluating at the same time both the effects.

11.4 Electronic Noses As discussed above, the lack of selectivity of many chemical sensors was considered as one of the main problems limiting their diff usion for practical applications. Nonetheless, observation of Nature offered a useful suggestion about the use of such devices. Investigations about olfaction receptors show that Nature strategies for odor recognition are completely different from those of analytical chemistry. The physiology of olfaction has made considerable advances, models of receptor mechanisms explaining the sensitivity to volatile compounds are now available, and the genes expressed by olfactive receptors are known [31]. Recent studies are also beginning to unveil the signal pathways leading from the generation of olfactory neuron signal to the conscious identification of odors [32]. Receptors were found to be rather unselective; each receptor senses several kinds of molecules, and each molecule is sensed by many receptors [33]. After this discovery, it was proposed that arrays of nonselective chemical sensors may show properties similar to those of natural olfaction [34]. After this conjecture, the possibility of developing artificial olfaction systems became possible, and such systems were soon dubbed as “electronic noses.” This denomination is currently given to any array of unselective chemical sensor coupled with some multicomponent classifier. Since the 1980s, almost all sensor technologies were used to build such systems. Odor classification properties of artificial systems were tested on several different fields proving that electronic noses could be in principle used to replace human olfaction in practical applications such as food quality and medical diagnosis [35]. The features of electronic noses are fundamentally dependent on the sensing properties of the artificial receptors. The possibility having some versatile tool to tailor the sensitivity and selectivity of sensors is of primary importance to make arrays capable of capturing either large or narrow ranges of chemicals, allowing for electronic nose application oriented optimizations. To this point of view, organic synthetic receptors offer an unlimited number of possibilities to assemble molecules endowed with differentiated sensing features.

11.5 The Application of Electronic Noses for Fish Freshness and Quality Measurement The composition of fish headspace is a source of information about its freshness. Previous investigations evidenced that the headspace composition is a result of the balance between the “fresh fish” odor and the microbial spoilage produced compounds [36]. The most important chemicals involved in the fresh fish odor are long-chain alcohols and carbonyls, bromophenols, and N-cyclic compounds. Their concentration and the presence of other compounds are rather typical of each species. On the other side, microbial spoilage produces short-chain alcohols and carbonyls, amines, sulfur compounds, and aromatic, N-cyclic, and acid compounds. The concentrations of these chemicals are directly correlated to the degree of spoilage. Among these compounds amines

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are considered as the typical markers for fish freshness detection. Standard analytical methods for volatile amines and also sensors for some specific amines have been used to inspect fish freshness. Nevertheless, amines become instrumentally appreciable only when spoilage processes take place. Minor contributions to the fish headspace come from contamination of the environment (e.g., petroleum in sea), from fish processing, and finally from products of lipid oxidation [37]. The number of compounds whose concentrations are only partially correlated makes this application particularly appealing for sensor arrays of partially selective chemical sensors. Such sensor arrangement consists in the application of a number of sensors characterized by a broad sensitivity toward species that are relevant for a certain application. When properly analyzed by pattern recognition methods, the data produced by a sensor array can classify samples according to some of their global features; in case of fishes, typically according to the freshness or more precisely according to the balance between fresh and spoilage produced compounds. In recent years attempts to use electronic nose technology to track the spoilage processes occurring in fishes have been reported in numerous articles. Most of these are feasibility studies, showing the ability of the electronic nose to track the different spoilage levels occurring at different storage times. Instruments based on different sensor technologies have been used, such as metal-oxide chemoresistor sensors [38–40], MOSFET sensors [41], amperometric sensors [42], conducting polymer sensors [43], quartz microbalance sensors [44,45], and optical indicators [46]. In addition, hybrid electronic noses were used combining different sensor technologies such as QMB and amperometric sensors [47]. In order to understand the potential of electronic noses to detect fish freshness, let us discuss a simulation of a case study. In Figure 11.3 the time evolution of the major families of volatile compounds found in the headspace of fishes is shown. Data are extrapolated from an investigation by Strachan and Nicholson [48]. Let us consider the use of an array of sensors absolutely selective for each individual family of compounds mentioned in Figure 11.3. Each sensor then provides a signal proportional to the concentration of each family. Sensor data can be conveniently represented by a principal component analysis (PCA) scores plot. PCA is a data analysis method allowing the representation of a multidimensional dataset in a reduced dimensionality space, for example, a plane. The representation plane is determined as that where the data variance is maximized and then the statistical properties of the dataset are, as much as possible, preserved [49]. Results shown in Figure 11.4 demonstrate a continuous progress after the 8th day, but the behavior at the beginning is absolutely nonlinear, with a super impression of 6th and 1st days. Apparently, with an array of selective sensors it is not possible to distinguish between fresh and flat fishes. The sensitivity of chemical sensors is not immediately related to the molecular family but rather to the interaction mechanism. In this regard, it is more realistic to consider an array of sensors specific for a single interaction mechanism. An imperfect application of this method was demonstrated with engineered polymer-coated QMB [50]. In gas chromatography, the interaction between polymers and volatile compounds is often described by the linear sorption energy relationship (LSER) model [51]. In LSER, five different kinds of interactions are considered: dispersion, polarity, dipolarity, hydrogen bond basic, and acid. Since LSER was fruitfully used to model polymer-based chemical sensors [52], let us consider an array specific for each LSER interaction and one compound for family. Analyzing the data with PCA the plot of Figure 11.5 is obtained. As a result, the progress of spoilage is less linear with respect to Figure 11.4, and fresh, flat, and sweet conditions are hardly identified. This result is rather surprising because fish spoilage is in general expected to be a linear and somewhat straightforward process; nonetheless, the chemical complexity of the problem, the accumulation of some compound, and the decrease in others result in a nonlinear problem. The same nonlinearity is observed with electronic noses; Figure 11.6

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Amines Aromatics Fresh fish alcohols Fresh fish carbonyls Short-chain alcohols Sulfides

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Figure 11.3 Time evolution of the major families of volatile compounds in fish headspace. The typical sensorial description is also reported.

shows the scores plot of a partial least-squares discriminant analysis model related to an array of metalloporphyrin-coated QMBs. The experiment was related to COD fishes, and original data were previously published [53]. Results are qualitatively similar to those shown in Figure 11.5, with a folding back of the spoilage process in the representation plane. This feature that can be interpreted as a failure of the electronic nose is likely due to an intrinsic nonlinearity of the studied problem. Nonetheless, humans provide a more reliable identification of fish freshness. It is important to consider that sensorial methods of freshness appraisal involve the use of sight (to evaluate the skin appearance and the color and the global aspect of eyes), tactile (to test the flesh firmness and elasticity), and olfaction (to smell the gill odor) [54], and the use of only one sense (e.g., olfaction) provides several errors of evaluation. As a consequence, in order to measure the quality of fish instrumentally, an integration of instruments is necessary, each able to capture different aspects of fishes. The fusion of multi-instrumental information can then be treated as the descriptors provided by a trained panel providing a sort of artificial quality index [55]. The possibility of developing a multisensor device to measure and/or estimate fish freshness with a combination of instrumental techniques (electronic noses, spectroscopic methods, texture meters, image analyzers, color meters, and devices measuring electrical properties) has been illustrated in different applications related to cods [56,57], sardines [58], and groupies [59].

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Scores plot 2 30 1.5 2 4 6

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Figure 11.4 PCA scores plot of a simulated experiment where sensors selective for the compound family in Figure 11.3 are used. Data show the impossibility of distinguishing the spoilage process in the first 6 days and an abrupt change between 6th and 8th days. Scores plot 1.5 10 12

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Figure 11.5 PCA scores plot of data related to a virtual array of sensors, each specific for a single interaction mechanism among those modeled by LSER.

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Handbook of Seafood and Seafood Products Analysis Scores plot

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Figure 11.6 PCA score plot of metalloporphyrin-coated QMB; data are related to cod fish fillets, and labels indicate the storage days in ice.

11.6

Conclusions

The conversion of chemical information into electric signals that can be measured, stored, analyzed, transmitted and integrated with other data can be performed by several different technologies. These technologies are sometimes equivalent in terms of performances, and for some specific applications, one technology may outperform the others. It is important in any application to design the optimal sensor array to determine quality and quantity of the relevant chemical species and to select sensors optimizing sensitivity and resolution. In this regard, the application of arrays of sensors can greatly improve the performance in terms of prediction of quality and freshness. Chemical sensors are an almost mature technology for many practical applications. In the case of fish and seafood freshness and quality determination, all the actors of the food chain (producers, processors, and consumers) are potential users of chemical sensor technology. Each step of the food chain has peculiar needs that a proper chemical sensor approach can in principle contribute to satisfy. As an example, at producer level the increment in quality and yield, at processors level the screening of quality of incoming products to optimize the processing and to sort processed food, and finally at consumer level, the control of quality and safety both on the market and at home. All these applications require instruments able to work on-site. Food-related sites are usually highly contaminated from the point of view of odor. At the current state of the art, sensors are

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not able to distinguish between background and relevant odor, so that the performance of the sampling of an application is difficult. From this perspective, portable systems without any conditioning of sample are of limited use for fish inspection. Let us imagine, for instance, measuring the odor of a fish in a typical storage room among dozens of stacks of fish crates. On the other hand, there are applications, interesting at industrial level, where existing chemical sensors can be specialized, in terms of sampling and data presentations, in order to fulfill user requirement. For this a strong cooperation between sensor developers and end users is necessary in order to optimize practical solutions. At this level a correct and careful analysis of user needs and expectations and an education effort toward the users are important to disseminate the intrinsic novelty carried by sensor systems such as those widely belonging to the class of artificial olfaction. It is also important that developers and users are aware of the intrinsic limit of information that is carried by the volatile part of a food. For instance, it is important to consider that sensory analysis is almost never confined to only olfactory perception. Actually, synesthetic action among the senses is required to form a full judgment over a certain food sample. As an example, in fish analysis, quality index, linearly correlated with the days in ice, is calculated considering at the same time visual, tactile, and olfactory perceptions. This suggests that to fully reproduce the perceptions of humans with artificial sensors, the electronic nose has to be compared and integrated with instruments providing information about visual aspects, texture, and firmness. This opens a further novel investigation direction involving again researchers from different areas, confirming that interdisciplinarity is the most strong added value for food analysis.

References 1. Coultate, T.P. Food: The Chemistry of its Components, RSC Press, Cambridge, U.K., 2002. 2. Olafsdottir, G. et al. Methods to evaluate fish freshness in research and industry, Trends Food Sci. Tech., 87, 258, 1997. 3. Bremner, H.A. Toward practical definitions of quality for food science, Crit. Rev. Food Sci., 40, 83, 2000. 4. Fraden, J. Handbook of Modern Sensors, AIP Press, New York, 2004. 5. D’Amico, A. and Di Natale, C. A contribution on some basic definitions of sensors properties, IEEE Sens. J., 1, 183, 2001. 6. Alberty, R., Physical Chemistry, John Wiley & Sons, New York, 1982. 7. Madou, M. and Morrison, S. Chemical Sensing with Solid State Devices, Academic Press, San Diego, CA, 1989. 8. Barsan, N., Koziej, D., and Weimar, U. Metal oxide based gas sensor research: How to? Sens. Actuators B, 121, 18, 2007. 9. Comini, E. Metal oxide nano crystals for gas sensing, Anal. Chim. Acta, 568, 28, 2006. 10. Roy, S. and Basu, S. ZnO thin film sensors for detecting dimethyl- and trimethyl-amine vapors, J. Mater. Sci. Mater. Electron., 15, 321, 2004. 11. Egashira, M., Shimizu, Y., and Takao, Y. Trimethylamine sensor based on semiconductive metaloxides for detection of fish freshness, Sens. Actuators B, 1, 108, 1990. 12. Hammond, J. et al. A semiconducting metal-oxide array for monitoring fish freshness, Sens. Actuators B, 84, 113, 2004. 13. Heeger, A.J. Semiconducting and metallic polymers (Nobel lecture), Angew. Chem. Int. Ed., 40, 2591, 2001. 14. Persaud, K. Polymers for chemical sensing, Mater. Today, 8, 38, 2005.

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15. Du, W.X. et al. Microbiological, sensory, and electronic nose evaluations of yellowfin tuna under various storage conditions, J. Food. Prot., 64, 2027, 2001. 16. Olafsdottir, G., Martinsdóttir, E., and Jónsson, E.H. Rapid gas sensor measurements to predict the freshness of capelin (Mallotus villosus). J. Agric. Food Chem., 45, 2654, 1997. 17. Ballantine, D.S. et al. Acoustic Wave Sensors, Academic Press, San Diego, CA, 1997. 18. Brunink, J. et al. The application of metalloporphyrins as coating material for quartz microbalance based chemical sensors, Anal. Chim. Acta, 325, 53, 1996. 19. Battiston, F.M. et al. A chemical sensor based on a microfabricated cantilever array with simultaneous resonance-frequency and bending readout, Sens. Actuators B, 77, 122, 2002. 20. Lundstrom, I. et al. A hydrogen sensitive MOS field effect transistor, Appl. Phys. Lett., 26, 55, 1975. 21. Winquist, F. et al. Modified palladium metal-oxide semiconductor structure with increased ammonia gas sensitivity, Appl. Phys. Lett., 43, 839, 1983. 22. Andersson, M. et al. Development of a ChemFET sensor with molecular films of porphyrins as sensitive layers, Sens. Actuators B, 77, 567, 2001. 23. Takulapalli, B. et al. Electrical detection of amine ligation to a metalloporphyrin via a hybrid SOIMOSFET, J. Am. Chem. Soc., 130, 2226, 2008. 24. Tozawa, H., Enokihara, K., and Amano, K. Proposed modification of dyer’s method for trimethylamine determination in cod fish, Technical Conference on Fish Inspection and Quality Control, Halifax (Canada), 15–25 July, 1969. 25. Paquit, A. et al. Development of a smart packaging for the monitoring of fish spoilage, Talanta, 102, 466, 2007. 26. Gauglitz, G. Optical sensing looks to new field. Trends Anal. Chem., 25, 748, 2006. 27. Rakow, N. and Suslick, K. A colorimetric sensor array for odour visualization, Nature, 406, 710, 108, 705, 2000. 28. Rakow et al. Molecular recognition and discrimination of amines with a colorimetric array, Angew. Chem. Int. Ed., 44, 4458, 2005. 29. Filippini, D., Svensson, S., and Lundström, I. Computer screen as a programmable light source for visible absorption characterization of (bio)chemical assays, Chem. Commun., 240, 2003. 30. Filippini, D. et al. Chemical sensing with familiar devices, Angew. Chem. Int. Ed., 45, 3800, 2006. 31. Buck, L. and Axel, R. A novel multigene family may encode odorant receptors: A molecular basis for odor recognition, Cell, 65, 175, 1991. 32. Friedrich, R.W. and Stopfer, M. Recent dynamics in olfactory population coding, Curr. Opin. Neurobiol., 11, 468, 2001. 33. Sicard, G. and Holley, A. Receptor cell responses to odorants: Similiarities and differences among odorants, Brain Res., 292, 283, 1984. 34. Persaud, K. and Dodds, G. Analysis of discrimination mechanisms in the mammalian olfactory system using a model nose, Nature, 299, 352, 1982. 35. Röck, F., Barsan, N., and Weimar, U. Electronic nose: Current status and future trends, Chem. Rev., 108, 705, 2008. 36. Josephson, D., Lindsay, R., and Olafsdottir, G. Measurement of volatile aroma constituents as a means for following sensory deterioration of fresh fish and fishery products; in Seafood Quality Determination Symposium, D. Kramer, L. Liston (eds.), 10–14, November 1986, Elsevier, Amsterdam, the Netherlands, 1986. 37. Ólafsdóttir, G. and Fleurence, J. Evaluation of fish freshness using volatile compounds: Classification of volatile compounds in fish, in Methods to Determine the Freshness of Fish in Research and Industry, Proceedings of the Final meeting of the Concerted Action “Evaluation of Fish Freshness” AIR3 CT94 2283. Nantes, November 12–14, 1997. International Institute of Refrigeration, 55–69. 38. Ólafsson, R. et al. Monitoring of fish freshness using tin oxide sensors, in Sensors and Sensory Systems for an Electronic Nose, Gardner, J.W., Bartlett, P.N., (eds.), Kluwer, Dordrecht, the Netherlands, 1992, p. 257.

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39. Schweizer-Berberich, P.M., Vaihinger, S., and Göpel, W. Characterisation of food freshness with sensor arrays, Sens. Actuators, 18, 282, 1994. 40. Ólafsdóttir, G. et al. Prediction of microbial and sensory quality of cold smoked Atlantic salmon (Salmo salar) by electronic nose, J. Food Sci., 70, 563, 2005. 41. Haugen, J.E. and Undeland, I. Lipid oxidation in herring fillets (Clupea harengus) during ice storage measured by a commercial hybrid gas-sensor array system, J. Agric. Food Chem., 51, 752, 2003. 42. Ólafsdóttir, G., Martinsdóttir, E., and Jónsson, E.H. Rapid gas sensor measurements to predict the freshness of capelin (Mallotus villosus), J. Agric. Food Chem., 45, 2654, 1997. 43. Du, W.X. et al. Potential application of the electronic nose for quality assessment of salmon fillets under various storage conditions, J. Food Sci., 67, 307, 2002. 44. Di Natale, C. et al. Recognition of fish storage time by a metalloporphyrins-coated QMB sensor array, Meas. Sci. Technol., 7, 1103, 1996. 45. Zhao, C.Z. et al. Assay of fish freshness using trimethylamine vapor probe based on a sensitive membrane on piezoelectric quartz crystal, Sens. Actuators B, 81, 218, 2002. 46. Alimelli, A. et al. Fish freshness detection by a computer screen photoassisted based gas sensor array. Anal. Chim. Acta, 582, 320, 2006. 47. Olafsdottir, G., Di Natale, C., and Macagnano, A. Measurements of quality of fish by electronic noses, in Quality of Fish from Catch to Consumer: Labeling, Monitoring and Traceability, Luten, J.B., Oehlenschlager, J., and Olafsdottir, G., (eds.), Wageningen Academic Publishers, Wageningen, the Netherlands. 2003, 225. 48. Strachan, N. and Nicholson, F. Gill air analysis as an indicator of cod freshness and spoilage, Int. J. Food Sci. Tech., 27, 261, 1992. 49. Johnson, R. and Wichern, D. Applied Multivariate Statistical Analysis, Prentice Hall Inc., Englewood Cliffs, NJ, 1982. 50. Hierlemann, A. et al. Polymer based sensor array and mulicomponent analysis for the detection of hazardous organic vapours in the environment, Sens. Actuators B, 26, 126, 1995. 51. Grate, J. and Abrahams, H. Solubility interactions and the design of chemically selective sorbent coatings for chemical sensors and arrays. Sens. Actuators B, 3, 85, 1991. 52. Houser, E. et al. Rational materials design of sorben coatings for explosives: Applications with chemical sensors, Talanta, 54, 469, 2001. 53. Di Natale, C. et al. Comparison and integration of different electronic noses for freshness evaluation of cod-fish fillets, Sens. Actuators B, 77, 572, 2001. 54. Luten, J.B. and Martinsdottir, E. QIM an European tool for fish freshness evaluation in the fishery chain, in Methods to Determine the Freshness of Fish in Research and Industry, Proceedings of the Final Meeting of the Concerted Action “Evaluation of Fish Freshness” AIR3 CT94 2283. Nantes, November 12–14, 1997. International Institute of Refrigeration, 287. 55. Di Natale, C. Data fusion in Mustec: Towards the definition of an artificial quality index, in Quality of Fish from Catch to Consumer: Labeling, Monitoring and Traceability, Luten, J.B., Oehlenschlager, J., and Olafsdottir, G., (eds.), Wageningen Academic Publishers, Wageningen, the Netherlands, 2003, 273. 56. Olafsdottir, G. et al. Multisensor for fish quality determination, Trends Food Sci. Technol., 15, 86, 2004. 57. Kent, M. et al. A new multivariate approach to the problem of fish quality estimation, Food Chem., 87, 531, 2004. 58. Macagnano, A. et al. A model to predict fish quality from instrumental features, Sens. Actuators B, 111, 293, 2005. 59. Di Natale, C. et al. Ubiquitous chemical sensing and optical imaging for ubiquitous environments, IEEE International Conference on Robotics and Automation, Rome, April 10–14, 2007.

Chapter 12

Physical Sensors and Techniques Ruth De los Reyes Cánovas, Pedro José Fito Suñer, Ana Andrés Grau, and Pedro Fito-Maupoey Contents 12.1 Sensors for Quality Assessment .....................................................................................170 12.2 The Importance of Quality Control—Advances in the Online Control Techniques ....................................................................................................................170 12.3 New Technologies for Online Control ..........................................................................171 12.3.1 Ultrasounds—Acoustic Spectroscopy ..............................................................172 12.3.2 Visible Spectroscopy .........................................................................................173 12.3.3 IR Spectroscopy ...............................................................................................173 12.3.4 RF Spectroscopy—Impedance Spectroscopy ...................................................174 12.3.5 Microwave Spectroscopy—Dielectric Spectroscopy .........................................174 12.3.6 Advantages and Benefits of Microwave Methods .............................................175 12.4 Overview of Microwave Theory .................................................................................... 176 12.5 Applications of Microwave Technology in the Assessment or the Control of Processes ...................................................................................................................179 12.5.1 Determination of Moisture Content ................................................................180 12.5.2 Freshness and Salting/Desalting Process Quality Control of Fish and Seafood, by Microwaves: Methods and Equipments ........................................182 12.6 Conclusions ...................................................................................................................184 References ................................................................................................................................184

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12.1 Sensors for Quality Assessment A food quality sensor is a device that can respond to some physical or chemical property or properties of food and transform the response(s) into a signal, often an electric signal. This signal provides direct information about the quality factor(s) to be measured or may have a known relation to the quality factors. Usually, sensors are classified according to their mode of use: online, at-line, or off-line. Online sensors operate directly in the process, and they give a real time signal, which relates to the quality factors. Thus, an online sensor has the advantage of giving an immediate quality measurement and provides possibilities for regulating the process by adjustments. At-line sensors are devices to be used for instance in split-flow measurements, requiring reagent additions or equilibrations/reaction times. They often have short-response times (minutes or seconds) and also allow process corrections. Off-line sensors are laboratory devices, responding within hours or days. Traditionally the on/at-line quality control was restricted to external properties (weight, size, color, etc.) that can be measured by a simple balance or by a sophisticated video camera, and the internal properties were determined off-line by destructive and time-consuming technologies. This chapter tries to show the increasing growth of new and efficient online and at-line control methods that can provide important information about the internal quality of foods, focusing on the seafood sector advances.

12.2

The Importance of Quality Control—Advances in the Online Control Techniques

Quality control is essential in the food industry, and efficient quality assurance is becoming increasingly important. Since consumers expect good shelf life and high-safety products with an adequate ratio of quality–price, the food industry is progressively investing more and more capital in quality control, research, and development, as well as in machinery for the separation of products by their varying degrees of quality (i.e., the calibration lines for fruit processing). However, quality in food products is very difficult to define. Consumers perceive the quality of a product on the basis of a feeling of satisfaction that some sensory properties produce in them, such as color, taste, ease of consumption, or flavor. Th is perception is used to choose the product one wishes to buy. In this way, different physical and chemical parameters related to the quality of foodstuffs have been selected [1]. The acquisition of these parameters that characterize the abstract concept of “quality perceived by the consumer” leads to the development of the necessary technology for application in the classification of products. Existing techniques in food quality assessment, either instrumental or sensory evaluation, can provide reliable information about food quality; however, these techniques are destructive, timeconsuming, and unsuitable for online application. Because of that, traditionally, the development of in-line calibrators was restricted to external properties (weight, size, etc.). This was due to the absence of nondestructive technologies that would allow the product classification by its properties (internal properties). Therefore, quality control in manufacturing lines was limited to destructive off-line analyses that determine the acceptance or disposal of much of the production of the day, as a result of not being able to perform online nondestructive measures that would correct the manufacturing process in real time.

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New analytical techniques have been (and they are still being) developed to study the quality of complex food materials and to monitor the properties of foods during processing; these techniques can provide new quality control systems of the internal (and external) properties of foods that act in real time and in a nondestructive way. This kind of system not only permit an assessment of quality in terms of their properties but also, with the appropriate hardware and software, allow input from the manufacturing line with information obtained from the measurement of quality parameters selected (feedback); therefore it is possible to apply to the product under development the necessary corrective measures while it is still in the manufacturing line. Thus, it is able to obtain a final product that will always be within the margins of quality predetermined. These systems will reach three milestones, the new sensors’ concept of being easy-to-use, an excellence in accuracy, and low cost in the sensor’s compounds. In addition to the requirements of consumers, food inspectors require good manufacturing practices, safety, labeling, and compliance with the regulations. In this way many new food safety concepts and key quality parameters have arisen during the last decade: Hazard analysis critical control points (HACCP), total quality management (TQM), ISO 9000 Certifications, traceability, and authentication all require improved control methods. In addition, they all call for intime and online sensors for control, new data systems, warning systems, tight feedback loops for automation of the production, and so forth. Further, food producers are increasingly asking for efficient control methods, in particular through online or at-line quality sensors, first to satisfy the consumer and regulatory requirements and second to improve the production feasibility, quality sorting, automation, and reduction of production cost and production time (increased throughputs). The great challenge is indeed to focus on the real time and online sensors and data systems surveying processes and products, controlling the automated process and the raw material stream, sensing the final product quality, and typing the product labels, nutritional and health information, and much more. Concretely, the safety and quality of fishery products has been of particular concern in recent years. With the increasing globalization of fishery product sales, processors, consumers, and regulatory officials have been seeking improved methods for determining freshness and quality [2]. It is necessary to stress that fish quality is a complex concept involving a whole range of factors, which for the consumer include, for example, safety, nutritional quality, availability, convenience and integrity, freshness, eating quality, the obvious physical attributes of the species, size, and product type [3,4]. A study performed by Consumers Union found that more than one-quarter of the fish samples tested were on the brink of spoilage [5]. Information about handling, processing, and storage techniques, including time/temperature histories that can affect the freshness and quality of the products, is very important for the partners in the chain. One of the most unique characteristics of fish as food is that it is a highly perishable commodity. Consequently, time passed after catch and the temperature “history” of fish are very often the key factor determining the final quality characteristics of a fish product [6].

12.3 New Technologies for Online Control The quality of almost all the industrial processes depends on the modification of a few parameters, which are commonly structural, physical, or chemical properties, such as water content for drying processes. In general, these properties need slow and destructive methods to be controlled, but online methods are required for industrial quality control.

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Given the premise that online control requires a nondestructive method, which, moreover, must act in real time and without producing permanent effects on the food; it is almost imperative to resort to elastic (sonic) waves such as ultrasounds or to nonionizing electromagnetic radiation, such as radio frequency (RF), microwave, thermal and near-infrared (NIR), and visible. The interaction between wave radiation and matter as a function of wavelength or frequency is called spectroscopy. The spectroscopic techniques use the information found in the spectrum that is emitted for the food to predict certain of its qualities. It is also necessary to work at very low power in order to not cause permanent effects such as heating. When these waves pass through foods (or are refracted by them), some of their propagation parameters are modified. Thanks to advancing technology, the modification of these parameters can be measured in real time, fulfilling the initial premise. Normally the modification of any quality parameter is macroscopically correlated to the change in any wave parameter that can be controlled; below are cited some examples of the use of these new technologies in the quality control of foodstuffs. Ultrasonic velocity in fish tissues, chicken, and raw meat mixtures can be related to its composition using semiempirical equations [7]. Visible (and near UV) transmittance method has been investigated to inspect the internal quality (freshness) of intact chicken egg [8]; NIR measurements are widely used in the food industry to determine the sugar content in fruits [9]; impedance measurements (RF) can determine salt and water content in salmon filets [10]; and dielectric measurements at microwave frequencies can be used to analyze water activity [11] and water content [12,13] in foodstuffs; The salt and water content are related to dielectric properties of cod at microwave frequencies [14–16]. It is impossible to address all these techniques with precision, the reason is, in this chapter, we concentrate on electromagnetic methods at microwave frequencies. Nevertheless, the other techniques that enable online control have been briefly commented on below, exposing their main disadvantages and highlighting the advances in the field of seafood.

12.3.1 Ultrasounds—Acoustic Spectroscopy Ultrasonic is a rapidly growing field of research, which is finding increasing use in the food industry for the analysis of food products. Ultrasound is a form of energy generated by sound (really pressure) waves of frequencies that are too high to be detected by human ear, i.e., above 16 kHz [17]. Ultrasound attenuation spectroscopy (acoustic spectroscopy) is a method for characterizing properties of fluids and dispersed particles. Ultrasound imaging is a versatile, well-established, and widely used diagnostic tool. This technique encompasses a wide range of imaging modes and techniques that use the interaction of sound waves with living tissues to produce an image of the tissues or, in the case of Doppler-based modes, determine the velocity of a moving tissue. Ultrasound, when propagated through a biological structure, induces compressions and depressions of the medium particles, and a high amount of energy can be imparted. Depending on the frequency used and the sound wave amplitude applied, a number of physical, chemical, and biochemical effects can be observed, which enable a variety of applications [18,19]. Highfrequency, low-energy diagnostic ultrasounds are used as a nondestructive analytical technique for quality assurance and process control with particular reference to physicochemical properties such as composition, structure, and physical state of foods [20]. For fish samples, Suvanich et al. [21] published a report on how the ultrasonic velocity measurements show potential for analyzing fish composition. The main disadvantage of ultrasound is that the energy propagates poorly through a gaseous medium. It is virtually impossible for


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ultrasound to pass through air; thus, ultrasound transducers must have airless contact with the sample during examinations [22]. This complicates the noncontact measurements.

12.3.2 Visible Spectroscopy In recent years, the usefulness of visible spectroscopy/near infrared spectroscopy (VS/NIRS) has been researched for many quality aspects [23–25]. This technique measures the reflectance of light from the product in the visible and NIR wavelength range; the visible spectrum is a function of the entire structure of the compound rather than specific bonds. Other information should be used in conjunction with visible spectra in determining the specific properties of interest. For example, the freshness of cod was estimated by Heia et al. [26] using the visible wavelengths only; the main disadvantage of this method is that only the surface of the sample is examined.

12.3.3 IR Spectroscopy In the recent years, NIR technology has been widely developed as an analytical tool. NIR spectroscopy is based on the absorption of electromagnetic radiation at wavelengths in the range 780–2500 nm. NIR spectra of foods comprise broad bands arising from overlapping absorptions corresponding mainly to overtones and combinations of vibration modes involving C–H, O–H, and N–H chemical bonds [27]. This makes it very feasible for measurements to be made in organic and biological systems. Focusing on fish products, Uddin et al. [28] applied NIR spectroscopy to assess the end point temperature (EPT) of heated fish and shellfish meats; a multispectral imaging NIR transflectance system was developed for online determination of moisture content in dried salted codfish [29]. A rapid, NIR spectroscopic method has been developed by Zhang and Lee [30] to directly determine free fatty acids (FFA) in fish oil and for the assessment of mackerel quality. All these techniques have been gradually implemented as monitoring systems in food processing [31], but their use is limited by their low penetration in the product (it depends on the wave length, but it is measured in terms of tenths of a millimeter [32] and is dependent on less-precise reference methods [27]. The most popular IR spectroscopy is the NIR one, but it is not the only one. Mid-infrared (MIR) and Raman spectroscopy have high structural selectivity and contain more of the type of information needed in structural elucidation studies. MIR spectroscopy concerns the region of the spectrum lying between 4,000 and 400 cm−1 (2,500–25,000 nm). When radiation with energy corresponding to the MIR range interacts with a molecule, the energy at defined frequencies can be partially absorbed. The region of the electromagnetic spectrum under consideration in Raman spectroscopy is similar to that in MIR, but it involves a scattering process. Raman spectroscopy is based on the shift of an excited incident beam of radiation that results from inelastic interactions between the photons and the sample molecules. In the fish sector, Karoui et al. [33] applied MIR spectroscopy combined with chemometric tools to determine whether fish has been frozen–thawed. Marquardt and Wold [34] concluded that Raman spectroscopy might be a useful tool for rapid and nondestructive analysis of fish quality. Most industrial processes require the measurement of temperature. The far IR, which is also called thermal infrared (TIR) refers to electromagnetic waves with a wavelength of between 3.5 and 20 micrometers, and it is able to provide thermal information. Thermal infrared imagers translate the energy transmitted in the infrared wavelength into data that can be processed

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into a visible light spectrum video display. Thermography (infrared; thermal scans) uses specially designed infrared video or still cameras to make images (called thermograms) that show surface heat variations. This technology has a number of applications, for example, recent studies conducted by Fito et al. [35] lay the groundwork for the use of TIR image for the control of the optimum drying time in a citrus line. Focusing on fish industry, Jacobsen and Pedersen [36] developed a method based on infrared measurement of temperature changes in cold-water prawns during the glazing process studied in a small-scale controlled experiment. The method is thus remote and physically based on the heat transfer between prawns and glazing water.

12.3.4

RF Spectroscopy—Impedance Spectroscopy

Radio frequency is an electromagnetic radiation within the range of 3 Hz to 300 GHz. This range corresponds to the frequency of alternating current electrical signals used to produce and detect radio waves. Different techniques have been developed for quality control based on the response of foods to waves in the RF region. The technique called “bioelectrical impedance analysis” (BIA) is highly effective for measuring human body composition such as fat content, lean muscle, or total water [37] and nutritional status [37,38] and there is abundant supporting literature from medical studies demonstrating the effectiveness of the approach. This technique works at 50 kHz and is also an accurate predictor of the composition of fish [39,40] as the amount of water or proportion of fat tissue to lean tissue is correlated to BIA measurements through regression equations built on multiple measurements of control groups [41]. Impedance spectroscopy measures the dielectric properties (see Section 12.4) of a “food material” as a function of frequency; this term usually applies to the range of RF frequencies, sometimes extended to low microwaves. Impedance spectroscopy has been widely used to estimate the physiological state of various biological tissues [42,43]. In studies of a biological tissue, it is of great importance to establish an appropriate equivalent circuit model to relate the measured data to the physical and physiological properties. A number of spectroscopic methods in RF have been used quite recently to measure the quality-determining properties of frozen fish [44,45]. Haddock muscle showed significant changes in its dielectric properties during rigor mortis at frequencies between 1 Hz and 100 kHz [46]. In quality control of fish, the principal method of data analysis of impedance results has been to calculate indices with the measurements conducted at one or two frequencies [44,47]. With living tissues and in the postmortem period, impedance data have been analyzed by regression at each measured frequency and at several selected frequencies, by Cole-Cole analysis, and so on [48], but multivariate techniques of data analysis are still not widely used. The main disadvantages of RF for online monitoring are related to the physical size of its hardware, which is very voluminous and difficult to manage; moreover, interactions with metals and other materials can be problematic, and ionic conduction effects (i.e., due to dissolved salts) are highly significant (masking other effects).

12.3.5 Microwave Spectroscopy—Dielectric Spectroscopy The actual state of art of microwave technology permits measuring in real time and in a nondestructive way most of the parameters that are related to quality control. For instance, in the late


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sixties, microwave sensors emerged as a plausible solution for real-time, nondestructive sensing of moisture content in a variety of materials [49–51]. Moreover, in recent years, the price of microwave components has dropped drastically because of a surge in demand from the wireless telecommunications sector. This, with new developments in solid-state and planar circuit technologies, provides an opportunity to develop reasonably priced microwave/RF sensors. Therefore, the application of microwave technologies to food quality control is a growing interest for the industry. Until recently, the interest of the food industry in microwave applications had been fi xed mainly in dielectric heating. These applications appeared in the years following the end of the Second World War, but the development of microwaves stopped due to technological reasons and the high cost of investment. At the beginning of the 1980s, the possibilities of microwave applications and their considerable advantages were recognized, and microwave ovens become more popular. This increase in the use of domestic microwave ovens gave rise to a reduction in the cost of the relatively high-power magnetron. However, the cost of these elements increases exponentially when the power is on an industrial scale [35]. Presently, domestic microwave ovens are universally accepted by consumers, and other microwave heating applications are widely used in industry; baking, drying, blanching, thawing, tempering, and packaging are the most important. Therefore, considerable experience has now been accumulated in this field and can be used in the design of sensor systems based on microwaves. These sensors are viable and affordable for online control in food industrial processes. Dielectric spectroscopy measures the dielectric properties (see Section 12.4) of a “material” as a function of frequency; this term usually applies to the range of microwave frequencies, sometimes extended to high RF. Dielectric spectroscopy is considered to be a very useful tool in food quality determinations, because, as will be explained in Sections 12.4 and 12.5, dielectric properties of biological tissues are closely correlated with water content and the aggregation state of it. Furthermore, the dielectric properties depend not only on water binding in foods but also on its composition. The interplay between molecular composition, presence of ions, electrical charges on proteins, and pH variations leads to a complex dielectric spectrum regulated by several phenomena. Dielectric properties are also related to structure, and the structural organization and composition of a muscle makes it a highly anisotropic dielectric material. This dielectric anisotropy was modeled by Felbacq et al. [52] to provide insight into microwave–muscle interactions. It tends to decrease during ageing or process-related cellular degradation. The main theoretical aspects of microwaves are treated in Section 12.4. In Section 12.5 some interesting applications of microwave technology in quality control are cited.

12.3.6 Advantages and Benefits of Microwave Methods A very important benefit of microwave sensing is that the bulk property (i.e., moisture or density) is determined, in contrast to surface determination provided, for example, with infrared (IR) or NIR techniques. This is particularly important in monitoring operations, for example, drying, where moisture gradients exist in the material; variations in moisture can exist within a few microns of the surface, but their effects are substantially reduced or insignificant at microwave frequencies. Another decided advantage is logistical flexibility in installation. With a wide variety of sensors from which to choose, placement can be on conveyors or in hoppers, shakers, pipes,

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chutes, and so on. Installation is generally minimally intrusive. Moreover, results can be obtained almost in real time, because the measurement time ranges from a few milliseconds to one second. A further advantage is that microwave radiation is noncontaminating and environmentally safe at power levels typically used for online sensing. Human exposure is usually less than that from common consumer electronic devices such as cordless and cellular telephones. Finally, microwave sensors are insensitive to environmental conditions such as dust, color, or ambient light, vapors, and machine vibrations, in contrast to IR and NIR techniques.

12.4

Overview of Microwave Theory

Microwaves are a common designation for electromagnetic waves at frequencies between 300 MHz and 300 GHz. These waves travel through the free space with a given energy (E) and propagation parameters, which are mainly magnitude (A) and phase (q). When they find a different “dielectric material” (in this case, food), one part of the radiation is refracted and another one passes through it (see Figure 12.1). The amount of radiation refracted or transmitted by food as well as its new propagation parameters are governed by the dielectric properties of the material. Therefore, the measurement of these properties allows both the characterization of food and the control of the process (see Figure 12.1). In the communications argot, “materials” are usually divided into the categories of conductors, insulators, and dielectrics. “Dielectric materials” cover the whole spectrum of anything between conductors and insulators. Therefore, dielectrics can consist of polar molecules or nonpolar molecules, or very often both. According to this classification, foods are “dielectric materials” (or really an addition of dielectric materials) susceptible to be defined by their dielectric properties. Complex permittivity (e r) (Equation 12.1) is the dielectric property that describes food behavior under an electromagnetic field [53].

E1, A1, θ1

, θ3 E 3, A 3

E1, A1, θ1

, θ5 E 5, A 5

Material permittivity εr1 = ε΄r1 –j.ε˝r1 Natural or industrial process

Modified material permittivity εr2 = ε΄r2 –j.ε˝r2

E2, A2, θ2

Product characterization

E4, A4, θ4 Processes control (or monitoring)

Figure 12.1 Scheme of the possibilities of the measurement of dielectric properties in quality control applications.


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177

The real part of complex permittivity is called the dielectric constant (e′), and the imaginary ′′ ). The subscript r indicates that values are related to part is called the effective loss factor ( ε eff vacuum, and the variable is therefore dimensionless: ′′ εr = ε ′ − j ε eff

(12.1)

Under a microwave field, the charges of certain food components (water, salts, etc.) try to displace from their equilibrium positions to orientate themselves following the field, storing microwave energy that is released when the applied field stops. This behavior is called polarization; e′ denotes the material’s ability to store this electromagnetic energy (or the ability to be polarized). Only a ′′ is perfect dielectric can store and release wave energy without absorbing it. The parameter ε eff related to absorption and dissipation of the electric energy from the field. Such energy absorptions are caused by different factors that depend on structure, composition, and measurement ′′ can be expressed by Equation 12.2 [53]: frequency, thus ε eff ε ′eff = ε ′′d + ε ′′MW + ε ′′e + ε ′′a + σ/ε o ω

(12.2)

In this equation the last term is called ionic losses. The symbols s, e o, and w refer to material conductivity, vacuum permittivity, and angular frequency, respectively. Subscripts d, MW, e, and a indicate dipolar, Maxwell–Wagner, and electronic and atomic losses, respectively. The different contributing mechanisms to the loss factor of a moist material are schematically represented in Figure 12.2.

ε˝

+ i

+

+ –

+ –

–

–

+ –

+ – + + –

dw

MW a e

da

1.8E10 3E8 3E11 Radio frequency Microwaves IR AC L–M–K wave VHF dm

cm

mm

μm

log f (Hz) 3E14 V

UV

nm

Figure 12.2 Schematic representation of the different effects that contribute to effective loss factor (e″e ff ) along the electromagnetic spectrum (logarithmic scale). i, ionic losses; MW, Maxwell–Wagner effect; dw, dipolar losses of water; da, dipolar losses of isopropyl alcohol; a, atomic losses; e, electronic losses.

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Under a microwave field, molecules with an asymmetric charge distribution (permanent dipoles such as water) rotate trying to align themselves with the electric field, storing part of the wave energy [54]. The dipolar contribution to total losses is one of the most important at microwave frequencies due to the fact that water is an abundant and common component in foods. Otherwise, as frequency is increased (the highest microwave frequencies and above them), the electromagnetic field can affect smaller particles, inducing dipoles even in neutral molecules (atomic polarization) and neutral atoms (electronic polarization). Atomic and electronic losses have behavior similar to that of permanent dipolar losses. At RF and the lowest microwave frequencies, charged atoms and molecules (ions) are affected by the field. Such ions move trying to follow the changes in the electric field. In case ions do not find any impediment (aqueous solutions, conducting materials), ionic conductivity gives rise to an increment in effective losses. At these frequencies, the ionic losses are the main contributors to the loss factor (supposing ions to be present in the material). Foods are complex systems and usually present conducting regions surrounded by nonconducting regions, for example, foods with a cellular structure have cytoplasm (conducting region) surrounded by the membrane (nonconducting region). In these cases, ions are trapped by the interfaces (nonconducting regions) and, as the ion movement is limited, the charges are accumulated, increasing the overall capacitance of the food [55] and the dielectric constant (Maxwell– Wagner Polarization). This phenomenon is produced at low frequencies at which the charges have enough time to accumulate at the borders of the conducting regions. The Maxwell–Wagner losses curve vs. frequency has the same shape as the dipolar losses curve (see Figure 12.2). At higher frequencies, the charges do not have enough time to accumulate and the polarization of the conducting region does not occur. At frequencies above the Maxwell– Wagner relaxation frequency, both ionic losses and the Maxwell–Wagner effect are difficult to distinguish due to the fact that both effects exhibit the same slope (1/f ). Foods are multicomponent and multiphase systems; therefore, more than one mechanism contributes to the combined effects. Figure 12.3 shows different shape variations in effective loss factor curves vs. frequency for the case of combined dipolar and ionic losses. Type_0 represents a typical pure dipolar loss factor curve (without ionic contribution), s increases between type_0 and type_4 curves (the corresponding ionic contribution is marked in discontinuous trace), ε d″ max is the highest value of dipolar losses, and relaxation frequency is the inverse of relaxation time [53,16]. In general, foods are dielectric materials with high losses and, under a microwave field, they can absorb part of the wave energy. The power that can be dissipated in a given material volume ′′ by Equation 12.3, in which E is the electric field strength [53]: (Pv) is related to ε eff Pv = 2π f ε0 ε eff ·E 2 (W/cm3 )

(12.3)

The high-power dissipation in foods has given rise to numerous high-power heating applications that have been developed since the fi fties. The interest in improving heating applications has provided a great deal of knowledge on dielectric properties and wave parameter measurements. Th is detailed knowledge has been very useful in further research into new lowpower online sensors, which relate these properties or parameters to process variables of food industry.


