Biotechnology in Functional Foods and Nutraceuticals

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Author: Debasis Bagchi | Francis C. Lau | Dilip K. Ghosh

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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-13: 978-1-4200-8712-3 (Ebook-PDF) 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. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com


Dedicated to my dearest Harry and Benita Singh. —Debasis Bagchi To my mother with gratitude and affection. —Francis C. Lau To my wife, Sumita, and two sons, Rohit and Roneet, for their consistent help and support. —Dilip K. Ghosh



Contents Preface...............................................................................................................................................xi Editors ............................................................................................................................................ xiii Contributors ..................................................................................................................................... xv

PART I Biotechnology for the Enhancement of Functional Foods and Nutraceuticals Chapter 1

Advances in Biotechnology for the Production of Functional Foods .......................... 3 Yun-Hwa Peggy Hsieh and Jack Appiah Ofori

Chapter 2

Functional Foods and Biotechnology in Japan .......................................................... 29 Harukazu Fukami

Chapter 3

Basic and Clinical Studies on Active Hexose Correlated Compound........................ 51 Takehito Miura, Kentaro Kitadate, Hiroshi Nishioka, and Koji Wakame

Chapter 4

Biotechnology and Breeding for Enhancing the Nutritional Value of Berry Fruit ............................................................................................................. 61 Jessica Scalzo and Bruno Mezzetti

Chapter 5

Improving the Bioavailability of Polyphenols............................................................ 81 Tetsuya Konishi and M. Mamunur Rahman

Chapter 6

The Function of the Next Generation Polyphenol, “Oligonol” .................................. 91 Takehito Miura, Kentaro Kitadate, and Hajime Fujii

Chapter 7

Application of Biotechnology in the Development of a Healthy Oil Capable of Suppressing Fat Accumulation in the Body ......................................................... 103 Hiroyuki Takeuchi

Chapter 8

Effects of Nutraceutical Antioxidants on Age-Related Hearing Loss ..................... 113 Shinichi Someya, Tomas A. Prolla, and Masaru Tanokura

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PART II Chapter 9

Contents

The Impact of Genetic Modification on Functional Foods Increased Production of Nutriments by Genetically Engineered Bacteria .............. 127 Kazuhiko Tabata and Satoshi Koizumi

Chapter 10 Recent Advances in the Development of Transgenic Pulse Crops........................... 139 Susan Eapen Chapter 11 The Improvement and Enhancement of Phyto-Ingredients Using New Technology of Genetic Recombination............................................................ 157 Hisabumi Takase Chapter 12 Metabolic Engineering of Bioactive Phenylpropanoids in Crops ............................ 181 Kevin M. Davies Chapter 13 The Use of Biotechnology to Reduce the Dependency of Crop Plants on Fertilizers, Pesticides, and Other Agrochemicals ............................................... 197 Zeba F. Alam Chapter 14 Animal Biotechnology: Applications and Potential Risks ....................................... 219 Rama Shanker Verma, Abhilash, Sugapriya M.D., and Chithra R. Chapter 15 Application of Micro-RNA in Regenerative Nutraceuticals and Functional Foods ...................................................................................................... 251 Ji Wu, Huacheng Luo, Li Zhou, and Jie Xiang Chapter 16 Microbial Production of Organic Acids and Its Improvement by Genome Shuffling................................................................................................ 265 Takashi Yamada

PART III New Frontier in Food Manufacturing Process Chapter 17 Microalgal Biotechnology in the Production of Nutraceuticals ............................... 279 Niels-Henrik Norsker, Maria Barbosa, and René Wijffels Chapter 18 The Innovation of Technology for Microalgae Cultivation and Its Application for Functional Foods and the Nutraceutical Industry .............................................. 313 Akira Satoh, Masaharu Ishikura, Nagisa Murakami, Kai Zhang, and Daisuke Sasaki