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179

ε˝ 4

3 σ/ωε0 +

+ –

+ –

2

+ –

+

– +

– 1 εd˝ 0 log ( f )

Figure 12.3 Influence of salt content in systems with different proportions of dipoles (water) and ions (salts) in the shape of effective loss factor curve. Salt content increases in curves from 0 (water) and 4 (saturation). (Adapted from De los Reyes, R. et al., Medida de propiedades dieléctricas en alimentos y su aplicación en el control de calidad de productos y procesos, ProQuest (Ed.), 2007.)

12.5 Applications of Microwave Technology in the Assessment or the Control of Processes The applications of electromagnetic radiation in the microwave band are varied and cover broad fields, from the radar [56] and radiometry [57], to medical applications, such as the diagnosis of breast cancer [58] and other image applications. In addition, industrial applications have been developed, such as rubber vulcanization [59], soils, wood, and animal products disinfection [60–62], or food processing [63,64]. They are so many that some frequency bands have been reserved especially for industrial, scientific, and medical applications (ISM). These frequencies are detailed in Table 12.1. Microwave applications that are better known within the food industry are related to energy absorption and, therefore, are made at high power and usually at 2.45 GHz, which is the frequency often reserved in Europe for industrial applications. These applications are mainly used for heating, pasteurization, sterilization, dehydration, thawing, and scalding [65–67]. Recently, the application of microwaves in combination with warm air in drying of foods has been also studied, either during the whole drying process or in part of it [68,69]. Within this field, applications to the drying of fruits and vegetables are notable for their interest to the food industry [70,71]. However, as noted above, the development of the technology that brings this large number of applications has allowed the onslaught of new applications such as the assessment or the control of

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Handbook of Seafood and Seafood Products Analysis Table 12.1 Frequency Bands Reserved for Industrial, Scientific, and Medical Applications (ISM) Frequency (MHz)

Wave Longitude (cm)

433.92 ± 8

69.14

915 ± 13

32.75

2,450 ± 50

12.24

5,800 ± 75

5.17

24,125 ± 125

1.36

processes by microwaves in a nondestructive way (MNDT or MNDE) which is receiving a growing interest in the food industry. In these applications, very low power is used to avoid permanent effects in foods. As a result of that, the methods for determining dielectric properties have experienced a spectacular expansion within the field of the analysis of materials by microwaves, which until relatively recently, was exclusively associated with the design of electronic equipment. As has been explained before, the measurement of the dielectric properties can provide important information during industrial processes due to the relationships between food properties and electromagnetic parameters. This is because low-power microwaves change their parameters (amplitude, phase) according to the food properties, and this change can be measured in real time. This is the basic principle on which food-quality microwave sensors are based. Complex permittivity can be correlated with structural, physical, and chemical properties such as humidity, soluble solids content, porosity, characteristics of solid matrix, and density [16]. The changes in these properties are usually related with the treatments applied to foods throughout the industrial process; for instance, water losses in drying processes [72] or salt losses in desalting processes [14,15]. In addition, the structural changes produced in macromolecules, such as protein denaturalization, can occur during processing, leading to a modification of the dielectric properties [73]. For all these reasons, the measurement of dielectric properties can be used as a tool for online food process control. This section provides an overview of the most important microwave applications as techniques in food control.

12.5.1

Determination of Moisture Content

Water represents the main component of foods influenced by microwave energy and, therefore, nowadays most methods of determining moisture content are based on electrical properties. The determination of moisture based on electromagnetic parameters has been used in agriculture for at least 90 years and has been in common use for 50 years [12,74,75]. Diverse studies have been carried out relating the dielectric constant and loss factor with moisture in foods [76,74]. Further researches in this field have occurred during recent years. Trabelsi and Nelson [77] studied a method of moisture sensing in grains and seeds by measuring their dielectric properties. The reliability of the method was tested for soybean, corn, wheat, sorghum, and barley. The frequency used was 7 GHz with the free space technique. In the same year, the authors used the same technique at 2–18 GHz to determine the dielectric properties of cereal grains and oilseeds in order to predict the moisture content by microwave measurements [78]. This article presents a unified


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181

grain moisture algorithm, based on measurements of the real part of the complex permittivity of grain at 149 MHz using the transmission line method. Trabelsi and Nelson [79] reported the moisture in unshelled and shelled peanuts using the free space method at a frequency of 8 GHz. In 2005, Joshi [80] reported a technique for online, time domain, nondestructive microwave aquametry (US Patent numbers 6,204,670 and 6,407,555); this technique was used for determining moisture levels in substances such as seeds, soil, tissue paper, and milk powder. Plaza-González et al. [81] have published a report about a microwave sensor intended for online measurements of paper moisture. Since most efforts have been directed to the moisture determination of different materials, commercial meters for online moisture measurements have already been developed. These moisture meters are based on automatic online calculations of the reflected wave and dielectric permittivity, yielding physicochemical properties, such as moisture, chemical composition, and density, without affecting the product. For instance, Keam Holdem® Industry (Auckland, New Zealand) provides online moisture testing and analyzing systems. This manufacturer provides devices for measuring moisture in processed cheese, moisture and salt in butter, moisture and density in dried lumber and whole kernel grain, and fat-to-lean ratio in pork middles. A microwave moisture meter has also been developed for continuous control of moisture in grains, sugar, and dry milk in technological processes [82]. A consortium of companies from different countries, Microradar®, produces a commercial microwave moisture meter for measuring moisture in fluids, solids, and bulk materials based on this method. The enterprise KDC Technology Corporation (www.kdctech.com) provides microwave sensors for monitoring industrial processes and quality control. KDC sensors work in a wide range of applications such as monitoring moisture and density of manufactured wood and wood-based products, construction, and agricultural and processed food products. Patented contact (MDA1000) and noncontact (MMA-2000) sensors are used for online, continuous process monitoring of solids, particulates, and liquids or for in situ nondestructive testing/inspection. Another interesting application for online moisture measurement is a sensor for green tea developed by Okamura and Tsukamoto [72], which can measure moisture as high as 160%–300% on dry basis by use of microwaves at 3 GHz with a microstripline (Figure 12.4). A Guided Microwave Spectrometer (Thermo Electron Corporation, Waltham, MA) has been developed for online measurements of multiphase products. This guide is used to measure Microwave source Receiver Microstripline

Tea leaves

Electric field

Figure 12.4 Schema of a microstripline used for tea leaves moisture measurement. (Adapted from Okamura, S. and Tsukamoto, S., New sensor for high moist leaves in green tea production, in Proceedings of ISEMA 2005, Kupfer, K. (Ed.), MFPA an der Bauhaus-Universität Weimar, Weimar, Germany, 2005, 340–346.)

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moisture in raw materials such as corn, rice, soybeans, and in processed materials such as tomato paste and ground meat. It can also measure content of soluble solids, pH, viscosity, and acidity in orange juice, soft drinks, mayonnaise, and tomato products; fat in ground meats, peanut butter, and milk and other dairy products; salt in mashed potatoes and most vegetable products and, lastly, alcohol in beverages.

12.5.2

Freshness and Salting/Desalting Process Quality Control of Fish and Seafood, by Microwaves: Methods and Equipments

The dielectric properties of fish products have been measured by different authors [83–86]; nevertheless, the electromagnetic determination of quality parameters in muscle tissues is still a complex challenge due to its complex matrix, heterogeneous composition, and anisotropic disposition. It is important to point out that the limitation of most dielectric probes is the volume of the sample that interacts with the field. The volume has to be representative of the whole piece of fish, due to the fact that the electromagnetic parameters in this kind of tissue vary in a heterogeneous way. It has been reported that it is possible to predict the fat composition in fish using electromagnetic measurements [87]; this is because it is clearly related to the water content of the product, so that if one is known the other can be determined; this is the knowledge base of the “Torrymeter” mentioned later. Moreover, this author [88,89] has studied the determination of added water in fish using microwave dielectric spectra measurements. Measurements of dielectric properties have been tested and used during almost 40 years for quality grading and remaining shelf life determination of various fish. These investigations have been mainly focused on freshness and self-life evaluation and detecting fishes previously thawed. However, a number of research studies have been carried out to control or monitor the processing of fish products. In this field, De los Reyes et al. [14,15] verified the viability of an online measurement system using low-power microwaves to determine the desalting point of salted cod. Dielectric spectroscopy was performed on cod samples at different desalting stages and on its desalting solutions in order to find the appropriate measurement frequency. Figure 12.5 shows the dielectric spectra (e′ and e″) from cod loin samples (2 cm/side parallelepipeds) at desalting times (t) yielding from 15 min to 48 h. Optimum frequencies were selected from the spectrum, and dielectric properties data were related to other physicochemical properties of cod samples measured at the same desalting stages, such as moisture and salt content. Good correlations were found between salt content in cod samples and their loss factor values at 200 and 300 MHz. These results indicated the viability of developing an online control system for a cod desalting process. Polarimetric measurements, that is, with a linearly polarized electric field, make it possible to evaluate anisotropy. This method has been applied to assess fish freshness [90]. This is because, after death, muscle is not able to use energy by the respiratory system. Postmortem changes lead to a temporary rigidity of muscles, decreasing the water-holding capacity [91]. The level of glycogen stored in the animal at the time of slaughter affects the texture of the future marketed meat. For all these reasons, during rigor mortis the dielectric properties are expected to change. The “Intellectron Fishtester” [92], the “Torrymeter” (Distell.com), and the “RT-Freshtester” (RT rafagnatækni), represent instruments with increasing degrees of sophistication invented for fish-quality evaluation. Readings from all these instruments are based in the reflected dielectric properties of fish, because they decrease with storage time, almost following a straight line. Based on these rapid and nondestructive measurements, the “RT-Freshtester” allows automatic grading of 60–70 fish per min. Nevertheless, electrical properties of fish are not directly responsible for


ε΄, ε˝ 800

0.2 GHz 0.3 GHz

0.9 GHz 1.8 GHz 2.45 GHz

◾

183

10 GHz

700 600 500

ε˝ t

400 300 200 100

ε΄

t

0 1E + 08

1E + 09

1E + 10

Frequency

Figure 12.5 Dielectric spectra from cod samples at desalting times (t) yielding from 15 min to 48 h. The arrows beside t indicate the growth of the desalting time. Frequency axis is in the logarithmic scale, and broken lines mark the selected frequencies (0.2, 0.3, 0.9, 1.8, 2.45, and 10 GHz). (Adapted from De los Reyes, R. et al., Dielectric spectroscopy studies of “salted cod-water” systems during the desalting process, in Proceedings of the IMPI’s 40th Annual Symposium, 2006.)

sensory spoilage and it is, therefore, to be expected that numerous factors influence the relationship between such measurements and seafood spoilage. In fact, these instruments need calibration depending on the season and fish handling procedures, and they are unsuitable for grading frozen–thawed fish, partially frozen, that is, superchilled fish, fish chilled in refrigerated seawater, or for fish fillets. This and the high cost of the instruments limit their practical use in the seafood sector for freshness evaluation. However, electrical measurements can also be used to test if fish was previously frozen [2]. Kent et al. [93] studied the effect of storage time and temperature on the dielectric properties of thawed–frozen cod (Gadus morhua) in order to estimate the quality of this product. The same year, Kent et al. [94] developed a combination of dielectric spectroscopy and multivariate analysis to determine the quality of chilled Baltic cod (Gadus morhua). These researches yielded a prototype developed by SEQUID [95,96] for measuring and analyzing the quality of different seafood. The SEQUID project concentrated on the measurement of the dielectric properties of fish tissue as a function of time both in frozen and chilled storage. This project has shown that it is possible, using a combination of time domain reflectometry and multivariate analysis, to predict certain quality-related variables, both sensory and biochemical, with an accuracy comparable to existing methods. Kent et al. [97] have also reported a way to determine the quality of frozen hake (Merluccius capensis) by analyzing its changes in microwave dielectric properties. The above mentioned “Torrymeter” has been successfully improved as a sensor for measuring fish freshness as a result of these investigations. In further investigations, the SEQUID project has shown that it is possible to predict certain quality-related variables (with comparable accuracy to existing methods) using a combination of time-domain reflectometry at microwave and RF frequencies and multivariate analysis [98].

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12.6

◾


Conclusions

It is possible to implant reliable online sensors in fish industry both for determining the freshness as well as for monitoring processes (salting/desalting, thawing, etc.). The future of control in fish processing is the analysis of the physical and chemical properties using the dielectric signal at different frequencies, using multisensors. Multivariable knowledge of the process yields a modeling of the product.

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Chapter 13

Methods for Freshness Quality and Deterioration Yesim Ozogul Contents 13.1 Introduction ..................................................................................................................190 13.2 Sensory Methods ...........................................................................................................190 13.2.1 The European Union Freshness Grading (EU or EC Scheme) ..........................191 13.2.2 The Quality Index Method ..............................................................................191 13.2.3 The Torry Scheme ............................................................................................192 13.2.4 The Quantitative Descriptive Analysis .............................................................192 13.3 Physical Methods ..........................................................................................................194 13.3.1 Texture Analysis ...............................................................................................194 13.3.2 The Torrymeter ................................................................................................194 13.3.3 The Intellectron Fischtester VI .........................................................................195 13.3.4 The RT-Freshtester ...........................................................................................195 13.3.5 The Cosmos .....................................................................................................195 13.3.6 Electronic Nose ................................................................................................196 13.3.7 Near-Infrared Reflectance Spectroscopy...........................................................196 13.4 Chemical and Biochemical Methods .............................................................................197 13.4.1 ATP and Its Breakdown Products ....................................................................197 13.4.2 Biogenic Amines ..............................................................................................199 13.4.3 pH....................................................................................................................199 13.4.4 Total Volatile Basic Nitrogen........................................................................... 200 13.4.5 Trimethylamine .............................................................................................. 200 13.4.6 Dimethylamine ................................................................................................201 189

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13.4.7 Formaldehyde ..................................................................................................201 13.4.8 Lipid Oxidation Indicators ...............................................................................201 13.4.9 Lipid Hydrolysis .............................................................................................. 203 13.5 Microbiological Methods ............................................................................................. 203 References ............................................................................................................................... 204

13.1

Introduction

Seafood is generally considered to be a high-protein food, low in fat and saturated fat when compared with other protein-rich animal foods. It is well known that fish oil is the major and the best source of polyunsaturated fatty acids (PUFA), called omega-3 fatty acids, especially eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Scientific evidence suggests that omega-3 fatty acids are essential for normal growth and development throughout the life cycle and inhibit the formation of atherosclerotic plaques, prevent arrhythmias, and contribute to the prevention or amelioration of autoimmune disorders, Crohn’s disease, breast, colon and prostate cancers, rheumatoid arthritis, and particularly cardiovascular diseases [1–6]. The Nutrition Committee of the American Heart Association recommends consumption of any type of fish two or three times a week. Therefore, it is important to prevent their loss due to oxidation. Freshness is the most important attribute when assessing the quality of seafood and is of great concern in the seafood sector [7]. The quality of seafood degrades after death due to the chemical reactions [changes in protein and lipid fractions, the formation of biogenic amines and hypoxanthine (Hx)] and microbiological spoilage. As a result of these events, sensory quality of seafood deteriorates [8–13]. Seafoods are rich in PUFAs, which are susceptible to lipid oxidation. It leads to the development of off flavor and off odors in edible oils and fat-containing foods called oxidative rancidity [14,15]. Because of their high degree of unsaturation, they are less resistant to oxidation than other animal or vegetable oils [14]. This chapter summarizes methods used for evaluation of freshness and spoilage of seafood. As it is well known, no single instrumental method is reliable for assessment of the freshness and spoilage of seafood. However, chemical, microbiological methods along with sensory methods have been applied by commercial seafood companies and many researchers to ensure that the seafood products meet expectations of consumers. The current regulation of the European Community (1996) establishes principles based on sensory, chemical, and microbiological analysis to control and certify the quality warranty in the seafood field (Council Regulation No.: 2406/96). The shelf life of fish is affected by many factors such as handling, storage condition from catch to the consumers, the kind of fishing gear, bleeding, gutting methods, season, catching ground, age, and life cycle of fish affecting the nutritional quality, freshness, and safety of seafood. Therefore, estimation of remaining shelf life of fish should be made with caution [7].

13.2

Sensory Methods

Sensory evaluation is the most important method in freshness assessments. Sensory evaluation is defined as the scientific discipline used to evoke, measure, analyze, and interpret reactions to characteristics of food as perceived through the senses of sight, smell, taste, touch, and hearing [16]. Sensory evaluation provides rapid measurements of freshness of seafood. There has been a trend to standardize sensory evaluation as an objective assessment of freshness. Sensory characteristics of

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191

whole fish are clearly visible to consumers, and sensory methods are still the most satisfactory way of assessing the freshness quality since they give the best idea of consumer acceptance [17]. Freshness declines as storage life progresses until the product is no longer acceptable to consumers. The most appropriate method to assess freshness is a sensory panel. There are many factors affecting the measurement of sensory quality, including the sample under investigation, the assessment method, and the judges [18]. There are two types of sensory methods, subjective and objective. Subjective assessments of fish have been used for acceptability. They are often estimated generally using adjectives such as like/dislike or good/bad, which require subjective decisions. Fish freshness is most commonly determined by objective scoring based on organoleptic changes that occur as fish storage time is extended [19]. Objective scoring schemes require trained, expert judges, but the advantage is that panels can be small. These assessors individually use their appropriate senses (sight, smell, taste, and touch) to determine the level of each sensory characteristic in the defined grade standard appropriate for the seafood examined [20]. Subjective assessment, where the response is based on the assessor’s preference for a product, can be applied in the fields like market research and product development where the reaction of consumers is needed. Assessment in quality control must be objective [16]. Assessors must be trained and have clear and descriptive guidelines and standards to get reliable results for sensory analyses [21]. Sensory methods are also fast and nondestructive unless fish is cooked.

13.2.1

The European Union Freshness Grading (EU or EC Scheme)

The EU Freshness Grading was introduced for the first time in the Council Regulation No. 103/76 (for fish) and 104/76 (for crustaceans) and updated by decision No. 2406/96 (for some fish, some crustaceans, and only one cephalopod, the cuttlefish). The EU scheme is commonly accepted in the EU countries for freshness grading to market fish within the Union and generally carried out by trained personnel in auctions. Whole and gutted fish are assessed in terms of appearance of skin, eyes, gills, surface slime, belly cavity, odor, and texture of fish. There are four quality levels in the EC scheme, E (extra), A (good quality), B (satisfactory quality), where E is the highest quality and below level B (called Unfit or C) is the level where fish is discarded or rejected for human consumption. However, there are still some disadvantages; trained and experienced persons are required, since the scheme uses only general parameters for iced fish [16,22,23]. It does not take differences between species into account. In addition, it does not give information on the remaining shelf life of fish. A suggestion for renewal of the EU scheme can be seen in the Multilingual Guide to EU Freshness Grades for Fishery Products [24], in which special schemes for some fish species (whitefish, dogfish, herring, and mackerel) were developed.

13.2.2 The Quality Index Method The quality index method (QIM) has been suggested as an alternative to the EU scheme. The QIM, originally developed by the Tasmanian Food Research Unit in Australia [25] and improved further, is considered to be rapid and reliable to measure the freshness of whole fish stored in ice [21,22]. This method is based on significant sensory parameters (skin, slime, eyes, belly, odor, gills, etc.) for raw fish [25,26], and the characteristics listed on the sheet are assessed and appropriate demerit point score is recorded (from 0 to 3). The scores for all characteristics are summed to give the overall sensory score. Quality index (QI) is close to 0 for very fresh fish, whereas higher scores are obtained as the fish deteriorates [16,26]. There is a linear correlation between the sensory

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quality expressed as a demerit score and storage life on ice, which makes it possible to predict remaining storage life on ice. During spoilage, a higher score can be given for a single parameter [27]. This method is considered to be a relatively fast, nondestructive method based on direct observation of sensory parameters of fish and can also be specific for species. In addition, the QIM is suitable for early stage of storage of fish where other instrumental methods are not accurate [28]. Hyldig [29] indicated that the QIM is expected to become the leading reference method for the assessment of fresh fish within the European community. QIM Eurofish published a manual [21] containing QIM schemes for 12 fish species and information about how to use the QIM schemes (QIM-Eurofish 2004). The advantages of QIM are that it requires short training, is rapid and easy to perform, and is nondestructive and can be used as a tool in production planning and quality warranty work [27]. Rapid PC-based QIM is also available on the Internet at http://www.dfu.min.dk/QIMRS/qim_0202.htm. QIM Rating system software was developed for cod, herring, saithe, and redfish by the Danish Institute for Fisheries Research. Recently developed QIM schemes were presented for raw gilthead sea bream (Sparus aurata) [30], farmed Atlantic salmon (Salmo salar) [31], fresh cod (Gadus morhua) [32], common octopus (Octopus vulgaris) [33], herring (Clupea harengus) (Table 13.1) [34], brill, haddock, plaice, pollock, redfish shrimp, sole, and turbot (Scopthalmus rhombus, Melanogrammus aelefigus, Pleuronectes platessa, Pollachius virens, Sebastes mentella marinus, Pandalus borealis, Solea vulgaris, and Scopthalmus maximus, respectively) [21].

13.2.3

The Torry Scheme

In contrast to the QIM, the Torry Scheme was developed at the Torry Research Station for use with expert and trained judges. The most comprehensive scoring scheme to assess fish is the Torry Scheme [36]. It has been widely used in its original or modified forms. The Torry Scheme, often referred to as the Torry scale, is a descriptive 10-point scale and has been developed for lean, medium fat, and fat fish species. In this scheme, panelists evaluate the odor and flavor of cooked fillets. The scores are given from 10 (very fresh) to 3 (spoiled) (Table 13.2). The average score of 5.5 may be used as the limit for consumption [21].

13.2.4

The Quantitative Descriptive Analysis

Quantitative descriptive analysis (QDA) is used by a trained sensory panel to analyze the sensory attributes of products such as texture, odor, and flavor. QDA provides a detailed description of all flavor characteristics in a qualitative and quantitative way. The method can also be used for texture. The trained panel is handed a broad selection of reference samples and use the samples for creating terminology that describes all aspects of the product [16]. Descriptive words should be carefully selected, and the panelists trained should agree with the terms. Objective terms should be used rather than subjective terms. In QDA, the words for describing the odor and flavor of the fish can be categorized into two groups, positive and negative sensory parameters based on whether fish are fresh fish or fish at the end of the storage period [37]. Objective sensory methods are essential for quality control and estimation of shelf life of seafood. However, sensory methods are time consuming, expensive, trained personnel required, and not always practical for large-scale commercial purposes. Therefore, instrumental methods are also needed to satisfy the need for quality measurements in fish industry.

Methods for Freshness Quality and Deterioration Table 13.1 QIM Scheme for Sensory Evaluation of Herring Quality Parameter Whole fish

Appearance of skin

Blood on gill cover

Texture on loin

Texture of belly

Odor

Eyes

Appearance

Shape

Gills

Color

Odor

Description

Score

Very shiny

0

Shiny

1

Matte

2

None

0

Very little (10%–30%)

1

Some (30%–50%)

2

Much (50%–100%)

3

Hard

0

Firm

1

Yielding

2

Soft

3

Firm

0

Soft

1

Burst

2

Fresh sea odor

0

Neutral

1

Slight off odor

2

Strong off odor

3

Bright

0

Somewhat lusterless

1

Convex

0

Flat

1

Sunken

2

Characteristic red

0

Somewhat pale, matte, brown

1

Fresh, seaweedy, metallic

0

Neutral

1

Some off odor

2

Strong off odor

3

Sources: Modified by Jónsdóttir, S., Quality Standards for Fish: Final Report Phase II, Nordic Industrial Fund (in Danish), pp. 37–59, 1992; developed by Nielsen, D. and Hyldig, G., Food Res. Int., 37, 975, 2004. With permission.

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Table 13.2 Torry Score Sheet for Freshness Evaluation of Cooked Cod Fillets Odor

Flavor

Score

Initially weak odor of sweet, boiled milk, starchy, followed by strengthening of these odors

Watery, metallic, starchy. Initially no sweetness but meaty flavors with slight sweetness may develop

10

Shellfish, seaweed, boiled meat

Sweet and meaty characteristic

9

Loss of odor, natural odor

Sweet and characteristic flavors but reduced in intensity

8

Wood shavings, wood sap, vanillin

Neutral

7

Condensed milk, boiled potato

Insipid

6

Milk jug odors, reminiscent of boiled clothes

Slight sourness, trace of “off” flavors

5

Lactic acid, sour milk, TMA

Slight bitterness, sour, “off” flavors, TMA

4

Lower fatty acids (e.g., acetic or butyric acids) decomposed grass, soapy, turnip, tallow

Strong bitterness, rubber, slight sulfide

3

Source: Shewan, J.M. et al., J. Sci. Food Agric., 4, 283, 1953. With permission.

13.3

Physical Methods

13.3.1 Texture Analysis Texture analyses for seafood are extremely important in research, quality control, and product development in the seafood industry [38]. Fish muscle may become soft or mushy as a result of autolytic degradation or tough as a result of frozen storage [16]. Fish muscle has higher levels of indigenous proteases, which immediately begin to break down the proteins after the harvesting, during processing, improper handling storage, and cooking [39,40]. Texture includes the most common characteristics such as hardness, springiness, and chewiness of food. Among textural attributes, hardness is the most important to the consumer, deciding the commercial value of the meat [41]. Numerous mechanical methods have been used to measure texture; however, there is little agreement on which is the best method [42].

13.3.2

The Torrymeter

The Torry fish freshness meter “Torrymeter” was developed at Torry Research Station in Aberdeen, Scotland. Dielectric properties of fish are used for determination of freshness. Dielectric properties of fish skin and muscle alter in a systematic way during spoilage as tissue components degrade. These changes occurring at microscopic level are related to alterations in appearance, odor, texture, and flavor during spoilage and have been used as quality indicators since the first commercial version of the Torrymeter in 1970 [43]. A linear relationship was found between Torrymeter readings and sensory attributes for cod, Baltic herring, hake, blue whiting, flounder, mackerel,


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whole, iced gilthead sea bream, and farmed Senegalese sole [43–49]. However, Gelman et al. [50] found that the Torrymeter readings obtained from six species of different origin were poorly correlated with sensory evaluation. Inácio et al. [51] also studied the effect of washing with tap and treated seawater on the quality of whole scad (Trachurus trachurus) and found that Torrymeter and RT-Freshmeter readings were significantly (P < 0.05) lower in fish washed with seawater than fish washed with tap water or unwashed. This could be explained by seawater containing ions, which interfere with the reading of both instruments as they are based on electrical properties of skin. The skin of fish could be affected by osmolarity and contact with electrically charged particles [51]. Fat also has an effect on the dielectric properties of fish and tends to make observed Torrymeter values more variable [47]. The loss of skin and muscle integrity and deterioration of the skin caused by bruising during harvesting and packing operations would result in more variable Torrymeter values.

13.3.3 The Intellectron Fischtester VI The basic principles of Torrymeter (the United Kingdom) and the Intellectron Fischtester VI (Germany) are similar, measuring the electric properties (resistance, conductivity, and capacitance) of the fish flesh [52]. The electric properties of fish can change after death of the fish due to disruption of the cell membranes by autolysis. The method is based on conduction through skin and, therefore, works only on whole fish and fillets with skin on. Mechanical abuse and freezing can affect the readings. The Intellectron Fischtester VI gives reliable information about the days in ice and left of iced stored fish. It has also reported that there is a linear correlation between the instrument readings obtained on the day of harvest/catch and the date of spoilage [53]. The Fischtester readings can be used as an objective criterion for the state of freshness/spoilage together with sensory data across the fish chain.

13.3.4

The RT-Freshtester

Like Torrymeter and the Intellectron Fischtester VI, RT-Freshtester reflects dielectrical properties of fish, and readings from all instruments decrease with storage time. RT-Freshtester, fast and nondestructive, allows automatic grading of 60–70 fish/min. However, these instruments need calibration depending on sample preparation, season, fishing grounds, and fish-handling procedures. They are unsuitable for frozen or thawed fish, partly frozen such as superchilled fish, and fish chilled in refrigerated seawater [54].

13.3.5

The Cosmos

The “Cosmos” instrument developed by Japanese is applied for the evaluation of fish quality by determination of smell intensity. Like other instruments, the “Cosmos” instrument is handheld, portable, as well as rapid and nondestructive. Therefore, it could be used for evaluation of fresh and chilled fish in the seafood industry and on fishing vessels. Gelman et al. [50] found strong correlation between the organoleptic and Cosmos results for six species of fish and concluded that application of the “Cosmos” instrument for objective quantitative evaluation of fresh and chilled fish quality by determination of smell intensity appears to be practicable.

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13.3.6


Electronic Nose

Odor, the main indicator of fish freshness, has been analyzed by sensory panel or gas chromatography (GC). Since these kinds of analyses are both time consuming and expensive, an electronic nose called FreshSense was developed and distributed by Element-Bodvaki in Iceland and has been found to be a rapid, nondestructive method to measure volatile compounds, indicating spoilage of odors in seafood. FreshSense is based on a closed, static sampling system and electrochemical gas sensors, which are sensitive to volatile compounds. The most important chemicals involved in fresh fish odors are long-chain alcohols and carbonyls, bromophenols, and N-cyclic compounds. However, short-chain alcohols and carbonyls, amines, sulfur compounds, and aromatic, N-cyclic, and acid compounds are produced by microbial activity and lipid oxidation during storage of fish [55,56]. The concentrations of these compounds are related to the degree of spoilage. Different electronic noses have been employed for measurement of fish freshness. These are metal-oxide semiconductor gas sensors, electrochemical sensors (CO, H2O, NO, SO2, and NH3), thickness shear mode quartz resonators, semiconductor dimethylamine (DMA) gas sensor, and prototype solid-state–based gas sensor called the FishNose [57–62]. Olafsdottir et al. [63] studied the freshness of iced redfish and found that there was a good correlation between the response of CO sensor and QIM method for both air and modified atmosphere storage of redfish. Trggvadottir and Olafsdottir [64] also found that the response of all electronic sensor (CO, H2O, NO, SO2, and NH3) results for haddock from different seasons showed a similar trend. Studies on cod fillets and heads also gave similar results, and it was found that CO sensor showed the highest response [65]. It has been indicated that a combination of electronic nose systems based on different sensor technologies improved the performances compared with the single technology for the codfish fillets [66]. Fish freshness has also been evaluated by a computer screen photoassisted technique (CSPT)based gas sensor array. This technique is based on the fact that a computer screen can be easily programmed to show millions of colors, combining wavelengths in the optical range [56]. Previous optics-based electronic noses relied on absorbance and fluorescence. However, CSPT evaluates both effects [56,67–71]. Data analysis is important in electronic nose measurements, which determines the relation between sensor output patterns and the properties of the sample being analyzed [72]. The most frequently used methods are artificial neural networks (ANNs), chemometric analysis such as principal component analysis (PCA), and partial least-square regression (PLS-R).

13.3.7 Near-Infrared Reflectance Spectroscopy Near-infrared reflectance spectroscopy has been used in various analytical applications. The technique is characterized by speed and simplicity; it has the ability to measure numerous samples within a short time; it can be operated on-/at-line; and it is nondestructive, easy to handle, and requires little training of operators [73]. This method has been applied for determination of fat, water, and protein content in fish [74–78], free fatty acid (FFA) in fish oils [79,80], water-holding capacity of thawed fish muscle [81], and quality assessment of frozen minced red hake [82], cod caught by long line and gillnet [73], and thawed, chilled modified atmosphere packed (MAP) cod fillets [83]. Fourier transform infrared (FT-IR) spectroscopy is another technology that is a rapid, nondestructive, online industrial production chain. On the other hand, it requires too much handling of samples, causing changes in protein and muscle structure. Compared with FT-IR, diff use reflectance infrared Fourier transform (DRIFT) spectroscopy has advantages; that is, it is fast,


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its use is simple, sensitive, cheap, and requires a small amount of sample. For the first time, this technique has been applied to sardine muscle during iced storage, and it has been indicated that this spectroscopic technique is useful in assessing the freshness and quality of sardine during iced storage [84]. Traceability is becoming a method of providing safer food supplies and of connecting producers and consumers. Traceability can be defined as the history of a product in terms of the direct properties of that product and/or properties that are associated with that product once these products have been subject to particular value-adding processes [85]. The traceability system can also be used for the determination of fish freshness, recording the product temperature from the moment of catch. This alternative method could be cost effective and definitely more reliable.

13.4 Chemical and Biochemical Methods Chemical and biochemical methods for the evaluation of seafood quality are more reliable and accurate, since they eliminate personal opinions on the product quality. These objective methods should correlate with sensory quality, and the chemical compound that is determined should increase or decrease as microbial spoilage or autolysis progresses [16]. Currently, the most used method to evaluate fish freshness is to combine several measurements obtained from different methods and correlate the findings with sensory analysis [59]. The most used procedures for the objective measurements of seafood quality are given in the next sections.

13.4.1 ATP and Its Breakdown Products Rigor mortis occurs in postmortem muscle tissue and is associated with stiffness of muscle or flesh. This process results from breakdown of adenosine triphosphate (ATP), which is the main energy source for metabolic activity. It has been indicated that there is a correlation between nucleotide catabolism and loss of freshness. Nucleotide breakdown reflects both action of autolytic enzymes and bacterial action [16]. The sequences of nucleotide catabolism proceed as shown in Figure 13.1. The initial stage of the reaction catalyzed by endogenous enzymes takes place quickly, leading to accumulation of adenosine diphosphate (ADP) and inosine monophosphate (IMP). The oxidation Adenosine triphosphate (ATP) Adenosine diphosphate (ADP) Adenosine monophosphate (AMP) Inosine monophosphate (IMP) Inosine (Ino) Hypoxanthine (Hx) Xanthine (Xa) Uric acid (Uric)

Figure 13.1 shown.

In postmortem fish muscle, degradation of ATP proceeds according to the sequence

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of Hx to xanthine and uric acid is slower and is the result of endogenous enzyme activity or microbial activity [86]. The IMP is associated with fresh fish flavor, whereas inosine and Hx reflect poor quality [87]. The concentrations of ATP and its breakdown products have been used as indicators of freshness in many fish species [8,9,13,88–92]. The K value proposed by Saito et al. [93] is a biochemical index for fish quality assessment based on nucleotide degradation. The K value includes intermediate breakdown products, and it varies within species of fish [94,95]. Since adenosine nucleotides are almost converted to IMP within 24 h postmortem [96], Karube et al. [97] proposed the Ki value, which excludes ATP, ADP, and adenosine monophosphate (AMP). However, in some species ATP, ADP, and AMP remain even after 2 weeks [97]. With some species, the Ki value has been shown to increase very rapidly and then remain constant even though freshness quality continues to decrease greatly [98,99]. Therefore, the K value can be superior to the other values. The G value proposed by Burns et al. [100] was found to be superior to Ki value for iced Atlantic cod, although it was observed to decrease during the first 2 or 3 days of iced storage, before its subsequent increase. In addition, H values have been described by Luong et al. [101] as an index of freshness quality. The H value of iced Pacific cod was observed to increase steadily, indicating its superiority to Ki value [101]. Gill et al. [102] also proposed Fr value for yellow fin tuna. These results showed that measuring the concentration of single nucleotide degradation product to determine freshness quality of seafood is not appropriate, but measuring the concentration of ATP and its degradation products can be useful in determining freshness quality [20]. P value has been described by Shahidi et al. [103]. Determination of G and P values are useful with lean fish. However, it is difficult to obtain meaningful G and P values since fatty fish deteriorate due to rancidity [103]. It was reported that K and related values increased linearly (except Fr value) with storage time in turbot [91], European eel [13], and sea bream [104]. The rate of nucleotide degradation varies with species, body location (dark or white muscle), stress during capture, handling, season, and storage conditions [105,106]. Several methods have been proposed for the analysis of single or a combination of nucleotide catabolites, but the high-performance liquid chromatography (HPLC) method is the most reliable among them. The K, Ki, G, P, H, and Fr values are calculated by the procedures described by Saito et al. [93], Karube et al. [97], Burns et al. [100], Shahidi et al. [103], Luong et al. [101], and Gill et al. [102], respectively. The formulas are as follows: lno + Hx ⎡ ⎤ K (%) = ⎢ ⎥⎦ × 100 ATP + ADP + AMP + IMP + lno + Hx ⎣ lno + Hx ⎡ ⎤ K i (%) = ⎢ ⎥⎦ × 100 IMP + lno + Hx ⎣ lno + Hx ⎡ ⎤ G (%) = ⎢ × 100 ⎣ AMP + IMP + lno ⎥⎦ lno + Hx ⎡ ⎤ P (%) = ⎢ ⎥⎦ × 100 AMP + IMP + lno + Hx ⎣ Hx ⎡ ⎤ H (%) = ⎢ ⎥⎦ × 100 IMP + lno + Hx ⎣ IMP ⎡ ⎤ Fr (%) = ⎢ ⎥⎦ × 100 IMP lno Hx + + ⎣


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13.4.2 Biogenic Amines The concentration of biogenic amines has been reported to be a reliable method of measuring the quality of fish, depending on the species being examined [10,11,107,108]. The formation of biogenic amines results from microbial degradation during the later storage of fish, and the concentration of these increases with storage time [91,109,110]. Biogenic amines are generated by microbial decarboxylation of specific free amino acids in fish or shellfish tissue [111]. The most significant biogenic amines produced postmortem in fish and shellfish products are histamine, putrescine, cadaverine, tyramine, tryptamine, 2-phenylethylamine, spermine, spermidine, and agmatine. The importance of estimating the concentration of biogenic amines in fish and fish products is related to their impact on human health and food quality. Since the amines are produced by spoilage bacteria toward the end of shelf life of a fish, their levels are considered as indices of spoilage rather than freshness [112]. In addition, the disadvantages of using biogenic amines as an index of freshness quality are that their absence does not necessarily indicate a high-quality product [113]. Among the biogenic amines, histamine is potentially hazardous and the causative agent of histaminic intoxication [114]. The others especially putrescine and cadaverine have been reported to enhance the toxicity of histamine [115]. Consumption of seafood containing high amounts of these amines can have toxicological effects. These problems may be more severe in sensitive consumers who have a reduced mono- and diamine oxidase activity [116], the enzyme responsible for its detoxification. The hazardous concentrations of histamine are 5 mg/100 g and 20 mg/100 g fish—the legal limit for histamine set by the U.S. Food and Drug Administration [117] and the EU [118], respectively. The biogenic amine content of fish depends on fish species, free amino acid content [112], the presence of decarboxylase-positive microorganisms, the moment of capture, and stomach contents at death, since microbial flora vary seasonally [11]. By means of decarboxylation reactions, tyrosine produces tyramine, histidine yields histamine, and arginine leads to putrescine. Cadaverine is derived from lysine, tryptamine from tryptophan, and 2-phenylethylamine is derived from phenylalanine. Putrescine is also an intermediate of a metabolic pathway that leads to spermidine and spermine [119]. The QI and the biogenic amine index (BAI) were proposed by Mietz and Karmas [120] and Veciana-Nogues et al. [121] for determination of quality of fish, respectively. The formulas used were as follows: QI = (histamine + putrescine + cadaverine)/1 + (spermidine + spermine) BAI = (histamine + putrescine + cadaverine + tyramine) QI is based on the increases in putrescine, cadaverine, and histamine and decreases in spermine and spermidine during storage of fish, whereas BAI is based on increases in histamine, putrescine, cadaverine, and tyramine. There are various analytical techniques used to determine the concentration of biogenic amines, including thin-layer chromatography (TLC) [122,123], HPLC [120,124,125], GC [126,127], capillary zone electrophoresis (CZE) [128,129], and use of a biosensor [130–132]. Among these techniques, HPLC is mostly performed because of its sensitivity, reliability, and reproducibility.

13.4.3

pH

The pH is also an important parameter to show depletion in tissue and quality of flesh during storage. Process technology is influenced by rigor development, postmortem temperature, and pH [133]. Postmortem pH varies from 5.5.0 to 7.1 depending on season, species, and other factors

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[134,135]. Low pH is used as an indicator of stress at the time of slaughtering of many animals. Low initial pH is associated with higher stress before slaughtering [13,136–139]. Th is is caused by the depletion of energy reserves, mainly glycogen, with the production of lactate. Since the activity of enzymes depends on pH, it affects reactions taking place during storage of fish. A relatively low pH may cause a decrease in water binding to the myofibrils, affecting light scattering and the appearance of fish. Low pH also promotes oxidation of myoglobin and lipids [134].

13.4.4 Total Volatile Basic Nitrogen In seafood, total volatile basic nitrogen (TVB-N) primarily includes trimethylamine (TMA, produced by spoilage bacteria), ammonia (produced by deamination of amino acids and nucleotide catabolites), and DMA (produced by autolytic enzymes during frozen storage). The analyses of these indicators are considered unreliable because they reflect later stages of spoilage rather than freshness [140]. The European Commission (Council Regulation No. 95/149/EEC of March 1995) on fish hygiene specifies that if the organoleptic examination indicates any doubt as to the freshness of the fish, TVB-N should be used as a chemical check. The level of TVB-N in freshly caught fish is generally between 5 and 20 mg N/100 g muscle. However, the levels of 30–35 mg N/100 g muscle are considered the limit of acceptability for icestored cold-water fish [17,141]. Based on the results obtained from the literature, TVB-N level correlated with fish quality, as shown in a variety of fish such as European hake [142], Atlantic cod [143], sardine [12, 144], and European eel [13]. However, the level of TVB-N was not correlated with the time of storage of some fish species, such as frozen eel [145], turbot [92], pike perch [146], farmed gilthead sea bream [147], and hake [148]. Therefore, it could not be regarded as a good indicator of fish freshness and proved to be better as a spoilage index. It is well known that determination of TVB-N differs systematically according to the procedures used. The EC reference method for TVB-N determination, involving preliminary deproteinization with perchloric acid, was compared with two routine methods. The first one includes direct distillation of fish after adding magnesium oxide, whereas the second one includes the use of trichloroacetic acid instead of perchloric acid [149]. It was found that there was a good correlation between three methods, and direct distillation methods have been recommended as a rapid routine method.

13.4.5

Trimethylamine

The one type of spoilage caused by microorganisms often detected as a fishy odor is due to the decomposition of trimethylamine oxide (TMAO) via the enzyme TMAOase demethylase, as shown below: Following death of fish, bacteria act upon TMAO to produce TMA, which is considered to be the main cause of off odors in fish products [58,59]. Therefore, TMA is produced by the decomposition of TMAO due to bacterial spoilage and enzymatic activity [150,151], and it has been used as an indicator of marine fish spoilage: CH3 CH3 – N=O CH3 TMAO

CH3 CH3 – N CH3 TMA


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TMAO appears to be part of the system used for osmoregulation. The TMAO content of seafood varies with species, age, fish size, time of year, and environmental factors [152]. Seawater fish have 1–100 mg TMAO in every 100 g muscular tissue, whereas freshwater fish generally contain only 5–20 mg% [153]. TMA can be used as a spoilage indicator and not as an index of freshness. However, its usefulness depends on time of year, location of catching, stage of spoilage, type of storage and processing, and methods employed for analysis. TMA is not produced in a significant amount during the early stages of chilled storage of fish, but it appears after 3 or 4 days, after which the rate of production of TMA parallels the bacterial proliferation pattern [154]. Fresh fish has a very low amount of TMA with values less than 1.5 mg TMA/100 g in fresh cod, but values increase during spoilage. The fish is considered stale when the rate of TMA production is higher than 30 mg/100 g cod [155]. Several assays have been described for the determination of TMAOase activity in fish muscle [151,156,157]. Many analytical methods have been developed for the measurements of TMA, DMA, or TVB-N contents, including steam distillation [158], Conway microdiff usion and titration [159], colorimetric method [160], photometry [161], HPLC method [162], GC method [163, 164], a capillary electrophoresis method [165], semiconducting metal–oxide array [166], flowinjection-gas diff usion method [167], biosensor using flavin-containing monooxygenase type-3 [168], and solid-state sensors based on bromocresol green [169].