Contents

ix

Chapter 19 Production of Nattokinase as a Fibrinolytic Enzyme by an Ingenious Fermentation Technology: Safety and Efficacy Studies ................................................................................................. 331 Shinsaku Takaoka, Kazuya Ogasawara, and Hiroyoshi Moriyama Chapter 20 Synthesis of Antihypertensive GABA-Enriched Dairy Products Using Lactic Acid Bacteria ................................................................................................. 349 Kazuhito Hayakawa Chapter 21 Production of High-Quality Probiotics Using Novel Fermentation and Stabilization Technologies ....................................................................................... 361 Franck Grattepanche and Christophe Lacroix Chapter 22 Tracking the Careers of Grape and Wine Polymers Using Biotechnology and Systems Biology ................................................................................................ 389 John P. Moore and Benoit Divol Chapter 23 The Impact of Supercritical Extraction and Fractionation Technology on the Functional Food and Nutraceutical Industry............................................................407 Andrés Moure, Beatriz Díaz-Reinoso, Herminia Domínguez, and Juan Carlos Parajó Chapter 24 The Application of Nanotechnology to Functional Foods and Nutraceuticals to Enhance Their Bioactivities ................................................................................. 447 Ping-Chung Kuo

PART IV

Quality Assurance and Safety: Design and Implementation

Chapter 25 Enhancing the Nutritional Quality of Fruit Juices: Advanced Technologies for Juice Extraction and Pasteurization ....................................................................465 Robert D. Hancock and Derek Stewart Chapter 26 Probiotics: Health Benefits, Efficacy, and Safety ..................................................... 485 Nagendra P. Shah Chapter 27 Use of High Pressure Technology to Inactivate Bacterial Spores in Foods ........................................................................................ 497 Noriyuki Igura, Seiji Noma, and Mitsuya Shimoda


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Contents

PART V Legal, Social, and Regulatory Aspects of Food Biotechnology Chapter 28 Regulations of Biotechnology: Generally Recognized as Safe (GRAS) and Health Claims ....................................................................................................507 Ryan R. Simon, Earle R. Nestmann, Kathy Musa-Veloso, and Ian C. Munro Chapter 29 Global Food Biotechnology Regulations and Urgency for Harmonization ............. 531 Dilip K. Ghosh and Peter Williams

PART VI

Future of Biotechnology

Chapter 30 Future Strategies for the Development of Biotechnology-Enhanced Functional Foods and Their Contribution to Human Nutrition ............................... 545 Dilip K. Ghosh, Francis C. Lau, and Debasis Bagchi

Index .............................................................................................................................................. 549


Preface Biotechnology has been used thousands of years ago in the manufacturing of food products. The most ancient form of biotechnology, fermentation, involved the use of microorganisms such as yeasts for the production of wine, vinegar, and bread. Dairy products such as yogurt and cheese were produced by lactic acid bacteria and molds. Although these techniques are still used, the cultures that were used in ancient times have been modified to provide high-quality products with increased yield. Modern food biotechnology has evolved into a billion-dollar industry, with the promise of producing foods that provide functions beyond the basic nutrients they contain. These functional foods or nutraceuticals have become increasingly important to consumers who are interested in the health benefits of functional foods in the prevention of illness and chronic conditions. Biotechnology is a collection of biology-based technologies used mainly in agriculture, food science, and medicine. Agricultural biotechnology may involve the use of molecular and/or biochemical techniques to produce desired traits, while eliminating many unwanted traits in plants, through the use and manipulation of genetic information. In fact, agricultural biotechnology has been seriously affected by the new recombinant DNA technique that emerged in the 1970s. Genetic modification has significantly improved the yield, quality, and nutritional value of crop plants and animal products. It was estimated that approximately 13.3 million farmers in 25 countries were using agricultural biotechnology in 2009. This came at a time when the world sought science-based and consumer-focused approaches to solving the problem of feeding a growing population. In this respect, agricultural biotechnology is able to deliver resilient crops with enhanced yield even when they are grown in harsh environments. Animal biotechnology also plays an important role in agriculture today. Genetic modification is used to improve livestock selection and breeding. Moreover, animal genomics is utilized to provide optimal nutritional needs for animals to generate high-quality animal products such as meat, milk, and eggs. Overall, biotechnology helps in enhancing food manufacturing processes, improving food preservation, and ensuring food safety. Thus, biotechnology provides the necessary means for the development and improvement of bioactive components in functional foods and nutraceuticals. This book covers the various aspects of biotechnology in nutraceuticals and functional foods. The goal of the book is to provide readers with comprehensive reviews, by a panel of experts from around the world, focusing on state-of-the-art topics that are broad in scope yet concise in structure. This book is divided into six parts. The first part gives an overview of recent advances in biotechnology and their contribution to food science. The second part examines the impact of genetic modification on functional foods. The third part explores food manufacturing technology. The fourth part gives insight into quality assurance and safety of foods. The fifth part updates current views on legal, social, and regulatory aspects of food biotechnology. A final commentary concludes the book by offering an overview of future directions in the applications of biotechnology to functional foods and nutraceuticals.

xi © 2010 Taylor and Francis Group, LLC


Editors Debasis Bagchi received his PhD degree in medicinal chemistry in 1982. He is a professor in the Department of Pharmacological and Pharmaceutical Sciences at University of Houston College of Pharmacy, Houston, Texas. Dr. Bagchi is also senior vice president of Research & Development of InterHealth Nutraceuticals, Inc., Benicia, California. Dr. Bagchi is currently the president-elect of American College of Nutrition, Clearwater, Florida, and also serves as a distinguished advisor on the Japanese Institute for Health Food Standards, Tokyo, Japan and as chairperson on the Nutraceuticals and Functional Foods Division, Institute of Food Technologists, Chicago, Illinois. He serves as the vice-chair of the International Society for Nutraceuticals and Functional Foods (ISNFF). His research interests include free radicals, human diseases, carcinogenesis, pathophysiology, mechanistic aspects of cytoprotection by antioxidants, regulatory pathways in obesity and gene expression, and biotechnology. Dr. Bagchi has 271 peer-reviewed publications and numerous books. He has delivered invited lectures in various national and international scientific conferences, organized workshops, and group discussion sessions. He is a fellow of the American College of Nutrition, member of the Society of Toxicology, member of the New York Academy of Sciences, fellow of the Nutrition Research Academy, and member of the trichloroethylene (TCE) stakeholder Committee of the Wright Patterson Air Force Base, Dayton, Ohio. Dr. Bagchi is a member of the Study Section and Peer Review Committee of the National Institutes of Health, Bethesda, Maryland. He is also serving as editorial board member of numerous peer reviewed journals, including Antioxidants and Redox Signaling, Journal of Functional Foods, Cancer Letters, Journal of American College of Nutrition, The Original Internist, and other scientific and medical journals. He is currently serving as associate editor of the Journal of Functional Foods and Journal of American College of Nutrition. Dr. Bagchi received funding from various institutions and agencies, including the U.S. Air Force Office of Scientific Research, Nebraska State, Department of Health, Biomedical Research Support Grant from National Institutes of Health, National Cancer Institute, Health Future Foundation, The Procter & Gamble Company, and Abbott Laboratories. Francis C. Lau is currently a scientist at InterHealth Nutraceuticals Inc., Benicia, California. He obtained his BS degree in biochemistry from the University of Alberta, Edmonton, Canada. He went on to pursue his MS degrees in molecular biology and computer information systems from the University of San Francisco and the University of Houston, respectively. Dr. Lau obtained his PhD degree in neuroscience from the College of Veterinary Medicine at Texas A&M University. He then held a postdoctoral fellowship at the National Institutes of Health, where he was granted an Intramural Research Training Award. Prior to joining InterHealth Nutraceuticals, Dr. Lau held a position at the United States Department of Agriculture (USDA) Human Nutrition Research Center on Aging, where he studied the benefits of nutraceuticals and functional foods such as dietary antioxidants in memory and aging. He has published numerous scientific papers, invited reviews, and book chapters. He is a fellow of the American College of Nutrition. His recent research interests focus on the effects of nutraceuticals and functional foods on cardiovascular health, joint health, diabetes health, and on promoting healthy body weight. Dilip K. Ghosh received his PhD in biomedical science from the University of Calcutta (UoC), India. Previously, he held positions in Organon (India) Ltd., a division of Organon International, BV and AKZO-NOBEL, The Netherlands; HortResearch, New Zealand; USDA-ARS, HNRCA at Tufts University, Boston; and The Smart Foods Centre, University of Wollongong, Australia. He has been xiii © 2010 Taylor and Francis Group, LLC

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Editors

involved for a long time in drug development and functional food research & development and its commercialization in both academic and industry domains. He is a fellow of the American College of Nutrition and is also a member of several editorial boards. Currently, Dr. Ghosh is a director at nutriConnect (http://www.nutriconnect.com.au). His research interests include oxidative stress, bioactive, functional foods and their relationship with human health, and regulatory and scientific aspects of functional foods and nutraceuticals.