13.4.6

Dimethylamine

As mentioned earlier, fish contain TMAO, which is converted to TMA by bacteria in iced fish. During chilled or frozen storage of fish, when bacterial growth is inhibited, this reaction is replaced by a slow conversion by an enzyme to DMA and formaldehyde [16,150]. The formation of these products may cause severe quality changes or spoilage during prolonged frozen storage. The amount of DMA produced depends on species (except gadoid species, other species do not develop adequate amounts of DMA), the storage temperature, and time. DMA can be used as a spoilage index during frozen storage of some species such as frozen hake [170].

13.4.7

Formaldehyde

The formaldehyde content in seafood products is generally considered as nontoxic, but it can react with a number of chemical compounds such as amino acid residues, terminal amino groups, and low-molecular weight compounds, causing denaturation and cross-linking of proteins [171]. This reduces the solubility of myofibrillar proteins [172]. The formaldehyde content of frozen seafood is generally used as a spoilage index, especially in gadoid fish.

13.4.8

Lipid Oxidation Indicators

During processing and storage, enzymatic and nonenzymatic lipid oxidation occurs. A close relationship has been found between lipid damage and quality of the final product [173]. Fresh fish has a limited shelf life and is prone to deterioration, whereas fish can be stored in a frozen state for several months without severe changes in quality. The limiting factor of frozen storage in lean fish species is denaturation of proteins, which results in a dry and firm texture of the fish muscle [174]. However, lipid oxidation is the limiting factor in fatty fish species, resulting in rancidity.

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Handbook of Seafood and Seafood Products Analysis Initiation: Initiators (heat, light, trace of heavy metals) R+H

RH Propagation: O2 R

RO2

RO2 + RH

ROOH + R

ROOH

RO + OH

2ROOH

ROO + ROO + H2O

Termination: R+R

RR

R + ROO

ROOH

ROO + ROO

ROOH + O2

Figure 13.2 The autoxidation of fatty acids. (From Hamilton, R.J., in Rancidity in Foods, 3rd edn., Allen, J.C. and Hamilton, R.C. (Eds.), Chapman & Hall, London, U.K., 1994, pp. 1–22.)

Off taste and off odor are usually defined as rancidity. Seafood has highly unsaturated lipid content. Under chilled/frozen conditions, lipid oxidation compounds interact with proteins, leading to protein denaturation, nutritional losses, and modification of electrophoretic profiles of proteins [172,175–177]. Many factors affect the onset and development of rancidity (oxidative and hydrolytic degradation of lipids), including the degree of unsaturation of the oil, the type and concentrations of antioxidants, pro-oxidants, moisture content, oxygen availability, temperature, and degree of exposure to light [178–180]. Several chemical and physical techniques applied alone or together have been used to determine the degree of oxidation and hydrolytic degradation of lipids in edible oils. There are three steps in autoxidation of unsaturated fatty acids; initiation, propagation, and termination (Figure 13.2). Initiators (such as light, heat) convert RH to free radicals (initiation phase), and free radicals react with oxygen to produce peroxide radicals (ROO). The peroxide radical can attack another lipid molecule RH, resulting in peroxide (ROOH) and new free radical (propagation phase). Peroxides are not stable compounds, and they break down to aldehydes, ketones, and alcohols, which are the volatile products causing off flavor in products. The amount of reactive compounds increases gradually, and then the quantity of radicals and peroxides decreases, forming stable deterioration products (termination phase) [181,182]. Excess free radicals and peroxides in foods cause destruction of essential fatty acids and vitamins A, C, E, B6, thiamine, and pantothenic acid. Free radicals from oxidizing lipids can polymerize with proteins and destroy certain amino acids. Peroxides can also react with proteins and result in a decrease in their nutritional value. They also destroy pigments, produce toxins, and cause off flavor/odors [183]. Fish oil contains about 20% of their total fatty acids as long-chain PUFA, consequently, fish and fish oils are highly susceptible to the development of oxidative rancidity, unpleasant odors, off flavors, and taints [179,180]. The amount of hydroperoxides can be used as a measure of the extent of oxidation in the early stages. The hydroperoxide value is generally shortened to peroxide value (PV). The major chemical indicators for the determination of the extent of oxidative rancidity


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are anisidine value (AV), PV, TOTOX (2VP + AV), and thiobarbituric acid (TBA). PV measures primary products of lipid oxidation, which break down to secondary products of oxidation or react with proteins. Increase in the PV is most useful as an index of the earlier stages of oxidation; as oxidation proceeds the PV can start to fall. AV and TBA values measure the secondary products of lipid oxidation. During prolonged storage of seafood, PV, AV, and TBA values may increase, reach a peak, and decline [184,185]. Many methods have been employed for the measurements of lipid oxidation in foods as a means of determining the degree of damage [20,186]. However, there are some difficulties with common methods when quality has to be assessed, since oxidation products are unstable and react with biological amino constituents, such as proteins, peptides, free amino acids, and phospholipids, causing production of interaction products [187]. Analysis of these interaction products by fluorescence detection as a quality assessment index for frozen-stored sardine was studied by Aubourg et al. [188] and it was found that fluorescence detection of interaction compounds can provide an accurate method to assess quality differences during frozen storage of sardine. Cozzolino et al. [80] also reported that PLS-R and near-infrared (NIR) spectroscopy to monitor both oxidation and hydrolytic degradation of lipids in fish oil can be successfully employed.

13.4.9

Lipid Hydrolysis

Hydrolysis leads to hydrolytic rancidity and involves hydrothermal or enzymic (lipase) hydrolysis to FFA and other products. FFAs and their oxidation products would have an effect on muscle texture and functionality, since they interact with myofibrillar proteins and promote protein aggregation [189]. A gradual increase in FFA formation was obtained for all kinds of samples as a result of the frozen storage time for fatty fish such as tuna, sardine, European eel, horse mackerel [13,188,190,191], and lean fish such as blue whiting, haddock, cod [192,193], and also freshwater fish [194].

13.5

Microbiological Methods

Numbers and types of microbes present in foods are important indicators of safety and quality. Microbiological analyses of seafood involve testing for presence or absence of pathogens such as salmonellas and determination of numbers of colony-forming units (CFU) named “total viable counts (TVC)” or “aerobic plate count (APC),” or numbers of CFU of indicator organisms such as Enterobacteriaceae, coliforms, or enterococci [195]. Microbial assessments have been carried out to monitor the numbers of various groups of microorganisms during the production process as part of food safety objectives and also hazard analysis critical control point (HACCP) systems [196]. Spoilage of fish and fish products is a result of the production of off odors and flavors mainly caused by bacterial metabolites [197]. The numbers of specific spoilage organisms (SSOs) and the concentration of their metabolites can be used as objective quality indicators for determination of shelf life of seafood. It is possible to predict shelf life of seafood based on knowledge of initial numbers and growth of SSO. Microbial growth models can be used to determine the effect of various time/temperature combinations on shelf life of fish in production and distribution chain. It was indicated that the main requirements for shelf life predictions are to collect information about SSO, spoilage domain such as the range of environmental conditions over which a particular SSO is responsible for spoilage and spoilage level [198]. Mathematics models have been well established for the growth of spoilage bacteria such as Photobacterium phosphoreum, Shewanella putrefaciens

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[198], Brochothrix thermosphacta [199], Listeria monocytogenes [200], and Clostridium perfringens [201], which were shown to correlate with remaining shelf life of product and also correlated better than classical TVC measurements. Prediction of the remaining shelf life of seafood requires reliable estimates of the initial population of SSO, because it varies from batch to batch due to season, feeding, catching method, handling, and storage after catch [202]. Among the microbiological methods for determination of bacterial counts in a short time, impedance is the most promising [203]. Mathematical models along with impedance technique may provide reliable information on shelf life of seafood within 24 h. The change in electrical properties (impedance, conductance, and capacitance) due to the growth of microorganisms in the culture media has been used for the rapid estimation of total bacterial counts [204], coliforms [205], and Salmonella spp. [206]. The principle of the impedance measurement is based on the phenomenon that at a time point (i.e., detection time—DT) at which bacteria have grown to a population of approximately 107 CFU/mL or higher, an accelerating change in impedance (or conductance) will occur in the growth media. The decrease in impedance (or increase in conductance) is due to the breakdown of the substrate molecules in the media to smaller molecules (e.g., acids), which have more charges than the substrate itself [207]. Current microbiological culture methods rely on growth in culture media, followed by isolation, and biochemical and serological identification. These methods are laborious and time consuming, requiring a minimum of 1 or 5 days to recognize. These methods are also not appropriate for online processing of seafood. However, modern microbiological techniques [such as polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR)], antibody techniques [such as enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunomagnetic chemiluminescence (ELIMCL)], and oligonucleotide probes give results in 1 day or even less [209–213]. On the other hand, these methods have limitations in performing quantitative analyses, also lack in sensitivity, and are costly.

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134. Haard, N., The role of enzymes in determining seafood color, flavor and texture. In: Safety and Quality Issues in Fish Processing. H.A. Bremner (Ed.), Woodhead Publishing Limited, Cambridge, U.K., 2002, pp. 221–254. 135. Simeonidou, S., Govaris, A., and Vareltzis, K., Quality assessment of seven Mediterranean fish species during storage on ice. Food Res. Int., 30(7), 479–484, 1998. 136. Azam, K., Mackie, I.M., and Smith, J., The effect of slaughter method on the quality of rainbow trout (Salmo gairdneri) during storage on ice. Int. J. Food Sci. Technol., 24, 69–79, 1989. 137. Marx H., Brunner B., Weinzierl, W., Hofmann, R., and Stolle, A., Methods of stunning freshwater fish: Impact on meat quality and aspects of animal welfare. Zeitschrift fur Lebensmittel-Untersuchung Und-Forschung, 204A, 282–286, 1997. 138. van de Vis, J.V., Oehlenschläger, J., Kuhlmann, H., Münkner, W., Robb, D.H.F., and Schelvis-Smit, A.A.M., Effect of commercial and experimental slaughter of eels (Anguilla anguilla L.) on quality and welfare. In: Farmed Fish Quality. S.C. Kestin and P.D. Warriss (Eds.), Fishing News Book, Oxford, U.K., pp. 234–248, 2001. 139. Morzel M. and van de Vis, H., Effect of the slaughter method on the quality of raw and smoked eels (Anguilla anguilla L.). Aquacult. Res., 34, 1–11, 2003. 140. Rehbein, H. and Oehlenschlager, J., Zur zusammensetzung der TVB-N fraktion in sauren extrakten und alkalischen destillaten von seefishfillet. Archiv für Lebensmittelhygiene, 33, 44–48, 1982. 141. Huss, H.H., Fresh Fish: Quality and Quality Changes. Food and Agriculture Organization (FAO) of the United Nations, Rome, Italy, p. 132, 1988. 142. Perez-Villarreal, B. and Howgate, P., Composition of European hake, Merluccius. J. Sci. Food Agric., 40, 347–356, 1987. 143. Botta, J.R., Lauder, J.T., and Jewer, M.A., Effect of methodology on total volatile basic nitrogen (TVB-N) determination as an index of quality of fresh Atlantic cod (Gadus morhua). J. Food Sci., 49, 734–736, 750, 1984. 144. Ababouch, L.H., Souibri, L., Rhaliby, K., Ouadhi, O., Battal, M., and Busta, F.F., Quality changes in sardines (Sardina pilchardus) stored in ice and at ambient temperatures. Food Microbiol., 13, 123–132, 1996. 145. Özoğul, Y., Özogul, F., Özkütük, A.S., and Küley, E., Hydrolysis and oxidation of European eel oil during frozen storage for 48 weeks. Eur. Food Res. Technol., 224, 33–37, 2006. 146. Özyurt, G., Özogul, Y., Özyurt, C.E., Polat, A., Özogul, F., Gökbulut, G., Ersoy, B., and Küley, E., Determination of the quality parameters of pike perch Sander lucioperca caught by gillnet, longline and harpoon in Turkey. Fish. Sci., 73, 412–420, 2007. 147. Tejada, M. and Huidobro, A., Quality of farmed gilthead sea bream (Sparus aurata) during ice storage related to the slaughter method and gutting. Eur. Food Res. Technol., 215, 1–7, 2002. 148. Baixas-Nogueras, S., Bover-Cid, S., Veciana-Nogués, M., and Vidal-Carou, M., Chemical and sensory changes in Mediterranean hake (Merluccius merluccius) under refrigeration (6°C–8°C) and stored in ice. J. Agric. Food Chem., 50, 6504–6510, 2002. 149. Vyncke, W., Comparison of the official EC method for the determination of total volatile bases in fish with routine methods. Archiv für Lebensmittelhygiene, 47, 110–112, 1996. 150. Simeonidou, S., Govaris, A., and Vareltzis, K., Effect of frozen storage on the quality of whole fish and fillets of horse mackerel (Trachurus trachurus) and Mediterranean hake (Merluccius mediterraneus). Z Lebensm Unters Forsch A, 204, 405–410, 1997. 151. Nielsen, M.K., Havemeister, W., Rehbein, H., Sotelo, C.G., and Jørgensen, B.M., A sensitive trimethylamine-N-oxide aldolase assay in two steps without deproteinisation. J. Sci. Food Agric., 80, 197, 2000. 152. Hebard, C.E., Flick, G.J., and Martin, R.E., Occurrence and significance of trimethylamine oxide and its derivatives in fish and shellfish. In: Chemistry and Biochemistry of Marine Food Products. R.E. Martin, G.J. Flick, C.E. Hebard, and D.R. Ward (Eds.), Avi, Westport, CO, pp. 149–304, 1982. 153. Stansby, M.E. and Olcott, H.S., Composition of fish. Chapter 26. In: Industrial Fishery Technology. Reinhold Pub., New York, 1963.

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154. Lindsay, R.C., Flavour of fish. In: Seafoods Chemistry, Processing Technology and Quality. F. Shahidi and J.R. Botta (Eds.), Chapman and Hall, Great Britain, pp. 75–82, 1994. 155. Bonnell, A.D., Quality Assurance in Seafood Processing: A Practical Guide. Chapman and Hall, London, U.K., pp. 74–75, 1994. 156. Gill, T.A. and Paulson, A.T., Localization, characterization and partial purification of TMAO-ase. Comp. Biochem. Physiol., 71B, 49–56, 1982. 157. Joly, A., Cottin, P., Hanching, L., and Ducastaing, A., Trimethlyamine N-oxide demethylase (TMAO-ase) of saithe (Pollachius-virens) kidney-a study of some physicochemical and enzymatic properties. J. Sci. Food Agric., 59, 261–267, 1992. 158. Malle, P. and Tao, S., Rapid quantitative determination of trimethylamine using steam distillation. J. Food Protect., 50, 756–760, 1987. 159. Conway, Microdiffusion Analysis and Volumetric Error. Crosby Lockwood and Sons, London, U.K., 1947. 160. AOAC Official Method 971.14. Trimethylamine nitrogen in seafood. In: AOAC Official Methods of Analysis. AOAC, Arlington, VA, p. 7, 1995. 161. Zhi, Z.L., Rios, A., and Valcàrcel, M., Direct determination of trimethylamine in fi sh in the flow-reversal injection mode using a gas extraction sampling device. Anal. Chem., 67, 871–877, 1995. 162. Malle, P. and Valle, M., Assay of biogenic amines involved in fish decomposition. J. Assoc. Off. Anal. Chem., 79, 43–49, 1996. 163. Ann daCosta, K., Vrbanac, J.J., and Zeisel, S.H., The measurement of dimethylamine, trimethylamine, and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry. Anal. Biochem., 187(2), 234–239, 1990. 164. Veciana-Nogues, M.T., Albala-Hurtado, M.S., Izquierdo-Pulido, M., and Vida-Carou, M.C., Validation of a gas-chromatographic method for volatile amine determination in fish samples. Food Chem., 57, 569–573, 1996. 165. Timm, M. and Jørgensen, Bo M., Simultaneous determination of ammonia, dimethylamine, trimethylamine and trimethylamine-imageoxide in fish extracts by capillary electrophoresis with indirect UV detection. Food Chem., 76, 509–518, 2002. 166. Hammond, J., Marquis, B., Micheals, R., Oickle, B., Segee, B., and Vetelino, J., A semiconducting metal-oxide array for monitoring fish freshness. Sens. Actuat. B, 84, 113–122, 2002. 167. Dhaouadi, A., Monser, L., Sadok, S., and Adhoum, N., Validation of a flow-injection-gas diff usion method for total volatile basic nitrogen determination in seafood products. Food Chem., 103(3), 1049–1053, 2007. 168. Mitsubayashi, K., Kubotera, Y., Yano, K., Hashimoto, Y., Kon, T., and Nakakura, S., Trimethylamine biosensor with flavin-containing monooxygenase type 3 (FMO3) for fish-freshness analysis. Sens. Actuat. B, 103, 463–467, 2004. 169. Pacquit, A., Lau, K.T., McLaughlin, H., Frisby, J., Quilty, B., Diamonda, D., Development of a volatile amine sensor for the monitoring of fish spoilage. Talanta, 69, 515–520, 2006. 170. Rey-Mansilla, M. Del M., Sotelo, C.G., Aubourg, S.P., Rehbein, H., Havemeister, W., Jørgensen, B., and Nielsen, M.K., Localization of formaldehyde production during frozen storage of European hake (Merluccius merluccius). Eur. Food Res. Technol., 213, 43–47, 2001. 171. Nielsen, M.K. and Jørgensen, Bo M., Quantitative relationship between trimethylamine oxide aldolase activity and formaldehyde accumulation in white muscle gadiform fish during frozen storage. J. Agric. Food. Chem., 52, 3814–3822, 2004. 172. Sikorski, Z.E. and Kolakowska, A., Changes in protein in frozen stored fish. In: Seafood Proteins. Z.E. Sikorski, B.S. Pan, and F. Shahidi (Eds.), Chapman & Hall, New York, pp. 99–112, 1994. 173. Hsieh, R. and Kinsella, J., Oxidation of polyunsaturated fatty acids: Mechanisms, products, and inhibition with emphasis on fish. Adv. Food Res. Nutr. Res., 33, 233–341, 1989. 174. Mackie, I.M., The effects of freezing on flesh proteins. Food Rev. Int., 9, 575–610, 1993. 175. Castrillón, A., Álvarez-Pontes, E., García, M.T., and Navarro, P., Influence of frozen storage and defrosting on the chemical and nutritional quality of sardine (Clupea pilchardus). J. Sci. Food Agric., 70, 29–34, 1996.


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176. Undeland, I. and H., Lingnert, Lipid oxidation in fillets of herring (Clupea harengus) during frozen storage. Influence of prefreezing storage. J. Agric. Food Chem., 47, 2075–2081, 1999. 177. Saeed, S. and Howell, N.K., Effect of lipid oxidation and frozen storage on muscle proteins of atlantic mackerel (Scomber scombrus). J. Sci. Food Agric., 82, 579–586, 2002. 178. Chan, H.W.S., The mechanism of autoxidation. In: Autoxidation of Unsaturated Lipids. Food Science and Technology. Series of Monographs. H.W.S. Chan (Ed.), Academic Press, London, U.K., pp. 1–16, 1987. 179. Borquez, R., Kolle, W.D., Wolf, W., and Spie, W.E.L., A rapid method to determine the oxidation kinetics of n−3 fatty acids in fish oil. Food Sci. Technol., 30, 502–507, 1997. 180. Chantachum, S., Benjakul, S., and Sriwirat, N., Separation and quality of fish oil from pre-cooked and non pre-cooked tuna heads. Food Chem., 69, 289–294, 2000. 181. Deman, J.M., Principles of Food Chemistry. 2nd Edn. An AVI Book, United States, pp. 38–80, 1990. 182. Hamilton, R.J., Kalu, C., Prisk, E., Padley, F.B., and Pierce, H., Chemistry of free radicals in lipids. Food Chem., 60(2), 193–199, 1997. 183. Tucker, B.W., Overview of current seafood nutritional issues: Formation of potentially toxic products. In: Seafood Safety, Processing and Biotechnology. F. Shahidi, Y. Jones, and D.D. Kitts (Eds.). Technomic Publishing Company, Lancaster, PA, pp. 5–10, 1997. 184. Melton, S., Methodology for following lipid oxidation in muscle foods. Food Technol., 37(7), 111–116, 1983. 185. Aubourg, S.P., Piñeiro, C., and González, M.J., Quality loss related to rancidity development during frozen storage of horse mackerel (Trachurus trachurus). JAOCS, 81(7), 671–678, 2004. 186. Kim, R. and Labella, F., Comparison of analytical methods for monitoring autoxidation profiles of authentic lipids. J. Lipid Res. 28, 1110–1117, 1987. 187. Leake, L. and Karel, M., Nature of fluorescent compounds generated by exposure of protein to oxidizing lipids. J. Food Biochem., 9, 117–136, 1985. 188. Aubourg, S.P., Sotelo, C.G., and Pérez-Martín, R., Assessment of quality hanges in frozen sardine (Sardina pilchardus) by fluorescence detection. JAOCS, 75(5), 575–580, 1998. 189. Pacheco-Aguilar, R., Lugo-Sánchez, M.E., and Robles-Burgueňo, M., Postmortem biochemical and functional characteristic of monterey sardine muscle stored at 0°C. J. Food Sci., 65(1), 40–47, 2000. 190. Medina I., Sacchi, R., and Aubourg, S., A 13C-NMR study of lipid alterations during fish canning: Effect of filling medium. J. Sci. Food Agric., 69, 445–450, 1995. 191. Aubourg, S.P. and Ugliano, M., Effect of brine pre-treatment on lipid stability of frozen horse mackerel (Trachurus trachurus). Eur. Food Res. Technol., 215, 91–95, 2002. 192. Aubourg, S., Rey-Mansilla, M., and Sotelo, C., Differential lipid damage in various muscle zones of frozen hake. Eur. Food Res. Technol., 208, 189–193, 1999. 193. Aubourg, S. and Medina, I., Influence of storage time and temperature on lipid deterioration during cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) frozen storage. J. Sci. Food Agric., 79, 1943–1948, 1999. 194. Kaneniwa, M., Miao, S., Yuan, C., Lida, H., and Fukuda, Y., Lipid components and enzymatic hydrolysis of lipids in muscle of Chinese freshwater fish. J. Am. Oil Chem. Soc., 77(8), 825–831, 2007. 195. Corry, J.E.L., Hedgesb, A.J., and Jarvisc, B., Measurement uncertainty of the EU methods for microbiological examination of red meat. Food Microbiol., 24, 652–657, 2007. 196. ICMSF, Micro-organisms in Foods 7. Microbiological Testing in Food Safety Management. Kluwer, Amsterdam, the Netherlands, 2002. 197. Gram, L. and Huss, H.H., Microbiological spoilage of fish and fish products. Int. J. Food Microbiol., 33, 121–137, 1996. 198. Dalgaard, P., Modelling of microbial activity and prediction of shelf life of packed fresh fish. Int. J. Food Microbiol., 19, 305–318, 1995. 199. McClure, P.J., Baranyi, J., Boogard, E., Kelly, T.M., and Roberts, T.A., A predictive model for the combined effect of pH, sodium chloride and storage temperature on the growth of Brochothrix thermosphacta. Int. J. Food Microbiol., 19, 161–178, 1993.

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200. Carrasco, E., Valero, A., Pérez-Rodríguez, F., García-Gimeno, R.M., and Zurera, G., Management of microbiological safety of ready-to-eat meat products by mathematical modelling: Listeria monocytogenes as an example. Int. J. Food Microbiol., 114, 221–226, 2007. 201. Juneja, V.K., Huang, L., and Thippareddi, H.H., Predictive model for growth of Clostridium perfringens in cooked cured pork. Int. J. Food Microbiol., 110, 85–92, 2006. 202. Koutsoumanis, K.P., Taoukis, P., Drosinos, E.H., and Nychas, G.-J.E., Applicability of an Arrhenius model for the combined effect of temperature and CO2 packaging on the spoilage microflora of fish. Appl. Environ. Microbiol., 66, 3528–3534, 2000. 203. Koutsoumanis, K., Lambropoulou, K., and Nychas, G.-J.E., A predictive model for the non-thermal inactivation of Salmonella enteritidis in a food model system supplemented with a natural antimicrobial. Int. J. Food Microbiol., 49(1–2), 63–74, 1999. 204. Ogden, I.D., Use of conductance methods to predict bacterial counts in fish. J. Appl. Bacteriol., 61, 263–268, 1986. 205. Firstenberg-Eden, R. and Klein, C.S., Evaluation of a rapid impedimetric procedure for the quantitative estimation of coliforms. J. Food Sci. 48, 1307–1311, 1983. 206. Bullock, R.D. and Frodsham, D., Rapid impedance detection of salmonella in confectionery using modified LICNR broth. J. Appl. Bacteriol., 66, 385–391, 1989. 207. Wu, J.J., Huang, A.H., Dai, J.H., and Chang, T.C., Rapid detection of oxacillin-resistant staphylococcus aureus in blood cultures by an impedance method. J. Clin. Microbiol., 1460–1464, June 1997. 208. Amagliani, G., Giammarini, C., Omiccioli, E., Brandi, G., and Magnani, M., Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods. Food Control, 18, 1137–1142, 2007. 209. Landete, J.M., Rivas, B. de las, Marcobal, A., and Muñoz, R., Molecular methods for the detection of biogenic amine-producing bacteria on foods. Int. J. Food Microbiol., 117, 258–269, 2007. 210. Nowak, B., Müffling, T.V., Chaunchom, S., and Hartung, J., Salmonella contamination in pigs at slaughter and on the farm: A field study using an antibody ELISA test and a PCR technique. Int. J. Food Microbiol., 115, 259–267, 2007. 211. Lee, C.Y., Panicker, G., and Bej, A.K., Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLinkk NH microwell plate sandwich hybridization. J. Microbiol. Methods, 53, 199–209, 2003. 212. Gehring, A.G., Irwin, P.L., Reed, S.A., Tu, S., Andreotti, P.E., Akhavan-Tafti, H., and Handley, R.S., Enzyme-linked immunomagnetic chemiluminescent detection of Escherichia coli O157:H7. J. Immunol. Methods, 293, 97–106, 2004. 213. Lida, K., Abe, A., Matsui, H., Danbara, H., Wakayama, S., and Kawahara, K., Rapid and sensitive method for detection of Salmonella strains using a combination of polymerase chain reaction and reverse dot-blot hybridization, FEMS Microbiol. Lett., 114(2), 167–172, 1993.

Chapter 14

Analytical Methods to Differentiate Farmed from Wild Seafood Iciar Martínez, Inger Beate Standal, Marit Aursand, Yumiko Yamashita, and Michiaki Yamashita Contents 14.1 Introduction ..................................................................................................................216 14.2 Morphological Examination..........................................................................................216 14.3 Genetic Analysis ............................................................................................................217 14.3.1 Sample Preservation and DNA Extraction Methods ........................................217 14.3.2 DNA Markers ..................................................................................................218 14.4 Analysis of Proteins .......................................................................................................218 14.4.1 Sample Preservation......................................................................................... 219 14.4.2 Protein Extraction ........................................................................................... 220 14.4.3 Analysis of Proteins ......................................................................................... 220 14.5 Analysis of the Lipid Content ........................................................................................221 14.5.1 Sample Preservation ........................................................................................ 222 14.5.2 Lipid Extraction and Gas Chromatography .................................................... 222 14.5.3 1H NMR and 13C NMR Analyses ................................................................... 222 14.6 Stable Isotopes .............................................................................................................. 224 14.6.1 Sample Preservation ........................................................................................ 225 14.6.2 SNIF–NMR and IRMS .................................................................................. 225

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14.7 Trace Element Fingerprint............................................................................................ 226 14.7.1 Sample Preservation ........................................................................................ 226 14.7.2 ICP-MS ........................................................................................................... 227 14.8 Other Methods ............................................................................................................ 227 Acknowledgments ................................................................................................................... 227 References ............................................................................................................................... 228

14.1 Introduction The implementation of analytical methods to differentiate farmed from wild-produced seafood is important to ensure correct consumer information and avoid fraud: Information about the production method of seafood is obligatory in the EU (CR EC No 2065/2001 of October 22, 2001 laying down detailed rules for the application of CR EC No 104/2000 regarding informing consumers about fishery and aquaculture products) and similar laws apply in Japan (Law on Standardization and Proper Labeling of Agricultural and Forestry Products, JAS Law, of 1999), and the United States (The Federal Food, Drug, and Cosmetic Act and The Fair Packaging and Labeling). Correct information about the production method of seafood is also important, because farmed and wild organisms carry different hazards and are therefore submitted to different regulations and analytical controls. For example, commercially farmed specimens may contain residues of veterinary drugs whose presence is unlikely in wild seafood, but wild specimens may contain parasites harmful to humans, and these are seldom present in farmed seafood. The production method is also part of the information essential to fulfill the traceability of a product and, therefore, analytical methods should be made available to confirm it. Several methods have been successfully applied to differentiate farmed from wild seafood, including morphological examination, genetic analyses, analysis of the protein and lipid contents, as well as examination of the stable isotope and trace element profiles. Standards for organic farming are still under development in many countries. Although in this work no special mention is made to organic farming, the set of technologies to apply are basically the same as those described here. In particular, stable isotope analyses combined with fatty acid (FA) profiles have proven particularly useful when tested.1

14.2 Morphological Examination There are few publications and no official guidelines for the morphological differentiation of farmed and wild aquatic organisms. In small Salmo salar, 100% discrimination between farmed (AquaGen strain) and wild parr was achieved by examining the body form, shape of the head, size of the eyes and mouth, and length of the pectoral fin.2 Also in an earlier study,3 it was shown that the morphology of the head, fins, and caudal peduncles could be used for a total correct classification of wild, farmed, and sea-ranched S. salar parr. The later study showed that the environmentally induced phenotypic divergence increased with age and with the numbers of generations under domestication. In cod, the most prominent differences are the higher condition factor, larger liver, and smaller head4 as well as backbone malformations in farmed specimens. Farmed cod often present unattractive black lines consisting of layers of melanin-filled cells associated with blood vessels due to overabundance of copper in commercial feeds.5 The flesh of farmed cod sometimes presents

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a translucent grayish aspect, in contrast to the white opaque color of the wild, and the liver in farmed cod is much bigger than the liver of wild fish.6 However, classification based on morphological criteria demands the presence of the morphologic diagnostic characters, which are usually absent in many intermediate products as well as in the ready-to-eat dish, thus limiting its application.

14.3 Genetic Analysis Doyle et al.7 proposed that the genetic diversity of aquacultured stocks of fish should be maintained and their genetic impact on wild stocks minimized by using breeding programs designed to generate genetic diversity. If this policy had been followed, it would be relatively difficult to find markers for wild and farmed fish, since diversity would be one of the selected traits in the farmed fish. However, in most breeding programs the fish are indeed selected based on commercially interesting traits such as growth performance,8,9 resistance to diseases or to stress,10 and optimal adaptation to different environments.9 Genetic analyses have allowed the differentiation of wild from farmed fish populations in a variety of species,11–13 and a loss of rare alleles has usually been observed in the farmed populations.11,14–16 Hayes et al.17 suggested that it was possible to assign accurately a fish sampled from the market place to either the farmed population or the wild using either microsatellite or single nucleotide polymorphism (SNP) markers. Genomics analyses are dealt with in more detail in Chapter 4 of this handbook.

14.3.1 Sample Preservation and DNA Extraction Methods The sample should be extracted as soon as possible after sampling, in particular if it has a high enzymatic activity (for example if it contains the hepatopancreas in a crustacean). Delays and the use of preservations methods will diminish the quality and the yield of DNA. If the sample must be preserved, the best method is to freeze it in liquid nitrogen or in a biofreezer. Depending on the type of sample and its use, a normal freezer (−20°C) may also be used. For very long periods, we recommend preservation in 96% ethanol. To extract frozen samples we recommend to start the procedure before the sample is completely thawed, since enzymatic activity also takes place at subzero temperatures. Samples fi xed in ethanol must be allowed to dry completely, so that all the ethanol is evaporated, and then be rehydrated in water or in the extraction buffer. One requisite condition for any genetic analysis is the obtention of good quality DNA suitable for PCR amplification, which is the most common analysis. Many commercial kits, such as Qiagen, Dynal (Invitrogen), Nucleospin (Clontech), Amersham Biosciences (GE Healthcare), Wizard (Promega), GeneRelease (Bio Venture Inc.), E.Z.N.A Stool DNA Isolation Kit (United Bioinformatica Inc.), and others, give satisfactory results. Each kit is provided with a detailed description of how to use it. The basic steps in all DNA extraction methods include the inactivation of nucleases, for example by chelating divalent cations using EDTA and EGTA. Th is step is not necessary in samples preserved in ethanol, because the enzymes are inactivated by the fi xation. Then, the cells are opened (by heat treatment, sonication, or by the use of Proteinase K) and proteins are removed usually by incubation with Proteinase K. The DNA is then separated from the contaminating cellular components by salt precipitation, chloroform extraction, treatment with Chelex,18 or gel filtration, and then recovered by ethanol or isopropanol precipitation. The DNA

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pellet is usually washed at least once with 70% ethanol, the alcohol is allowed to evaporate, and the DNA is reconstituted in double-distilled sterile water or in a slightly alkaline buffer (50 mm Tris–EDTA, pH 8.0). Three more methods that have reputedly produced good quality DNA suitable for amplification, and also for forensic studies, from food matrices include the use of hexadecyl–trimethyl ammonium bromide (CTAB),19 the Chelex method,18 and the salt extraction method.20,21 When using the salt extraction method with heavily degraded samples, we have found it helpful to leave the tubes after the first precipitation of DNA with isopropanol in a freezer at −20°C or at −80°C for a few hours before centrifugation (Marian Martinez de Pancorbo, University of the Basque Country, Spain, personal communication). In these samples the pellet may be practically invisible, so the next step, which is washing the pellet with 50% ethanol, must be performed very carefully not to lose the sample. On other occasions, however, it is possible to amplify the DNA of a sample by simply dehydrating it and placing a small amount directly into the PCR amplification mixture. This method has been successfully used by the authors of this paper (unpublished results) and by Bucklin and Kochert22 with whole individuals of Calanus.

14.3.2

DNA Markers

In recent years, several countries, such as Norway, the United States, China, India, Japan, and others, have started programs to map the whole genome of some species, including oysters, salmonids (salmon, trout, Arctic charr), cod, tilapia, catfish, shrimp, and bass. The outcome of these programs is already producing lists of genetic markers linked to traits of interest, which may be used to identify the strains of the farmed individuals that display an increased frequency of the desired traits.23 In principle, any marker, whether microsatellites, SNPs, genomic, or mitochondrial, has the potential to be useful to differentiate farmed from wild specimens of a given species.11,13,15,17 However, which markers and how many of them are necessary to differentiate a wild from a farmed specimen are completely dependant on the species and the breeding stock and need to be examined on an individual basis. It is worth mentioning, however, that the Norwegian company GenoMar has patented a method to trace back farmed individual Atlantic salmon, tilapia, Atlantic halibut, cod, and sea bass, by using a series of SNP and microsatellite polymorphisms by PCR and by oligonucleotide ligation assay (OLA).24 The method requires that all parent fish of the brood stock are DNA typed as well as all the individuals under examination. In the future, DNA analyses may be performed using chips that permit the determination in one fast step of many characteristics simultaneously, including the species, traits, breeding stock, and production method. An additional advantage is that it is possible to use robots for many of the steps (DNA preparation, sample preparation, etc.), which further increases the amount of samples that can be processed.

14.4 Analysis of Proteins No clear protein marker has been identified to discriminate farmed from wild seafood. However, protein markers commonly used for genetic analyses have the potential to be used as markers for farmed or wild, since some alleles are more frequent in one group than in the other.14 In addition, differences in the protein pattern of liver25 and muscle26,27 tissues between farmed and wild salmon and cod have been reported.


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Industrial fish farming is a relatively new activity compared with farming of land animals. Although feeds and breeding conditions need to be developed and optimized for each species, it is common to apply directly to new species those conditions that have proven successful for other species, and then modify them depending on the results. Thus, neither feeds nor breeding conditions may be optimal for farming, and this may induce stress in the farmed animals, which is reflected in the composition of their organs.25–27 In addition, the depletion of the wild stocks of pelagic fish and the high price of feeds based on fish meal and oil, which would be a natural diet, have prompted the development of feed formulations based on vegetable oils and proteins.28–31 The source of protein in teleost fish is very important, and they require high levels of dietary protein (30%–60%), which they use as their preferred energy source.32 Unfortunately, the amino acid profiles of plant proteins do not meet the essential amino acid requirements of fish, and feed diets based on plant protein require supplementation with synthetic amino acids.33 Moreover, it has been shown that components in fish feeds may contain very high levels of metals (Cu, Zn, Fe, Mg, and Ca)34 and that vegetable meals may contain antinutritional factors (protease inhibitors, lectins, antigenic proteins, etc.)29 that may have adverse effects on fish.33,35,36 Martin et al.25 attributed the alteration in the protein expression in the liver of rainbow trout to the presence of antinutritional factors in feeds containing soy protein. Using proteomic analysis, these authors identified 33 differentially expressed proteins, including heat shock proteins, several enzymes, and structural and FA-binding proteins. The authors noted a downregulation of some structural proteins in fish-fed soy proteins, attributed to the fish’s increased requirement for energy metabolism. In addition, several enzymes involved in anabolic metabolism were downregulated in fish fed the diet rich in soybean meal, indicating increased emphases on catabolism relative to anabolism in the fish fed this diet. Interestingly, Olsson et al.,27 also registered the altered expression of five enzymes implicated in the glycolytic pathway and citric acid cycle in farmed cod. Texture is an important quality attribute of the fish flesh. Soft texture, usually considered negative, is more common in stressed and in farmed than in wild fish.37 Martinez et al.26 examined the protein expression in skeletal muscle of farmed and wild cod by high-resolution twodimensional electrophoresis and found differences between the two, which were attributed to increased proteolytic activity in the muscle of the farmed compared with the wild cod. Johnston et al.38 found that the reason for the softening in this species did not seem to be the faster growth of the farmed fish, and they hypothesized that the greater concentration of insoluble collagen present in wild salmon may contribute to their firmer texture. Some enzymatic systems that may be responsible for the muscle softening are metalloproteases and collagenases, lysosomal cathepsins, neutral calcium-activated calpains, and the proteasome. However, no study has identified yet the main system/s responsible for the soft texture in farmed fish or the spots that may be used as markers to discriminate farmed from wild fish. Proteins and proteomic analyses are dealt with in more detail in a different chapter of this handbook.

14.4.1 Sample Preservation The optimal case would be when the extraction of proteins can take place on the sample immediately after the experimental treatment, that is, with no preservation at all. When this is not possible, a preservation procedure that minimizes the modifications (denaturation, aggregation, loss of functional groups, and proteolysis) of the proteins in the sample should be chosen. Optimal methods include fast freezing and frozen storage using temperatures as low as possible. Thus, optimal freezing would be achieved immediately after excision by submersion in liquid nitrogen and storage at −80°C or by freezing and storing directly at −80°C. For short periods of time, −20°C

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may be acceptable. It should be noted that any preservation procedure will alter the protein profile in the sample, and, therefore, one should be very careful when comparing samples preserved and stored under different conditions.

14.4.2

Protein Extraction

There are many methods for extraction of proteins, depending on the proteins one wishes to examine. Since current studies are still trying to identify markers, we focus on the use of techniques with the potential to identify such markers. Proteomic techniques have a clear advantage in this field. The first step in proteomic analyses is to extract as many proteins as possible from the sample. However, due to the great diversity and properties of the proteins contained in the edible tissues of seafood, as well as the different degrees of processing to which the sample may have been submitted (freezing, cooking, etc.), the optimal extraction procedure for any given sample must be determined empirically. It is common to use several buffers with increasing concentration of chaotropic agents (urea, thiourea), detergents (CHAPS, Triton X-100, SDS), and reducing agents (b-mercaptoethanol, dithiothreitol (DTT), tributylphosphine) to solubilize the widest possible spectrum of proteins. Some authors have claimed that the use of DNase I and RNase in the extraction buffer increases the number of spots in the gels. In our experience, this may be because some commercial preparations of these enzymes are contaminated with proteases. We therefore recommend not to use such enzymes. In addition to published works,25–27 BioRad39 and GE Healthcare Amersham have some excellent manuals about protein extraction and analysis. The use of protease inhibitors should always be considered: use of some inhibitors and cocktails may help to preserve the sample during the extraction procedure, but they will hamper the study of protease activities that may be relevant in some other works. Their use should be evaluated for each particular study.

14.4.3 Analysis of Proteins As already mentioned, there are many methods suitable for protein analyses. The choice of method depends on the protein and the property one wishes to examine, and there are special protocols for each application. Proteomics permits the separation of many proteins (often thousands) from a complex protein mixture in one step, and, therefore, its application is widespread in many fields. The proteins are separated first according to their pI in 3% polyacrylamide gels in which a pH gradient is created using a mixture of ampholytes. The optimal pH range to choose depends on the sample, but 3–10 are commonly used for wide screenings. Afterward, the strip containing the proteins separated by their pI is loaded on top of the second-dimension SDS-PAGE gel, usually 8%–20% or 12% PAGE, for a wide screening. Both first and second-dimension gels can be purchased as precast, ready to use gels from several companies. After separation, the gels can be stained by Coomassie Blue (low sensitivity but compatible with mass spectrometry (MS) analysis necessary for subsequent peptide fingerprinting and sequencing), silver (high sensitivity, but not all protocols are compatible with MS), and fluorescent labeling or staining (of intermediate sensibility and also compatible with MS). The pictures of the gels containing similar samples of wild and farmed specimens obtained after scanning are compared using adequate software (such as Bionumerics or PDQuest) to identify differentially expressed spots that are then excised from the gels, destained, reduced, alkylated, and digested, usually with trypsin. The tryptic fragments are cleaned from contaminants, and peptide mass fingerprinting of the digests is then


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usually performed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. The proteins are afterward identified by searching in databases (National Centre for Biotechnology Information, NCBI) using suitable software (MASCOT, ProteinLynx Global SERVER), which uses MS data to identify proteins from primary sequence databases. The whole procedure is described in detail by Martin et al.25 and reviewed by Granvogl et al.41 The workload can be reduced by using precast gels and automated procedures with suitable software and robotic stations (sample and gel handling and staining, identification of diagnostic spots, spot cutters, etc.). However, due to frequent errors in the automated spot identification procedure (because of imperfect spot separation and identification caused by overlapping of spots, very different staining intensities, etc.), the final identification and assignment of the spots in the gels must be performed visually by trained personnel. This is probably the most time consuming step of the whole procedure. Proteomic analysis is a complicated procedure necessary to identify the biological markers. Once the diagnostic proteins are identified, however, the whole process can be greatly simplified by targeting only the biomarkers: raising or synthesizing antibodies targeting those proteins in order to use them in several formats. For example, lateral flow strip tests permit in situ easy and fast screening of seafood samples; and ELISA format will permit the routine analyses of many samples, for example, in control laboratories. Development of protein chips will facilitate the simultaneous screening of many targets and samples.