Contributors Abhilash Department of Biotechnology Stem Cell and Molecular Biology Laboratory Indian Institute of Technology Madras Chennai, India

Susan Eapen Nuclear Agriculture and Biotechnology Division Bhabha Atomic Research Centre Mumbai, India

Zeba F. Alam Spectrum Institute of Science and Technology (Pvt.) Ltd Colombo, Sri Lanka

Hajime Fujii R&D Division, Bio Chemical Branch Amino Up Chemical Co., Ltd Kiyota-ku, Sapporo, Japan

Debasis Bagchi College of Pharmacy University of Houston Houston, Texas

Harukazu Fukami Department of Bioscience and Biotechnology Kyotogakuen University Kyoto, Japan

Maria Barbosa Bioprocess Engineering group Wageningen University and Research Wageningen, The Netherlands

Dilip K. Ghosh Nutriconnect Castle Hill, Sydney, Australia

Chithra R. Department of Biotechnology Stem Cell and Molecular Biology Laboratory Indian Institute of Technology Madras Chennai, India Kevin M. Davies The New Zealand Institute for Plant and Food Research Limited Palmerston North, New Zealand Beatriz Díaz-Reinoso Departamento Enxeñería Química Universidade de Vigo, Edificio Politécnico Ourense, Spain

Franck Grattepanche Laboratory of Food Biotechnology Institute of Food Science and Nutrition Zürich, Switzerland Robert D. Hancock Plant Products and Food Quality Programme Scottish Crop Research Institute Invergowrie, Dundee, United Kingdom Kazuhito Hayakawa Yakult Central Institute for Microbiological Research Tokyo, Japan

Benoit Divol Institute for Wine Biotechnology Stellenbosch University Matieland, South Africa

Yun-Hwa Peggy Hsieh Department of Nutrition, Food and Exercise Sciences Florida State University Tallahassee, Florida

Herminia Domínguez Departamento Enxeñería Química Universidade de Vigo, Edificio Politécnico Ourense, Spain

Noriyuki Igura Laboratory of Food Process Engineering Kyushu University Fukuoka, Japan xv


xvi

Masaharu Ishikura Life Science Business Division Yamaha Motor Co., Ltd Shizuoka, Japan Kentaro Kitadate Bio Chemical Branch and

Contributors

Takehito Miura Bio Chemical Branch and Scientific and Research Advisory Unit Research and Development Division Amino Up Chemical Co., Ltd Sapporo, Japan

Scientific and Research Advisory Unit Research and Development Division Amino Up Chemical Co., Ltd Sapporo, Japan

John P. Moore Institute for Wine Biotechnology Stellenbosch University Matieland, South Africa

Satoshi Koizumi Bioprocess Development Center Kyowa Hakko Bio Co., Ltd Ibaraki, Japan

Hiroyoshi Moriyama Laboratory of Pharmacotherapeutics Showa Pharmaceutical University Tokyo, Japan

Tetsuya Konishi Department of Functional and Analytical Food Sciences Niigata University of Pharmacy and Applied Life Sciences Niigata, Japan

Andrés Moure Departamento Enxeñería Química Universidade de Vigo, Edificio Politécnico Ourense, Spain

Ping-Chung Kuo Department of Biotechnology National Formosa University Taiwan, Republic of China Christophe Lacroix Laboratory of Food Biotechnology Institute of Food Science and Nutrition Zürich, Switzerland Francis C. Lau InterHealth Research Center Benicia, California Huacheng Luo School of Life Science and Biotechnology Shanghai Jiao Tong University Shanghai, China Bruno Mezzetti Department of Environmental and Crop Science Università Politecnica delle Marche Ancona, Italy


Ian C. Munro Cantox Health Sciences International Mississauga, Ontario, Canada Nagisa Murakami Life Science Business Division Yamaha Motor Co., Ltd Shizuoka, Japan Kathy Musa-Veloso Cantox Health Sciences International Mississauga, Ontario, Canada Earle R. Nestmann Cantox Health Sciences International Mississauga, Ontario, Canada Hiroshi Nishioka R&D Division, Bio Chemical Branch Amino Up Chemical Co., Ltd Sapporo, Japan Seiji Noma Laboratory of Food Process Engineering Kyushu University Fukuoka, Japan

Contributors

xvii

Niels-Henrik Norsker Bioprocess Engineering group Wageningen University and Research Wageningen, The Netherlands

Mitsuya Shimoda Laboratory of Food Process Engineering Kyushu University Fukuoka, Japan

Jack Appiah Ofori Department of Nutrition, Food and Exercise Sciences Florida State University Tallahassee, Florida

Ryan R. Simon Cantox Health Sciences International Mississauga, Ontario, Canada

Kazuya Ogasawara Japan Bio Science Laboratory Co., Ltd. Ibaraki-shi, Osaka, Japan Juan Carlos Parajó Departamento Enxeñería Química Universidade de Vigo, Edificio Politécnico Ourense, Spain Tomas A. Prolla Department of Genetics and Medical Genetics University of Wisconsin Madison, Wisconsin M. Mamunur Rahman Department of Functional and Analytical Food Sciences Niigata University of Pharmacy and Applied Life Sciences Niigata, Japan Daisuke Sasaki Life Science Business Division Yamaha Motor Co., Ltd Shizuoka, Japan Akira Satoh Life Science Business Division Yamaha Motor Co., Ltd Shizuoka, Japan Jessica Scalzo New Zealand Institute for Plant and Food Research Limited Hawke’s Bay Research Centre Havelock North, New Zealand Nagendra P. Shah Victoria University, Melbourne, Victoria, Australia


Shinichi Someya Departments of Genetics and Medical Genetics University of Wisconsin Madison, Wisconsin and Department of Applied Biological Chemistry University of Tokyo Tokyo, Japan Derek Stewart Plant Products and Food Quality Programme Scottish Crop Research Institute Invergowrie, Dundee, United Kingdom Sugapriya M.D. Department of Biotechnology Stem Cell and Molecular Biology Laboratory Indian Institute of Technology Madras Chennai, India Kazuhiko Tabata Bioprocess Development Center Kyowa Hakko Bio Co., Ltd Ibaraki, Japan Shinsaku Takaoka Japan Bio Science Laboratory Co., Ltd Ibaraki-shi, Osaka, Japan Hisabumi Takase Kyoto Gakuen University Sogabe, Kyoto, Japan Hiroyuki Takeuchi Department of Food and Nutrition Toyama College Toyama, Japan Masaru Tanokura Department of Applied Biological Chemistry University of Tokyo Tokyo, Japan

xviii

Contributors

Rama Shanker Verma Department of Biotechnology Stem Cell and Molecular Biology Laboratory Indian Institute of Technology Madras Chennai, India

Jie Xiang School of Life Science and Biotechnology Shanghai Jiao Tong University Shanghai, China

Koji Wakame R&D Division, Bio Chemical Branch Amino Up Chemical Co., Ltd Kiyota-ku, Sapporo, Japan

Takashi Yamada Chemical Management Center National Institute of Technology and Evaluation (NITE) Tokyo, Japan

René Wijffels Bioprocess Engineering group Wageningen University and Research Wageningen, The Netherlands Peter Williams Smart Foods Centre, School of Health Sciences University of Wollongong Wollongong, New South Wales, Australia Ji Wu School of Life Science and Biotechnology Shanghai Jiao Tong University Shanghai, China


Kai Zhang Life Science Business Division Yamaha Motor Co., Ltd Shizuoka, Japan Li Zhou School of Life Science and Biotechnology Shanghai Jiao Tong University Shanghai, China

Part I Biotechnology for the Enhancement of Functional Foods and Nutraceuticals



in Biotechnology 1 Advances for the Production of Functional Foods Yun-Hwa Peggy Hsieh and Jack Appiah Ofori CONTENTS 1.1 1.2

Introduction ..............................................................................................................................3 Biotechnology for the Production of Plant-Based Functional Foods .......................................5 1.2.1 Biofortification with Essential Micronutrients .............................................................5 1.2.1.1 Vitamin A ......................................................................................................8 1.2.1.2 Iron .................................................................................................................8 1.2.1.3 Zinc .............................................................................................................. 10 1.2.2 Biofortification with Phytochemicals ......................................................................... 10 1.2.3 Modification of Macronutrients .................................................................................. 11 1.2.3.1 Oils ............................................................................................................... 11 1.2.3.2 Proteins ........................................................................................................ 14 1.2.4 Production of Hypoallergenic Foods .......................................................................... 16 1.2.5 Reduction of Antinutrients ......................................................................................... 17 1.3 Biotechnology for the Production of Animal-Based Functional Foods ................................. 17 1.3.1 Meat Products ............................................................................................................. 18 1.3.1.1 In Vitro Meat ................................................................................................ 18 1.3.1.2 Meat with a Modified FA Profile ................................................................. 18 1.3.2 Dairy Foods ................................................................................................................ 19 1.3.2.1 Milk for the Lactose-Intolerant Population ................................................. 19 1.3.2.2 Milk with Enriched Antimicrobial Protein, Lysozyme ...............................20 1.3.2.3 Milk with an Improved FA Profile .............................................................. 21 1.4 Final Remarks ......................................................................................................................... 22 References ........................................................................................................................................ 22