14.5 Analysis of the Lipid Content The analysis of the triglyceride (TG) fraction, in particular when combined with stable isotope composition (see the following paragraph), has often given correct classification of farmed and wild specimens.42,43 The changes in the FA composition of the TG fraction following changes in the composition of the diet have been explained using a dilution model.44 Frequently, the total amount of TGs alone may be used as a criterion to differentiate farmed from wild fish, because farmed fish usually have a much higher content than wild ones.45–48 In addition, the FA profile of TGs reflects that of the feed,44 and this FA fingerprint has often been successfully used49–52 as a diagnostic to identify the production method. The FA profile of vegetable oils (such as corn, cottonseed, linseed, olive, palm, rapeseed, soybean, and sunflower) used as partial substitutes for marine oils in fish feeds53–57 have in common a very low or undetectable amount of the long-chain omega-3 polyunsaturated fatty acids (PUFAs) C20:5n3 (EPA) and C22:6n3 (DHA) characteristic from fish oils, and often a single FA may account for about 50% of the total FA content in these oils. The FA composition of fish oils is more complex, that is, there are more FAs present in detectable amounts, and it is seldom that only one of them makes more than 25% of the total. As in vegetable oils, which FA is more abundant is species dependant. C16:0 and C18:1n9 are relatively abundant in all fish oils, but C22:1 (several isomers) is relatively more abundant in Coho salmon, capelin, herring, and sand eel and C22:6n3 is more abundant (over 10%) in Atlantic and Coho salmon, sand eel, and sardines than in capelin, herring, or menhaden.53,55–58 Specific FAs are selectively retained or used. In Atlantic salmon for example, it has been shown that there was selective deposition and retention of C22:6n3, so that the concentrations in flesh were higher than in the diet, whereas C18:1n9, C22:1n11, C18:2n6, and C18:3n3 were selectively metabolized.59 Other studies in the same species showed that the flesh had higher levels of C18:1n9 and C22:6n3 and less C20:5n3 than the feeds.60 The FAs C18:1n9 and 18:2n6 may act as markers

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for vegetable oils, and in particular, the latter seems to be the most persistent after a dietary switch to fish oil diet, and even after the levels of C20:5n3 and C22:6n3 were restored to the original high levels, the ratio n3/n6 was not fully restored.52,61 As indicated by Refsgaard et al.,62 one must always take into account the very wide variation in the concentrations of lipid components that can be found in apparently homogeneous populations of farmed salmon, which, together with the special feed formulations used for organic farming and the fact that escaped farmed fish and wild fish eating around farms may display intermediate lipid profiles,63,64 may contribute to the difficulty of performing correct classifications as wild/ farmed based only on the FA composition.

14.5.1 Sample Preservation Due to the high levels of PUFAs, it is particularly important to exercise care when working with marine lipids: it is recommended to use low temperature (work in ice or in a cold room) and avoid or minimize exposition to air and light in order to prevent lipid hydrolysis, oxidation, and polymerization. Fresh samples should be kept wrapped in air- and light-tight containers and stored at low temperatures. If freezing is required, it is best to use as low a temperature as possible, that is, −80°C, and an inert atmosphere.

14.5.2 Lipid Extraction and Gas Chromatography Procedures for lipid extraction are described in another book chapter of this series. For detailed descriptions of the analysis of fish samples, the reader is directed to several publications46,50,52 that give detailed descriptions of the procedure.

14.5.3

1

H NMR and 13C NMR Analyses

High-resolution nuclear magnetic resonance spectroscopy (HR-NMR) has emerged as a popular technique in the analysis of foodstuff, including fish and fish products. NMR spectroscopy exploits the magnetic properties of certain nuclei: nuclei that contain odd numbers of protons or neutrons have an intrinsic magnetic moment and angular momentum. The most commonly measured nucleus is 1H (the most receptive isotope at natural abundance), but NMR is applicable to any nucleus possessing spin (e.g., 2H, 13C, 15N, 14N, 19F, 31P, 17O, 29Si, 10B, 11B, 23Na, 35Cl, 195Pt). NMR spectroscopy can be used to identify functional groups, since in a one-dimensional spectrum each peak is produced by those nuclei placed in an identical local chemical environment. The spectrum is often used to obtain information about the number and type of molecules in a mixture. HR-NMR has been particularly valuable in the study of marine lipids, because it provides multicomponent information and can be applied nondestructively.65 NMR gives a fingerprint of the sample analyzed, which may be used as a rapid profiling technique. The most commonly used HR-NMR techniques in wild/farmed classification are 1H NMR and 13C NMR, both of which are able to detect a range of metabolites in a nontargeted way. The major advantage of 1H NMR spectroscopy compared with 13C NMR is the higher sensitivity and thereby shorter acquisition times per experiment. On the other hand, 13C NMR has a greater range of chemical shifts, which leads to less overlapping of signals, and is the preferred tool in lipid analysis when interpretation of


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spectra is the goal. Multivariate methods are frequently applied to study differences among NMR spectra.66,67 1H NMR has been used to perform quantitative measurements of total n-3 FAs and of the levels of DHA.68,69 This analysis can be carried out with a high degree of automation and gives a rapid fingerprint (2–5 min) of the lipid profile. 1H NMR has also been applied to differentiate between wild and farmed salmon and sea bream of different origins.70,71 13C NMR gives information about FA composition of fish72 and the positional distribution of PUFAs in triacylglycerols and phospholipids,73 which is of value for authentication purposes. Both HR 1H and 13C NMR, in conjunction with chemometrics, have allowed the differentiation between wild and farmed salmon74 and cod51 of different origins. The most commonly used solvent in the analysis of neutral lipids is deuterated chloroform (i.e., 99.8% CDCl3), which is easily evaporated, leaving the sample ready for analysis. Typically, a sample size of 50–100 mg of lipid in 0.5–0.8 mL solvent is used, although the optimal sample size depends on the instrument. Tetramethylsilane (TMS) is usually added as a chemical shift and intensity reference. Standardized procedures should be followed to ensure repeatability and comparability,75 and it is important that all the samples contain the same volume. 13C NMR and 1H NMR spectra are fi rst obtained by Fourier transformation of the resulting free-induction decay (FID) function after applying a prospective line-broadening function. Normally, phasing and baseline correction are applied but no zero fi lling, because it may interfere with the multivariate data analysis. Typically, the chemical shift scale is referred to the shift of TMS or indirectly to TMS by the peaks from chloroform at 7.28 ppm for 1H NMR and by the triplet of CDCl3 at 77.0 ppm for 13C NMR. Factors that affect the exact chemical shift of NMR signals include the type of solvent used, pH, interactions with metal ions, hydrogen bondings, and other intermolecular interactions.76 In some studies, a semiquantitative 13C NMR approach has been chosen, due to the fact that quantitative measurements require a considerable longer experimental time. However, even though the signal intensities within each spectrum are not quantitative, the relative intensities for corresponding signals across different spectra are comparable. The assignment of spectral resonances gives information about the chemical composition of the samples, but it is not necessary for classification purposes. The application of multivariate statistics to NMR spectral data increases the potential of the technique considerably. Both the area/ intensities of peaks and full spectra can be input for multivariate analysis. When full spectra are used, they are normally converted to ASCII or JCAMP file formats. Regions without signals or unwanted signals are removed before multivariate analysis.66 Potential problems about inconsistencies in ppm values between samples in the data analyses should be solved by manual alignment or data pretreatment methods.77 Regarding reproducibility issues, the whole procedure from sample preparation to analysis by a data exploration technique can be affected by factors unrelated to the sample characteristic of interest.75 Small differences in experimental conditions, such as instabilities in apparatus, temperature variations, inhomogeneities in the applied magnetic field, or differences in relative concentrations of the samples analyzed, may lead to erroneous classification.77 It is advisable to check that all spectra have acceptable linewidth and lineshape after the NMR analysis. It is expected that in the future the use of flow injection systems, ideally for screening many samples with short acquisition time, will increase the sample throughput significantly, although this approach has still not been widely used for authentication purposes.71 Another technique that in the future may be used more often is the analysis of intact tissue by high-resolution magic angle spinning (HR-MAS).78

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14.6 Stable Isotopes The variation in the abundance of the stable isotopes of carbon, nitrogen, and oxygen has been proposed as a method suitable for food authentication.79 Carbon exists as two stable isotopes: 12C (abundance 98.89%) and 13C (1.11%); nitrogen as two: 14N (99.63) and 15N (0.37%); and oxygen as three: 16O (99.759%), 17O (0.037%), and 18O (0.204%). Since a molecule containing heavier isotopic forms has stronger chemical bonds, the abundance of stable isotopes varies among different compounds. In addition, a kinetic fractionation occurs, because the enzymatic reaction rates on substrates that contain the lighter isotopic forms are faster than in reactions involving the heavier isotopic forms. Thus, the abundances of the stable isotopes differ between substrate and product. Moreover, since the physical properties of molecules containing heavier isotopic forms are different, equilibrium reactions also lead to a fractionation of the isotopic forms. A significant kinetic fractionation is already found in the initial fi xation of carbon dioxide in photosynthesis: the isotopic signature of C3 plants (plants that form a three-carbon compound as the first stable intermediate in the incorporation of CO2, mostly broadleaf plants and plants in the temperate zones) shows a higher degree of 13C depletion than the C4 plants (where the CO2 is converted first into a four-carbon organic acid: these plants are mostly found in warm sunny regions, typically tropical grasses, such as maize, although many broadleaf plants are also C4). For example, while typical d13C mean values of C3 plants may be −26/−28‰, C4 plants may have d13C mean values of −12/−14‰.80 The natural isotopic abundance largely varies depending on the chemical forms. Some atmospheric gases, such as CO2, N2, and O2, exhibit limited variation. In contrast, N2O and CH4 exhibit wide isotopic variation, and they reflect both significant isotopic fractionation by microbes and the different biological substrates producing these gases. The isotopic abundances in animal tissues and animal food products are the summation of the feeds ingested throughout all their life, plus the kinetic fractionations occurring in animal metabolism. For example, the 13C/12C ratio for both milk fat and cheese protein give information on the type of forage fed to the cows.81 This is because the 13C/12C ratio depends almost exclusively on the photosynthetic mechanism used by the plants for CO2 fi xation. Differences in the 15N/14N ratio also result essentially from diet. Usually animal products become enriched in the heavier isotope (15N and 13C), depending on their diet and their position in the trophic chain: the higher its position in the trophic chain, the higher the proportion of the heavier isotope.82 Dempson and Power83 examined the potential of using stable isotopes of carbon and nitrogen 13 (d C and d15N) by isotope ratio mass spectrometry (IRMS) to identify escaped farmed Atlantic salmon. Samples of muscle tissue of wild salmon were significantly more enriched in nitrogen (d15N: mean = 12.75; SD ± 0.38‰) but depleted in lipid-corrected carbon (d13C: mean = −20.51; SD ± 0.23‰) than the aquaculture specimens, resulting in a complete separation of the two groups. Aursand et al.50 were also able to correctly classify Atlantic salmon according to their geographic origin and production method by using four FA compositions (C16:0, C16:1n-9, C18:1n-9, and C22:1n-9) together with the overall isotope ratio 2H/1H of the fish oils and three deuterium molar fractions obtained by site-specific natural isotope fraction studied by NMR (SNIF–NMR). Using the d15N of choline and the d18O of total oil, Thomas et al.52 were able to classify correctly according to the production method, 171 Atlantic salmon specimens originating from three continents and 15 different geographic regions. Introducing the percentage of C18:2n6 as a third variable in their model, they were also able to correctly classify the fish according to their geographic origin. Bell et al.46 were equally successful classifying sea bass using the FA profile, d13C of individual FA,


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d13C and d18O of total muscle oil, and d15N of the glycerol choline fraction of flesh phospholipids. Molkeltin et al.1 were able to differentiate wild, organic, and commercially farmed Atlantic salmon measuring d13C and d15N by IRMS in raw fillets. The research of Sweeting et al.84 has helped to understand the nitrogen isotopic variations in fishes, since these authors assessed the effects of body size, experimental duration, and environmental conditions on fish tissue. The assumption that fractionation was independent of body mass was upheld for muscle and heart tissue but not for liver. Interestingly, the d15N values of heart and liver were also affected by environmental temperature, probably reflecting the metabolic functions of these tissues and their associated turnover rates.

14.6.1 Sample Preservation It is very important not to contaminate the sample during handling. For example, it must not be washed in the laboratory after collection (which may alter the O and H profile of the sample). The C and O isotopic profiles of fish tissues may be altered if CO is used for stunning or killing, and so on. Usually, the collected tissue samples are dried at a constant temperature of approximately 50°C for 48 h, pulverized to a fine powder using a ball mill grinder, and stored in glass desiccation vials until analyzed.

14.6.2 SNIF–NMR and IRMS Two methods are used to assess stable isotopes: SNIF–NMR and IRMS.48 NMR techniques have been described previously. An advantage of SNIF–NMR over IRMS is that it produces a distinct isotopic fingerprint giving information on the frequency of each isotope in a given molecule and the position of the isotope in the molecule, whereas IRMS gives only an average value of the isotopic forms in the molecule. However, SNIF–NMR can only be applied to the few isotopomers possessing spin, whereas IRMS can be applied to all except 12 elements. The light elements, such as carbon, nitrogen, and oxygen isotopes, are typically determined with a gas isotope rationing mass spectrometer. The instrument consists of an ionizing source, a flight tube with a magnet, and a detector to measure the different isotopic species. The element is converted to a gaseous form to be analyzed by the mass spectrometer, thus hydrogen is introduced as H2, carbon as CO, nitrogen as N2, and oxygen as CO2. The gas is introduced in the mass spectrometer and is ionized by removal of an electron in the ion source. The ionized gas is then introduced in the flight tube under vacuum or carried by helium; the paths of isotopic species are deflected by the magnet by an angle that is a direct function of their mass over charge ratio, the ions are finally detected at the detector, and the abundance ratios of the heavy and light isotopic species are then calculated. Approximately 1 mg of dried, ground tissue is used in the simultaneous analysis of stable C and N isotopes.85,86 To facilitate comparisons between specimens with differing lipid contents, d13C values are normalized for lipid content following techniques developed by McConnaughey and McRoy87 and validated by Kline et al.88 Stable isotope ratios are expressed in delta (d) notation with measurements consisting of parts per thousand difference (‰) between the isotopic ratio of a sample relative to an international standard, as follows: d = (R sample/R standard − 1) × 1000‰, where R is the heavy:light isotopic ratio of the sample or standard, respectively (for carbon 13C:12C, for nitrogen 15N:14N). Enriched samples contain relatively more of the heavier isotopes. All international standards are

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set at 0‰ by convention. Carbonate rock from the Pee Dee Belemnite formation89 and nitrogen gas in the atmosphere90 are used as the standards for carbon and nitrogen, respectively.

14.7

Trace Element Fingerprint

Sometimes, farmed and wild specimens of the same species have different geographic distributions, and often the geographic origin of both farmed and wild seafood may be of relevance for its safety, quality, and price. Multivariate trace elemental analysis is increasingly used as a technique to differentiate the geographic origins of foodstuff.91 In the case of fish, otolith chemistry is used as a recorder of time and environmental conditions.92–94 Otolith chemistry is useful for identifying the natal origin and assessing the relative contribution of different nursery areas to mixed adult stocks. Thus, in addition to DNA-based species identification techniques, multivariate trace elemental analysis is expected to be helpful in determining whether the fish was farmed and its geographic distribution. Biochemical analytical techniques using multiple elemental analysis, as well as vitamin K and its metabolites, have been used to differentiate the geographical distribution of origin of farmed Japanese eel. Recently, false labeling problems were encountered in which imported live Japanese eels from Taiwan were illegally sold as being of Japanese origin. The origins of farmed and wild eel collected from different regions in Japan, Taiwan, and China were compared by analyzing the trace and heavy metal contents in the muscles to determine the differences among the fish farms for cultured eels and also to identify the river where wild eels had been caught.95 By using ICP-MS analysis the sensitivity in the determination of rare trace elements can be increased from the nM to pM level. Rare trace elements taken from the environment, such as uranium, lead, cadmium, and vanadium, were shown to be of relevance to determine the origin of eels. The same research group (Yamashita et al., unpublished data) examined the trace element composition of the muscle and shell of littleneck clams collected in Japan, Korea, and China, and they found distinct patterns for each of the three origins. Thirteen elements were shown to be the most diagnostic. Multivariate analysis showed that differences in elemental composition in the muscle between Japanese and imported clams were mainly due to two factors: Factor 1, attributable to cobalt, copper, and strontium levels and Factor 3, attributable to manganese and vanadium levels. In addition, cadmium and arsenic levels in the muscles of clams from China and Korea were higher than those of clams from Japan, with the exception that clams from Miyagi had high arsenic content. Therefore, multiple elemental analysis could also be used in this case to identify imported clams from China and Korea.

14.7.1 Sample Preservation As for stable isotope analysis, it is very important to avoid contaminating the sample during sampling, handling, and analysis, in particular since the analysis may detect contaminants at the pM level. All implements and containers should be cleaned with 0.5 M nitric acid and rinsed with Milli-Q ultrapure deionized water. Each sample should be separated from the tissues using ceramic knives and scissors and Teflon-coated tweezers to avoid contamination of metals, and it must be accurately weighed with a microbalance. The sample may be stored in a centrifuge tube at a temperature of −40°C or lower until analyzed. The first step in the analysis is the digestion of the sample: 0.1–1 g of tissue samples are placed into 50 mL Teflon tubes and 8–16 sample volumes of a mixture of concentrated trace-metal-grade nitric acid/hydroperoxide mixture (5:3) is added. The


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digestion may be carried out by placing the tubes in a microwave oven (for example, Multiwave 3000 Microwave Oven, Perkin-Elmer), and the resulting digest is a clear liquid with a yellow tint. Afterwards, an internal standard mixture is added, the samples are diluted to a final volume of 50 mL in Digitube (SCP Science, Canada), and stored at room temperature until use.

14.7.2 ICP-MS Multielement determination of trace elements is usually measured by inductively coupled plasma mass spectroscopy (ICP-MS).96 Samples digested as described above are introduced by pneumatic nebulization into a radio frequency plasma, where energy transfer processes cause desolvation, atomization, and ionization. The ions are extracted from the plasma through a differentially pumped vacuum interface and are separated on the basis of mass-to-charge ratio by a quadrupole mass spectrometer that has a minimum resolution capability of 1 atomic mass unit (amu) peak width at 5% peak height. Ions transmitted through the quadrupole are detected by continuous dynode electron multiplier assembly, and the ion information is processed by a data handling system. To initiate the proper operating configuration of the instrument and data system, the mass calibration and resolution are checked using diluted metal solutions as standards. For internal standardization, five internal standards are used: Sc, Y, In, Tb, and Bi. To verify that the instrument is properly calibrated on a continuous basis, a calibration blank and calibration standards are used as surrogate test samples after every 10 analyses. If the measured concentration deviates from the true concentration by more than 10%, the instrument is recalibrated, and the last 10 samples are analyzed again. For the determination of mercury, which suffers from severe memory effects, the total mercury concentration is determined by cold vapor atomic absorption spectrometry,97 using an automatic mercury analyzer (Hiranuma HG-200, Japan). Each solution (5 mL) of the microwave samples is applied to the atomic absorption spectrophotometer.

14.8

Other Methods

Depending on the species, there might be specific requirements that may be targeted to identify the production method. In the farming of salmon for example, the use of carotenoids is allowed, but although the diet of wild salmon contains astaxanthin, most artificial feeds contain a mixture of canthaxanthin and astaxanthins of different origins (both natural and synthetic). Analysis by chiral chromatography can be used to identify a chiral form (the meso form 3R,3′S) that does not occur naturally and can therefore be used as a marker for farmed salmon.42,98 However, much of the astaxanthin used in fish feed nowadays is produced from cultured microalgae or from krill,98 making this approach more unreliable than it used to be.

Acknowledgments This work was carried out with the financial support of the EU-STREP Project Sigma Chain: “Developing a Stakeholders’ Guide on the Vulnerability of Food and Feed Chains to Dangerous Agents and Substances” Contract No FOOD-CT-2004-506359, the Norwegian Research

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Council, SINTEF Fisheries and Aquaculture, and by grants from the Fisheries Research Agency and the Ministry of Agriculture, Forestry and Fisheries of Japan.

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22. Bucklin, A. and Kochert, T.D., Source regions for recruitment of Calanus finmarchicus to Georges Bank: Evidence from molecular population genetic analysis of mtDNA, Deep Sea Res. II, 43, 1665, 1996. 23. Delghandi, M., Mortensen, A., and Westgaard, J.I., Simultaneous analysis of six microsatellite markers in Atlantic cod (Gadus morhua): A novel multiplex assay system for use in selective breeding studies, Mar. Biotechnol., 5,141, 2003. 24. Lie, O. et al., Verification of food origin based on nucleic acid pattern recognition, Genomar, Asa (NO), EP1472366, http://www.freepatentsonline.com/EP1472366A2.html, 2004. 25. Martin, S.A.M. et al., Proteomic sensitivity to dietary manipulations in rainbow trout, Biochim. Biophys. Acta, 651, 17, 2003. 26. Martinez, I., Slizyte, R., and Dauksas, E., High resolution two-dimensional electrophoresis as a tool to differentiate wild from farmed cod (Gadus morhua) and to assess the protein composition of klipfish, Food Chem., 102, 504, 2007. 27. Olsson, G.B. et al., Metabolic disorders in muscle of farmed Atlantic cod (Gadus morhua), Aquacult. Res., 38, 1223, 2007. 28. Gomes, E.F. et al., Replacement of fish meal by plant-proteins in diets for rainbow trout (Oncorhynchus mykiss)-effect of the quality of the fish-meal based control diets on digestibility and nutrient balance, Water Sci. Technol., 31, 205, 1995. 29. Kaushik, S.J. et al., Partial or total replacement of fish meal by soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout, Oncorhynchus mykiss, Aquaculture, 133, 257, 1995. 30. Burel, C. et al., Potential o f plant-protein sources as fish meal substitutes in diets for turbot (Psetta maxima): Growth, nutrient utilisation and thyroid status, Aquaculture, 188, 363, 2000. 31. Carter, C.G. and Hauler, R.C., Fish meal replacement by plant meals in extruded feeds for Atlantic salmon, Salmo salar L., Aquaculture, 185, 299, 2000. 32. Cowey, C.B., Protein and amino acid requirements: A critique of methods, J. Appl. Ichthyol., 11, 199, 1995. 33. Krogdahl, A., Lea, T.B., and Olli, J.L. Soybean proteinase inhibitors affect intestinal trypsin activities and amino acid digestibilities in rainbow trout (Oncorhynchus mykiss), Comp. Biochem. Physiol., 107A, 215, 1994. 34. Olsson, G.B. et al., Gelatinolytic activity in muscle of farmed and wild Atlantic cod (Gadus morhua) related to muscle softening, in Seafood from Fish to Dish, Quality, Safety and Processing of Wild and Farmed Fish, Luten, J.B. et al., Eds., Wageningen Academic Publishers. the Netherlands, 2006, 161. 35. Vielma, J. et al., Influence of dietary soy and phytase levels on performance and body composition of large rainbow trout (Oncorhynchus mykiss) and algal availability of phosphorus load, Aquaculture, 183, 349, 2000. 36. Francis, G., Makkar, H.P.S., and Becker, K., Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish, Aquaculture, 199, 197, 2001. 37. Roth, B., Slinde, E., and Arildsen, J., Pre or post mortem muscle activity in Atlantic salmon (Salmo salar). The effect on rigor mortis and the physical properties of flesh, Aquaculture, 257, 504, 2006. 38. Johnston, I.A. et al., Fast growth was not associated with an increased incidence of soft flesh and gaping in two strains of Atlantic salmon (Salmo salar) grown under different environmental conditions, Aquaculture, 265, 148, 2007. 39. Bio-Rad. 2-D Electrophoresis for Proteomics: A Methods and Product Manual, Garfin, D. and Heerdet, L., Eds., http://www.biorad.com/LifeScience/pdf/Bulletin_2651.pdf, 2001. 40. GE Healthcare. 2-D Electrophoresis. Principles and Methods. Handbook 80-6429-60AC. (http://www1. gelifesciences.com/aptrix/upp00919.nsf/Content/2A3643B6787 885E0C 12570BE000DC671/$file/ 80642960.pdf, 2004. 41. Granvogl, B., Ploscher, M., and Eichacker, L.A., Sample preparation by in-gel digestion for mass spectrometry-based proteomics, Anal. Bioanal. Chem., 389, 991, 2007.

230 ◾ Handbook of Seafood and Seafood Products Analysis 42. Martinez, I., Revision of analytical methodologies to verify the production method of fish, in Seafood from Fish to Dish, Quality, Safety and Processing of Wild and Farmed Fish, Luten, J.B. et al., Eds., Wageningen Academic Publishers, the Netherlands, 2006, p. 541. 43. Martinez, I., Evaluation of the profile of lipids as a tool to discriminate wild from farmed salmon, 52 p. SEAFOODplus report 6.3.8. ISBN 978-87-7075-001-1, 2006. 44. Jobling, M., Are modifications in tissue fatty acid profiles following a change in diet the results of dilution? Test of a simple dilution model, Aquaculture, 232, 551, 2004. 45. Olsson, G.B. et al., Seasonal variations in chemical and sensory characteristics of farmed and wild Atlantic halibut (Hippoglossus hippoglossus), Aquaculture, 217, 191, 2003. 46. Bell, J.G. et al., Discrimination of wild and cultured European sea bass (Dicentrarchus labrax) using chemical and isotopic analyses, J. Agric. Food Chem., 55, 5934, 2007. 47. Grigorakis, K., Compositional and organoleptic quality of farmed and wild gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) and factors affecting it: A review, Aquaculture, 272, 55, 2007. 48. Martinez, I., Authenticity assessment based on other principles: Analysis of lipids, stable isotopes and trace elements, in Fishery Products: Quality, Safety and Authenticity, Oehlenschläger, J., and Rehbein, H., Eds., Blackwell Publishing, p. 388, 2009. 49. Chen, I. et al., Differentiation of cultured and wild sturgeon based on fatty acid composition, J. Food Sci., 60, 631, 1995. 50. Aursand, M., Mabon, F., and Martin, G.J., Characterization of farmed and wild salmon (Salmo salar), JAOCS, 77, 659, 2000. 51. Standal, I.B. et al., Discrimination of cod liver oil according to wild/farmed and geographical origins by GC and 13C NMR, JAOCS, 85, 105, 2008. 52. Thomas, F. et al., Determination of origin of Atlantic salmon (Salmo salar): The use of multiprobe and multielement isotopic analyses in combination with fatty acid composition to assess wild or farmed origin, J. Agric. Food Chem., 56, 989, 2008. 53. Gunstone, F.D., The Lipid Handbook, 2nd ed., Gunstone, F.D., Harwood, J.L., and Padley, F.B., Eds., Chapman & Hall, London, U.K., 1994. 54. Henderson, R.J., Bell, J.G., and Park, M.T., Polyunsaturated fatty acid composition of the salmon (Salmo salar L.) pineal organ: Modification by diet and effect on prostaglandin production, Biochim. Biophys. Acta, 1299, 289, 1996. 55. Dosanjh, B.S. et al., Influence of dietary blends of menhaden oil and canola oil on growth, muscle lipid composition, and thyroidal status of Atlantic salmon (Salmo salar) in sea water, Fish Physiol. Biochem., 19, 123, 1998. 56. Grisdale-Helland, B. et al., Influence of high contents of dietary soybean oil on growth, feed utilization, tissue fatty acid composition, heart histology and standard oxygen consumption of Atlantic salmon (Salmo salar) raised at two temperatures, Aquaculture, 207, 311, 2002. 57. Torstensen, B.E., Froyland, L., and Lie, Ø., Replacing dietary fish oil with increasing levels of rapeseed oil and olive oil—Effects on Atlantic salmon (Salmo salar L.) tissue and lipoprotein lipid composition and lipogenic enzyme activities, Aquacult. Nutr., 10, 175, 2004. 58. Aursand, M., Standal, I.B., and Axelson, D.E., High-resolution C-13 nuclear magnetic resonance spectroscopy pattern recognition of fish oil capsules, J. Agric. Food Chem., 55, 38, 2007. 59. Bell., J.G. et al., Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism, J. Nutr., 131, 1535, 2001. 60. Nichols, P.D., Mooney, B.D., and Elliot, N.G., Nutritional Value of Australian Seafood II, CSIRO Marine Research and FRDC, Hobart, Tasmania, Australia, 198pp. ISBN 1-876-996-07-2, 2002. 61. Bell, J.G. et al., Rapeseed oil as an alternative to marine fish oil in diets of post-smolt Atlantic salmon (Salmo salar): Changes in flesh fatty acid composition and effectiveness of subsequent fish oil “wash out,” Aquaculture, 218, 515, 2003.


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62. Refsgaard, H.H.F., Brockhoff, P.B., and Jensen, B., Biological variation of lipid constituents and distribution of tocopherols and astaxanthin in farmed Atlantic salmon (Salmo salar), J. Agric. Food Chem., 46, 808, 1998. 63. Skog, T.E. et al., Salmon farming affects the fatty acid composition and taste of wild saithe Pollachius virens L., Aquaculture Res., 34, 999, 2003. 64. Fernandez-Jover, D. et al., Changes in body condition and fatty acid composition of wild Mediterranean horse mackerel (Trachurus mediterraneus, Steindachner, 1868) associated to sea cage fish farms, Mar. Environ. Res., 63, 1, 2007. 65. Gribbestad, I.S., Aursand, M., and Martinez, I., High resolution 1H magnetic resonance spectroscopy of whole fish, fillets and extracts of farmed Atlantic salmon (Salmo salar) for quality assessment and compositional analyses, Aquaculture, 250, 445, 2005. 66. Lindon, J. C., Holmes, E., and Nicholson, J. K., Pattern recognition methods and applications in biomedical magnetic resonance, Prog. Nucl. Magn. Reson. Spectrosc., 39, 1, 2001. 67. Alam, T. M. and Alam, M. K. Chemometric analysis of NMR spectroscopy data: A review, Annu Rep. NMR Spectrosc., 54, 41, 2005. 68. Aursand, M., Rainuzzo, J., and Grasdalen, H., Quantitative high-resolution 13C and 1H nuclear magnetic resonance of fatty acids from white muscle of Atlantic salmon (Salmo salar), J. Am. Oil Chem. Soc., 70, 971, 1993. 69. Sacchi, R. et al., Proton nuclear magnetic resonance rapid and structure-specific determination of w-3 polyunsaturated fatty acids in fish lipids, J. Am. Oil Chem. Soc., 70, 225, 1993. 70. Masoum, S. et al., Application of support vector machines to 1H NMR data of fish oils: Methodology for the confirmation of wild and farmed salmon and their origins, Anal. Bioanal. Chem., 387, 1499, 2007. 71. Rezzi, S. et al., Classification of gilthead sea bream (Sparus aurata) from 1H NMR lipid profiling combined with principal component and linear discriminant analysis, J. Agric. Food Chem., 55, 9963, 2007. 72. Aursand, M. and Grasdalen, H., Interpretation of the 13C NMR spectra of omega-3 fatty acids and lipid extracted from the white muscle of Atlantic salmon (Salmo salar), Chem. Phys. Lipids, 62, 239, 1992. 73. Aursand, M., Jørgensen, L., and Grasdalen, H., Positional distribution of n-3 fatty acids in marine lipid triacylglycerols by high-resolution 13C nuclear magnetic resonance spectroscopy, J. Am. Oil Chem. Soc., 72, 293, 1995. 74. Aursand, M. and Axelson, D.E., Origin recognition of wild and farmed salmon (Norway and Scotland) using 13C NMR spectroscopy in combination with pattern recognition techniques, in Magnetic Resonance in Food Science: A View to the Future, Webb, G.A. et al., Eds., RSC Books, London, U.K., 2001, p. 227. 75. Defernez, M. and Colquhoun I.J., Factors affecting the robustness of metabolite fingerprinting using 1H NMR spectra, Phytochemistry, 62, 1009, 2003. 76. Fan, T.W.M., Metabolite profiling by one-and two-dimensional NMR analysis of complex mixtures, Prog. Nucl. Magn. Reson. Spectrosc., 28, 161, 1996. 77. Forshed, J., Schuppe-Koistinen, I., and Jacobsson, S.P., Peak alignment of NMR signals by means of a genetic algorithm, Anal, Chim. Acta 487, 189, 2003. 78. Aursand, M., Gribbestad, I.S., and Martinez, I. Omega-3 fatty acid content of intact muscle of farmed Atlantic salmon (Salmo salar) examined by 1H MAS NMR spectroscopy, in Handbook of Modern Magnetic Resonance Modern Magnetic Resonance, Part 1: Applications in Chemistry, Biological and Marine Sciences. Webb, G.A., Ed., Springer, Amsterdam, the Netherlands, 2006, 931. 79. Dennis, M., Recent developments in food authentication, Analyst, 123, 151R, 1998. 80. De Niro, M.J. and Epstein, S., Carbon isotopic evidence for different feeding patterns in two hyrax species occupying the same habitat, Science, 201, 906, 1978. 81. Camin, F. et al., Application of multielement stable isotope ratio analysis to the characterization of French, Italian, and Spanish cheese, J. Agric. Food Chem., 52, 6592, 2004.

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Chapter 15

Smoke Flavoring Technology in Seafood Vincent Varlet, Thierry Serot, and Carole Prost Contents 15.1 Introduction ................................................................................................................. 234 15.2 Smoke Flavoring Process .............................................................................................. 234 15.2.1 Liquid Smokes................................................................................................. 236 15.2.2 Smoke Oils ...................................................................................................... 237 15.2.3 Smoke Powders ............................................................................................... 237 15.2.4 Smoke By-Products ......................................................................................... 237 15.3 Use of Smoke Flavorings .............................................................................................. 238 15.4 Chemical Composition of Liquid Smokes .................................................................... 239 15.5 Role of SF Process Parameters in Volatile Compounds Generation .............................. 240 15.6 Organoleptic Roles of Volatile Compounds of SF .........................................................241 15.6.1 Role of Volatile Compounds of SF in the Odor ............................................... 241 15.6.2 Role of Volatile Compounds of SF in the Flavor .............................................. 245 15.6.3 Role of Volatile Compounds of SF in the Texture ............................................ 246 15.6.4 Role of Volatile Compounds of SF in the Aspect and Color ............................ 246 15.7 Role of Volatile Compounds of SF in Preservation ........................................................247 15.8 Polycyclic Aromatic Hydrocarbons............................................................................... 248 15.8.1 Properties and Toxicology ............................................................................... 248 15.8.2 Extraction and Analysis Methods of PAH in SF and Seafood Treated by SF ...................................................................................................249 15.9 Legislative Aspects.........................................................................................................249 15.9.1 European Regulations on PAH Found in SF ....................................................249 233

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15.9.2 European Regulations on PAH Concentration in Food Treated by SF ....................................................................................250 15.10 Conclusion ..................................................................................................................250 References ................................................................................................................................251

15.1

Introduction

Smoking is the oldest food preservation technique. Coupled to salting and drying steps, it allows decreasing microorganism activity. Moreover, wood smoke phenolic components are known to be antioxidants. Simultaneously, wood smoke imparts desired organoleptic characteristics such as smoky flavor. However, the smoking process has two main inconveniences: the production of carcinogenic contaminants—polycyclic aromatic hydrocarbons (PAHs)—during the incomplete pyrolysis of wood used to produce smoke and the release of smokes in the atmosphere. In United States where 75% of smoked foods are treated by liquid smokes, liquid smokes are commercialized since the end of the nineteenth century. This kind of smoke flavoring (SFs) appears as an alternative to the smoking process as it is carried out in Europe. Indeed, the use of liquid smokes avoids the release of smokes, allows a better control of PAH in the final product, and provides a higher diversity of smoked food [1]. Today, between 20% and 30% of European smoked food is treated by liquid smokes. SFs are widely used in the meat industry, and their uses in the seafood industry are increasing, especially thanks to the industrial benefits brought about by their use. By comparison with the smoking technology, the use of SF allows an easier storage (SF bottles versus wood logs), a better preservation of the combustible, and reduced risk of accidents due to fire. This industrial process leads to more homogenous products smoked with a repeatable intensity and provides an easier cleaning of the smokehouse. However, the legislation and the organoleptic quality of SF and products treated by SF constitute critical points that show the necessity of better improvement and harmonization of this technology.

15.2 Smoke Flavoring Process The first liquid SF was developed and patented by the Kansas pharmacist Wright in the late of nineteenth century [2]. SFs are obtained by the condensation of wood smoke and can be further fractionated, purified, or concentrated. The main woods used for smoke production are oak, beech, hickory, and marple, that is, mainly hard woods. Indeed, the chemical composition of soft woods is responsible for the generation of higher quantities of contaminants as PAH. The fractionation of smoke condensates allows obtaining a high diversity of SFs (powders, oils, aqueous solutions, etc.) with a wide range of organoleptic qualities, which give rise to new perspectives in the food industry [3]. It also allows the reduction of the PAH final concentrations. A simplified version of SF processes is presented in Figure 15.1. Wood sawdust is pyrolyzed in a furnace with low oxygen content. The smoke is filtered to eliminate particules and condensed. The gaseous smoke can be cooled down by water or by organic solvents. The combustible gases are recycled and directed to the furnace. After condensation, the crude smoke condensates are separated in three phases: a water insoluble heavy oil by-products phase, a water-soluble phase, and a water insoluble tar phase. The heavy oil by-products, obtained after a settling out time (several days) of the smoke condensates in the settling tank, are recycled and directed also to

Recycled combustible gases

Recycled heavy oil by-products

Settling tank

Figure 15.1 Diagram of fabrication of SFs.

Wood dust by-products

Patented furnace

Condensing tower

Filter stage one

Filter stage two

Dryer/blender

Oil exchange system

Further processing

Smoke powders

Smoke oils

Aqueous smokes

Smoke Flavoring Technology in Seafood ◾ 235

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Smoke extracts

Primary products

Liquid smokes

Smoke distillates

Smoke oils

Aqueous flavours

Soluble aqueous flavours Concentrates of liquid smoke

Buffered aqueous flavours

Smoke powders

Figure 15.2 SFs from primary products.

the furnace because they cannot be used for human consumption. However, a purified extract of the high-density water insoluble tar phase can be used for the production of SFs and is called primary tar fraction (PTF). The water-soluble phase leads to primary smoke condensates (PSC). Therefore, smoke condensates obtained from PTF and PSC are named primary smoke products (PP). Different SFs (liquid smokes, smoke oils, smoke powders, or smoke by-products) can be obtained after different steps of filtration, separation, or drying/blending of these PP. They are presented in Figure 15.2.

15.2.1

Liquid Smokes

Different kinds of liquid smokes are available: aqueous flavors, concentrates of liquid smokes, soluble aqueous flavors, and buffered aqueous flavors. Aqueous flavors, due to their low pH, can be used directly or diluted for applications requiring lower concentrations [4]. They can be employed in sauces or marinades of seafood products. This form of SF is especially used for the smoky taste that it confers to the food. Concentrates of liquid smokes consist of concentrated versions of aqueous flavors and require lower usage quantities. They are especially used when the final water rate in the treated food must be low. They are employed to confer smoky organoleptic qualities (tastes, flavors) and also the characteristic aspect and color of smoked food. Soluble aqueous flavors are aqueous flavors that contain an emulsifier such as polysorbates, allowing better water solubility. They are used when intermediate product dispersion is required as in brine. Finally, buffered aqueous flavors are partially neutralized or buffered aqueous flavors. These products have a pH greater than 4 and can also be added to the brine.

Smoke Flavoring Technology in Seafood

15.2.2

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237

Smoke Oils

Smoke oils are made by blending liquid smokes with vegetable oils, most frequently in 90:10 (v/v) proportions. They are less acidic than aqueous flavors and allow to exhibit more complex smoky tastes. However, these SFs are not used much in seafood industry, because smoke oils are especially employed in food preparations such as emulsions. As seafood emulsions are not very common, smoke oils can be only used in preparations such as taramas, fish sauces, or fish oils.

15.2.3 Smoke Powders Smoke powders are obtained by blending liquid smokes and dry powder carriers such as maltodextrines or barley and corn flours and drying them [5]. These smoke powders can be added to salt used for salting steps or to dehydrated sauces or soups elaborated from seafood products. Smoke powders can also be rehydrated and used in brine as liquid smokes. The final composition of smoke powders must be known in order to avoid the presence of allergens or other nonrequired additives such as nitrited salts. Nitrited salts, generally forbidden in seafood industry according to the countries, can be added to the smoke powders used in the meat industry to improve simultaneously the storage of food and to confer smoky characteristics to the final product. A reaction between the phenolic compounds of SFs and these nitrited salts or powders can lead to a nitrosation and to nitrophenols. These molecules can increase the generation of carcinogenic nitrosamines, consequent to the reaction between amino acids and nitrite. Therefore, smoke powders used in the meat industry should be different from those used in seafood industry in order to avoid nitrited salts in seafood treated with smoke powders. Therefore, it is very important to consider the salting step made with common salt mainly authorized for seafood and the curing technology made with a salt treated by nitrite and nitrate authorized for meat. We must distinguish dry salting and wet salting. Dry salting (or dry curing if nitrited salts are used) is made with dry salt deposited directly on food. Wet salting (or curing if nitrited salts are used) is made with brines spread on food or in which the food is dipped. Therefore, in seafood industry, the liquid smokes and smoke powders can be added to salt or in brine but not smoke oils. As smoke oils, smoke powders are mainly used to confer smoky tastes to the final product, whereas liquid smokes are used for the characteristic smoky odor and color of smoked products.

15.2.4 Smoke By-Products Smoke by-products are constituted by smoke extracts and smoke distillates [6]. Smoke extracts are produced by way of more or less selective extraction of smoke constituents directly from the smoke aerosol (by countercurrent circulation of water or organic solvents) or from the PP. Smoke distillates are obtained by the fractionating distillation of PP. The distillation is commonly performed with steam water at atmospheric pressure. Smoke by-products constitute more complex SFs. Consequently, their uses are really characteristic of a product and cannot be employed for a wide variety of food due to their typical organoleptic qualities. Consequently, hundreds of smoke by-products are available, but their uses are specific to a food: smoke aromatic preparations can be produced to treat certain kinds of meat and cannot be used for fishes for example. Indeed, the organoleptic qualities can vary in a high range changing the food matrix. Today, smoke manufacturers can control their products and can create smoke by-products whose uses are recommended for a kind of fish. SFs for herring, salmon, and so forth, are present in the market.

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The development of synthetic SFs must be also noticed. The progress made during the last decades in elucidating the chemical composition of wood smoke gave rise to attempts aiming at producing SF, composed entirely of synthetic compounds or partly from a liquid smoke base [7]. However, the synthetic SFs created are not sufficiently similar to real wood smoke or to SFs. Besides, SFs are so easy to produce that it would not be profitable to create synthetic SFs when natural ones are available at a cheap price.

15.3 Use of Smoke Flavorings There are four techniques to incorporate or deposit SFs in or on seafood products: showering, drenching/soaking, direct addition, and atomization. Showering is a technique currently used in North America, especially used for meat products, but it is also employed in the seafood industry. Water-based composed SFs such as soluble aqueous flavors or buffered aqueous flavors are commonly used in this technique, because to guarantee the homogeneity of the SFs during the treatment and to prevent the settling out of smoke condensates in water, an emulsifier must be added in the SFs. The final organoleptic qualities (color, taste, etc.) are dependant of the dilution of SFs in water according to proportions varying between 20% and 25% for SFs and 75% and 80% for water. Diluted SFs fall by gravity through perforated plates on the hung products. Liquid smoke solution is therefore recycled and filtered and the concentration is readjusted. Drenching/soaking is the opposite of showering technique, because the products are immersed in the SFs solution instead of pouring the SFs solution on the products. Products are dipped in SFs solution for short periods (from 5 to 60 s). Soluble aqueous flavors or buffered aqueous flavors are mainly used. They provide a better water solubility and prevent the heterogeneity of layer formation on the product surface or the product separation during storage. Direct addition consists in the incorporation of SFs during the fabrication of the food products. SFs can be incorporated directly with the ingredients during the formulation or through injection needles when the shape of the product cannot be modified. According to the final product, there are different carriers of SFs. Smoke powders are preferred when water use is impossible as in dehydrated mixes. Smoke oils are preferred for lipidic emulsions or lipidic sauces, but aqueous liquid smokes are the most used SFs in this technique. Indeed, liquid smokes are also employed in the curing brine, which can be injected into the product as for the salting step. Finally, atomization of SFs consists in the vaporization of liquid smokes, mainly liquid smoke concentrates on the products in a smokehouse. SF is sprayed with air under pressure through special nozzles and forms a wood smoke mist in the cell of smoking. The mist obtained is constituted of small droplets with a similar size as in real wood smoke, that is, between 15 and 20 mm. Therefore, from the granulometry point of view, vaporized liquid smoke is similar to real wood smoke, and this technique appears as an alternative to the smoking process. However, wood smoke is composed by a gaseous phase formed by the most volatile compounds, which carries a particulate or dispersed phase [8]. The mist generated is composed only by small droplets and there is no gaseous phase. From a physical point of view, the composition of liquid smoke mist is not similar to real wood smoke. This difference constitutes a critical point in the liquid smoking status, especially in the labeling of the smoked products. Indeed, in numerous European countries, products treated by liquid smoke atomization are considered as flavored and not smoked. In France, meat treated by this process is considered as smoked but « smoked by liquid smoke » must appear on the package. Other devices have been optimized in order to generate a similar physical composition of


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239

wood smoke with liquid smoke atomization. The SF is sprayed on a surface at high temperature, which favors the vaporization of SF [9]. Therefore, it creates a gaseous phase, but the optimization of the parameters to have a similar particulate or dispersed phase is not easy. In seafood industry, liquid smoke atomization is the most used technique of SF. The volume of liquid smoke mist is controlled by the number of nozzles and the smokehouse size. The adjustments are carried out on the flow of liquid smoke from the tank owing to a temporization on the liquid admission and on the flow of air under pressure. The smokehouse must be hermetically closed during atomization. The moisture control is essential, first to control the drying of the product and second, to favor the deposition of smoke components. In fish smoking, the drying step is necessary to prepare the surface of the fillets. This step must take into account the initial water rate of the raw material and the composition of the final product. The surface must present a beginning of protein coagulation, which confers a subtle glossy and sticky aspect. Important moisture favors the smoke penetration and strong smoky organoleptic characteristics, whereas weak moisture gives to the product a good color but a weaker smoky taste. According to the liquid smoke used, ventilation must be planned in order to reduce the moisture. Therefore, the methods of production and the possibilities of applications of SF are very wide. A high knowledge of the biochemical composition of the wood used and the parameters of the combustion are essential to generate SF. Similarly, a good knowledge of the food matrix to be treated is required to apply SF in the best conditions and to reach the expected organoleptic qualities controlled by SF chemical composition.

15.4

Chemical Composition of Liquid Smokes

The chemical composition of SF depends on the composition of the wood raw material used and especially the relative amounts and structure of its main components: two polysaccharides namely cellulose and hemicellulose and lignin. The SF composition can be complexified by the addition of spices and aromatic herbs [10]. The role of pyrolysis parameters as pyrolysis temperature, wood moisture, airflow, and air moisture are also essential in the SF final composition. The pyrolysis of cellulose initiates the hydrolysis of glucose followed by dehydration to 1,6-anhydroglucose (betaglucosan) and finally to acetic acid and its homologues, water, and sometimes small quantities of furans and phenolic compounds. The compounds generated from hemicellulose pyrolysis depend on the nature of the wood. Indeed, hemicellulose in hardwood (nonconiferous woods) is mainly constituted by pentosans whereas hemicellulose in softwood (coniferous woods) is mainly composed by hexosans. Hemicellulose pyrolysis leads to furan and its derivatives and aliphatic carboxylic acids. The thermal decomposition of pentosans provides a higher amount of furans than hexosans, which decompose to form alpha cellulose and provide a higher amount of PAH, hence the limitation of the use of softwoods for smoking. The wood polysaccharides lead to methanol, methanal, formic acid, acetaldehyde, hydroxyacetaldehyde, acetic acid, furfural and homologues, furanones, and various anhydroglucopyranoses (mostly levoglucosans) [11]. Glucuronic acids decompose to carboxylic acids, hence the high acidity of liquid smokes. Finally, lignin thermal decomposition provides compounds considered as most important for the smoke flavor, such as alkyl phenolic compounds and derivatives like phenolic ethers with methoxy groups in ortho position (guaiacol derivatives, predominant in softwoods) and in para position (syringol derivatives predominant in hardwoods). The main characteristics that permit the differentiation of hardwoods and softwoods are the guaiacol:syringol (G/S) and guaiacol:phenol (G/P) ratios. Hardwoods lead to G/S and G/P ratios, respectively, of 1.5 and 2. The pyrolysis of lignin can also lead to alkyls aryls

240 ◾


ethers from lignans, lignin dimers, and trimers [12], but they have a weak impact on the smoky flavor of SF and food processed with SF [13]. Therefore, the main organoleptically active volatile compounds generated during the pyrolysis process can be sorted in three groups of molecules: the phenolic compounds, known as the smoky skeleton of SF, the furannic derivatives, and the enolones derivatives [14].

15.5

Role of SF Process Parameters in Volatile Compounds Generation

Except the wood type that influences the smoke quality strongly [15], the wood granulometry and moisture, the pyrolysis temperature, the velocity, and humidity of air constitute key parameters of SF composition. The generation of volatile compounds is dependent on the wood pyrolysis temperature [16]. After the water release (close to 120°C–150°C), exothermic reactions of pyrolysis of wood components occur between 200°C and 250°C for hemicellulose, between 280°C and 320°C for cellulose, and 400°C for lignin [17]. According to the pyrolysis process, different groups of compounds are formed. The rate of carbonyl compounds increases gradually with the temperature from 200°C to 600°C. The rate of acids is higher for temperature lower than 300°C and decreases after 300°C with the increase in temperature. From 200°C to 600°C, the quantity of phenolic compounds increases with a maximum close to 500°C and decreases after 500°C. However, differences can be observed depending on the molecules. For example, phenol amount is multiplied by two between 450°C and 650°C, whereas syringol quantity is tripled. A temperature of 450°C–500°C was reported to lead to the best composition for the creation of carbonyls, furannic compounds, and phenolic compounds [10,18,19]. PAH must also be surveyed, because their contents in smoke or in food increase from 400°C to 1000°C. As the best pyrolysis temperature to obtain the required volatile compounds are between 380°C and 500°C, it seems difficult to generate the desired organoleptic volatile compounds without PAH contaminants. A step of filtration is almost obligatory to avoid these contaminants. Indeed, steps of SF purification through filters or apolar solvent washes are often required to decrease the PAH levels. The wood moisture appears as the second important parameter [20]. The high moisture allows to reduce the wood combustion efficiency. Therefore, a lower temperature is reached and allows increasing the generation of smoke volatile compounds and minimizing PAH formation. The use of hardwood, with a lower water rate than that in softwood, is recommended because it burns slower. An optimal moisture is planned in the industry between 17% and 20%, whereas a rate between 20% and 30% has been reported as optimal to reduce the emission of particules [11]. The air velocity indirectly influences the SF composition by the modification of pyrolysis temperature or smoke temperature [21,22]. A slow combustion is reached with weak air velocity. Air moisture is also very important and must be set in adequation with air velocity to keep the water rate constant in the air during the combustion. Lower concentrations of oxygenated compounds have been found to be caused by an oxygen depletion during combustion [20]. Wood granulometry can also influence SF composition, because it plays a role in the pyrolysis temperature. The combustion is faster when the granulometry of the wood raw material is important [23,24]. Then, the diversity of settings of pyrolysis parameters can explain the diversity of organoleptic volatile compounds and the diversity of qualities of SF. The manufacturer can choose SF according to the required result on the organoleptic characteristics of the final product. Due to their


◾

241

chemical composition, the diversity of SF causes diverse consequences on the texture, odor, taste, color, and preservation of the product.

15.6

Organoleptic Roles of Volatile Compounds of SF

15.6.1 Role of Volatile Compounds of SF in the Odor Even if the concentrations of odorant volatile compounds in SF can be various, two main classes of odor-impact molecules can be defined: phenolic compounds and carbonyl compounds, which gather furannic and enolone derivatives. Phenolic compounds are known to constitute the odorant “smoky” skeleton of wood smoke and smoked fish. They are the major compounds in SF with a wide range of odorant thresholds (Table 15.1) [14,28,29]. Some of them have very low odorant thresholds, making them odorant at low concentrations. Many studies have indicated that phenolic compounds present in the vapor phase of smoke may be odor-active compounds [30–32]. Phenolic compounds of medium volatility have been considered as the most important odorant molecules. The medium-boiling fraction (91°C–132°C) composed of isoeugenols, syringol, and methylsyringol has a pure and characteristic smoky flavor [10]. These observations have been recently corrected [14,33,34]. Phenolic compounds of low-boiling fraction (60°C–90°C) composed mainly of phenol, cresols, guaiacol, and alkylguaiacol may also contribute to imparting a smoky flavor to smoked fishes [13,34] (Table 15.2). The role of syringol is important, but it may not be the main contributor to wood smoke flavor. Similarly, phenolic compounds are not sufficient to explain the SF role in smoked fish odors. A much more complex mixture of compounds is responsible for the characteristic aroma of smoked fishes [35]. Table 15.1 Odorant Thresholds of Various Phenolic Compounds Phenolic Compounds

Odorant Thresholds in Water (μg/L)

References

Phenol

5900

[25]

o-Cresol

650

[25]

m-Cresol

680

[25]

p-Cresol

55

[26]

Guaiacol

3–21

[25]

4-Methylguaiacol

90

[25]

4-Ethylguaiacol

50

[27]

4-Vinylguaiacol

3

[26]

Vanilline

20–200

[25]

Syringol

1850

[25]

Eugenol

6–30

[26]

Ethylvanilline

100

[25]

LRI (DB5)

859

865

875

890

904

920

925

970

992

1036

1052

1068

1093

1110

1130

Compounds

Furfural

4-Methylpyridine

Furfuryl alcohol

2,6-Dimethylpyridine

2,4-Hexadienal

2-Methyl-2-cyclopenten1-one

2-Acetylfuran

5-Methylfurfural

Phenol

2-Hydroxy-3-methyl-2cyclopenten-1-one

2,3-Dimethyl-2cyclopentenone

o-Cresol

p-Cresol

Guaiacol

2,6-Dimethylphenol

MS, LRI, STD

MS, LRI, STD

MS, LRI, STD

MS, LRI, STD

MS, LRI, STD

MS, LRI

MS, LRI, STD

MS, LRI, STD

MS, LRI, STD

MS, LRI, STD

MS, LRI

MS, LRI

MS, LRI, STD

MS, LRI

MS, LRI, STD

Means of Identificationa

Chemical, burnt, spicy/ woody

Smoked, vanilla, ink

Animal, spicy, burnt

Chemical, spicy, burnt,

Spicy, wood fire, roasty

Cooked, spicy

Marine, metallic, chemical, mushroom

Cooked, earthy, green

Cooked vegetable, potato

Cooked potato, green

Cooked vegetable, fatty

Roasty, green, milk

Cooked/soup, chemical

Green, milk

Smoke, green

Odorant Attributes Given by the Judgesb

(3)

8

8

8

6

7

7

(4)

7

6

(5)

6

7

Number of Judgesc

(2)

7

6

5

3

4

4

(2)

6

5

(2)

4

4

Average Intensity d

(1.27 ± 0.75)

360.45 ± 172.07

74.18 ± 37.53

49.74 ± 27.62

17.48 ± 8.94

23.64 ± 18.44

65.55 ± 39.97

(24.63 ± 13.50)

20.22 ± 9.34

8.37 ± 3.98

(1.33 ± 1.53)

4.27 ± 2.90

42.17 ± 26.25

16.55 ± 9.12

124.24 ± 63.04

Mean ± SDe

Table 15.2 Odorant Characteristics and Concentrations of the Most Potent Odorant Volatile Compounds in Salmon Fillets Treated by Liquid Smoke


1132

1140

1147

1160–1180

1192

1247

1266

1282

1287

1307

1330

1330

1365

1370

1382

1400

2,3,4Trimethylcyclopenten-1one

3-Ethyl-2-hydroxy-2cyclopentenone

1,2-Dimethoxybenzene

2,4- and 2,5Dimethylphenol/ (E)-2-nonenal

4-Methylguaiacol

2,3-Dimethoxytoluene

(E)-2-Decenal

3,5-Dimethoxytoluene

4-Ethylguaiacol

Indanone

4-Vinylguaiacol

(E,E)-2,4-Decadienal

Syringol

Eugenol

4-Propylguaiacol

1,2,3-Trimethoxy-5methylbenzene

MS, LRI

MS, LRI, STD

MS, LRI, STD

MS, LRI, STD

MS, LRI, STD

MS, LRI, STD

MS, LRI

MS, LRI, STD

MS, LRI

MS, LRI

MS, LRI

MS, LRI, STD

MS, LRI, STD

MS, LRI

MS

MS

Cooked, earthy

Green, spicy, vanilla

Spicy, smoke, clove

Burnt rubber, spicy

Oily, green, fatty

Smoke, green, spicy

Sawdust, rotten, burnt

Green, smoke, vanilla, clove

Burnt, green, chemical

Spicy, green, milk

Cooked vegetable, fatty, green

Candy, spicy, smoked

Cucumber, violet, spicy, smoked

Ashes, green

Solvent, medicinal

Cooked, green, spicy

(5)

8

8

8

7

(3)

7

8

(5)

6

7

7

8

6

7

6

(2)

5

5

5

5

(2)

4

6

(3)

3

4

5

6

3

4

5

◾

(continued)

(2.15 ± 1.15)

15.21 ± 7.86

36.51 ± 18.17

44.61 ± 22.91

8.82 ± 6.72

(3.24 ± 1.95)

2.87 ± 1.71

86.85 ± 40.97

(6.25 ± 4.15)

4.26 ± 1.79

6.62 ± 4.12

482.15 ± 243.13

18.96 ± 10,46

11.16 ± 5.50

10.49 ± 6.36

17.25 ± 10.35

Smoke Flavoring Technology in Seafood 243

1423

1473

1527

1615

1680

(Z)-Isoeugenol

(E)-Isoeugenol

2,3,5-Trimethoxytoluene

4-Allylsyringol

8-Heptadecene

MS, LRI

MS, LRI

MS, LRI

MS, LRI, STD

MS, LRI, STD

Means of Identificationa

Animal, roasty, chemical

Smoke, rotten

Spicy, woody

Clove, green, roasty

Burnt rubber, spicy

Odorant Attributes Given by the Judgesb

6

7

(4)

7

6

Number of Judgesc

4

4

(2)

4

3

Average Intensityd

6.87 ± 2.68

1.23 ± 0.39

(20.55 ± 8.48)

24.81 ± 11.35

7.40 ± 3.77

Mean ± SDe

e

d

c

b

a

Means of identification: MS, mass spectrum (identified using the mass spectra of the compounds), LRI, linear retention index (when the LRI of the identified compound corresponds to the LRI in the literature), STD, standard (when the retention time, spectrum, and odor description of an identified compound correspond to the retention time, spectrum, and odor description of the injected standard of this compound). When only MS is available for identification, it must be considered as an attempt of identification. The odor given corresponds to the odor detected by the judges during olfactometric analysis for its retention time but not surely due to the compound that we try to identify. Number of judges (out of eight) who have detected an odor. Average intensity of the eight judges is rounded to the nearest whole number. An intensity between 5 and 5.5 is rounded to 5 and an intensity between 5.5 and 6 is rounded to 6 (1 = very weak odor, 9 = very strong odor intensity). In micrograms equivalents of dodecane per 100 g of smoked salmon. Means of three fillets.

Note: Frequency of detection, odor intensity, and quantities of odor-active compounds detected by fewer than six judges are indicated in parenthesis.

Source: Varlet, V. et al., J. Agric. Food Chem., 55, 4518, 2007.

LRI (DB5)

Compounds

Table 15.2 (continued) Odorant Characteristics and Concentrations of the Most Potent Odorant Volatile Compounds in Salmon Fillets Treated by Liquid Smoke



◾

245

Carbonyl compounds have also been reported as contributors to the smoky aroma of wood smoke. A polyfunctional carbonyl subfraction was isolated from wood smoke and possessed a caramellic/burnt sugar aromatic note [36]. Two categories of carbonyl compounds can be differentiated: furannic compounds and enolone derivatives. Furfural and homologues exhibit cooked/roasty aromatic notes. Furannic compounds were thought to contribute to soften the heavy smoky aromas associated with phenolic compounds [37,38]. More recently, furannic compounds were found to play a role in cold smoke odors of liquid smoke or fishes treated by liquid smoke [14,34]. Enolone derivatives are compounds derived from cyclopentenone. They were isolated early from wood smoke and described as grassy, sometimes cooked, and seem to contribute little to overall aroma, because they are not the compounds mainly detected in SF and seafood treated by SF odors by sensory analysis [37]. However, as the other minor odor-active compounds, if they do not have a strong individual influence, they may contribute in mixture to the overall odor. The determination of the role of SF components in the final product odor is complex due to the odorant interactions that can occur between the odor-active compounds. Synergic or masking effects are possible and make the final odor complex. Furthermore, reactions between liquid smoke compounds and the components of the matrix can occur through Maillard and Strecker reactions. The amino acids from the seafood matrix and the carbonyl compounds from the SF can generate furannic compounds and nitrogen-containing compounds with roasty/smoky aromatic notes [19,39]. Moreover, the physical state of SF can also influence the aroma, and the odorant contribution of odor-active compounds cannot be the same if the SF is in the form of powders, liquids, or oils. The oils used in smoke oils can soften the smoke aroma.

15.6.2 Role of Volatile Compounds of SF in the Flavor Phenolic compounds were shown as the major contributors of the smoky flavor [35]. The taste thresholds of some phenolic compounds were determined [40] and showed a high diversity between the molecules, but taste was not investigated as much as odor. For several decades, it was commonly admitted that syringol derivatives impart a smoky odor and guaiacol derivatives contribute to a smoky flavor. Recently, guaiacol derivatives and more generally the phenolic compounds of low-boiling fraction molecules (60°C–90°C) have been shown to cause the odor. As for the assessment of the odor, the determination of the effects of compounds of SF on the flavor is complex, and very little information is available. However, early works performed on individual phenolic compounds have identified the impact of guaiacol on the smoky flavor, whereas syringol and 4-methylguaiacol showed the same but lower effect than guaiacol [40]. Concerning the bitter taste, 4-methylguaiacol perception was superior to that of syringol and guaiacol. The high-boiling fraction of phenolic compounds (133°C–200°C) was described with an acid and chemical property that was judged of poor quality. Then, the same compounds responsible for the odor should be involved in the flavor that SF confers to seafood. Sensory analysis performed on standards confirmed the importance of guaiacol and o-cresol in the smoky flavor and dimethylphenol, 4-methylguaiacol, and isoeugenol in spicy/sweet flavor [41]. However, phenolic compounds are not the only flavor-active compounds. The fractionation of a commercial liquid smoke preparation evaluated by a sensory panel concluded that the phenol fraction was essential but not complete from a sensory standpoint [42]. The results of this fractionation are given in Table 15.3.

246 ◾ Handbook of Seafood and Seafood Products Analysis Table 15.3

Sensory Taste Intensities of Liquid Smoke Fractions Fractionb

Taste Property

a b

a

1

2

3

4

5

6

Smoke taste intensity

6

7

3

11

4

10

Tarry taste intensity

3

1

2

0

6

1

Chemical taste intensity

1

1

3

0

1

0

Acidulous taste intensity

1

2

3

0

0

0

Intensity scale: 0 = below threshold; 11 = highest value. 1, distilled at 67°C–90°C; 2, distilled at 91°C–132°C; 3, distilled at 133°C– 200°C; 4, phenolic subfraction; 5, terpene subfraction; 6, whole liquid smoke.

Other compounds such as enolone derivatives could also play a role in the SF flavor. Studies on standards have shown that cyclotene was a flavor-active compound [41]. According to their concentrations found in the different SF, furannic compounds could also have an effect on the flavor [43].

15.6.3 Role of Volatile Compounds of SF in the Texture The texture of smoked products is due to coagulation of proteins. Formaldehyde, which has been reported in wood smoke and smoked meat [41] but not in smoked fishes until now, can react with proteins. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side chains of arginine, cysteine, histidine, and lysine residues [44]. Formaldehyde seems to be involved in the texture of smoked fishes and to be responsible for the layer at the dried surface of fishes [45]. The acidic aqueous SF can also increase the coagulation of proteins and act on the texture. SFs under powder or oil forms do not act on the surface texture, because they are added in the product during its fabrication. However, they could play a role in the inner texture of the product.

15.6.4 Role of Volatile Compounds of SF in the Aspect and Color The color of seafood treated by SF can derive from physical and chemical reactions. Indeed, smoke condensates are colored mainly due to phenolic compounds, which have brown/yellow characteristic color. Their physical deposition of SF on seafood can confer its color to the product, which can vary from golden yellow to dark brown according to the nature of the wood, the eventual dilutions of SF, and the intensity of the process. However, Maillard and Strecker compounds can also be responsible of the color of the smoked product [45]. In the liquid smoking process, the product must also be placed in a dry and hot ambient atmosphere for short periods in order to favor color formation. Thus, the deposition of Maillard compounds leads to a darker color of fish flesh [46]. A brief drying after smoke absorption can cause a higher level of dehydration and lead to higher amounts of Maillard products. After scission and dehydration, melanoidines could be created by polymerization through aldolic condensations. These


◾

247

compounds give to the final product a brown color, but no information is available concerning this pathway [47]. Carbonyl-amino reactions as Maillard reaction could play a main role in smoked food. Protein-bound lysine, the most prevalent essential amino acid in fish, because of its terminal amino group, is considered as a major source of the amino components in such reactions, but a loss in arginine and histidine is also observed. Glycolic aldehyde, methylglyoxal, and 2-oxopropanal are considered to be important color precursors [6,24]. A part of the fi nal color could derive from phenolic compounds with aldehyde function. Coniferaldehyde and syringaldehyde are considered to be irreversibly bound to proteins and to contribute orange tints to the products [24]. Finally, the glossy aspect noticeable on certain smoked products is the result of reactions between phenolic compounds and aldehydes [48]. They lead to resinous substances (phenoplasts). The polymerization is favored by the heat, and the degrees of reticulation of the molecule vary as a function of time [49].

15.7

Role of Volatile Compounds of SF in Preservation

Smoking process is the oldest preservation technique because of the antimicrobial and antioxidants properties of wood smoke. Food industries are working to develop new applications of smoke condensates, which could contribute to product safety by controlling the growth of foodborne pathogens. Studies on the antimicrobial activity of some smoke condensates have revealed very variable effects on the growth of microorganisms [50]. The antioxidant compounds of wood smoke condensates are those with an active phenolic function. The antioxidant behavior is increasing with the temperature of the boiling point of the phenolic compounds [44]. The most active compounds are polyhydroxyphenolic compounds such as pyrogallol and resorcinol. Among monohydroxyphenolic compounds, the antioxidant properties depend on the radical located in the para position from the hydroxy group as in 4-methylguaiacol, 4-vinylguaiacol, or 4-propenylsyringol. The antioxidant activity of guaiacol, syringol, 4-methylsyringol, and 4-vinylsyringol is lower. An oxidant molecule acts by electronic capture and can trouble the preservation of the product by the initiation of lipid oxidation. The phenolic compounds can give an electron to stabilize the oxidant molecule and with their ringlike structure and mesomeric forms, phenolic compounds can easily support the lack of electrons. A synergic effect has been shown between high-boiling point phenolic compounds and oxidized phenolic compounds and it prolongs the antioxidant action [44]. However, there is a critical concentration that must not be overcome to avoid an inversion of antioxidant effect that can become prooxidant. Concerning the antimicrobial effect of wood smoke condensates, it seems that phenolic compounds and carboxylic acids play an inhibitory role, especially against bacteria [51]. Carbonyl compounds and esters are nearly not implied, and hydrocarbons are not influential. As in odor and flavor, the activity of compounds must take into account the synergic or antagonist effects in mixture; that is why some researchers have concluded the absence of relation between the inhibitory effect of essential oils and their phenolic content. Therefore, phenolic compounds and carboxylic acids, alone or in synergy, could be responsible for most of the antimicrobial properties.


15.8

Polycyclic Aromatic Hydrocarbons

15.8.1

Properties and Toxicology

PAHs are well known as being food contaminants and carcinogens [52]. As they can be absorbed by animals, they are considered as environmental pollutants and can contaminate the human feed raw material [53,54]. However, home cooking and industrial food processes represent the major source of human contamination [55]. PAHs are considered as carcinogenic contaminants of processed food [56], particularly smoked food [57,58]. These compounds have been studied for several years, especially benzo[a]pyrene (B[a]P) [59]. B[a]P is the first PAH whose toxicity and carcinogenicity was assessed from the observations of Sir Percival Plott in 1775 at St Bartholomew hospital of London about cancer of the scrotum of the chimney sweepers. Therefore, it is used as the leading substance to illustrate PAH contamination. PAHs are formed by the incomplete burning of carbon-containing material. In SF, PAHs are generated during smoke production by wood pyrolysis. PAHs comprise fused aromatic rings made up of carbon and hydrogen atoms: up to four fused benzene rings, they are considered as heavy PAHs; more toxic than light PAHs, which are constituted by less than four benzene rings. Hundreds of individual PAHs may be formed and released during the process of incomplete burning of the wood. Owing to their lipophilic properties (log Kow between 4 and 7), PAHs can cross the biological membranes and accumulate in tissues. They are considered as carcinogenic contaminants, because their catabolism leads to poly-hydroxy-epoxy-PAH suitable for binding to DNA adducts, hence their toxicity (Figure 15.3). Therefore, the uses of SF in food industrial processes must be ruled out in order to guarantee food

O DNA adducts OH OH B[a]P 7,8-dihydrodiol-9,10-epoxyde

Benzo[a]pyrene:B[a]P

Glutathion S OH OH OH

O

OH OH B[a]P 7,8-catechol

OH

OH B[a]P 7,8-dihydrodiol

O B[a]P 7,8 epoxyde

OH

S

B[a]P glutathion conjugate

COOH O O OH

O

O

OH

OH

OH OH

B[a]P sulfo-conjugate

Detoxification products

Figure 15.3 Benzo[a]pyrene metabolization.

B[a]P glucuronide


◾

249

safety avoiding PAH contamination. In the 1980s, the U.S. Environmental Protection Agency (US-EPA) identified a list of 16 PAHs as the most frequently found [60]. Among them, eight light PAHs were considered as environmental contaminants, with a weak toxicity but high concentrations in the samples analyzed. The eight heavy PAHs left were shown as being carcinogenic or mutagenic contaminants and gave rise to serious health concern. Indeed, even if they were found in weak quantities, these PAHs were considered as toxic at low levels.

15.8.2 Extraction and Analysis Methods of PAH in SF and Seafood Treated by SF The quantification of PAHs in SF and seafood treated by SFs is performed in two steps: an extraction step and the analysis step, which combines a separation step and a detection step. The extraction step must integrate the composition of the matrix. In the case of liquid matrices as liquid smoke, a liquid–liquid solvent extraction is often used [61–63]. Apolar solvents or mixes of apolar and semipolar solvents are used to extract the maximum of PAHs. In the case of solid seafood treated by liquid smoke, solid–liquid extraction can be carried out. However, PAHs are often coextracted with fat matter, which can disturb the extraction, cause chromatographic coelutions, and lead to mistakes in the identification. Therefore, purification and/or delipidation steps such as saponification are often applied to reduce the fat matter rate of samples [64]. Purification was especially performed on an alumina or silica column, but solid-phase extraction (SPE) cartridges are now more frequently employed. The nature of the SPE cartridge phase is linked to the extraction method and the biochemical composition of the initial matrix. Other extraction devices have been developed to investigate PAH in smoked food such as accelerated solvent extraction (ASE) [65], supercritical fluid extraction (SFE) [66] or solid-phase microextraction (SPME) [67], and stir bar sorptive extraction (SBSE) [68] but not on liquid smokes or seafood treated by SF. Although all steps are important, the analysis step is the most critical point. For the separation of the PAHs extracted from SF or seafood treated by SF, gas chromatography and liquid chromatography are the most used techniques [55]. Gas chromatography is coupled to mass spectrometry [58,69], flame ionization detector (FID) [60], or tandem mass spectrometry [70], and liquid chromatography is coupled to ultraviolet or fluorimetric detector [59–63]. Parameters of the chromatographic separation and detection must be adjusted to avoid coelutions with interferences from lipids. Moreover, the chromatography must be sufficiently efficient to separate isomers of PAH, because they do not have the same toxicity. Thus, it is essential to quantify only the toxictargeted compounds. Several devices are therefore developed to optimize the analysis, such as bidimensional chromatography at the gaseous phase (GC/GC) [71] or liquid phase (LC/LC) [72], but, to our knowledge, they have not been applied to SF or seafood treated by SF.

15.9

Legislative Aspects

15.9.1 European Regulations on PAH Found in SF In 2003, a European regulation set the maximum contents of PAH in the primary products (PP) of smoke condensates used for the production of SF, that is, PSC and PTF. In both condensates, the concentrations of benzo[a]anthracene (B[a]A) and benzo[a]pyrene (B[a]P) must not exceed 20 and 10 mg/kg of liquid smoke, respectively [73]. This harmonization was necessary to homogenize the legislation about SFs. For example, in Italy, the maximum levels of B[a]A and B[a]P were set at 20 and 10 mg/kg of liquid smoke, respectively [74].

250 ◾


However, the 2003 maximum values must be reviewed again, because these values were set for PP and not SF, whereas food is treated by SF and not directly by PP. The high maximum values authorized in PP do not seem well adjusted with the weak final PAH contamination of SF. Indeed, the PAH contamination reached in SF is largely below the values authorized in PP [60,69,75]. However, according to the origin of SF and industrial manufacturers, important differences in PAH concentrations are noticeable [57,63] which justifies controls and regulations. Moreover, the toxicity of other heavy PAHs was recently demonstrated and the monitoring of these PAHs was recommended by a European regulation published in 2005 [76]. Therefore, it is legitimate to wonder if the exclusive monitoring of the B[a]A and B[a]P in PP is adequate to illustrate the PAH contamination of SF.

15.9.2 European Regulations on PAH Concentration in Food Treated by SF In 1988, a European regulation set the maximum content of B[a]P in foodstuffs treated by SF at 0.03 mg/kg. As for SF, the PAH contamination was only set for B[a]P [77]. This value is very low compared to those authorized in PP. Th is fact can be understood by the use of smoke condensates in flavoring quantities, that is, very small amounts. However, the vaporization of SF in a smokehouse causes a loophole in the legislation. In certain countries such as France, for meat industry, atomization of liquid smoke in a smokehouse is considered as a smoking process but the maximum level of PAH must not overcome that of flavoring legislation, that is, 0.03 mg/kg. Indeed, this process can also be considered as a flavoring of the surface of the product, as drenching or showering. Therefore, it is paradoxical to apply flavoring regulations to the smoking process. Indeed, the smoking regulations set a maximum B[a]P value of 5 mg/kg of smoked fishery products and smoked crustaceans, excluding bivalve molluscs, brown meat of crab, and head and thorax meat of lobster and similar large crustaceans [76,78]. This value is the result of the necessary harmonization between the national laws of European countries [79]. Thus, atomization of liquid smoke must be lower than 0.03 mg/kg and leads often to noncompliant smoked products. Indeed, SFs are used in higher quantities than those employed in flavoring processes. However, if it is considered as smoking technique, the respective legal B[a]P amount is 5 mg/kg. In this case, atomization of liquid smoke would constitute the smoking technique, leading to less PAH contaminated food by comparison with the traditional smoking techniques [80].

15.10

Conclusion

A wide range of SFs and uses of SFs are now available to flavor seafood products. Moreover, SFs appear as a safe alternative to smoking techniques. The content of PAH in SFs and in the final product can be better controlled than during traditional smoking. Moreover, it leads to lower PAH contents. Finally, the liquid smoking process decreases the emissions of PAH compounds to the environment. All these benefits could help to reconsider the status of atomization of liquid smoke and the maximum PAH contents related. Nevertheless, it can lead to problems of labeling, but it could also initiate an international consideration of labeling of smoked and flavored food. The main criticism that can be formulated against SF is the lack of control of the final organoleptic qualities of such processed food. Therefore, it is necessary to better control the composition of SFs and to improve knowledge about the influence of the pyrolysis parameters (wood nature,


◾

251

wood size, temperature, moisture, etc.). However, the optimization of SFs effects on food products must be done avoiding PAH generation. Finally, the traceability of SF must be improved, which could contribute to give to the SF a less processed characteristic. Indeed, in France, SFs are forbidden for the smoking of organic products from aquaculture, whereas SFs are produced from natural wood. The problem can come from the emulsifiers that are sometimes added in SFs. Besides, according to allergic people and religious groups, the processed food cannot be consumed, and today no information is available.

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NUTRITIONAL QUALITY

III

Chapter 16

Composition and Calories Eva Falch, Ingrid Overrein, Christel Solberg, and Rasa Slizyte Contents 16.1 Introduction ................................................................................................................. 258 16.2 Nondestructive Analysis of Total Proximate Composition............................................. 258 16.3 Lipids ............................................................................................................................267 16.3.1 Nutritional Aspects ..........................................................................................267 16.3.2 Methods for Determination of Total Lipids .....................................................267 16.3.3 Nondestructive Methods ................................................................................. 268 16.3.4 Comparison of Methods ................................................................................. 269 16.4 Proteins ........................................................................................................................ 269 16.4.1 Nutritional Aspects ......................................................................................... 269 16.4.2 Methods for Protein Determination .................................................................270 16.4.3 Determination of Total Nitrogen .....................................................................270 16.4.4 Direct Methods for Soluble Protein Determination .........................................270 16.4.5 Nondestructive Analysis of Proteins ................................................................ 273 16.5 Determination of Carbohydrate Content ..................................................................... 273 16.6 Determination of Water Content ..................................................................................274 16.7 Calories .........................................................................................................................274 16.7.1 Direct Measurement of Energy ........................................................................274 16.7.2 Indirect Measurements of Energy.....................................................................275 16.7.3 Food Composition Tables and Databases .........................................................276 References ................................................................................................................................276

257


16.1

Introduction

The proximate composition in most fish and shellfish is primarily water, proteins, and lipids. In fish meat these constituents make up about 98% of the total mass, and the other minor constituents include carbohydrates, vitamins, and minerals [1]. Proximate data on different fish species are collected in databases such as the FishBase (www.fishbase.org); however, the chemical composition of fish generally varies due to seasons, geographical locations, stages of maturity, and sizes, and so on. Therefore, to ensure obtaining data on the exact proximate composition, analysis should be performed on the specific samples. There are several methods available to analyze the major components in seafood and the main methods along with their advantages and limitations are presented in Table 16.1 and further discussed in the text below. Section 16.7 deals with the different methods to determine and calculate calories in fish and shellfish.

16.2 Nondestructive Analysis of Total Proximate Composition Analysis of each nutrient separately is time-consuming and requires a diverse set of equipments. Methods for simultaneous determination of the major components are therefore valuable. Nearinfrared spectroscopy (NIR) is the most common method for such analysis and is therefore comprehensively presented in this chapter. NIR has been found to be a reliable, rapid, and easy to perform nondestructive analysis for simultaneous determination of the major components in fish. The development of NIR in food analysis started with the development of analysis of cereal grains and oilseeds in Canada [2]. As well as increased efficiency of the Canadian wheat segregation program, the adoption of NIR testing resulted in a total cost saving of CAN$ 2.5 m per year and a saving for the environment by replacing the Kjeldahl system, which involves concentrated sulfuric acid and heavy metal catalysts, by the chemical-free NIR method. When Williams was running the program for the Canadian Grain Commission, 600,000 Kjeldahl analyses were conducted per year and incidentally producing 47 ton of caustic waste in the process. The first instruments on the market were filter instruments measuring in reflectance mode. During the 1980s monochromator instruments were developed, making it possible to measure over the whole NIR spectrum and not only on a small number of selected filters. The NIR spectrum is defined between the wavelength 800 and 2500 nm, but the available detectors cover a smaller range; the silicon detector covers the range 400–1100 nm, an indium gallium arsenic covers the range 800–1700 nm, and a lead sulfide, the range 1100–2500 nm. The NIR radiation interacting with a sample may be absorbed, transmitted, or reflected depending on the interaction with NIR wavelength and physical status of the sample as transparent or nontransparent. Diff use transmittance measurements are usually carried out in the 800–1100 nm region of the spectrum, where the weak absorptions enable useful data to be obtained using sample thickness of 1–2 cm of samples such as meat, cheese, or whole grain. However, in this spectral region the spectrum of the transmitted light is very compact and no single peaks are visible, making it difficult to use this spectral range before the development of multivariate calibration technology, such as the introduction of partial-least squares (PLS) by Martens in 1982 [3]. The spectral data will be reduced by principal component analysis, and then one can perform a linear regression on the principal components. The end result is a calibration equation from which the constituent of interest is calculated from a linear combination of spectral data.

Reflection, transflection, or transmission of nearinfrared light (850–1700 nm)

Measurement of ultrasonic velocity

Samples are placed in an electromagnetic field and electric conductivity is measured

Ultrasound

Total body electrical conductivity (TOBEC)

Principle

NIR/NIT

Total Proximate Composition

Methods

May obtain data on water, lipids, and protein content noninvasively, nondestructive, can be used on live fish

Rapid, nondestructive, precise, fully automated, and can be performed online

Rapid method simultaneously analyzing fat, water, and protein. Nondestructive and can be used on live fish.

Advantages

Need more research

Few articles on fish composition

Specific for different species, physiological and physical states can affect values of conductivity, can be nonsensitive, expensive

Ultrasonic properties of tissue depend on composition and temperature

For reflectance instruments (surface analysis) some drawbacks such as interference by starch and lipids, displacement of reflectance spectrum by moisture content, and disturbance by particle size in samples

Calibrations need to be made against reference methods. Different calibrations for different species and organs. Calibrations require skilled personnel. The equipments are relatively expensive.

Drawbacks

(continued)

[21,172–174]

[17–20,173]

[11,133]

[4–6,8]

Selected References

Table 16.1 Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products

Composition and Calories ◾ 259

Extraction of minced samples generally using chloroform and methanol as solvent Gravimetric determination

Extraction of minced samples by solvents in automatic systems (Soxhlet, (SoxTech, fexICA), Fosslet, etc.)

The sample is dried and from the water content found, the fat content can be calculated theoretically by the formula Fat% = 80 − water %

Manual solvent extraction

Automatic solvent extraction

Microwave drying

Chemical Extractions:

Total Lipid Determination

Nuclei of atoms in a sample provide spectra when the sample is exposed to a magnetic field

Principle

A simple and inexpensive method

No use of chemicals

No laboratory facilities are required

Automatic, less exposure to chemicals (compared with manual solvent extraction)

Possibilities to further characterize the lipids extracted

Provides high total lipid yield

Rapid, nondestructive (See under NMR below)

Advantages

Precision level may be dependent on sample (maturity stages of the fish, lipid content, location of lipids, processing)

Physical and chemical changes might occur during examination

Requires laboratory facilities

May discriminate structured fat (such as phospholipids)

Requires well-trained laboratory personnel

Destructive technique

Use of health hazard chemicals

Time-consuming


Excellent for determination of fat and water content or even distinguish lipid classes and water properties. Need of sample specific calibrations

Weaknesses due to quantification of proteins without combining with destructive methods

Drawbacks

[46]

[43]

[26–31]

[13–15,22,55,57]

Selected References

Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products

◾

Nuclear magnetic resonance (NMR)

Methods

Table 16.1 (continued)

260 Handbook of Seafood and Seafood Products Analysis

Transmitted or transflected Near Infrared light (800–1700 nm).

See NMR above

NIR/NIT

Low-field NMR

NMR mouse

Determination of water by analyzing the dielectric properties using a microwave strip (calculation of lipids as for the drying method).

Fat meters

Nondestructive Methods:

Nondestructive and rapid. The NMR mouse is rapid, portable (small size), and nondestructive and allows in vivo measurements

Allows in vivo measurements

Some portable instruments are available

Broad range of applications, may also provide other nutrient data in the same analysis

Nondestructive and rapid

Allows in vivo measurements

Relatively inexpensive, rapid, easy, nondestructive, and portable

No laboratory facilities are required

Traditional low-field instruments require withdrawal of homogeneous samples for analysis (invasive)

Expensive, requires specific calibrations

See NIR/NIT above

Most suitable for neutral lipid determination

Needs to be calibrated for the individual species

Precision level may be dependent on sample (maturity stages of the fish, lipid content, location of lipids, processing)

(continued)

[14–16,22,50,55]

[4–6,8]

[46,51–52]


High temperature combustion and detection of N by thermal conductivity detector

Dumas combustion method

Direct Protein Determination on Soluble Proteins

Sample digestion followed by neutralization, distillation, trapping of ammonia, and titration with acid

Kjeldahl

Total Nitrogen Determination

Proteins

Principle

Rapid, inexpensive to use and sensitive to low concentrations of proteins

Rapid, easy to perform, inexpensive to use, safe (no chemical exposure), and environmentally friendly

Widely used internationally, standard method for comparison, high precision and good reproducibility, independent of physical state of sample

Advantages

Most samples must undergo steps of sample preparation before they can be analyzed. Absorbance depends on the type of protein analyzed

Limited to soluble proteins

High initial costs. Difficult to obtain the accurate protein concentration

Does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein, interference by nonprotein nitrogen compounds, time-consuming, low sensitivity, hazardous, potentially toxic chemicals are used

Drawbacks

[53, 74,133]

[53,67,120,133]

Selected References


◾

Protein Determination

Methods



A violet-purplish color is produced when copper(II) ions interact with peptide bonds under alkaline conditions. Absorbance at 540 nm

Copper(II) ions in alkaline solution react with protein to form complexes, which react with the Folinphenol reagent, and reaction products are detected between 500 and 750 nm

The protein and dye complex causes a shift in the absorption maximum of the dye from 465 to 595 nm. The amount of absorption is proportional to the protein present

Measurement of UV absorption (280 nm)

Biuret method (Alkaline copper reagent test)

Lowry protein assay

Dye-binding (Bradford) method

Near-UV absorption

Rapid, nondestructive, no addition of reagents required

Rapid, easy to perform, high sensitivity, and internationally accepted

High sensitivity and easy to perform

Negligible interference from materials that absorb at lower wavelengths, technique is less sensitive to protein type: it utilizes absorption involving peptide bonds that are common to all proteins, easy to perform

Low sensitivity, interference by UV-absorbing compounds (nucleic acids and nucleotides), depends on amino acid composition

Color formation and binding depend on proteins present, protein-dye complex adsorbs on glass surface, interference from common laboratory chemicals, variation of binding capacity for different batches of commercial grade dyes

Standard curve is nonlinear. Unstable reagents are used. Other compounds can interfere, color development depends on amino acid composition

Relatively low sensitivity compared with other UV-visible methods. High amounts of endogenous proteases may cause errors, interference from ammonia, buffers salts, detergents

[133]

(continued)

[68,131,133,176,177]

[67,133]

[133,175]


Hydrolysis, derivatization, chromatographic separation, and detection of amino acids with UV absorbance, fluorescence

Highperformance chromatography (HPLC)

Absorption at 780–2500 nm

Transmitted or transflected Near-infrared light (800–1700 nm)

Infrared absorption

NIR/NIT

Nondestructive Determination

Measurement of UV absorption

Principle

See NIR/NIT above

Rapid, nondestructive, multicomponent analysis

Faster than ion exchange chromatography, quantifies amino acids, value for net protein, option to quantify free amino acids, sensitive

Rapid, nondestructive, no addition of reagents required, high sensitivity, low dependency of signal response on amino acid composition, low interference from nucleic acids and nucleotides

Advantages

See NIR/NIT above

Strong interference by water, influence by lipids and sample particle size, complex calibration

Most methods do not include all amino acids, hydrolysis destroys some of the amino acids. Derivatization agents: OPA: no derivatization with secondary amino acids. FMOC: less soluble, might interfere

Interference by oxygen and UV-absorbing compounds (buffer, salts)

Drawbacks

[133]

[90–92,96,97, 108,178]

[132,133]

Selected References


◾

Far-UV absorption

Methods



The sample is dried until constant weight (e.g., 12 or 24 h) and water evaporated is determined.