1.1 INTRODUCTION The old adage “you are what you eat” derives from the idea that you must eat good food in order to be fit and healthy. The role of diet in health promotion and disease prevention has been acknowledged for centuries, based on experience and epidemiological data. Recent research has revealed the important role that diet plays in preventing and/or slowing the progression of major chronic diseases such as cancer, diabetes, and cardiovascular diseases (CVDs). This has heightened popular awareness of the importance of diet in well-being (Barnes and Prasain, 2005). Diet has thus become a core component of public health plans geared toward preventing premature chronic diseases, promoting healthier aging, and maintaining optimum health throughout life. 3 © 2010 Taylor and Francis Group, LLC

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Biotechnology in Functional Foods and Nutraceuticals

This belief in the health benefits of foodstuffs is the basis for the current surge in interest in nutraceuticals and functional foods (Milner, 2002). Food has been used as a pharmaceutical agent to treat diseases since time immemorial and the pharmaceutical use of food is the basis of the concept of nutraceuticals (Klein et al., 2000). The term nutraceutical, a combination of “nutrition” and “pharmaceutical,” is generally defined as “a food or part of a food that provides medicinal and health benefits, including the prevention and/or treatment of a disease” (Ramaa et al., 2006). There is less agreement about a definition of functional foods; they have been defined in various ways, but at present there is no universally accepted definition. Nor is there likely to be, as foods which are not currently considered to be functional foods may come to be regarded as such in the future and functional food is therefore technically only a concept (Roberfroid, 2002). Functional foods have been broadly defined as “foods similar in appearance to conventional foods, which are consumed as part of a normal diet and have demonstrated physiological benefits and/or reduce the risk of chronic disease beyond basic nutritional functions” (Clydesdale, 1997). Although there is no clearcut distinction between functional foods and nutraceuticals, the basic difference between them is in the form in which they are presented. Functional foods are “foods” similar in appearance to conventional foods, or they may even be conventional foods, consumed as part of a usual diet, demonstrated to have physiological benefits and/or to reduce the risk of chronic disease beyond basic nutritional functions (Health Canada, 2002). Nutraceuticals, on the other hand, are dietary supplements that supply a concentrated version of a postulated bioactive agent extracted from a food, and are presented in a “nonfood matrix,” often in the form of capsules or tablets and utilized with the aim of promoting health in dosages that exceed those naturally present in foods (Espin et al., 2007). Largely as a result of consumers’ growing awareness of the health benefits of food, there has been a huge increase in the demand for nutraceuticals and functional foods. Modern agricultural and food manufacturing practices are orientated toward the production of value-added crops and manufactured food products that are not only nutritious, wholesome, and palatable, but which also have health enhancing and disease preventing benefits (Hsieh and Ofori, 2007). However, beyond this demand for foods with health enhancing qualities, consumers are looking for natural products. Consequently, not only are the ingredients themselves used to enhance food required to be natural but also the processes from which they are derived. Among the processes currently utilized for food production, biotechnology seems to offer a powerful way to produce all kinds of desired food materials while at the same time fulfilling the criteria of being natural (Senorans et al., 2003). The Convention on Biological Diversity (CBD) defines biotechnology as “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use” (CBD, 2000). Traditionally, biotechnology has been used to manufacture food products for more than 8000 years: examples include bread, alcoholic beverages, cheese, yoghurt, vinegar, and other foodstuffs produced using the enzymes inherent in various microorganisms. In recent years new techniques have become available and these forms of modern biotechnology are commonly referred to as “genetic engineering” or, from the scientific perspective, “recombinant DNA technology.” The CBD defines modern biotechnology as “the application of (a) in vitro nucleic acid techniques, including recombinant DNA and direct injection of nucleic acid into cells or organelles or (b) fusion of cells beyond the taxonomic family that overcome natural physiological reproductive or recombination barriers and that are not techniques used in traditional breeding and selection” (CBD, 2000). As in food production, a major revolution has resulted from the use of modern biotechnology in agriculture. In plant breeding, biotechnology can be used to introduce new characteristics with specific benefits into plants in a far more selective, controlled, and precise manner than is possible using traditional plant breeding techniques. Early applications focused on the production of high yielding crop varieties to meet the demands of an ever-growing world population through the provision of crop varieties with improved agronomic qualities such as drought, disease, and insect resistance, but there may also be additional benefits. Insect-resistant corn (Bt corn) sustains relatively minimal insect damage and is therefore much less prone to infection by fungi and molds compared to non-insect-resistant (non-Bt) corn.