Drying by irradiation

Volumetric analysis of water after boiling in toluene

See NIR/NIT above

See NMR above

Air or vacuum drying

Infrared drying Microwave drying

Dean and Stark method

NIR/NIT

NMR

Water Determination

Possible to distinguish between free and bounded water

See NIR/NIT above

Faster than oven drying methods

Shorter analysis time compared with air and vacuum drying. For the microwave method it is possible to analyze many samples simultaneously

Simple to use and inexpensive equipments required

Calibrations are needed and knowledge on chemometry is an advantage

See NIR/NIT above

Uses health hazard chemicals (toluene)


Risk of overheating

Long analysis time. Air drying (101°C) may lead to thermal damage, Vacuum drying: may be difficult to keep uniform temperature distribution in the oven

[152,178]

[151]

[40]

[151]


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Near-infrared transmittance (NIT) instruments are particularly suitable to the analysis of fish. Generally, the sample has to be minced, and it is usually possible to run several subsamples. The results are averaged to obtain more representative spectral data from the sample. The spectral data are then used to perform multivariate calibrations against the chemical or physical data. The same spectral data will be used against the different selected variables, so one can simultaneously predict, for example, water, fat, and protein content from the same spectral data as accurately as the traditional “wet” chemical methods [4]. To analyze directly on a fillet one needs an interactance probe; this involves illumination and detection at laterally separated points on the sample’s surface. It is normally accomplished using a fiber-optic probe in which one set of fiber-optic bundles carries the incident radiation and another carries the reflected radiation. Due to the striped structure of fish muscle, it is necessary to have a large interactance probe, usually two times 2 cm. With this type of probe it is possible to make analysis directly on the fillet, without previous mincing, but with a slightly lower accuracy [4–6]. Portable instruments are now available [7], and successful results are also obtained for whole fish [5] and for live fish [8]. Instead of a conventional monochromator, instruments are now also made with diode arrays, making it possible to measure the whole spectrum at the same time and in that way reducing the time for measurement, making online analysis possible [8]. NIR absorption will change with temperature and calibration, and NIR measurements must therefore be made on samples with approximately the same temperature [9]. Moreover, the measurements are affected by texture and whether the sample has been frozen and thawed [10,11]. Due to the requirement of extensive sample specific calibrations, the analysis should be performed by skilled personnel [12]; however, once calibrated the analysis is easy to perform. Nuclear magnetic resonance (NMR) is another nondestructive technique that enables determination of fat and water, and recent studies have shown that it might be possible to also gain data on protein levels in dried samples [13]. The low-field NMR instruments commonly in use require withdrawal of cylindrical samples of 10–40 mm diameter for analysis [14,15]. The method is fast, accurate, and easy to use when the calibrations are performed. A new handheld portable NMR instrument (NMR mouse) has recently been developed [16,14], and it enables an analysis time of less than 20 s and can even be used in vivo on living fish [14]. Less common methods for nondestructive analysis of proximate composition in fi sh are ultrasound techniques [17–20], the total body electrical conductivity (TOBEC) technique [21], and magnetic resonance imaging (MRI) [22]. The ultrasound method is rapid, automated, and can be used online, and empirical equations have been developed to relate the ultrasonic velocity to composition [17]. A weakness in this method is the variations in ultrasonic properties of fi sh tissue due to temperature [17]. For nonfatty fi sh, the solid nonfat content can be determined from a single measurement; however at least two temperatures are suggested during analysis of fat and solid nonfat in fatty tissue [17]. In the TOBEC method the live fish is placed in a low-frequency electromagnetic field, and the distinct electrical characteristics of body fat and fat free tissue provide the proximate data [21]. MRI can provide valuable information on proximate composition and distribution of chemical constituents in fish samples [14]; however, these imaging instruments are expensive and are used primarily in certain research laboratories. Calculation of fat content by measuring the water content is possible with cheap, robust instruments (see below), but they can be used only when the protein content is stable.

Composition and Calories

16.3

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267

Lipids

16.3.1 Nutritional Aspects Marine lipids contain the omega-3 fatty acids such as C20:5n-3 (EPA) and C22:6n-3 (DHA) with well-documented beneficial health effects [23–25]. These fatty acids are found in all parts of the fish and are constituents of different lipid classes such as phospholipids, triacylglycerols, lysophospholipids, partial glycerides, esters, and free fatty acids. Marine lipids are the only source of EPA and DHA, and extraction and utilization of these fatty acids is a major industry. The market shares for higher value applications such as food ingredients, health care products, and medicine are increasing owing to the supply to aquaculture business.

16.3.2 Methods for Determination of Total Lipids The lipid content in fish can be determined by several different methods varying in efficiency, total lipid yield, accuracy, skill requirement, and cost. The main methods are shown in Table 16.1 ranging from organic solvent extraction, microwave drying, to nondestructive techniques. Fish lipids are generally composed of polar and neutral lipid compounds. Although the triacylglycerols dominate in the lipid classes of fatty fish such as the pelagic species, the phospholipids are the main lipid class in lean white fish species. In addition, other derivatives of fatty acids (partial glycerides, free fatty acids, esters etc.), sterols, fat-soluble vitamins, and carotenoids are found in fish and comprise the large group called total lipids. Chemical methods: Traditional methods for determination of total lipids are generally based on solvent extraction followed by gravimetric determination. The lipid yield obtained is highly dependent on the solvent system, and using a combination of polar and nonpolar solvents it is possible to extract the total lipids and not only the free lipids such as triacylglycerols. Differences in lipid yield among the methods are claimed to correlate with the extraction efficiency of the more tightly bounded polar lipids such as phospholipids [26]. A combination of chloroform, methanol, and water is most often used for manual extraction of total lipids in fish [27,28]. The methanol penetrates the tissue while the chloroform dissolves the fat. The samples are first homogenized and after several extraction steps, followed by evaporation of solvents, the total lipids are gravimetrically determined. The Bligh & Dyer method (B&D) was originally used on fish muscle and less solvent volumes were used compared with the Folch method. A comparison between the Folch and B&D method has previously shown that the B&D method underestimates the lipid yield when the lipid content in fish muscle is above 2%, whereas no significant differences are found at lower levels [29]. Modifications of the B&D method are widely reported in the literature [30,31], although these specific modifications are rarely described in detail [29]. One recent study demonstrated that a modified B&D method using NaCl and electrolyzed cathode water gave higher lipid yield compared with the conventional method [32]. Generally, the crude lipids extracted by B&D compose a broad range of lipid classes, and the method demonstrates a high efficiency in extracting both polar and neutral lipids. However, parameters such as solvent ratio, order of solvent addition, and number of extraction steps are important parameters that affect the lipid yield and might be individually suited for specific sample material differing in lipid class composition. An example is the increased lipid yield obtained when using higher amounts of methanol, which was explained by a better extraction of phospholipids in a study by Smedes and Askland [31].

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Due to the high lipid yield generally obtained by the B&D method, it has been widely used as a reference to test the efficiency of other methods, and it is particularly used in research laboratories. Additionally, this extraction allows the successive characterization of lipids such as lipid classes (tri-, di-, and monoacylglycerols, free fatty acids, phospholipids etc.), lipid oxidation products, and fatty acid composition. Hence, manual extraction is relatively time-consuming, requires laboratory facilities, and the solvents used are toxic to humans and environment. Less toxic solvents are used in some studies [31,33–37] without achieving the same lipid yield as that obtained by using the traditional solvents. Solvent extraction of animal tissues in general and procedures for preparation of samples are comprehensively discussed by Christie [38] and by the same author in the Lipid Library Website (http://www.lipidlibrary.co.uk/topics/extract2/index.htm). Another commonly used method for solvent extraction of fatty fish species is the ethylacetate method [39] without the use of expensive equipment. The method even specifies what part of the fish should be included in the analysis. Ethylacetate has replaced the health-harmful benzene that was used in the early extractions. Among the automatic solvent extraction techniques, the Soxhlet method [40] and modifications of this method have been most widely used for determination of total lipids in fish. The sample is lyophilized before solvent extractions, removal of solvents, and gravimetric determination [41]. Petroleum ether and diethyl ether are the most common solvent used but the use of hexane and acetone are also reported in some studies [41,26]. The original Soxhlet method was developed by Soxhlet in 1879. This was originally a time-consuming method (16 h); however, today, there are more rapid methods available based on the same principle with commercial instrumentation such as the SoxTec equipment. New developments in this field are continuously reducing the analysis time, and a new microwave-integrated Soxhlet may run samples in less than an hour [42]. Lipid content can also be determined without the use of chemicals such as in the microwave drying method. This is a simple and inexpensive method that indirectly calculates the lipid content from the water content analyzed [43]. The principle behind this method is a reported reverse intercorrelation between water and lipid content in clupeid fish [43–45] calculated from the following formula: Fat content% = 80% − water content % [43]. Limitations in this method lie particularly in the lack of fitness of the intercorrelation between water and lipids during different maturity stages for the fish [46] and also variations between different locations in the fish [46–50]. Furthermore, this intercorrelation is affected by processing, particularly heat treatment, that might reduce the water content.

16.3.3 Nondestructive Methods The intercorrelation between water and lipids in fish is also applied as the principle for the nondestructive portable Fat Meters developed by Kent [44,51–52]. The sample is irradiated by microwaves with a microwave strip, the water is measured by the dielectric properties, and the lipid content is then calculated. These instruments (Fish Fat Meters and Torry Fat Meters) are calibrated for a range of fish species [45], and they are simple to use. However, these methods share some of the same limitations as those in the microwave drying method such as the lack of fitness during spawning, and additionally, the accuracy of the Fat Meters has also been reported to be dependent on the lipid content in the fish [46]. Although the Fat Meter is limited to determining fat and water content, methods such as NIR spectroscopy may simultaneously determine the content of lipids, proteins, and water from the surface of the sample in a few seconds [4,53]. The NMR technique has particularly been applied


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in quantification of lipids in fish [15,46,54–56], and the low-field NMR can distinguish between different lipid classes [57]. When increasing the field strength to high-resolution NMR, a range of different lipid constituents can be detected [58,59]. The ultrasound velocity technique has provided data that enable classification of salmon muscle into low, medium, and high fat [20]. See earlier section in this chapter for further information on these methods.

16.3.4 Comparison of Methods Nondestructive and rapid techniques are of particular importance for fatty fish such as herring, mackerel, and some farmed fish species. The lipid content in these species usually shows large variation, and analysis results are valuable on board the fishing vessel or processing plant for sorting into groups based on their lipid content. Vogt et al. [43] who compared the lipid yield obtained by Torry Fat Meter, NIR, the microwave method and a modified Soxhlet, found that the NIR- and microwave methods were closest to the reference solvent extraction (R 2 = 0.90). A high correlation (R 2 = 0.96) has been found between ethyl acetate extraction and NIT analysis of whole minced capelin [60], and another study [46] demonstrated a good correlation between NIR and solvent extraction in specific locations of the fish (middle part of fish and fi llet skin side) (R 2 = 0.80–0.93). NMR measurements, in the same study, showed a good correlation with the solvent extraction when the analysis was performed on minced samples. Generally, the solvent extraction techniques obtain the higher yield, which might be explained by the contribution of other lipid classes than triacylglycerols, such as polar lipids and sterols that are not always included in the rapid analyses. However, readings from the Fat Meter have been reported to show higher yield than reference values in samples of herring [61], which might be explained by the variation in the intercorrelation between water and lipids. Th is same study demonstrated a bigger difference between the methods at higher lipid content in the samples. Higher variation between methods are reported when analyzing lean fish compared with fatty fish high in unpolar lipids [26]. The statement of what is the most suitable method for lipid determination is highly dependent on the applicability and what criteria are the most important for the analysis such as accuracy, robustness, time of analysis, use of solvents, and portability, and so on.

16.4

Proteins

16.4.1 Nutritional Aspects Due to its favorable content and balance of essential and nonessential amino acids, fish protein is regarded to be of high nutritive value. Seafood proteins are also highly digestible, which adds to the understanding that digestibility of raw fish meat is in the range 90%–98% and that of shellfish about 85% [62]. Protein and amino acid requirements vary through life and are generally higher among young growing children compared with adults [63,64]. These nutritional aspects are more comprehensively described in other chapters in this book. Fish and marine invertebrate tissue contains from about 11%–24% (ww) crude protein depending on species, nutritional conditions, and the type of muscle. Although amino acid composition might vary among different types of tissue, there is a high similarity in the same tissue among species as pointed out by Mambrini and Kaushik [65]. The total body composition of amino acids shows high similarity among various cultured fish species [66].

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16.4.2


Methods for Protein Determination

Several of the most important methods for protein determination in food date from the late 1800s (Dumas, Nessler’s reagent, Biuret, Kjeldahl, Folin-Ciocalteau, and Dye binding) [67]. Quantification of total protein in fish and fish products can be determined by total organic nitrogen followed by conversion into crude protein or by a set of direct methods.

16.4.3

Determination of Total Nitrogen

Determination of proteins by analysis of total nitrogen (N) multiplied by a specific factor is a common procedure in fish analysis [68]. The N content of food is commonly determined using the Kjeldahl [69] or the Dumas [70] methods. Kjeldahl includes digestion of material and quantifies only N that is transformable to NH4+ using titration, colorimetry, or an ion-specific electrode [71]. In the Dumas method, all N is converted to N2 through combustion using a nitrogen element analyzer. Generally, the Dumas method gives higher N values than the Kjeldahl method [72–74], and a Kjeldahl-N to Dumas-N ratio of 0.80 for fish has been calculated [71]. The conversion factor for N was originally 6.25, based on average nitrogen content in different proteins of 16%, which might not be suitable for all protein sources, as they vary in amino acid composition. Generally, studies on fish have shown lower values with a more specific conversion factor of 5.8 presented for fish filet [75,76], and a factor of 4.94 (nitrogen to net protein) for protein estimates for fish and fish products are suggested by Salo-Väänänen and Koivistoinen [77]. More specific conversion factors based on the N content in isolated proteins are frequently applied for different categories of food [78]. Salo-Väänänen and Koivistoinen [77] showed that the true conversion factor was 5%–20% lower than the general 6.25 in a line of food products. Moreover, up to 40% variations were found in a comparison study of the 6.25 factor against foodspecific factors or sum of amino acids [79]. These differences indicate a significant contribution of nitrogen from other than amino acids or protein structures. Large amounts of those compounds are found in fish and fish products, probably due to both natural composition and degradation products [77]. These other N contributions might originate from nucleic acids, nucleotides, trimethylamine n-oxide (TMAO), free amino acids, or others. Contributions of N from products such as urea might appear in sharks, skates, and rays. There are, however, options to separate protein N from nonprotein N by precipitation and filtration after solvent extraction if required [80]. The nitrogenous compounds that do not originate from proteins can also be separated using methods such as ion-exchange chromatography (IEC), gas chromatography (GC), thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) [81,82].

16.4.4

Direct Methods for Soluble Protein Determination

Protein is amino acids linked together via peptide bonds, and quantification of these amino acids might give more accurate values for protein estimates [68,77,83]. The term “net protein” is often used for those values that are corrected for added water during analysis. There are options to exclude or include the free amino acids during sample preparations, or they have also been analyzed separately using HPLC methods [84–86]. A more extensive description of various methods and techniques used in protein analyses are covered by Owusu-Apenten [67]. Acid hydrolysis followed by amino acid quantification such as by HPLC [87–90] or the more traditional IEC [89,91–93] are direct and specific methods for protein determination. During IEC, the derivatization of amino acids takes place postcolumn in most methods using, for


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example, ninhydrin [94,89] or O-phthalaldehyde (OPA) [95]. Common derivatization reagents for quantification of amino acids in HPLC methods are OPA [90,96] and 9-fluorenylmethyl chloroformate (FMOC) [96], which are often used in combination with 2-mercaptoethanol, ethanethiol [90], or 3-mercaptopropionic acid [90,96]. An additional derivatization agent 2-(9-anthryl)ethyl chloroformate showed good correlation with the use of FMOC and lower detection limits for amino acids when analyzed in UV absorbance due to better spectral properties of the produced chromophore [97]. Other derivatization reagents are discussed in Sarwar and Botting [91] and in Fekkes [92]. In HPLC methods both pre- and postcolumn derivatizations are used with variable mobile phases based on methanol and acetonitrile. The reaction time, choice of solvents, and the concentration of 2-mercaptoethanol determine the efficiency of the reaction between OPA and amino acids with influence on quantification of the amino acids [90] (generally, 2-mercaptoethanol should be kept in the lower concentration range for optimization of the method [90]). OPA does not react with secondary amino acids, and FMOC is, among others, less soluble and might create interference reactions, but by combining those both, the primary and secondary amino acids can be detected [98]. Further optimization of this approach and adding an online dialysis step have improved the method with separation of 25 amino acids, and quantification of most of them [96]. Hyp (hydroxyproline), which is primarily found in connective collagenous tissue [99], might otherwise be quantified through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [100,101] or N2-(5-fluoro-2,4-dinitrophenyl)-l-valine amide [102]. Alternative methods are the spectrophotometric determination of Hyp as a measure of collagen [103] or collagen/gelatin in fish skin [104], the latter using a modified spectrophotometric method for Hyp determination by Bergman and Loxley [105]. The destruction of Trp (tryptophan) during hydrolysis in hydrochloric acid can be omitted by replacing with a line of others, including methane sulfonic acid containing 3-(2-aminoethyl) indole [106,107]. Enhanced signal of tyrosine, phenylalanine, and Trp has also been obtained using online photolysis with chemoluminescence methods in the HPLC system [108]. A more comprehensive overview of alternative methods for quantification of Trp is otherwise reviewed by Molnar-Pearl [109] and includes both alkali hydrolyses along with more complex derivatization and detection methods. During amino acid determination with the HPLC methods, detection of Cys (cysteine/ cysteine) might require special procedures during extract preparations such as iodoacetic acid [110] or 3,3′-dithiodipropionic acid as used in Glencross et al. [111]. Some nitrogenous compounds such as nucleic acids and amines, the latter originating mainly from microbial decarboxylation of amino acids in food such as putrescine, cadaverine, spermidine, spermine, tyramine, and histamine [112], can also be separated using methods such as HPLC [113,114] and reverse-phase HPLC [115,116]. Amino acid determination is often used in nutritional studies on fish, and requirements are frequently determined after analysis using IEC or HPLC methods [111,117–119], or alternatively 13C-NMR after extraction has been applied in such studies [120]. Quantification of the individual amino acids in HPLC methods is based on standards (amino acids) and use of an internal analytical standard such as a-butyric acid (ABA), responses to those, and molecular weight make the basis for calculating the amino acids. The protein values are calculated as the sum of all amino acids corrected for water added during hydrolyses, and the free amino acids might be removed through the extraction procedure or analyzed separately. Proteins can also be determined by a number of spectrophotometric methods. Some of these analyses are based on the ability of proteins to absorb (or scatter) light, whereas in other analyses, proteins are chemically or physically modified to absorb (or scatter) light. Due to variation of amino acid composition in proteins, most of these methods give results that can be different from absolute protein concentrations [83].

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Methods where proteins are chemically or physically modified for determination (colorimetric assays) can also be divided in to two groups: dye-binding reaction and redox reaction with proteins [121]. In the redox spectrophotometric methods, analyses are based on reaction with Folin reagent, and the following methods could be mentioned: Biuret reaction [122], Lowry protein method [123], and bicinchoninic acid (BCA) assay [124]. In the Biuret reaction Cu(II) with proteins in alkaline medium is reduced to Cu(I), which binds to protein forming a Cu(I)–peptide complex with purplish-violet color [121]. The same principle is used in BCA assay, where Cu(I) is detected by reaction with BCA, which gives an intense purple color [125]. One of the most popular methods in this group is the Lowry protein method [123], which is initially based on the Biuret reaction, where peptide bonds react with Cu(II) in alkaline medium to produce Cu(I). Later Cu(I) reacts with the Folin reagent. The reaction gives a strong blue color [83]. The intensity of color partly depends on the amount of Tyr and Trp in samples but can also be influenced by other components such as N-containing buffer or carbohydrates [121]. The amounts of proteins in sardine determined by the Lowry method were comparable to those determined by Kjeldahl method [121]. The Lowry method is suitable for protein extracts such as actomyosin, which is an important component in surimi-based products [126]. However, the BCA assay is shorter compared with the Lowry method (where two steps are needed), more flexible and stable in alkaline conditions, and has a broad linear range. The BSA assay can also be interpreted by the usual chemical components such as EDTA, thiols, reducing sugars, hydrogen peroxide, or phospholipids [121,125]. The dye-binding spectrophotometric assay is based on the reaction between acid dye and positively charged amino acid residues in proteins [121]. In acidic conditions, the created insoluble complexes are removed and the unbound dye is determined by measuring its absorbance. The amount of protein is proportional to the amount of bound dye. Coomassie dye in acidic conditions binds to proteins and creates complexes that influence a color shift from a maximum from 465 nm to 595 nm, using the Bradford method [127]. Absorbance of Coomassie dye-protein complex is measured at 595 (575–615) nm, because the difference between the two forms of the dye is greatest in this area. Within the linear range of the assay (∼5–25 mg/mL), the protein amount is proportional to bounded Coomassie [127]. This method is suitable for determination of extractability of proteins [128] or protein content in extracts [129–131]. Th is technique is simple, sensitive, and uses shorter analysis time compared with the Lowry method. Moreover, the dye-binding assay is less affected by reagents and nonprotein components from biological samples [132]. Proteins in solution can be quantified in a simple spectrophotometric analysis by near- or farUV absorbance [133,134]. Absorption in the near UV by proteins depends mostly on the content of Tyr and Trp and less on the amount of phenylalanine (Phe) and disulfide bonds. This absorbance measurement is simple, sensitive, needs no reagents, and the sample is recoverable [133,134] Crude protein extracts or individual fractions of proteins [135] can be measured at 280 nm. Disadvantages of the method include interference with other components such as nucleic acid, which absorbs in the same wavelength region [133]. Far-UV absorption can also be used for determination of protein content: peptide bonds absorb in the area with the maximum at about 190 nm. Different proteins give a small variation in absorbance, and the method can be considered as accurate for protein determination. However, oxygen also absorbs at these wavelengths, and to avoid interference, measurements at 205 nm is used. It should also be mentioned that components such as carbohydrates, salts, lipids, amides, phosphates, and detergents interfere [133,134].


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16.4.5 Nondestructive Analysis of Proteins Recently, other advanced and nondestructive methods have become more common for determining protein. NIR is one of these [4,53], and it was originally developed for protein analysis and has since that time been developed and calibrated for a range of fish species. Low-field NMR is generally not suitable for protein determination in a nondestructive manner. See earlier text for more information on the nondestructive techniques.

16.5 Determination of Carbohydrate Content Carbohydrates are often classified into three broad groups: sugars (mono- and disaccharides), oligosaccharides (three to nine monosaccharides) and, polysaccharides (more than nine). The content of carbohydrates in fish muscle is low [136,137] and is further influenced by conditions experienced before and during capture, which may lead to depletion of glycogen stores and thereby a decrease in the carbohydrate level. Under anoxic conditions postmortem, glycogen will continue to be metabolized, resulting in increased lactic acid along with reduced pH and eventually a gradual loss of the sweet, meaty character of fresh fish. Some marine invertebrates on the other hand are characterized by a high content of carbohydrates; up to 10.2% and 12.5% total sugars can be found in subcuticular tissue of spiny lobster and blue crab, respectively, with the highest amounts of glucose followed by galactose and mannose [138]. Glycogen stores of scallops are highly dependent on season (temperature, food availability, and lifecycle), and highest levels are usually reached after the summer period [139], showing levels up to 23%–25% glycogen of dry weight of adductor muscle [139,140]. Seasonal variations of glycogen content in mussels (Mytilus edulis) are also high, showing values in the range 4%–37% of tissue dry weight [141,142]. Among the line of methods suitable for seafood, the amount of total carbohydrates in shellfish can be determined by using the phenol-sulfuric acid procedures described by Dubois et al. [143] as used for scallop (Pecten maximus) in Maguire et al. [144] and silver carp in Gnaiger and Bitterlich [144]. This method is based on hydrolysis of polysaccharides and does not measure all sugar molecules in the materials equally accurately, because the carbohydrates are absorbed at different maximum wavelengths and in addition differ in the ability to form the chromogenes formed in the method. If measurements are performed at 488 nm and a standard curve is prepared using glucose, this will lead to a possible underestimation in the case of chemical characteristics of monosaccharides deviant from glucose. This relatively simple method is often used, because it gives a good estimate of total carbohydrates in tissue that contain 10% or more of hexose polymers [145]. Glycogen from seafood can also be determined after preparation of solution of glucose units using a range of assay kits for glucose followed by colorimetric determination (Boehringer Mannheim, Cayman chemicals, Biovision or others), as described for Abalone tissue using a combination lipid and glucose extraction method in studies of Allen et al. [146]. Glycogen levels in small amounts of tissue can additionally be analyzed using the anthrone methods with spectrophotometric determinations [147–149], which have been demonstrated as useful for scallop [150]. Carbohydrates are frequently calculated and expressed as total carbohydrates by difference, which is the remainder after subtraction of moisture, crude protein, total fat, and ash and includes fibers if present in the analyzed material. An excellent overview of definitions and internationally used carbohydrate tag names along with applicable analytical procedures for food in general is given by Munro and Burlingame [151].

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16.6 Determination of Water Content Water content in fish can be determined by simple drying methods. Using conventional air ovens, a common practice has been to dry the sample at 105°C for 12 h, which by experience has shown satisfactory drying of fish and fish products. To ensure complete drying, the sample can be dried to constant weight. Other methods [40] refer to 101°C for 24 h by conventional ovens and 70°C for 24 h using vacuum ovens. The sample is weighed in a container, and after heating the sample is cooled and weighed again. The water content is determined by the following formula: Water content (%) =

(Weight of wet material − weight of dried material) × 100 Weight of wet material

Infrared and some microwave ovens may allow an analysis time of 1–2 h [152]. Further, the new nondestructive methods such as NIR/NIT, NMR, or Fatmeter, which are described previously in this chapter, may be used for fast determination of water, and the low-field NMR technique can even distinguish between free and bounded water [15,153]. In a volumetric method (Dean & Stark), the samples are boiled in toluene before measuring the volume of water. This method is relatively fast but uses toluene, which is hazardous to health [152].

16.7

Calories

The energy content of food is generally given in kilocalories (kcal) and kilojoules (kJ), which have a conversion factor of 1 kcal = 4.184 kJ. Seafood show variable composition of proteins and fat, and energy content is dependent on this distribution, which often might also be highly influenced by seasonal variations. In a seasonal study of 35 fish and shellfish species, Soriguer et al. [154] found a substantial variation in biochemical composition, where even mackerel known as fatty type of fish, in parts of the year could be classified within the lean fish category. The lipid level in particular has high significance for the calorie content of fish, with implications for calculations in dietary studies and databases; this is important to bear in mind when these are used.

16.7.1 Direct Measurement of Energy The gross energy content of food (measured as heat of combustion, kcal/g) may be determined directly by using a bomb calorimeter (micro- or macromethods), which includes burning food with oxygen in an insulated container of constant volume [155,156]. The heat is adsorbed in water, and the energy is determined from the mass of water, its temperature rise, and its specific heat. Dichromate wet oxidation with potassium dichromate is also sometimes used as a direct method, giving rise to slightly lower energy values in fish samples than when measured by bomb calorimetric methods [157,158]. Food composition databases are not based on direct measurements of gross energy, because those are not equal to energy requirements [159]. Instead the metabolizable food energy is used, which accounts for the energy in food remaining after losses through the feces, gas, urea, and the body surface [160].


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16.7.2 Indirect Measurements of Energy The energy released by oxidation of protein, fat, and carbohydrate is the basis for sets of conversion factors. The Atwater general factor system is the foundation for the most frequently used systems for energy conversion [161], which originates from combustion with adjustments for losses in digestion, absorption, and excretion of urea. The Atwater general energy conversion values are 4.0 kcal/g for proteins, 9.0 kcal/g for lipids, and 4.0 kcal/g for carbohydrates (calculated by difference, i.e., subtracting water, ash, proteins, and lipids). Originally no differences were determined between the fiber and available digestive carbohydrates, but exploring more specific heat of combustion led to factors of 3.75 kcal/g when used for monosaccharides and 4.2 kcal/g for polysaccharides, with application in the Atwater system [162]. However, the specific conversion factor used for carbohydrates in shellfish is 4.11 kcal/g [163]. For other food material, energy factors for dietary fiber have been developed, taking into account availability, provided also by the microorganisms in the colon giving values recommended by FAO [164] of 8.0 kJ/g (2.0 kcal/g). A more specific set of factors for energy conversion were developed due to different combustion rates and digestibility of various sources of proteins and fats and additional impact caused by processing. The specific set of factors presented in Merrill and Watt [163,165] arrived at 4.27 kcal/g for protein and 9.02 kcal/g for fat in meat and fish. It is, however, important to consider the choice of analytical methods regarding conversion of proteins to calories. Both the variable nonprotein N and the variations in amino acid composition in different protein sources might have implications on the calculated energy levels if based on N analysis (see above). When energy contributions from proteins are set, the most accurate method will be as the sum of amino acids (free and protein bound). Alternatively, Kjeldahl or Dumas techniques are used with more source-specific conversion factors such as those used by Jones [166] or others, when these are known. In terms of conversion to energy, the more specific conversion factor of 5.65 kcal/g for protein was suggested [167] and tested in combination with direct energy measurements for use with fish tissue, resulting in slightly higher values compared with bomb calorimetric methods [157]. Calculation of energy contribution from fat might include analysis of fatty acids with total fat calculated as triacylglycerol equivalents [160]. For fatty fish muscle the factor 0.90 is used in conversion of total fat to total fatty acids, whereas 0.70 is used for white fish muscle [169]. Gravimetric methods are also used for energy calculations, which (depending on methods used; see above) would include weight of the additional lipid components that are not transformed to energy, per se. The calorie content of extracted lipids (methanol/chloroform extraction) from fish tissue as found by microcalorimetric methods suggests the use of a lower energy conversion factor such as 8.49 kcal/g [157]. Gross energy levels obtained from bomb calorimetry might deviate from energy when based on analysis and conversion factors due to the lipid calculations. A high level of lipids in tissue is usually accompanied with high energetic content by both methods. However, with high levels of sterols, the gross energy by bomb calorimeter can be higher than the metabolic energy level calculated from the analysis by use of conversion factors. Th is method deviation was pointed out for low-lipid squid samples by Krishnamoorthy et al. [169]. In the study of feed, fish, and feces by Henken et al. [158] three different methods for calculating energy content were compared (I, dichromate wet oxidation; II, bomb calorimeter; or III, chemical analyses followed by conversion factors 5.65, 9.45, 4.2 kcal/g [proteins:fat:carbohydrates]). Proteins were calculated with N*6.25, fat analyzed by Soxhlet with hexane extraction, and carbohydrates calculated by difference. Agreements were obtained in methods II and III and lower energy values were obtained with method I. Inadequate protein oxidation by dichromate method

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[170] was solved by correction factors but still resulted in lower values in fish, feed, and feces compared with bomb calorimetry or direct analyses followed by conversion factors. In recent years the field of nutrition has become highly complex due to developments in both analytical and physiological methods. A variety of different analytical methods are in use along with various sets of conversion factors, which again are based on their own specific analytical methods. In scientific work it is particularly important to specify methods and calculations made in the presented results. Standardization of analytical methods and energy conversion factors might improve the use of nutrient databases for energy calculation.

16.7.3 Food Composition Tables and Databases Food composition databases are practical tools providing a line of useful information on foodrelated subjects. For the users it is convenient to find further links, reports, published works, nutrient composition tables, and so forth, through a database. Researchers are requested to make relevant publications available through these pages, adding to the up-front knowledge in the area. When food databases contain original analytical results, the values can be trusted to represent more accurate levels and are more useful for governmental and research purposes. There are several general databases available to the public both on international, regional, and national levels such as those of The International Network of Food Data Systems (FAO/INFOODS), United States Department of Agriculture (USDA), Pacific Island Food Composition Tables (PIFCT), and German Nutrient Database (BSL). The user groups for food databases are among others found within the groups of food researchers and industry, dieticians, epidemiological and health researchers, and national and governmental authorities. National and regional food composition tables are important, because they may reveal specific dietary traits of subpopulations important for health and epidemiological research. Differing nutritional definitions are also common as with different sets of energy conversion factors, which is important to be aware of when food tables are used. Databases as such FishBase provide specific tables for seafood such as proximate data and energy levels of different organs and ecological data of harvested species in specific regions. However, the databases might have a potential for improvement with regard to expected variability in the composition of food items, which might be due to seasonal variations, variations experienced during the growth, production phase, or as influenced by storage or processing conditions. Additionally, processed food with many ingredients is complex, some nutrients are labile, and constituents such as fat and moisture might be added and/or removed during food preparations. As it might be practically impossible to obtain the full detailed composition, there is selection of constituents in food tables. Most databases contain 10–25 food groups [160], but some also contain more than 100 nutrients and food components such as the Nutrition Data System for Research (NDS-R) in the United States [171]. Skills and knowledge in the analytical methods on which the values are based on, advantages, and drawbacks in the table values are required.

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109. Molnar-Perl, I., Tryptophan analysis in peptides and proteins, mainly by liquid chromatography, Review. J. Chromatogr, A, 763, 1–10. 1997. 110. Campanella, L., Crescentini, G., and Avino, P., Simultaneous determination of cysteine, cystine and 18 other amino acids in various metrices by high performance liquid chromatography, J. Chromatogr. A, 833, 137–145, 1999. 111. Glencross, B., Hawkins, W., Evans, D., Rutherford, N., McCafferty, P., Dods, K. and Sipsas, S., Assessing the implications of variability in the digestible protein and energy value of lupin kernel meals when fed to rainbow trout, Oncorhynchus mykiss, Aquaculture, 277, 251–262, 2008. 112. Vacha, F., Krizek, M., and Pavlieck, T., Content of biogenic amines in common carp (Cyprinus carpio). In: Towards a Predictable Quality. Aquaculture Europe ‘99. Trondheim, Norway, August 7–10, 249–250, 1999. 113. Ozogul, F., Taylor, K.D.A., Quantick, P., and Ozogul, Y., Biogenic amine formation in Atlantic herring (Clupea herengus) stored under modified atmosphere packing using a rapid HPLC method, Int. J. Food. Sci. Tech., 37, 515–522, 2002. 114. Korös, A, Hanczkó, R., Jámbor, A., Qian, Y., Perl, A., and Molnár-Perl, I., Analysis of amino acids and biogenic amines in biological tissues as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate derivatives by high-performance liquid chromatography A deproteinization study, J. Chromatogr. A, 1149, 46–55, 2007. 115. Krause, I., Bockhardt, A. Neckermann, H., Henle, T., and Klostermeyer, H., Simultaneous determination of amino acids and biogenic amines by reversed-phase high-performance liquid chromatography of the dabsyl derivatives, J. Chromatogr. A, 715, 67–79, 1995. 116. Pereira, V., Pontes, M., Camara, J.S., and Marques, J.C., Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldehyde derivatization procedure, J. Chromatogr. A, 1189, 435–443, 2008. 117. Berge, G.E., Sveier, H., and Lied, E., Nutrition on Atlantic salmon (Salmon salar); the requirements and metabolic effect of lysine, Comp. Biochem. Physiol. Part A, 120, 477–485, 1998. 118. Refstie, S., Bakke-McKellop, A.M., Penn, M.H., Sundby, A., Shearer, K.D., and Krogdahl, Å., Capacity for digestive hydrolysis and amino acid absorption in Atlantic salmon (Salmo salar) fed diets with soybean meal or inulin with or without addition of antibiotics, Aquaculture, 261, 392–406, 2006. 119. Espe, M., Lemme, A., Petri, A., and El-Mowafi, A., Assessment of lysine requirement for maximal protein accretion in Atlantic salmon usin plant protein diets, Aquaculture, 263, 168–178, 2007. 120. Conceicao, L.E.C., Grasdalen, H., and Dinis, M.T., A new method to estimate the relative bioavailability of individual amino acids in fish larvae using C-NMR spectroscopy, Comp. Biochem. Physiol. Part B, 103–109, 2003. 121. Sozgen, K., Cekic, S.D., Tutem, E., and Apak, R., Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium, Talanta, 68(5), 1601–1609, 2006. 122. Noll, J.S., Simmonds, D.H., and Bushuk, W.C., A modified biuret reagent for the determination of protein, Cereal Chem., 52, 610–616, 1974. 123. Lowry, G.H., Rosenbraugh, R.J., Farr, A.L., and Randall, R.J., Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193, 263–275, 1951. 124. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk, D.C., Measurement of protein using bicinchoninic acid, Anal. Biochem., 150, 76–85, 1985. 125. Walker, J.M., The bicinchoninic acid (BCA) assay for protein quantitation, In: The Protein Protocols Handbook, 2nd edn., Ed., Walker, J.M., Humana Press Inc., Totowa, NJ, 2002. 126. Liu, R., Zhao, S.-M., Xiong, S.-B., Qui, C.-G., and Xie, B.-J., Rheological properties of fish actomyosin and pork actomyosin solutions, J. Food Eng., 85, 173–179, 2008. 127. Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248–254, 1976.


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128. Sarkardei, S. and Howell, N.K., The effects of freeze-drying and storage on the FT-Raman spectra of Atlantic mackerel (Scomber scombrus) and horse mackerel (Trachurus trachurus), Food Chem., 103, 62–70, 2007. 129. Benjakul, S. and Bauer, F., Physicochemical and enzymatic changes of cod muscle proteins subjected to different freeze-thaw cycles, J. Sci. Food Agr., 80, 1143–1150, 2000. 130. Hultmann, L. and Rustad, T., Iced storage of Atlantic salmon (Salmo salar)–effects on endogenous enzymes and their impact on muscle proteins and texture, Food Chem., 87, 31–41, 2004. 131. Sovik, S. L. and Rustad, T., Effect of season and fishing ground on the activity of lipases in byproducts from cod (Gadus morhua). LWT-Food Sci. Technol., 38, 867–876, 2005. 132. Kruger, N.J., The Bradford method for protein quantitation. In: The Protein Protocols Handbook, 2nd edn, Ed., Walker, J.M., Humana Press Inc., Totowa, NJ, 2002. 133. Aitken, A. and Learmonth, M.P., Protein determination by UV absorption. In: The Protein Protocols Handbook, 2nd edn., Ed., Walker, J.M., Humana Press Inc., Totowa, NJ, 2002. 134. Van Camp, J. and Dierckx, S., Proteins. In: Handbook of Food Analysis, Physical Characterisation and Nutrient Analysis, Volume I, 2nd edn., Revised and expanded, Ed., Leo, M.L., Nollet, F. Marcel Dekker, Inc., New York, 2004. 135. Watts, M., Munday, B.L., and Burke, C.M., Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau, Fish Shellfish Immun., 11, 491–503, 2001. 136. Mendel, B., Kemp, A., and Myers, O.K., A colorimetric micro-method for the determination of glucose, Biochem. J., 56, 639–646, 1954. 137. Schultz, M., Liese, A.D., Mayer-Davis, E.J., D’Agostino, R.B., Fang, F., Sparks, K.C., and Wolever, T.M., Nutritional correlates of dietary glycaemic index: New aspects from a population perspective, Br. J. Nutr., 94, 397–406, 2005. 138. Kimura, S., Studies on marine invertebrate collagens. V. The neutral sugar composition and glucosylated hydroxylysine contents of several collagens, Bull. Jpn. Soc. Sci. Fish., 38, 1153, 1972. 139. Ansell, A.D., Storage and utilization of reserves in Pectinid bivalves with particular reference to the adductor muscle. Proceedings of the Scallop Workshop, Brest, France 8–13 May, 17pp, 1978. 140. Epp, J., Bricelj, V.M., and Malouf. R.E., Seasonal partitioning and utilization of energy reserves in two age classes of the bay scallop Argopecten irradians (Lamark), J. Exp. Mar. Biol. Ecol., 121, 113–136, 1988. 141. Zwann, A. de. and Zandee, D.I., Body distribution and seasonal changes in the glycogen content of the common sea mussel Mytilus edulis, Comp. Biochem. Physiol. A: Physiology, 43(1), 53–58, 1972. 142. Dare, P.J. and Edwards, D.B., Seasonal changes in flesh weight and biochemical composition of mussels (Mytilus edulis L.) in the Conwy estuary, North Wales, J. Exp. Mar. Biol. Ecol., 18(2), 89–97, 1975. 143. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F., Colorimetric method for determination of sugars and related substances, Anal. Chem., 18, 350–356, 1956. 144. Maguire, J.A., Fleury, P.G., and Burnell, G.M., Some methods for quantifying quality in the scallop Pecten maximus (L.), J. Shellfish Res., 18(1), 59–66, 1999. 145. Gerhardt, P., Murray, R.G.E., Wood, W.A., and Krieg, N.R., Chemical analysis. In: Methods for General and Molecular Bacteriology, 2nd edn., Am. Soc. Microbiol., pp. 518–520, 1994. 146. Allen, V.J., Marsden, I.D., Ragg, N.L.C., and Gieseg, S., The effects of tactile stimulants on feeding, growth, behaviour, and meat quality of cultures Blackfoot abalone, Haliotis iris, Aquaculture, 257, 294–308, 2006. 147. Carroll, N.V., Longley, R.W., and Roe, J.H., The determination of glycogen in liver and muscle by use of anthrone reagent, Department of Biochemistry, School of Medicine, George Washington University, Washington, D.C., J. Biol. Chem., 583–593, 1955. 148. Templeton, M.C., Microdetermination of glycogen with anthrone reagent, Northwestern University, Dept. Medical Anatomy, Publn no. 658, 670–672, 1961. 149. Van Handel, E., Estimation of glycogen in small amounts of tissue, Anal. Biochem., 11, 256–265, 1965.