Advances in Biotechnology for the Production of Functional Foods

5

As a consequence, the level of toxins such as aflatoxin, which is carcinogenic for both humans and livestock, produced by these pathogens is much lower in Bt corn than in non-Bt corn. These crops with improved agronomic qualities are referred to as the first generation of genetically modified (GM) crops, the benefits of which are not always immediately apparent to consumers. More recent advances in modern biotechnology have been concerned with the production of crops and food animals endowed with additional nutritional and health benefits that are more obvious to the consumer. Even conventional breeding programs have begun to shift towards the production of crops with enhanced health benefits, partly as a result of the reservations that certain individuals and populations have regarding GM foods. These benefits involve improvements in food quality and safety, as well as providing consumers with foods that are specifically designed to be more nutritious and beneficial to health (Chassy et al., 2004). As this chapter will demonstrate, functional foods not only address the serious problem of global malnutrition but can also provide consumers with a wide range of palatable food options on grocery shelves for the treatment or prevention of ailments. Examples of selected crops genetically engineered to enhance their nutritional content are listed in Table 1.1. A representative selection of the functional food materials that have been developed to date will be examined in turn, including the technologies, the rationale behind their development, and their benefits, as well as future goals.

1.2 1.2.1

BIOTECHNOLOGY FOR THE PRODUCTION OF PLANT-BASED FUNCTIONAL FOODS BIOFORTIFICATION WITH ESSENTIAL MICRONUTRIENTS

Micronutrient deficiency is a major global health problem, with over two billion people in the world today estimated to be lacking in key vitamins and minerals, particularly vitamin A, zinc, iron, and iodine (WHO/WFP/UNICEF, 2001). Biofortification of staple foods, which involves the genetic engineering of foods to provide varieties that contain higher than normal amounts of healthpromoting nutrients, is seen as a new and better approach to combating nutritional deficiencies, and is likely eventually to replace traditional techniques such as supplementation and fortification (Haas et al., 2005). Biofortification is an attractive option, as traditional methods suffer from serious shortcomings, including the high costs involved and the difficulty of enabling individuals in remote areas to gain access to these improved foods. An effective supplementation and fortification program is dependent upon political stability, which is often lacking in the areas where such nutritional intervention programs are most needed. Also, although fertilization of crops to increase mineral concentrations in the edible portions does indeed lead to increases in leaf mineral concentrations and improved yield, this does not always appreciably increase the mineral concentrations in the fruit, seed, or grain. In addition, fertilizers can be costly, both economically and environmentally, and must be reapplied at intervals (White and Broadley, 2005). Another advantage of biofortification is that it does not require a change in behavior by either farmers or consumers (IFPRI, 2002). While concerted international efforts have been made for decades to alleviate micronutrient malnutrition, transgenic approaches can complement ongoing breeding efforts and provide urgently needed biofortified crops to feed the burgeoning world population with nutritious food (Mayer et al., 2008). Rice is unquestionably the most significant food crop in the world, with over 50% of the world’s population depending on rice as their daily staple food (Zimmermann and Hurrell, 2002). A great deal of work has gone into breeding better varieties of rice to keep pace with growing demand. Since rice is the staple food for most of the developing world, improving its nutritional value through biofortification can also help address the problem of malnutrition (Bajaj and Mohanty, 2005). Efforts to improve the nutritional value of other staple food crops such as wheat, corn, and cassava through biofortification continue as a part of programs to address global malnutrition, which predominantly affects the poorest regions of the world where these crops are consumed as staples. Biofortification of these staple crops has mainly focused on increasing vitamin A, zinc, and iron levels, because