284 ◾ Handbook of Seafood and Seafood Products Analysis 150. Beltran-Lugo, A.I., Maeda-Martinez, A.N., Pacheco-Aguilar R., and Nolasco-Soria, H.G., Seasonal variations in chemical, physical, textural, and microstructural properties of adductor muscles of Pacific lions-paw scallop (Nodipecten subnodosus), Aquaculture, 258, 619–632, 2006. 151. Munro, J. and Burlingame, B., Carbohydrates and related food components: INFOODS tagnames, meanings, and uses, J. Food Comp. Anal., 9, 100–118, 1996. 152. Christie, R., Kent, M., and Lees, A., Microwave and infra-red drying versus conventional oven drying methods for moisture determination in fish flesh, Int. J. Food Sci. Technol., 20(2), 117–127, 1985. 153. Aursand, I.G., Gallart-Jornet, L., Axelson, D., Erikson, U., and Rustad, T., Water distribution in brine salted cod (Gadus morhua) and salmon (Salmo salar): A low field NMR study, J. Agric. Food Chem., 56, 6252–6260, 2008. 154. Soriguer, F., Serna, S., Valverde, E., Hernando, J., Martin-Reyes, A., Soriguer, M., Pareja, A., Tinahones, F., Esteva, I., Lipid, protein, and calorie content of different Atlantic and Mediterranean fish, shellfish, and molluscs commonly eaten in the south of Spain, Eur. J. Epidemiology, 13, 451–463, 1997. 155. Miller, D.S. and Payne, P.R., A ballistic bomb calorimeter, Br. J. Nutr., 13, 501–508, 1959. 156. Brown J.C., Faulks, R.M., and Livesey, G., Developing an international food energy system, Food Technology International (Europe), 29–33, 1993. 157. Craig, J.F., Kenley, M.J., and Talling, J.F., Comparative estimations of the energy content of fish tissue from bomb calorimetry, wet oxidation and proximate analysis, Freshwater Biol., 8, 585–590, 1978. 158. Henken, A.M., Lucas, H., Tijssen, P.A.T., and Machiels, M.A.M., A comparison between methods used to determine the energy content of feed, fish and faeces samples, Aquaculture, 58, 195–201, 1986. 159. Charrondiere, U.R., Chevassus-Agnes, S., Marroni, S., Burlingame, B., Impact of different macronutrient definitions and energy conversion factors on energy supply estimations, J. Food. Comp. Anal., 17, 339–360, 2004. 160. FAO, Food energy-methods of analysis and conversion factors. Report of a technical workshop, Food and nutritional paper 77. Rome. http://www.fao.org/es/ESN/nutrition/requirements_pub_en.stm. 2003. 161. Atwater, W.O. and Woods, C.D., The chemical composition of American food materials. United States Department of Agriculture Office of Experiment Stations, Bulletin 28. Washington, DC, Government Printing Office, 1896. 162. Southgate, D.A.T. and Durnin, J.V.G.A., Calorie conversion factors. An experimental reassessment of the factors used in the calculation of the energy value of human diets, Br. J. Nutr., 24, 517–535, 1970. 163. Merrill, A.L. and Watt, B.K., Energy value of foods, basis and derivation, Agriculture Handbook No.74. U.S. Department of Agriculture, Washington DC, 1973. 164. FAO/WHO, Carbohydrates in human nutrition, Report of a joint FAO/WHO expert consultation, Rome, 1997. FAO Food and Nutrition Paper 66. Rome. 1998. 165. Merrill, A.L. and Watt, B.K., Energy value of foods, basis and derivation, Agriculture Handbook. No. 74. United States Department of Agriculture, Washington, DC, 1955. 166. Jones, D.B., Factors for converting percentages of nitrogen in foods and feeds into percentages of proteins, United States Department of Agriculture Circular No. 183, Washington, DC, 1931, updated in 1941. 167. Broody, S., Bioenergetics and Growth, Reinhold Publishing Corporation, New York, 1945. 168. Exler, J., Kinsella, J.E., and Watt, B.K., Lipids and fatty acids of important finfish. New data for nutrient tables, J. Am. Oil Chem. Soc., 52, 154–159, 1975. 169. Krishnamoorthy, R.V., Venkataramiah, A., Lakshmi, G.J., and Biesiot, P., Caloric densities of shellfish meat and fats, J. Agric. Food. Chem., 27(5), 1125–1127, 1979. 170. Newell, R.I.E., An evaluation of the wet oxidation technique for use in determining the energy content of seston samples. Can. J.Fish. Aquat. Sci., 39, 1383–1388, 1982.


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171. Schakel, S.F., Maintaining a nutrient database in a changing marketplace: Keeping pace with changing food products–A research perspective, J. Food Comp. Anal., 14, 315–322, 2001. 172. Freese, M. and Hamid, M.A.K., Lipid content determination in whole fish using ultrasonic pulse backscatter. In: Ultrasonic Symposium Conference, 1974, Ed., De Klerk, J., Publisher IEEE, 69–76. 173. Lantry, B.F., Stewart, D.J., Rand, P.S., and Mills, E.L., Evaluation of total body electrical conductivity to estimate whole body water content of yellow perch (Perca flavescens) and alewife (Alosa pseudoharengu), Fish. Bull., 97, 71–79, 1999. 174. Novinger, D. C. and Martinez Del Rio, C., Failure of total body electrical conductivity to predict lipid content of brook trout, North Am. J. Fish. Manag., 19(4), 3, 1999. 175. Hancz, C., Milisits, G., and Horn, P., In vivo measurement of total body lipid content of common carp (Cyprinus carpio L.) by electrical conductivity, Arch. Tierzucht–Archives of Animal Breeding 46, 397–402, 2003. 176. Turgut, H., Drawbacks in the use of the Biuret method for determination of the same protein in differently treated fish samples, Food Chem., 4, 161–165, 1979. 177. Shono, N.I. and Baskaeva, E.M., Bradford’s method of determining protein: Application, advantages and disadvantages, Lab Delo., 4, 4–7, 1989. 178. Southgate, D.A.T., Availability of and needs for reliable analytical methods for the assay of foods, Food and Nutr. Bull., 5(2), 30–39, 1983. 179. Molnar-Perl, I. Derivatisation and chromatographic behaviour of the o-phthalaldehyde amino acid derivatives obtained with various SH-group containing additives, J. Chromatogr. A, 913, 283–302, 2001. 180. Jørgensen, B.M. and Jensen, K.N., Water distribution and mobility in fish products in relation to quality. In: Modern Magnetic Resonance, Part 1: Applications in Chemistry, Biological and Marine Sciences, Webb, Ed., G.A., 905–908, 2006, Springer, Dordercht, the Netherlands.

Chapter 17

Essential Amino Acids M. Concepción Aristoy and Fidel Toldrá Contents 17.1 Introduction ................................................................................................................. 287 17.2 Sample Preparation for the Analysis of Seafood Essential Amino Acids ....................... 288 17.2.1 Sample Preparation for Free Essential Amino Acid Analysis ........................... 288 17.2.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis................................................................................................... 289 17.3 Seafood Essential Amino Acid Analysis........................................................................ 290 17.3.1 High-Performance Liquid Chromatographic Methods.....................................291 17.3.1.1 Cation Exchange Chromatography ..................................................291 17.3.1.2 Reversed-Phase High-Performance Liquid Chromatography .......... 292 17.3.2 Gas Liquid Chromatographic Methods ........................................................... 298 17.3.3 Capillary Zone Electrophoretic Methods ........................................................ 298 17.3.4 Mass Spectrometry .......................................................................................... 299 17.4 Conclusions .................................................................................................................. 300 References ............................................................................................................................... 300

17.1 Introduction Amino acids are the basic components of the muscle protein structure of seafood. However, not all proteins have the same nutritional value, because protein quality strongly depends on its amino acid composition and digestibility.1 Fish and, in general, seafood proteins are considered as highquality proteins because of their balanced content in amino acids, especially in all the essential amino acids necessary for physical and mental well-being. Amino acids may also be found in free form, which contribute to fish taste and indirectly to aroma 2,3 by generation of volatile 287

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compounds through Maillard reactions and Strecker degradations.4 Branched-chain essential amino acids (valine, isoleucine, and leucine), sulfur-containing amino acids (methionine and cystine/cysteine), and aromatic amino acids (phenylalanine and tyrosine) are the most important from this point of view. Free amino acids initiate important changes at early postmortem and during storage and can be very useful as quality indices of processing and storage.5–10 Thus, the analysis of essential amino acids in seafood is important for the evaluation of both the nutritive value and the sensory quality of seafood. In this chapter, methods for the analysis of amino acids in seafood, especially of those considered essentials, are described. Special attention is also devoted to the analysis of the sulfur amino acid cysteine for several reasons: (1) the high reactivity of its thiol group, which confers numerous biological functions to this amino acid (precursor to the antioxidant glutathione), (2) its ability to cross-link proteins, which increases the protein stability in the harsh extracellular environment by conferring proteolytic resistance, and so forth, or (3) its Maillard reaction with sugars yielding characteristic flavors. Although classified as nonessential, in rare cases, cysteine may be essential for infants, the elderly, and individuals with certain metabolic disease or who suffer from malabsorption syndromes. A more detailed description of amino acid methods of analysis may be found in the work of Aristoy and Toldrá.11

17.2

Sample Preparation for the Analysis of Seafood Essential Amino Acids

Free or total essential amino acids are analyzed from the whole amino acid profile. Sample preparation will depend on whether free or total essential amino acids have to be analyzed.

17.2.1 Sample Preparation for Free Essential Amino Acid Analysis Sample preparation for free essential amino acids includes their extraction and the cleanup or deproteinization of the extract. The extraction consists in the separation of the free amino acid fraction from the insoluble portion of the matrix (fish muscle). It is usually achieved by homogenization of the ground sample in an appropriate solvent by using a Stomacher, Polytron, or by means of a simple stirring in warm solvent. The extraction solvent can be hot water, 0.01–0.1 N hydrochloric acid solution, or diluted phosphate buffers. In some cases, concentrated strong acid solutions such as 4% of 5-sulfosalicylic acid,12,13 5% of trichloroacetic acid,14 6% of perchloric acid,15 or a rich alcohol-containing solution (>75%) such as ethanol16–18 or methanol19,20 have been successfully used as extraction solvents, with the additional advantage that proteins are not extracted and, then, there is no need for further cleaning up of the sample. Once homogenized, the sample is centrifuged at more than 10,000 g under refrigeration (4°C) to separate the supernatant from the nonextracted materials (pellet) and filtered through glass wool to retain any fat material remaining on the surface of the supernatant. Sample cleanup is necessary to eliminate proteins and polypeptides by means of the deproteinization process, which can be achieved through different chemical or physical procedures. Several chemical methods include the use of concentrated strong acids such as phosphotungstic (PTA), sulfosalicylic (SSA),13,18,21 perchloric (PCA),16,22 trichloroacetic (TCA),23–25 and picric

Essential Amino Acids

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(PA)26–28 acids or organic solvents such as methanol, ethanol, or acetonitrile.22 Under these conditions, proteins precipitate by denaturation, whereas free amino acids remain in solution. Some physical methods consist in centrifugation through cutoff membrane filters (1,000, 5,000, 10,000, 30,000 Da) that allow free amino acids through while retaining large compounds.18,29,30 All these methods give a sample solution rich in free amino acids but free of proteins. Differences among all these chemical and physical methods are caused by several aspects such as differences in the cutoff molecular weight, recovery of amino acids, compatibility with derivatization (pH, presence of salts, etc.), or separation method (interferences in the chromatogram, etc.), and so forth. A good choice may be the use of 0.6 N PCA, which is easily neutralized by the addition of KOH or potassium bicarbonate, to rend insoluble potassium perchlorate, which is easily separated by centrifugation, resulting in a very simple deproteinization procedure with no interferences. The use of organic solvents, by mixing two or three volumes of organic solvent with one volume of extract, has also given very good results,31–33 with amino acid recoveries around 100% for all them. An additional advantage is the easy evaporation to concentrate the sample. Some comparative studies have been published on these deproteinization techniques.22,34

17.2.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis The total essential amino acid profile is usually requested, because it gives information on the nutritional value of fish meat. Proteins must be hydrolyzed into their constituent amino acids before the analysis. The most common method used for complete hydrolysis of proteins is acid digestion. Typically, samples are treated with constant boiling 6 N hydrochloric acid in an oven at around 110°C for 20–96 h. Digestion at 145°C for 4 h has also been proposed.35–38 These temperatures in such acidic and oxidative medium may degrade some amino acids. Nitrogen atmosphere and sealed vials are required during the hydrolysis to minimize the degradation. The hydrolysis may be accomplished using either liquid-phase or vapor-phase methods. Liquid-phase, where the hydrochloric acid contacts the sample directly, is well suited to hydrolyze large amounts or complex samples. When limited amounts of sample are available, the vapor-phase hydrolysis method is preferred to minimize contaminants coming from aqueous 6 N hydrochloric acid. In the vapor-phase hydrolysis method, the tubes containing the samples are located inside large vessels containing the acid. Upon heating, only the acid vapor comes into contact with the sample, thus excluding nonvolatile contaminants. In both cases, liquid phase or vapor phase, oxygen is removed and substituted by nitrogen or other inert gas, creating an appropriate atmosphere inside the vessels to ensure low amino acid degradation. Therefore, a system capable of alternative air evacuating/inert gas purging to get a correct deaeration inside is valuable.39 Some commercial systems are available. One of them is the Pico-Tag Workstation that includes special vessels (flat-bottom glass tubes) fitted with a heat-resistant plastic screw cap equipped with a Teflon valve, which permits the alternative air evacuating/inert gas purging, also disposes of an oven to accomplish the hydrolysis.40,41 The use of microwave technology for the hydrolysis has been assayed by some authors.39,42 Sample manipulation (sample evaporation to dryness, addition of constant boiling hydrochloric acid and additives, and performance under vacuum) is similar to that of a conventional oven, but the duration of the treatment is shorter (less than 20 min). Hydrolysis may be improved by optimizing the temperature and time of incubation41 or with the addition of amino acid oxidation protective compounds. The presence of appropriate antioxidants/scavengers during hydrolysis can prevent losses of the most labile amino acids, all of them

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essential amino acids, such as tyrosine, serine, threonine, methionine, and tryptophan. Thus, protective agents currently used, up to 1% phenol or 0.1% sodium sulfite, improve the recovery of nearly all of these amino acids except tryptophan and cysteine. Tryptophan is often completely destroyed by hydrochloric acid hydrolysis, although considerable recoveries have been found if no oxygen is present. Some additives have been proposed to protect tryptophan against oxidation as is the case of thioglycolic acid.38 Alkaline hydrolysis instead of acid hydrolysis is also proposed (see below). Cyst(e)ine is partially oxidized during acid hydrolysis yielding several adducts: cystine, cysteine, cysteine sulfinic acid, and cysteic acid making its analysis rather difficult. The previous performic acid oxidation of cysteine to cysteic acid, in which methionine is also oxidized to methionine sulfone,36,43–50 improves cysteine (and methionine) recoveries, making the posterior analysis easier. The use of alkylating agents to stabilize the previous hydrolysis of cysteine constitutes a valid alternative. Good recoveries have been achieved by using 3-bromopropionic acid,51 3-bromopropylamine,52 4-vinyl pyridine53,54 or 3,3¢-dithiodipropionic acid.30,55,56 As can be observed in this section, no single set of conditions will yield the accurate determination of all essential amino acids. In fact, a compromise of conditions offers the best overall estimation for the largest number of amino acids. In general, the 22–24 h acid hydrolysis at 110°C (vapor-phase or liquid-phase hydrolysis) with the addition of a protective agent like 1% phenol, yields acceptable results for the majority of amino acids, being enough for the requirements of any food industry. Additionally, when the analysis of cyst(e)ine would be necessary, adequate hydrolysis procedure as the performic acid oxidation before the hydrolysis is a good alternative. When high sensitivity is required, the pyrolysis from 500°C for 3 h57 to 600°C overnight58 of all glass material in contact with the sample is advisable as well as the analysis of some blank samples to control the level of background present. The optimization of conditions for hydrolysis based on the study of hydrolysis time and temperature, acid-to-protein ratio, presence and concentration of oxidation protective agents, importance of a correct deaeration, and so on, has been extensively reported in papers35,36, 41,59,60 and books.61,62 An alternative to acid hydrolysis is the alkaline hydrolysis with 4.2 M of either NaOH, KOH, LiOH, or BaOH, with or without the addition of 1% (w/v) thiodiglycol for 18 h at 110°C, which is recommended by many authors47,58,63–66 for a better tryptophan determination. A third way to hydrolyze proteins is enzymatic hydrolysis by proteolytic enzymes such as trypsin, chymotrypsin, carboxypeptidase, papain, thermolysin, or pronase. This option is chosen to analyze specific amino acid sequences or single amino acids because of their specific and well-defined activity.67–69

17.3

Seafood Essential Amino Acid Analysis

The analysis of individual amino acids needs a previous separation of all others, unless a very selective way of detection is used. The separation of the individual amino acids in a mixture requires very efficient separation, such as chromatographic (liquid or gas chromatography (GC)) or capillary electrophoresis (CE) techniques. The choice mainly depends on the equipment available or personal preferences, because each possible methodology has advantages and drawbacks. Before or after this separation, amino acids used to be derivatized to allow their separation or to enhance their detection. Derivatization is a usual practice in amino acid analysis. The effect of a derivatizing agent is evaluated based on the following aspects: (1) It must be able to react with both primary and secondary amino acids, (2) give a quantitative and reproducible reaction,


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(3) yield a single derivative of each amino acid, (4) have mild and simple reaction conditions, (5) have the possibility of automation, (6) have good stability of the derivatization products, and (7) have no interferences due to by-products or excess of reagent. It must be remarked that the use of sufficient amount of reagent is of special importance when dealing with biological samples, because reagent-consuming amines, although unidentified, are always present.12 Two types of derivatives are obtained depending on the chosen separation and/or detection technique. The first type are derivatives that enhance amino acid detection in liquid media, and they include derivatives for spectroscopic or for electrochemical detection. The formed derivatives will be separated by high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) as it is important to choose the most adequate derivative, because their spectral (high-ultraviolet (UV) absorbing or fluorescence properties) or electrochemical characteristics will affect the sensitivity and selectivity of detection. The second type are derivatives that allow gas chromatographic amino acid separation by increasing their volatility and temperature stability.

17.3.1

High-Performance Liquid Chromatographic Methods

HPLC is the preferred technique to analyze amino acids. Amino acids, in their native form, absorb at 210 nm and thus cannot be used for spectroscopic detection, as it is a very unspecific detection wavelength. Only three amino acids (phenylalanine, tyrosine, and tryptophan) have a chromophore moiety that confers a suitable maximum absorbance for more specific UV detection (280 nm for tyrosine and tryptophan and 254 nm for phenylalanine). Tryptophan also possesses native fluorescence (l ex = 295 nm, l em = 345 nm), which facilitates a more selective detection.68 Thus, the spectroscopic detection of amino acids requires their previous derivatization to obtain an UV absorbing or fluorescent molecule. The derivatization reaction can be performed after separation of the amino acids (postcolumn derivatization) or before separating them (precolumn derivatization). Although postcolumn techniques should be run online for maximum accuracy, precolumn techniques can be run either offline or online. The HPLC techniques to analyze amino acids are cation exchange and reversed-phase (RP) chromatography and are described in Sections 17.3.1.1 and 17.3.1.2.

17.3.1.1 Cation Exchange Chromatography This methodology is based on the amino acid charge, and thus the underivatized amino acids are separated using sulfonated polystyrene beads as the stationary phase and aqueous sodium citrate buffers as the mobile phase. The elution involves a stepwise increase in both pH and sodium or lithium ion concentration. Under these conditions, the more acidic amino acids elute first, and those with more than one primary amino group or possessing a guanidyl residue elute at the end of the chromatogram. The original method required two separate columns and needed about 4 h to achieve a complete analysis. After separation, amino acids were converted into colored ninhydrin derivatives for spectrophotometric (colorimetric) detection. The classical procedure has been improved with a new polystyrene matrix that offers better resolution power due to smaller particle size, speed, pellicular packaging, and better detection systems. The latest generation of Moore and Stein amino acid analyzers also use o-phthaldialdehyde (OPA),15,38,70 fluorescamine, or 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole postcolumn derivatization to obtain highly fluorescent derivatives with enhanced sensitivity, permitting 5–10 pmol sensitivity as standard. Nevertheless, recent improvements of the ninhydrin derivatization method71–73

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together with the low sensitivity requirements of fish amino acid analysis still make this method the most used.42,74 Nowadays, the separation times for the 20 amino acids naturally occurring in fish proteins take around 1 h and somewhat longer (2 h) for physiological amino acids.75,76 There are other reports of applying this technique to the amino acid analysis in food and tissues. 66,70,77 After separation, the derivatizing reagent is pumped into the effluent from the column system, through a mixing manifold, followed by a reaction coil, and finally the derivatized amino acids reach an online detector system. This method has been employed in the classical Moore and Steintype commercial amino acid analyzers. Obviously, the main drawback of this type of derivatization method is the required additional equipment: another pump to introduce the reagent as well as mixing and sometimes heating devices. Another disadvantage is the peak broadening produced by the dead volume introduced behind the column. Although this broadening may not affect when using standard-bore columns with flow rates above 1 mL/min, postcolumn derivatization is not suitable for narrow-bore HPLC. There are many manufacturers (Beckman, Amersham Biosciences, Biotronik, Dionex, Hitachi, LKB, Pickering, Kontron, etc.) who offer integrated commercial systems including the column, buffer system, and an optimized methodology with the advantage of ease of use and reliability. The advantage of this method is the accurate results for all known sample types (food, tissues, biological fluids, feed, plants), which makes it a reference method for amino acid analysis. In this way, each new methodology must contrast its results with those obtained by cation exchange chromatography (CEC). The main drawbacks of this methodology are the high cost of the ion exchange amino acid analyzer and its maintenance, the highly complex mobile phase composition, and the long time of analysis.

17.3.1.2 Reversed-Phase High-Performance Liquid Chromatography RP-HPLC has been widely used, because it requires only a standard equipment that can be shared by different types of analysis. This fact and the proliferation of precolumn derivatizing agents have stimulated the development of RP-HPLC methods to analyze amino acids in all kind of matrices (food, plants, biological fluids, and tissues). Precolumn amino acid derivatization may be necessary to confer hydrophobicity to the amino acid molecule, making it adequate for partition based on chromatography, but, also, the formed molecule improves sensitivity and selectivity at the detection by allowing the spectroscopic (UV or fluorescent) detection of amino acids. The resulting system is simpler and cheaper compared with the combination of cation-exchange plus postcolumn derivatization and permits choosing among a great number of possible methodologies, with which many of them have been marketed. To choose the most appropriate method some aspects must be taken into account such as the following: the disposable detector (fluorescence or UV), possibility of automation of the derivatization reaction (in the autosampler), the analysis requirements for free or hydrolyzed amino acids or required sensibility, time for sample preparation and amino acids separation, or the stability of formed derivatives, (i.e., some difficulties to analyze some essential or sulfur-containing amino acid derivatives). The most usual derivatizing agents for tissue amino acids are described below. Phenylisothiocyanate (PITC): This methodology involves the conversion of primary and secondary amino acids to their phenylthiocarbamyl (PTC) derivatives, which are detectable at UV (254 nm) with detection limits around 5–50 pmol. All PTC-amino acids have similar response factors, which constitutes an advantage. The PTC-amino acids are moderately stable at room temperature for 1 day and much longer when kept under frozen storage, especially in a dry condition.


◾ 293

The methodology is well described in the literature.29,78–80 Sample preparation is quite tedious: it requires a basic medium (pH = 10.5), which is achieved by the addition of triethylamine and includes several drying steps, the last one being the elimination of the excess of reagent that may cause some damage to the chromatographic column. It is important to ensure a basic pH to get adequate derivatization recoveries, which is more critical when amino acids from acid hydrolysis are analyzed, because no buffer is used during the reaction. The reaction time is less than 10 min even though 20 min are recommended for a complete reaction.29,78–80 The chromatographic separation takes around 20 min for hydrolyzed amino acids and 60 min for physiological. Both examples applied to the analysis of total amino acids from hake and free amino acids from salmon are shown in Figures 17.1 and 17.2, respectively. Sarwar et al.81 reported a modification of the method in which the analysis of 27 physiological amino acids could be performed in 22 min (30 min including equilibration). The only limitation is the determination of PTC cystine that gives a poor linearity, which makes the quantitation of free cystine nonfeasible with this method.82 The selection of the column is critical to get a good resolved separation especially when the analysis of physiological amino acids is involved. Moreover, the residual PITC reagent left after evaporation will cause damage to the column package, as some columns are more resistant than others. This method is available as a commercially prepackaged system named Pico-Tag (Waters Associates, Milford, Massachusetts), which includes the analytical column, standards, and solvents. 700 Ala 600 Gly Absorbance at 254 nm (mAU)

500 Glu 400

lS Lys

300 Asp

Arg

Leu

200 Ser

Thr

Pro

100 OHpro

Tyr

His

Val

Met lle

Phe

0 0

2

4

6

8

10

12

14

16

18


Figure 17.1 Reversed-phase HPLC chromatogram of PTC amino acids from hydrolyzed hake muscle. IS, internal standard nor-Leucine.

294

◾ Handbook of Seafood and Seafood Products Analysis 1400

1200

Glu


Ans 1000

800

Gly

600

Tau βAla His

400 Asp 200

Ala

Lys

Pro

OHpro

Tyr Arg

0

lS

Val

Thr

Ser

Met

Leu lle

Trp Phe Orn

Gln 0

Asn

10

20

30

40

50


Figure 17.2 Reversed-phase HPLC chromatogram of PITC-free amino acids from salmon muscle extract. Tau, taurine; Ans, anserine; IS, internal standard nor-Leucine.

4-Dimethyl-aminoazobenzene-4′-sulfonyl chloride (Dabsyl-Cl): This reagent was first described in 1975 for use in amino acid analysis.83,84 Detection is by absorption in the visible range, presenting a maximum from 448 to 468 nm. The high wavelength of absorption makes the baseline chromatogram very stable with a large variety of solvents and gradient systems. Detection limits are in the low picomole range.58,84 Derivatives are very stable (weeks) and can be formed from both primary and secondary amino acids. The reaction time is around 15 min at 70°C and takes place in a basic medium with an excess of reagent. Reaction efficiency is highly matrix dependent and variable for different amino acids, because it is especially affected by the presence of high levels of some chloride salts.33 To overcome this problem and obtain an accurate calibration, standard amino acid solution should be derivatized under similar conditions. By-products originating from an excess of reagent absorb at the same wavelength and thus they appear in the chromatogram. Nevertheless, Stocchi et al.58 obtained a good separation of 35 dabsyl-amino acids and by-products in a 15 cm C18 column packed with 3 mm particle size. Commercial System Gold/Dabsylation Kit™ uses this technique (Beckman Instruments, Palo Alto California, United States). 1-Dimethylamino-naphthalene-5-sulfonyl chloride (Dansyl-Cl): Dansyl-Cl reacts with both primary and secondary amines to give a highly fluorescent derivative (l ex = 350, l em = 510 nm) although UV (l = 250 nm) detection may also be used. The dansylated amino acids are stable for 1 day85 or until 7 days when kept at −4°C86 and protected from light. The sample derivatization is rather simple, and only needs a basic pH, around 9.5, and a reaction time of 1 h at room temperature (in the dark), 15 min at 60°C,87 or even 2 min at 100°C. However, the reaction conditions


◾ 295

(pH, temperature, and excess of reagent) must be carefully fi xed to optimize the product yield and to minimize secondary reactions.86,88 Even so, this will commonly form multiple derivatives with histidine, lysine, and tyrosine. Histidine gives a very poor fluorescence response (10% of the other amino acids), reinforcing the poor reproducibility of its results.82 Another problem is the large excess of reagent needed to assure a quantitative reaction. This excess is hydrolyzed to dansyl sulfonic acid, which is present in excess as it is highly fluorescent and probably interferes into the chromatogram as a huge peak. On the contrary, this methodology reveals excellent linearity for cystine and also cystine-containing short-chain peptides.82,89 9-Fluorenylmethyl chloroformate (FMOC): This reagent yields stable derivatives (days) with primary and secondary amines. The derivative is fluorescent (l ex = 265 nm; l em = 315 nm) and is detected at the femtomole range. The major disadvantage is due to the reagent, itself or hydrolyzed, as it is highly fluorescent and then, the excess may interfere in the chromatogram and for this reason it must be extracted (with pentane or diethyl ether) or converted into noninterfering adduct before injection. The first option was included in the automated AminoTag method90 developed by Varian (Varian Associates Limited). In the second option, the reaction of the excess of reagent with a very hydrophobic amine as 1-adamantylamine (ADAM) gives a late-eluting noninterfering peak.91 This method is preferred because the addition of ADAM is more easily automatized. The reaction time is fast (45–90 s) and does not require any heating. In order to obtain reliable and precise results, reaction conditions, such as FMOC/amino acid ratio, as well as reaction time, have to be optimized very carefully. An automated precolumn derivatization routine, which includes the addition of ADAM, is of great advantage, because it guarantees the repeatability of parameters. Tryptophan adducts do not fluoresce and histidine and cyst(e)ine adducts fluoresce weakly. o-Phthaldialdehyde (OPA): This reagent reacts with primary amino acids in the presence of a mercaptan cofactor to give highly fluorescent 1-alkylthio-2-alkyl-substituted isoindols.92,93 The fluorescence is recorded at 455 or 470 nm after excitation at 230 or 330 nm, and the reagent itself is not fluorescent. OPA derivatives can be detected by UV absorption (338 nm) as well. The choice of the mercaptan can affect derivative stability, chromatographic selectivity, and fluorescent intensity;94–96 2-mercaptoethanol, ethanethiol, and 3-mercaptopropionic acid are the most frequently used. The derivatization is fast (1–3 min) and is performed at room temperature in alkaline (pH 9.5) medium. OPA amino acids are not stable; this problem is overcome by standardizing the time between sample derivatization and column injection by automation. This is relatively easy because the reaction is fast and no heating is necessary. Nowadays, many automatic injectors are programmable and able to achieve automatic derivatizations. Some reports have been published proposing several ways of automation,97,98 and some of them have been patented and commercially marketed (AutoTag OPA from Waters Associates). One of the main disadvantages of this procedure is the inability of OPA to react with secondary amines, which is not the case with any essential amino acid. The yield with lysine and cysteine is low and variable. The addition of detergents like Brij 35 to the derivatization reagent seems to increase the fluorescence response of lysine.99 In the case of cysteine, several methods have been proposed before derivatization. These methods include the conversion of cysteine and cystine to cysteic acid by oxidation with performic acid or carboxymethylation of the sulfhydryl residues with iodoacetic100,101 or the formation of the mixed disulfide S-2-carboxyethylthiocysteine (Cys-MPA) from cysteine and cystine, using 3,3′-dithiodipropionic acid55 and incorporated by Godel et al.94 into the automatic sample preparation protocol described by Schuster.32 In these methods, cysteine and cystine are quantified together. Another proposal102 consists of a slight modification in the OPA derivatization method by using 2-aminoethanol as a nucleophilic agent and altering the order of the addition of reagents in the automated derivatization procedure.32

296

◾


6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC): It reacts with primary and secondary amines from amino acids, peptides, and proteins, yielding very stable derivatives (1 week at room temperature) with fluorescent properties (l ex = 250 nm; l em = 395 nm), which are separated by RP-HPLC. UV detection (254 nm) may also be used. Sensitivity is in the femtomole range, making them very adequate for biochemical research.103 The main advantage of this reagent is that the yield and reproducibility of the derivatization reaction are scarcely interrupted by the presence of salts, detergents, lipids, and other compounds naturally occurring in biological samples and foods. Furthermore, the optimum pH for the reaction is in a broad range, from 8.2 to 10. Both facts facilitate sample preparation. The excess of reagent is consumed during the reaction to form aminoquinoline (AMQ), which is only weakly fluorescent at the amino acid derivatives detection conditions and does not interfere in the chromatogram. Reaction time is short, 1 min, but 10 min at 55°C would be necessary if a tyrosine monoderivative is required, because both mono- and di-derivatives are the initial adducts from tyrosine. The fluorescence of tryptophan derivative is very poor, and UV detection at 254 nm may be used for its analysis. In this case, the AMQ peak is very large at the beginning of the chromatogram and may interfere with the first eluting peaks (see Bosch et al.50). The chromatographic separation of these derivatives has been optimized for the amino acids from hydrolyzed proteins, and the separation of physiological amino acids is improved. Cystine and cysteine may be analyzed after their conversion to cysteic acid (CisH) by performic acid oxidation, because the resulting CisH is well separated inside the chromatogram. Figure 17.3A shows the separation of hydrolyzed amino acids from salmon, whereas Figure 17.3B shows the same sample but submitted to a performic acid oxidation before the hydrolysis in which the CisH peak appears by 7.5 min. The methodology has been marketed as a prepackaged AccQ Tag kit (Waters Corporation, Milford, Massachusetts, United States). Some of these derivatives are also susceptible to electrochemical detection, because they are molecules with electroactive functional groups. Indeed, OPA/mercaptoethanol or OPA/sulfite104,105 in addition to fluorescent properties possesses electroactivity (750 mV) and PITC106 has again the advantage of reacting with secondary amines. Electrochemical detection consists in one electrode or an array of electrodes mounted in a cell with an applied potential difference. Any electrical measure, such as current, potential, conductance, or charge, is related to the analyte concentration. Only amino acids with aromatic rings or sulfur-containing side chains are sufficiently electrochemically active to be detected by this method.107,108 If the choice of the derivative reaction is a challenge, the choice of the RP column is not an easy subject because of the great variability of commercially available RP columns. The most used column packaging consists of alkyl-bonded silica particles, mainly octadecylsilane. However, the selectivity obtained with each trademark column is different due to the particular chemistry employed in their manufacture rendering different density of bonded-phase coverage on the silica particle and hydrophobic behavior and, as a consequence, different selectivity. The presence of residual uncapped silanol groups on the silica surface, accessible to sample molecules, can cause unwanted tailing of peaks (especially for the basic amino acids). In these cases, the addition of a strong cation (i.e., triethylamine) to the mobile phase can overcome the problem. Nowadays, columns are more carefully manufactured with these silanol groups blocked or inaccessible by steric impediment avoiding the tailing. Due to these variables, different selectivity may be found among same columns, even those made by the same manufacturer. Only columns manufactured in the same batch are guaranteed to give the same selectivity if the rest of parameters are fi xed. It means that when transferring a published method to a particular set of samples, it will be necessary to readjust the chromatographic conditions to get a good separation of all amino acids.

Essential Amino Acids 200

◾

297

Leu

(A)

150 Val

lle Phe

αAba

100

Lys

Ala Glu

NH3 Arg Asp Thr Gly Ser His

Fluorescence (% FS)

50

Met Tyr Pro

0 1MeHis

βAla

200 (B)

150

100

MeS 50

CysH 0

0

5

10

15

20

25

30

35


Figure 17.3 Reversed-phase HPLC chromatogram of AQC amino acids from hydrolyzed salmon muscle (A) without and (B) after performic acid oxidation. 1MeHis, 1-methylhistidine; aAba, a aminobutyric acid used as internal standard; CysH, cysteic acid; MeS, methiomine sulfone.

Typical analytical column dimensions are 15 cm (for hydrolyzed amino acids) or 25–30 cm (for physiological amino acids), packed with 5 mm particle size or shorter columns (10 or 15 cm length) when packed with less than 3 mm particle size. Mobile-phase requirements consist in the ability to dissolve the sample while keeping it transparent to the detection system. Mobile-phase composition combines an aqueous buffered phase

298 ◾


with an organic phase constituted by acetonitrile and/or methanol and/or tetrahydrofuran. The buffer may be constituted by less than 100 mM concentration of acetate or phosphate. A finely adjusted binary (most used) or ternary gradient elution is often necessary when the overall amino acid profile from hydrolyzed and, especially, physiological amino acids has to be analyzed.

17.3.2

Gas Liquid Chromatographic Methods

The extremely high-resolution capacity is the main advantage of GC, in comparison with liquid chromatographic techniques, especially since the capillary columns appeared. Gas liquid chromatography (GLC) is not often used for the determination of amino acids from tissues or foods. Some applications67,109,110 comparing GLC with cation exchange chromatography reported different conclusions when analyzing some hydrolyzed food samples. Nevertheless, the technique is very efficient and it is worth mentioning the separation of 32 nonprotein amino acids from edible seeds, nuts, and beans111 or other results obtained in honey,112 milk,113 and cheese.114 In their analysis by GLC, the amino acids must be converted to volatile and thermostable molecules. Reactions consist of two stages: an esterification with an acidified alcohol followed by N-acylation with an acid anhydride in an anhydrous medium. The detector used is the flame ionization detector (FID), which is universal and the most widely used, whereas thermionic-N-P (NPD) or flame photometric detector (FPD) are selective toward organic compounds containing phosphorous and nitrogen, which are much more sensitive than FID for such compounds. The NPD was used by Buser and Erbersdobler115 and FPD by Kataoka et al.116 to analyze phosphoserine, phosphothreonine, and phosphotyrosine. The main advantages of these detectors are their high sensitivity and wide linear range. In many cases, GLC has been combined with mass spectrometry (MS) for detection and identification, especially in the analysis of D isomers,113,117–119 where the separation was achieved by using chiral-GC stationary phases. Recently, a very fast GC analysis of physiological amino acids, capable of separating 50 compounds, including amino acids, dipeptides, and amines, has been developed. This methodology has been patented as EZ:faast and commercialized by Phenomenex (Torrance, California, United States). The method yields a full amino acid profile (33 amino acids) in 15 min including a 7 min extraction-derivatization step plus 8 min for the gas chromatographic separation. Protein removal is not required, and the derivatives are stable and ready for GC/FID, GC/NPD, or GC and LC with MS detection. Described applications are available for the analysis of physiological amino acids in blood, plasma, and urine matrices but not in tissues in which the presence of natural dipeptides, anserine, carnosine, and balenine may complicate the amino acid analysis. GLC is, in summary, a very highly efficient technique adequate for the amino acid analysis, although applications on meat samples are scarcely described. GLC is not very expensive because no solvent is used, and the equipment is very versatile and usually available in any analytical laboratory.

17.3.3

Capillary Zone Electrophoretic Methods

Capillary zone electrophoretic technique is extremely efficient for the separation of charged solutes.120,121 The high efficiency, speed, and low amount of sample make this technique very interesting when compared with classical electrophoresis and chromatographic techniques. The difficulty of separating amino acids by this technique relies on their structure. Amino acids constitute a mix of basic, neutral, and acidic constituents, and even though a particular pH can significantly


◾ 299

improve the resolution of one kind, it is likely to cause overlap with the others. Under the conditions of electro-osmotic flow in CE, the species with different charge can be simultaneously analyzed but with serious doubts in their adequate resolution. CZE shows poor ability for the separation of neutral compounds, which constitutes an important limitation of this technique. Terabe et al.122,123 introduced a modified version of CZE in which surfactant-formed micelles were included in the running buffer to provide a two-phase chromatographic system for separating neutral compounds together with charged ones in a CE system. This technique has also been termed micellar electrokinetic capillary chromatography (MECC or MEKC).124 Basic theoretical considerations on this technique125 and its food applications126 are described elsewhere. With few exceptions,127–131 derivatization is used to improve separation, to enhance UV detection, or to allow fluorescence or electrochemical132 detection of amino acids. Good separations have been reported for precapillary derivatized amino acids with dansyl-Cl,125,133–135 PITC,136 phenylthiohydantoin,137,138 and OPA139 compared with the separation of OPA-amino acid derivatives by CZE with normal and micellar solutions, showing that higher efficiency is obtained by the MECC methods with sodium dodecyl sulfate (SDS) as micelle-forming substance. SDS is indeed the most used additive to form micelles in this kind of analysis, although other additives such as dodecyltrimethylammonium bromide,138 Tween 20,140 or even urea141 have been assayed. Other additives commonly used in this analysis are organic modifiers (acetonitrile, isobutanol, methanol, tetrahydrofurane, etc.). The effect of these additives on the electro-osmotic mobility and electrophoretic mobility of the micelle has been studied.141–143 The CE coupled to electrospray ionization (ESI) MS (CE-ESI-MS) allows direct amino acid analysis without derivatization,131 and thus, 19 amino acids were analyzed by CE-ESI-MS in only 17 min with a minimal sample preparation and no matrix interference. This report includes the optimization of important parameters like the choice of a volatile electrolyte (1 M formic acid) for the electrophoresis, compatible with MS, and the composition and flow rate of the sheath liquid to obtain the best sensitivity. When sensitivity is the target, it is relatively easy to analyze low picomol levels of OPA derivatives in micellar solutions by using a conventional fluorometric detector,139 which is usually enough for food analysis or an LIF (laser-induced fluorescence) detector,136,144,145 when looking for more selective and sensitive detectors with a wide linear dynamic range (3 orders of magnitude) to cover new high-sensitivity applications (chiral analysis, o-tyrosine analysis, biomedical or pharmaceutical research, etc.) and instrumentation (CE, microcolumn liquid chromatography, etc.). Some reviews covering high-sensitivity detection following CE have been published.146,147

17.3.4

Mass Spectrometry

MS is based on the conversion of components of a sample into rapidly moving gaseous ions, which can be resolved on the basis on their mass-to-charge ratios that are characteristic of each ion and allow its identification. The identification of the 22 protein amino acids may not be a problem, although this detector may be used for more complex identifications as in d- and l-isomer mixtures, nonprotein amino acids, and so forth. Unfortunately, the high cost of purchase and maintenance of mass spectrometers has inhibited their more widespread use in the food industry and/or food control. Nevertheless, reports in the literature of its applications are increasing rapidly. Mass spectrometer detectors were first connected to GC equipments. A good compatibility between both techniques, in particular when capillary columns were available, allowed a rapid development and the onset of these complementary techniques. Application in foods such as in the identification of nonprotein amino acids,21 chiral amino acids,113 o-tyrosine in chicken148 or pork149 tissues, and others,118,119 have been reported.

300 ◾


MS has also been used as a spectroscopic detector after HPLC or CZE, offering the additional advantage of analyzing the amino acids without derivatization, which means a minor sample manipulation, and, due to its high specificity, reduced problems related with matrix interferences or poor resolution between peaks. The connection of HPLC and MS detector is much more problematic than with GLC because of the incompatibility between both techniques (solvents from chromatography, high mobile-phase flow rate vs. vacuum). However, nowadays these difficulties have been overcome with the development of new interfaces, and the technique is widespread although it is still expensive. One of the main requirements for samples to be analyzed by MS is that analytes, amino acids in this case, must be ionized. Three types of ionization modes, atmospheric pressure microwave-induced plasma ionization (AP-MIPI), atmospheric pressure chemical ionization (APCI), and ESI, were compared by Kwon and Moini150 in relation to sensitivity. The best results were obtained by using AP-MIPI in conjunction with a dual oscillating capillary nebulizer.

17.4 Conclusions To obtain the total essential amino acids profile of a given seafood, the most important factors to take into account are the resolution power and selectivity. The highest resolution is obtained by GC with the capillary column technique, but tedious and time-consuming sample derivatization is required. In general, cation exchange and postcolumn derivatization or RP-HPLC precolumn derivatization techniques are the preferred methods. The majority of published reports in which seafood amino acids are analyzed have used the cation exchange method. Nevertheless, RP-HPLC methods with precolumn OPA or PITC derivatization are very convenient methods to use, and many applications can be found in other matrices like cheese or meat, which may be consulted.151,152 A very careful control of the derivatization reactions and chromatographic conditions are necessary for a consistent and reproducible analysis. Since many peaks corresponding to protein and nonprotein amino acids, nucleosides, small peptides, and so on, may appear in the chromatogram, a complete resolution of the whole peaks is really difficult. Therefore, the analytical technique for a determined sample must be carefully chosen based on the literature. The convenience of purchasing commercially available kits must be evaluated. When amino acids from seafood proteins have to be analyzed, the first decision is the choice of the hydrolysis method. In general, acid hydrolysis with HCl 6 N (110°C for 22 h or 145°C for 4 h) with an oxidation protective agent, such as phenol, and taking care of avoiding the presence of oxygen with vacuum and nitrogen purging, is enough for the majority of purposes. Particular hydrolysis problems related with certain amino acids are described in Section 17.2.2. The requirements in resolution are not so exigent as those for physiological amino acids, because fewer peaks appear in the chromatogram. Any separation strategy may give good results, and, once again, the convenience of purchasing commercially available prepackaged kits should be considered.

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136. Arellano, M., Simeon, N., Puig, P. et al. Several applications of capillary electrophoresis for wines analysis. Quantitation of organic and inorganic acids, inorganic cations, amino acids and biogenic amines. J. Int. Sci. Vigne Vin. 31, 213–218, 1997. 137. Castagnola, D.V., Rossetti, D.V., Cassiano, L. et al. Optimization of phenylthiohydantoinamino acid separation by micellar electrokinetic capillary chromatography. J. Chromatogr. 638, 327–333, 1993. 138. Otsuka, K., Terabe, S., Ando, T. Electrokinetic chromatography with micellar solutions separation of phenylthiohydantoin-amino acids. J. Chromatogr. 332, 219–226, 1985. 139. Liu, J., Cobb, K.A., Novotny, M. et al. Separation of pre-column ortho-phthalaldehyde-derivatized amino acids by capillary zone electrophoresis with normal and micellar solutions in the presence of organic modifiers. J. Chromatogr. 468, 55–65, 1989. 140. Matsubara, N., Terabe, S. Micellar electrokinetic chromatography. Meth Enzymol. 270, 319–341, Part A, 1996. 141. Otsuka, K., Terabe, S. Effect of methanol and urea on optical resolution of phenylthiohidantoinDL-amino acids by micellar electrokinetic chromatography with sodium N-dodecanoyl-l-valinate. Electrophoresis 11, 982–984, 1990. 142. Chen, N., Terabe, S. A quantitative study on the effect of organic modifiers in micellar electrokinetic chromatography. Electrophoresis 16, 2100–2103, 1995. 143. Chen, N., Terabe, S., Nakagawa, T. Effect of organic modifier concentrations in micellar electrokinetic chromatography. Electrophoresis 16, 1457–1462, 1995. 144. Jin, D.R., Miyahara, T., Oe, T. et al. Determination of d-amino acids labelled with fluorescent chiral reagents, R(−)and S(+)-a-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3benzoxadiazoles, in biological and food samples by liquid chromatography. Anal. Biochem. 269, 124– 132, 1999. 145. Novatchev, N., Ulrike, H. Evaluation of amino sugar, low molecular peptide and amino acid impurities of biotechnologically produced amino acids by means of CE. J. Pharm. Biomed. Anal. 28, 475–486, 2002. 146. Novotny, M.V., Cobb, K.A., Liu, J. Recent advances in capillary electrophoresis of proteins, peptides and amino acids. Electrophoresis 11, 735–749, 1990. 147. Issaq, H.J., Chan, K.C. Separation and detection of amino acids and their enantiomers by capillary electrophoresis: A review. Electrophoresis 16, 467–480, 1995. 148. Karam, L.R., Simic, M.G. Formation of o-tyrosine by radiation and organic solvents in chicken tissue. J. Biol. Chem. 265, 11581–11585, 1990. 149. Miyahara, M., Nagasawa, T., Izumi, K. et al. New LASER fluorometric detection for ortho-tyrosine in gamma-irradiated phenylalanine solution and pork. Food Irrad. Jap. 34, 3–8, 1999. 150. Kwon, J.Y., Moini, M. Analysis of underivatized amino acid mixtures using high performance liquid chromatography/dual oscillating nebulizer atmospheric pressure microwave induced plasma ionisation-mass spectrometry. J. Am. Soc. Mass. Spectrom. 12, 117–122, 2001. 151. Aristoy, M.C., Toldra´, F. Essential amino acids. In: Handbook of Muscle Foods Analysis. Nollet, L.M.L., Toldra´, F. (Eds.), CRC Press, Boca Raton, FL, 2009, 385–397. 152. Aristoy, M.C., Toldra´, F. Essential amino acids. In: Handbook of Dairy Foods Analysis. Nollet, L.M.L., Toldra´, F. (Eds.), CRC Press, Boca Raton, FL, 2009, 9–32.

Chapter 18

Antioxidants Nick Kalogeropoulos and Antonia Chiou Contents 18.1 Introduction ..................................................................................................................310 18.1.1 Oxidation and Its Implications.........................................................................310 18.1.1.1 Oxidative Stress and Its Implications ...............................................310 18.1.1.2 Lipid Peroxidation............................................................................311 18.1.1.3 Marine Lipid Oxidation ...................................................................311 18.1.2 Antioxidants.....................................................................................................311 18.2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems ..........................................................................................................313 18.3 Antioxidants in Seafood and Seafood Products .............................................................313 18.3.1 Antioxidant Enzymes .......................................................................................313 18.3.2 Ascorbic Acid ...................................................................................................314 18.3.2.1 Ascorbic Acid Functions ..................................................................314 18.3.2.2 Ascorbic Acid Analysis .....................................................................314 18.3.2.3 Occurrence of Ascorbic Acid in Marine Organisms ......................... 315 18.3.3 Vitamin E ........................................................................................................ 315 18.3.3.1 Vitamin E as an Antioxidant............................................................316 18.3.3.2 Vitamin E Determination ................................................................316 18.3.3.3 Occurrence of Vitamin E .................................................................317 18.3.4 Carotenoids ......................................................................................................317 18.3.4.1 Antioxidant and Other Functions of Carotenoids ............................317 18.3.4.2 Carotenoid Determination ...............................................................318 18.3.4.3 Occurrence of Carotenoids ..............................................................318

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18.3.5 Ubiquinone ......................................................................................................319 18.3.5.1 Function of Ubiquinone ..................................................................319 18.3.5.2 Determination of Ubiquinone .........................................................319 18.3.5.3 Occurrence of Ubiquinone...............................................................319 18.3.6 Other Endogenous Antioxidants ..................................................................... 320 18.4 Added Antioxidants ..................................................................................................... 320 18.4.1 Synthetic Antioxidants .................................................................................... 320 18.4.2 Natural Antioxidants .......................................................................................321 References ................................................................................................................................321

18.1

Introduction

Antioxidants evolved together with the emergence of photosynthesis by cyanobacteria, more than 2 billion years ago, as a defense against oxygen toxicity.1 Indeed cyanobacteria and the laterevolved green plants, being exposed to the oxygen they produce, are rich in antioxidants such as vitamins C and E, polyphenols, and carotenoids. Humans and most animals cannot synthesize the majority of these antioxidants and depend on the dietary intake from plant consumption. This is also followed in the marine environment, where antioxidants are mainly produced by photosynthetic organisms and are consequently transported through the trophic web.

18.1.1 Oxidation and Its Implications Oxidation is the transfer of electrons from one atom to another and represents an essential part of aerobic life, since oxygen is the final electron acceptor in the electron flow system that produces energy. When the electron flow becomes uncoupled (transfer of unpaired single electrons), generation of free radicals occurs, that is, electrically charged compounds that seek out and capture electrons from other compounds in order to neutralize themselves. Although the initial attack causes the neutralization of the free radical, another free radical is generated in the process, resulting in a chain reaction. Until subsequent free radicals are deactivated, thousands of free radical reactions may occur within a few seconds. Common free radicals in biological systems are the socalled reactive oxygen species (ROS), which include among others superoxide anion radical (O2•−), hydrogen peroxide (H2O2), nitric oxide (NO•), peroxyl (ROO•), alkoxyl (RO•), and hydroxyl (OH•) radicals, and the so-called reactive nitrogen species (RNS), such as peroxynitrite (ONOO−). ROS production in organisms is related to both the basal metabolism and the influence of environmental factors2; if ROS are not immediately intercepted by antioxidants, they may oxidize several cell components.

18.1.1.1

Oxidative Stress and Its Implications

Oxidative stress occurs when the prooxidant–antioxidant balance becomes too favorable to the prooxidants. Under conditions of oxidative stress, lipids, nucleic acids, and proteins may be damaged by reactive oxidants. Aging,3 pollution,4 and environmental stress5,6 have been reported to cause oxidative stress to fish or bivalves, resulting in an increased antioxidant activity and antioxidants

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loss or oxidation product development. There is increasing evidence that oxidative stress is implicated in the pathogenesis of many inflammatory and degenerative diseases and conditions.3,7

18.1.1.2

Lipid Peroxidation

A very damaging effect of oxidant reactive intermediates is lipid peroxidation, in which polyunsaturated fatty acids on lipid molecules are attacked and oxidized. This can be especially damaging to lipid-rich cell membranes. The health implications of tissue lipid oxidation are numerous and well documented.8 Studies on the pathological significance of dietary lipid oxidation products have indicated that some lipid oxidation products have cytotoxic, mutagenic, carcinogenic, atherogenic, and angiotoxic effects. Lipid oxidation is a complex procedure induced by oxygen in the presence of initiators such as light, heat, free radicals, and metal ions. Three reaction pathways have been proposed: (1) nonenzymatic chain autoxidation, (2) nonenzymatic and nonradical photooxidation, and (3) enzymatic oxidation. In the first two cases a combination of reactions involving 3O2 and 1O2 occurs. Nonradical photooxidation seems to be a minor reaction compared with the 3O2-induced radical chain autoxidation. Autoxidation occurs through a three-phase process, that is, initiation, propagation, and termination.9

18.1.1.3

Marine Lipid Oxidation

Compared with other food lipids, marine lipids are relatively more susceptible to oxidation, because of their high degree of unsaturation.10 Lipids deteriorate in seafood products during processing, handling, and storage, being the major cause of the development of off-flavor compounds and rancidity as well as a number of other reactions that reduce the shelf life and nutritive value of food products. Lipid oxidation of omega-3 polyunsaturated fatty acids (PUFA)-rich food products results in the development of particularly unpleasant off flavors, for which the human sensory apparatus has a low threshold,11 and unhealthy compounds that reduce their shelf life and nutritive value.

18.1.2

Antioxidants

Antioxidants are defined as any substance that when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate.12 In biological systems various biochemical defense mechanisms, including enzymatic systems and nonenzymatic antioxidants, protect the cellular components from oxidative damage. Moreover, antioxidants supplied by foods, mainly of plant origin, are essential for counteracting oxidative stress. For food systems, antioxidants are molecules that protect macromolecules from being oxidized.13 Antioxidants counteract oxidation in two different ways: They protect lipids from oxidation initiators, preventive antioxidants, and they stall the propagation phase, chain-breaking antioxidants.9 Preventive antioxidants hinder ROS formation or scavenge species responsible for oxidation initiation (O2•−, 1O2, etc.). Chain-breaking antioxidants intercept radical oxidation propagators (LOO•) or participate in halting radical chain propagation. Nevertheless, antioxidants often act via more than one mechanism that combines different types of antioxidant activity. The major components of the antioxidant defense system together with their proposed mechanisms of action are presented in Table 18.1. In general, antioxidants are

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Table 18.1 Major Components of Antioxidant Defense System and Proposed Mechanism of Action Antioxidant Species

Mechanism of Action

Enzymes Catalase

ROS detoxification (reduction of H2O2 to water)

Glutathione peroxidase

ROS detoxification (reduction of H2O2 to water)

Superoxide dismutase (SOD)

ROS detoxification (removal of superoxide radical)

Thioredoxin

ROS detoxification (reduction of peroxides)

Metal Ion Sequestration Transferrin

Transient metal chelators (chelates Fe)

Albumin

Transient metal chelators (chelates Fe, Cu)

Ceruloplasmin

Transient metal chelators (chelates Cu)

Ferritin


Lactalbumin


Phytochelatins

Transient metal chelators (chelates Cd, Zn, Cu)

Low Molecular Mass Ascorbic acid (exogenous)

Chain-breaking antioxidant, regenerates oxidized vitamin E

Carotenoids (exogenous)

1

Coenzyme Q (endogenous)

Synergistic to vitamin E

Urate (endogenous)

Scavenges NO2

Phospholipids (endogenous)

Transient metal chelators, ROS detoxification (hydroperoxides), synergistic to vitamin E

Polyphosphates, EDTA, citric acid

Transient metal chelators

Polyphenols (exogenous)

Transient metal chelators (the ones with o-diphenolic structure), chain-breaking antioxidants, regenerate oxidized vitamin E

Vitamin E (exogenous)

Chain-breaking antioxidant, scavenges peroxyradicals, 1O2 quencher

Bilirubin, 2-oxo acids, sex hormones melatonin, lipoic acid, carnosine, anserine, melanins (endogenous)

Compounds with proven antioxidant activity in vitro, but uncertain in vivo

O2 quenchers, chain-breaking antioxidants

Source: Adapted from Willcox, J.K. et al., Crit. Rev. Food Sci., 44, 275, 2004.

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effective at very low concentration levels. At higher levels most of them behave as prooxidants possibly due to their involvement in the initiation reactions.

18.2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems Analytical techniques developed for some of the common endogenous or exogenous antioxidants range from qualitative detection by color-developing reactions to semiquantitative and quantitative determinations by means of spectroscopic, voltammetric, polarographic, and chromatographic methods, the latter including paper, thin-layer, column chromatography and the more sophisticated gas chromatography (GC), and high-performance liquid chromatography (HPLC) alone or combined with mass spectroscopy. The available methods have been reviewed by Rajalakshmi and Narasimhan,15 Griffiths et al.,16 and Laguerre et al.9 The determination of specific waterand fat-soluble antioxidants is discussed in Sections 18.3.2 and 18.3.3. The available methods for monitoring the antioxidant capacity in biological and food systems in vitro or in vivo were recently reviewed by MacDonald-Wicks et al.17 and Wood et al.18 In reviewing methods and tests for the assessment of lipid oxidation, Kolanowski et al.19 concluded that, for quality control of fish oil and fish oil-containing foods, the correlation of instrumental and sensory methods with multivariate data analysis should be followed.

18.3

Antioxidants in Seafood and Seafood Products

In living organisms, oxidative damage to macromolecules is controlled by two types of antioxidant systems, one represented by enzymes and the second represented by low molecular mass compounds, such as ascorbic acid, tocopherols, carotenoids, coenzyme Q, glutathione (GSH), bilirubin, thiols, and uric acid, together with traces of phenolic compounds. These antioxidants act in a concerted way to protect sensitive molecules such as the unsaturated fatty acids from oxidation.

18.3.1

Antioxidant Enzymes

Catalases are metal-containing enzymes, widely distributed in aerobic cells that help in preventing the accumulation of H2O2 within cells. Catalase activities ranged between 386 and 1523 mmol/ min/g tissue in several Atlantic fish and was higher in liver, kidneys, spleen, and heart, and in red muscle compared with that in white.20 Superoxide dismutases (SOD): SODs are metalloproteins, with Mn, Cu, Zn, or Fe in their active site, which act as primary preventive inhibitors and catalyze the dismutation of superoxide anion (O2•−) by reducing one O2•− to H2O2 and oxidizing another one to O2.1 Cu/Zn SOD were purified from marine fish tissues, whereas Fe SOD were purified from red algae, blue-green algae, and algae.21 In several species of teleosts, cephalopods, and crustaceans from the Mediterranean sea, Cu/Zn-SOD activities ranged between 1.9 and 9.7 U/mg of protein,22 whereas in nine Atlantic fish species total SOD values ranged between 157 and 796 U/g fish and Mn-SOD ranged between 45 and 751 U/g.20

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Glutathione peroxidase (GPx): GPx is a selenium-containing enzyme found in animals, plants, and some bacteria. It catalyses the reduction of hydrogen (H2O2) or lipid peroxides (LOOH) while oxidizing 2 mol of reduced GSH. In several Atlantic fish species, GPx ranged between less than 0.03 and 0.23 mmol/min per g fish, with higher activities observed in kidneys, spleen, and heart,20 whereas in Mediterranean fish, cephalopods, and crustaceans, GPx activities ranged between 0.16 and 0.40 U/mg of protein.22 Glutathione reductase (GR): The ratios of GSH/GSSG in cells are normally kept high by the reduction of GSSG back to GSH, which is catalyzed by GR. GR activities were around 84 mU/mg protein in the digestive gland and the gill of mussels Perna perna.5 Glutathione S-transferases (GST): GSTs are involved in the metabolism of xenobiotics and/or oxidized components in the liver.23 GST activities of 129.6 and 630.7 U/mg protein in the digestive gland and gill of the mussel P. perna were reported.5 Peroxiredoxins (Prx): Prxs are ubiquitous enzymes that can be simultaneously both antioxidants and modulators of signal transduction and may be the most important H2O2-removal systems in animals, bacteria, and possibly plants.24

18.3.2 Ascorbic Acid 18.3.2.1

Ascorbic Acid Functions

Ascorbic acid (AA), the most active water-soluble antioxidant, is ubiquitous in eukaryotic cells of both plants and animals. Crustaceans and most fish lack the ability to synthesize AA 25; therefore, they are dependent on dietary supply of vitamins. Most of the AA physiological functions are related to its ability to act as an electron donor,21 and it exerts its properties by neutralizing toxic peroxides and by stabilizing free radicals, thus protecting lipids, proteins, and other biocomponents from oxidative damage.26 Ascorbic acid is noted for its complex multifunctional effects; depending on conditions, AA can act as antioxidant, prooxidant, metal chelator, and reducing agent or as oxygen scavenger.27 In aquatic organisms AA is involved in several physiological functions including growth, development, reproduction, wound healing, collagen synthesis, response to stressors, tyrosine metabolism, metal ion metabolism, protection of cells from oxidative damage, and the regeneration of vitamin E in its metabolically active form. In fish, vitamin C is known to play important roles in improving immune response and resistance to infectious diseases,28 resisting stress,29 and oxidation, while additionally it has a positive effect on the wound healing process.30 Besides AA, its reversibly oxidized form, dehydroascorbic acid (DHAA), also exists in various dietary and biological samples. Additionally l-ascorbate 2-sulfate (AAS) has been isolated from undeveloped cysts of brine shrimp and in tissues of trout.31

18.3.2.2

Ascorbic Acid Analysis

Owing to the importance of AA, many analytical techniques have been proposed for its determination. However, due to AA and DHAA reactivity and instability, many assays suffer from inattention to stability, sensitivity, specificity, and interferences if derivatization products (colorimetric) or retention times (chromatographic) are not verified for the compounds under investigation.32 The main assay types for AA determination are spectroscopic, reviewed by Zaporozhets and Krushinskaya,33 chromatographic—mainly GC and HPLC—recently reviewed by Oliveira and

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Watson,34 and enzymatic. Flow injection techniques35 as well as capillary electrophoresis36 are also employed for AA determination. Methods for DHAA determination, which provides information about the oxidative stress within an organism, were reviewed by Deutsch.37 HPLC assays are generally preferred for AA and DHAA determination in biological samples, as they have certain advantages against spectroscopic or enzymatic methods. Sample preparation–extraction: Sample processing involves removal of protein usually with metaphosphoric acid, which seems to provide the most efficient extraction in food samples while simultaneously preventing oxidation of AA.38 AA is stabilized against oxidation with reagents such as dithioerythritol or monosodium glutamate39 and EDTA. Conditions that promote degradation should be minimized, for example, by purging of samples with an inert gas, storing, if necessary, at no more than −70°C and using amber glass. Determination: Spectroscopic methods have been applied for the determination of AA and DHAA in fish like Atlantic halibut40 and striped bass.41 HPLC methods with electrochemical,22,42,43 ultraviolet (UV),44 and fluorescence45 detection have been reported for the determination of AA and/or DHAA in marine organisms. Electrochemical detection allows the determination of the reduced form only, whereas lack of sensitivity and specificity are the main deficiencies for UV detection. Fluorimetric detection is considered more sensitive and selective, allowing the determination of both AA and DHAA after postcolumn derivatization.38 Total vitamin C (AA plus DHAA) can be measured after oxidation of AA to DHAA, followed by derivatization to give a fluorescent compound. AA is usually oxidized by chemical methods based on active charcoal46 or iodine.47 o-Phenylenediamine (OPDA) and several derivatives47 have been the most used fluorescent reagents for AA determination. If measurement of DHAA is required, the most frequently used approach is to carry out reduction, involving agents like dithioerythritol, 2,3-mercaptopropan-1-ol and mercaptoethanol, or tris-(2carboxyethyl) phosphine hydrochloride.48 HPLC assays have been applied in the analysis of AA and DHAA in fish and crustaceans,22,31,42,45,49 shrimps,50 microalgae, and live food organisms.43

18.3.2.3

Occurrence of Ascorbic Acid in Marine Organisms

Muscles of wild and cultivated marine and freshwater fish generally contain low amounts of vitamin C, usually not exceeding 1 mg/100 g.22,45,51 Vitamin C is known to be concentrated in vital organs with active metabolism such as brain, liver, kidneys, and the gonads,49,52 confirming the hypothesis of the importance of ascorbic acid in preserving vital tissues from oxidation processes. The AA content of Mediterranean cephalopods and crustaceans was low, not exceeding 0.5 mg/100 g.22 In cysts of various batches and strains of Artemia, the ascorbic acid-2-sulfate (AAS) content ranged from 296 to 517 mg AAS/g dw.43 The ovaries of Litopenaeus Yannamei shrimps contained 35.1–46.1 mg AA/100 g,50 whereas fresh and canned sea urchin gonads contained 26.6 and 14.2 mg AA/100 g, respectively.46 Several species of microalgae commonly used in mariculture contained 1.0–4 mg AA/g d.w.43

18.3.3 Vitamin E The term vitamin E refers to a group of eight chemically related compounds possessing a 6-chromanol ring structure attached to a carbon side chain. Based on the structure of the side chain, these compounds are further classified as tocopherols (tocols) and tocotrienols, further designated as a-, b-, g-, and d-vitamers.

316 ◾

18.3.3.1


Vitamin E as an Antioxidant

Vitamin E is a natural, most effective lipid-soluble antioxidant and the principal membrane antioxidant in mammalian cells. Vitamin E antioxidant function, as a peroxyl radical scavenger that terminates chain reactions, is well known and well described.53,54 a-Tocopherol has been proposed to be a highly efficient antioxidant since (1) it reacts with peroxyl radicals faster than allowing the peroxyl radical to be involved in any other reactions, (2) it takes away the radical character from the oxidizing fatty acid and prevents it from further radical reactions, and (3) in the antioxidant reaction, a-tocopherol is turned into a fairly stable radical.54 Abstraction of the 6-OH hydrogen yields a tocopheroxyl radical. Tocopherol can be restored by reduction of the tocopheroxyl radical with redox-active reagents such as vitamin C, polyphenols, enzymes, or ubiquinol.54 In homogeneous solution phase autoxidation, the tocopheroxyl radical will react with a second peroxyl radical to give nonradical products. This second reaction leads to the destruction of tocopherol as an antioxidant. The relative antioxidant activity of different tocopherol homologs has been studied with much attention. According to Frankel,27 there are inconsistencies in these results, which can be attributed to the wide differences in substrates tested, the level of oxidation used in the tests, and the method used to analyze oxidation.

18.3.3.2 Vitamin E Determination Methods used for sample preparation and chromatographic analysis of tocopherols from various matrices have been reviewed.34,55,56 Vitamin E is not chemically bound to macromolecules, and using harsh reagents and conditions to liberate it (e.g., strong saponification) does not seem necessary, whereas on the other hand it can destroy the vitamins. However, efficient conditions are to be used for its release from lipophilic milieu, since vitamin E could be associated with other matrix components. Sample treatment for vitamin E analysis often includes saponification before extraction, frequently performed by heating with KOH in ethanol or methanol. Following saponification, the unsaponifiable components are extracted into an organic solvent. Several factors may interfere with the extraction of vitamin E from the saponification medium,55 among which are organic solvent, ethanol concentration, and the levels of lipids used in the digest. To overcome the oxidation of fat-soluble vitamins caused by saponification, the addition of antioxidants and internal standards is recommended. Vitamin E is commonly extracted using the Folch extraction with chloroform–methanol (2:1), acetone, diethyl ether, and Soxhlet extraction with a variety of solvents. Hexane, alone or with small amounts of more polar solvents such as ethanol and ethyl acetate, is the most frequently used extracting solvent. Alternative to liquid–liquid extraction the use of solid-phase extraction before HPLC analysis has been reported in the analysis of aquatic organisms.57 The use of supercritical fluid extraction for the determination of fat-soluble vitamins has also been reviewed.58 GC-flame ionization detection has been applied for the determination of vitamin since the early 1970s, but nowadays it is used at a lesser extent. HPLC separation of tocopherols provides a fast, simple, sensitive, selective, and more robust technique than GC. Separation of tocopherols is performed on both normal- and reversed-phase HPLC. Normal-phase HPLC via silica columns provides separation of all isomers, whereas b- and g-tocopherols are not easily separated in conventional reversed-phase columns; additionally, normal-phase HPLC operating with organic solvents allows high solubility of lipids. Reversed-phase C18 systems do not completely resolve b- and g-tocopherols, which may, however, be separated by a polymeric column. Nevertheless, when the separation of b- and g-tocopherols is not critical, C18 reversed-phase systems are preferred, since equilibration times are shorter, and better reproducibility is achieved. Electrochemical

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detection, fluorescence, UV absorbance detection, and light-scattering detection have been employed for determining tocopherols and related substances with sensitivities decreasing in the mentioned order.

18.3.3.3

Occurrence of Vitamin E

The vitamin E contents in the muscle tissue of 21 species of teleosts, 3 species of cephalopods, and 6 species of crustaceans caught from the central Mediterranean Sea has been found to range from 5.06 to 17.90 mg/kg, from 7.42 to 9.61 mg/kg, and from 5.77 to 11.70 mg/kg, respectively.59 In another study, the vitamin E level in 10 species of Mediterranean fish was reported to be 7.29 mg/kg.60 The total E vitamers (tocopherols and tocotrienols) of fish consumed in Hawaii61 has been found to range from 2.0 to 39.1 mg/kg wet weight. a-Tocopherol has been found to be the principal tocopherol in marine animals.61,62 Fish species have characteristic tocopherol levels in their tissue, which, since fish are unable to synthesize the vitamin, are related to diet. Considerable differences in a-tocopherol concentration have been reported between light and dark fish muscle, the latter presenting the higher values.51 Krill is a very good source of vitamin E (150 mg/kg tissue).63 Seasonal variations in tocopherol content of fish species62 and mussels64 have been reported. An age-dependent decline in lipophilic antioxidant levels has also been reported.59

18.3.4 Carotenoids Carotenoids are isoprenoid polyenes formed by joining eight isoprene units.65 A series of conjugated double bonds constitutes a chromatophore of variable length, resulting in characteristic yellow to red colors. Carotenoids may be divided into two major classes, based on the degree of substitution65: (1) highly unsaturated carotene hydrocarbons (C40H56) such as a-, b-carotene and lycopene, (2) xanthophylls, that is, oxygenated derivatives of carotene hydrocarbons. Carotenoids may occur in the free, esterified, or bound to macromolecules form. In invertebrates carotenoids may form stable complexes with proteins, that is, carotenoproteins, often with a different color than the original pigment.65 The red color of cooked crustaceans is produced by the release of the individual carotenoid (astaxanthin) from the carotenoproteins when denatured by the heat of cooking.

18.3.4.1

Antioxidant and Other Functions of Carotenoids

Among the proposed functions of carotenoids in aquaculture66 have been those of pigmentation, antioxidant functions, as a source of provitamin A, cellular protection from photodynamic damage, and enhancement of growth and reproductive potential. Some evidence suggests that these pigments may perform vital roles in growth and reproductive success in crustaceans. Dietary carotenoids are the sole biological precursors of retinoids in crustaceans. In terms of free-radical pathology, the most important biological functions of carotenoids appear to be their antioxidant nature, that is, their ability to quench free radical species such as 1O and 3O .65 Carotenoids may also act as chain-breaking antioxidants. There are at least three 2 2 possible mechanisms for the reaction of carotenoids with radical species: (1) radical addition, (2) electron transfer to the radical, or (3) allylic hydrogen abstraction.67 The antioxidant function of b-carotene complements the action of other antioxidants such as catalase, peroxidase, vitamin C, and vitamin E.21

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The relative antioxidant activity of carotenoids has been investigated in several studies. The presence of functional groups (hydroxyl, epoxide, or keto) and the type of ring located at the ends of the polyenoic chain can vary the rate of pigment oxidation. Carbonyl carotenoids such as canthaxanthin and astaxanthin are regarded as better free-radical scavengers than b-carotene. According to Naguib68 the higher antioxidant activity of astaxanthin compared with lutein, lycopene, and a- and b-carotenes could be attributed to an equilibrium with the enol form of the ketone astaxanthin in solution. In a complex system like seafood flesh, it was postulated that the higher hydrophilicity of astaxanthin compared with that of b-carotene provides a better contact to hydroperoxides and thus a more effective protection against lipid oxidation.69

18.3.4.2

Carotenoid Determination

Methods used for the analysis and chromatographic determination of carotenoids have been reviewed.70,71 The main problem associated with work on and manipulation of carotenoids is their instability, especially toward light, oxygen, and heat. During sample preparation and handling, precautions to minimize carotenoid oxidation should be taken (e.g., use of antioxidants, homogenization at low temperatures). The extraction procedures for seafood usually involve several solvents such as hexane isopropanol,72,73 methanol,74 and a mixture of methanol with other more apolar solvents e.g., chloroform; acetone alone or in combination with other solvents has also been used in crustaceans75,76 and salmonids77; supercritical fluid extraction has also been reported.58,78 In several cases alkaline saponification precedes carotenoid determination. Classical column chromatography, TLC, and spectroscopic methods were originally used for the determination of carotenoids. Nowadays, the most common method used in the analysis of carotenoids is HPLC employing various detection techniques. Both normal- and reversed-phase systems are used, either in isocratic or gradient elution modes, the latter generally preferred. Good separations of various carotenoids have been attained on a C-30 chemically bonded phase. UV/Vis detection is by far the most common used. Electrochemical, mass spectrometry, nuclear magnetic resonance detection, and Raman spectroscopy have also been used among others.70

18.3.4.3

Occurrence of Carotenoids

The occurrence of carotenoid species found in several seafoods and aquaculture has been reviewed by Shahidi et al.65 Although carotenoids are distributed in almost all living matter, their synthesis is restricted to plants and microorganisms; thus the presence of carotenoids in animal tissues is solely derived from their dietary intake. However, each species has its own carotenoid requirements, and each tissue even appears to have such specificity in the assimilation of carotenoids. Crustaceans, in general, contain the same major carotenoids among which are astaxanthin, canthaxanthin, and cryptoxanthin.65 The carapace and the ecdysial exoskeleton of crustaceans mainly contain astaxanthin and lutein; the recovery and quantification of carotenoids from several crustacean wastes have been the aim of several studies.72–74,76,79 The carotenoid content in the meat of the major marine crab from Indian waters was reported to be 3.4 mg/kg.75 The astaxanthin content of krill has been found in the range of 15–20 mg/kg tissue.63 In mollusks, various carotenoids have been identified.65 In fish, carotenoids are found in the skin, flesh, eggs, gonads, milt, liver, and eyes. Most of their skin and muscle pigments are xanthophylls.65 The color of flesh of salmon and trout is normally due to astaxanthin, while ovaries also contain high astaxanthin amounts; its concentration has been found to vary between 3 and 37 mg/kg.80,81 In the study of Czeczuga

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et al.82 the carotenoid content in the fillets of 22 species of fish from the fisheries of New Zealand ranged from 0.28 to 5.9 mg/kg. In the gonads of some of these fish species, the carotenoid content ranged from 0.38 to 1.03 mg/kg. The total carotenoid content of Sardina pilchardus from South Europe has been reported to be 10.5 mg/kg in the skin and muscle.83

18.3.5 Ubiquinone Coenzyme Q10 (also known as ubiquinone, coenzyme Q, and abbreviated at times to CoQ10, CoQ, Q10) is a benzoquinone, where the “Q” and the “10” in the name refer to the quinone chemical group and the 10 isoprenyl chemical subunits, respectively. The isoprenoid tail is found in various lengths (6–10 isoprene units) in plants and microorganisms.84 CoQ is found in two redox forms, and the redox cycle involves a reversible reduction to the respective ubiquinols (CoQH2).

18.3.5.1 Function of Ubiquinone In addition to its well-established function as a component of the mitochondrial respiratory chain, in recent years, ubiquinone has acquired increasing attention with regard to its function, in the reduced form (ubiquinol), as an antioxidant.85 Ubiquinone, partly in the reduced form, occurs in all cellular membranes as well as in blood serum and in serum lipoproteins. Ubiquinol efficiently protects membrane phospholipids and serum low-density lipoprotein (LDL) from lipid peroxidation, and, as recent data indicate, also mitochondrial membrane proteins, and DNA from free-radical–induced oxidative damage. Scientific evidence has indicated that ubiquinone may function together with tocopherols in protecting the function of biological membranes.86 Ubiquinol is the only known lipid-phase antioxidant that can be synthesized de novo in animal cells and for which an enzymatic mechanism in mitochondrial and microsomal electron-transport systems, which can regenerate the antioxidant ubiquinone form, exists.85

18.3.5.2

Determination of Ubiquinone

Extraction of ubiquinone is usually performed contemporaneously with other lipophilic compounds, using hexane after protein precipitation with methanol, ethanol,22,84,87,88 ethanol isopropanol,89 and tissue homogenization. Isopropanol has also been used as an extraction solvent.90 Several HPLC methods for the determination of total ubiquinone (the sum of ubiquinone and ubiquinol) have been described using electrochemical and/or UV detection.22,84,88,91

18.3.5.3 Occurrence of Ubiquinone Ubiquinone is widely distributed, as it is present in almost every cell of living organisms. The CoQ content varies in different organs, with the highest in energy-producing tissues, as the dark muscle of fish. The ubiquinone content of several food and marine organisms has been reported in several studies.22,51,84,87–89,91,92 The richest known food source of CoQ10 is the heart from mammal species.84,91 Concerning ubiquinone contents in fish, reported results show that fat fish flesh is the second most abundant source of CoQ10 after red meats.51,59,84,91 The ubiquinone content of pelagic fish flesh (mackerel and herring) was reported in the range of 12.3–67.7 mg/kg88; in the muscle of several Mediterranean species of teleosts, cephalopods, and crustaceans, the range of CoQ10

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content was 0.07–1.13, 0.10–1.06, and 0.21–1.15 mg/kg, respectively; the range of CoQ 10H2 content was 0.18–5.11, 0.27–7.18, and 2.11–8.37 mg/kg, respectively; CoQ 9 and CoQ 9H2 were also detected in some species of teleosts and cephalopods, at concentrations ranging from 0.10–2.5 to 0.18–6.35 mg/kg, respectively.22 The CoQ10H2 and CoQ10 levels in Mediterranean fish were reported to be 3.43 and 0.72 mg/kg, respectively.60 The total CoQ 10 (ubiquinone and ubiquinol) content in the muscle of fish and shellfish of Japanese diet has been found to be in the range of 1.8–130 and 1.7–4.9 mg/kg, respectively,90 in salmon, eel, and squid consumed in Japan, 3.8–7.6 mg/kg.92

18.3.6 Other Endogenous Antioxidants Glutathione: GSH is a tripeptide (Glu-Cys-Gly), existing in reduced GSH and oxidized (GSSG) forms. It is involved in free-radical scavenging, metabolism of toxic xenobiotic compounds, maintenance of thiol-disulfide status, and signal transduction.20 GSH provides a source of electrons that allow GPx to enzymically decompose hydrogen and lipid peroxides. Oxidized GSH, GSSG, is reduced back to GSH by GR, to maintain a high GSH/GSSG ratio, which could be considered as a quality parameter.22 GSH is usually determined enzymatically4,5 or by HPLC.22 Phospholipids: The antioxidant activity of phospholipids is mainly connected with their ability to function as synergists and metal chelators. Phospholipids exhibit synergism with tocopherols in fish oils.93,94 As phospholipids are involved in nonenzymic browning, they show higher antioxidant activity at elevated temperatures or where browning is considerable. Amino acids, amines, and peptides: Amines, peptides, and amino acids are known to exhibit antioxidant properties. Furthermore, amino acids are involved in nonenzymic browning reactions with carbonyls from oxidizing lipids, which lead to products with antioxidative properties.95 Protein hydrolysates from fish,96,97 and fish processing by-products,98 exert antioxidant and biological activities. Other antioxidants in fish and seafood: Aquatic plants like seagrass99 are known to possess antioxidant and anti-inflammatory activity. Phycocyanin, a blue-green algae pigment,100 the flesh and skin of hag fish101 and eel,102 and shrimp shell waste103 contain natural antioxidants capable of scavenging hydroxyl radicals. Phenolic antioxidants have been reported in green algae,104 microalgae and cyanobacteria,105 edible brown algae,106 and in mussels.6

18.4

Added Antioxidants

The naturally occurring antioxidants in seafood and seafood products impart a certain amount of protection by maintaining the balance between prooxidative and antioxidative factors. However, in postmortem conditions and during processing and prolonged storage, the endogenous antioxidants are consumed sequentially, and lipids are eventually oxidized,51,107 necessitating the addition of exogenous antioxidants to retard the onset of lipid oxidation and elongate the shelf life of seafood products.

18.4.1

Synthetic Antioxidants

Antioxidants allowed in food in the EU and the FDA are the calcium disodium salt of EDTA (E385), the phenolic antioxidants propyl gallate (E310), octyl gallate (E311), dodecyl gallate

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(E312), butylated hydroxyanisol (BHA, E320), butylated hydroxytoluene (BHT, E321), and tertbutylhydroquinone (TBHQ, E319), the more “natural” ascorbic acid (E300) and isoascorbic (erythorbic) acid (E315) and their salts, ascorbyl palmitate and stearate (E304), tocopherols (a-E307, d-E308, g-E309), and tocopherol concentrate (E306). However, there have been concerns about the possible negative health effects of synthetic antioxidants.108

18.4.2

Natural Antioxidants

Natural antioxidants isolated from aromatic herbs, tea, grapes, and seeds have recently gained interest as replacements for potentially toxic synthetic antioxidants.109,110 Natural antioxidants are readily accepted by the consumers, as they are considered to be safe, and no safety tests are required if they are components of foods that are consumed for centuries, whereas their main disadvantages are that (1) they are usually more expensive if purified and less efficient if not purified, (2) the properties of different preparations vary if not purified, (3) their safety is unknown, and (4) they may impart color, aftertaste or off flavor to the product.111 Several natural antioxidants have already been used in fish oils and fish products to retard oxidative deterioration. Dry oregano is effective in preventing oxidation in mackerel oil.112 Phenolic antioxidants from rosemary leaves have been successfully used in sardine oil, cod liver oil,113 horse mackerel, oil-in-water emulsions, and fish oils.114 Green tea polyphenols protect silver carp115 and fish oils113,116 from oxidation. Phenolic extracts from grape by-products were successfully applied in fish lipids and muscle.117–119 Polyphenols extracted from extra virgin olive oil retards oxidation of canned tuna,120 horse mackerel, and fish oils.114 Individual polyphenols, alone or in mixture with other antioxidants, are more effective than synthetic antioxidants in preventing oxidation of marine oils121,122 or frozen fish.123

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Chapter 19

Vitamins Young-Nam Kim Contents 19.1 Fat-Soluble Vitamins .................................................................................................... 328 19.1.1 Vitamin A and Carotenoids ............................................................................ 328 19.1.2 Vitamin D........................................................................................................332 19.1.3 Vitamin E ........................................................................................................332 19.1.4 Vitamin K ........................................................................................................333 19.2 Water-Soluble Vitamins................................................................................................ 334 19.2.1 Thiamin (Vitamin B1) ..................................................................................... 334 19.2.2 Riboflavin (Vitamin B2) ...................................................................................335 19.2.3 Niacin (Vitamin B3) ........................................................................................ 336 19.2.4 Vitamin B6 .......................................................................................................337 19.2.5 Folate ...............................................................................................................338 19.2.6 Vitamin B12 ..................................................................................................... 340 19.2.7 Pantothenic Acid ............................................................................................. 341 19.2.8 Biotin .............................................................................................................. 341 19.2.9 Vitamin C ....................................................................................................... 342 19.3 Summary ..................................................................................................................... 343 References ............................................................................................................................... 344

Vitamins are a group of complex organic compounds that are essential to normal functioning and essential metabolic reactions in the body. Vitamins are not used as a source of energy or as a source of structural tissue components but rather as cofactors or coenzymes in biochemical reactions. Vitamins are divided into two categories based on their solubility—those soluble in fat organic solvents are known as fat-soluble vitamins and those soluble in water are known as 327

328


water-soluble vitamins. Tables 19.1 and 19.2 list the concentrations of fat-soluble vitamins and water-soluble vitamins, respectively, in fish and seafood according to the U.S. Department of Agriculture National Nutrient Database [1]. The percentages of the daily value (DV) of vitamins estimated to be in 100 g of each seafood are included in Tables 19.1 and 19.2. The DV, established by the U.S. Food and Drug Administration, is a nutrient reference value intended to help consumers understand how foods fit into their overall diets [2]. Foods containing 20% or more of the DV of nutrients per reference amount are indicated to be “high,” “rich,” or “excellent” sources of the nutrients. Foods containing 10%–19% of the DV are categorized as “good” sources. Fish and shellfish are good to excellent sources of most of the B vitamins as defined by the U.S. Food and Drug Administration [3]. Fat-soluble vitamins A, D, and E are present in seafood in varying amounts, often in concentrations higher than those in other meats. Many species of fish and shellfish store high amounts of vitamins A, D, and E in their livers [4]. Little vitamin C is found in seafood, but it is considered a good to excellent source of B-complex vitamins. Generally, the methodologies used for determining the composition of various vitamins in seafood are the same as those used for other foods. Several high-performance liquid chromatography (HPLC) methods using various detections [5–9] have been proposed to determine several vitamins simultaneously. Internal standards are frequently used in the analytical methods for determination of vitamins. Several vitamins are lost or interconverted to their isomers in extraction and purification procedures during analyses. Various methods have been used for the determination of vitamins in foods. In-depth recent reviews of the methodologies for measuring vitamins are available [10,11].

19.1 Fat-Soluble Vitamins 19.1.1 Vitamin A and Carotenoids Vitamin A is a fat-soluble vitamin that is essential for humans and other vertebrates. Vitamin A as retinoids, primarily retinyl esters, is abundant in some animal-derived foods, whereas carotenoids are abundant in plant foods as pigments. Carotenoids are responsible for the color of fish and shellfish. Carotenoids cannot be synthesized by live animals but are obtained from their preys or aquafeeds, which have added carotenoids such as canthaxanthin and astaxanthin [12]. Because vitamin A and carotenoids are fat soluble, they are associated with the fat portion of foods. They are highly concentrated in fish liver oils, but small amounts are found in fish muscles or fillets. Fish are not a major source of this vitamin. In foods, retinyl esters and carotenoids are vulnerable to oxidation. Exposure to air, heat, and storage time also influence the destruction of vitamin A compounds. Thus, overcooking can cause loss of retinyl esters and provitamin A in foods. Reversed-phase HPLC followed by ultraviolet (UV) detection for retinoids and carotenoids is the most common method of analysis. HPLC methodologies are given in AOAC Official Methods 2001.13 and 2005.07 [13]. HPLC procedures for determination of retinoids and carotenoids in seafood [14–16] have also been published. During sample preparation and analysis, samples should be protected from heat, light, and oxidizing substances to avoid destructions and isomerizations of the retinoids and carotenoids. Antioxidants such as butylated hydroxytoluene (BHT), pyrogallol, or ascorbyl palmitate are used to prevent oxidation of retinoids and carotenoids. Alkali hydrolysis (saponification) is routinely used to extract retinoids and carotenoids from foods. Saponification removes chlorophylls, unwanted lipids, and other materials, which may interfere with chromatographic separation. Retinyl esters and carotenoid esters in foods are converted to retinol and carotenoids during saponification. However, the degradation and isomerization of

Vitamins ◾ TABLE 19.1

329

Concentrations of Fat-Soluble Vitamins in Selected Fish and Seafood Vitamin A

Food Product

IU/100 ga

Vitamin E

%DVb

Vitamin D

mg/100 gc

%DV

IU/100 gd

Vitamin K

%DV

μg/100 g

%DV

Catfish, raw

50

1

—e

—

500

125

—

—

Cod, raw

27

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