6


TABLE 1.1 Examples of Selected Staple Crops Genetically Engineered to Enhance Their Nutritional Content Target Nutrient

Enhancement

Technology

References

Rice Provitamin A

Increase in protein content and decrease in starch content

Ye et al. (2000), Paine et al. (2005)

Lucca et al. (2002)

Vasconcelos et al. (2003)

Lee et al. (2003)

Wu et al. (2003)

Maize Insertion of a construct SAG12-IPT gene consisting of the Young et al. (2004) cytokinin-synthesizing isopentenyl transferase (IPT) gene and the cysteine protease gene SAG (senescence associated gene) 12, into maize


Introduction of the entire b-carotene biosynthetic pathway into the rice endosperm through the insertion of two genes, phytoene synthesis (psy) and phytoene desaturase (crt 1), using Agrobacterium-mediated transformation Iron Increase iron content of the seeds by 2-fold compared to Insertion of ferritin (pfe), metallothioneinlike (rgMT) and phytase normal rice coupled with increased bioavailability of iron (phyA) genes into rice embryos through Agrobacterium-mediated in the seeds through enhanced iron absorption transformations. Pfe gene promotes accumulation of iron in the gene whereas the rgMT gene and phyA gene enhance the absorption of the iron and reduce the amount of the iron absorption inhibitor (phytic acid), respectively, thereby increasing bioavailability of the iron Zinc Increase in the zinc content of IR68144 (a conventionally A biolistic-mediated method using a construct of the plasmid bred high iron variety) from 34 ppm to amounts ranging pGPTV bar/Fer which encodes for the soybean ferritin protein from 36.2 to 55.5 ppm. High zinc content even after from Glycine max L, and the endosperm-specific promoter Glu polishing B-1 Sulfur containing Increase in the content of the sulfur containing amino acids, Insertion of a chimeric gene pGlu2S, encoding a precursor amino acids (cysteine methionine (29–76%), cysteine (31–75%), and crude polypeptide of the sulfur-rich seed storage protein, sesame 2S and methionine) protein content (0.64–3.54%) albumin (S2SA), into rice using Agrobacterium-mediated transformation Lysine Increase in lysine content of seeds by 0.9–6.6% Plasmid DNAs with tRNAlys genes coding lysine instead of glutamine (Gln), glutamic acid (Glu), and asparagine (Asn); or coding lysine at the chain termination codon, was introduced into rice callus through particle bombardment

Protein

Accumulation of carotenoids in the endosperm of rice

Multivitamins: vitamin A vitamin C folate Iron

Provitamin A

Protein

Methionine

Protein, zinc, and Iron

Increase in lysine content by 16.1–54.8% and a total protein A plasmid vector containing the sb401 gene under the control of a content by 11.6–39.0% storage protein promoter (P19z) that directs seed-specific expression was introduced into maize calli using microprojectile bombardment. A gene from potato which encodes a pollenspecific protein with high content of lysine Increase in vitamin A, vitamin C, and folate content of the Corn variety M37W was transformed by bombardment with metal transgenic corn by 112-, 6-, and 2-fold, respectively particles coated with five constructs made up of genes encoding enzymes in the biosynthetic pathways for β-carotene, vitamin C, and folate under the control of promoters Simultaneous expression of ferritin and phytase led to both Insertion of the plasmids pSF2 (expressing the soybean ferritin an increase in the overall iron content (20–70%) and its gene and pLPL–phyA (expressing the phytase gene) into maize bioavailability as a result of an increase in phytase callus by particle bombardment expression (up to 3 IU/g of seed) which reduced levels of phytic acid

Yu et al. (2005)

Naqvi et al. (2009)

Drakakaki et al. (2005)

Potato A total carotenoid increase of up to 2.5-fold and an increase Tuber-specific silencing of a key gene, lycopene ε-cyclase (LYC-e) Diretto et al. (2006) in β-carotene levels up to a maximum of 14-fold in potato in a branched competitive pathway in the biosynthesis of tubers carotenoids which is responsible for the production of lutein instead of carotenoids. The plasmid pBI33:As-e was introduced into potato (Desiree variety) through Agrobacterium-mediated transformation Increase in total protein content by 35–45% and an increase Plasmids were constructed from a gene that encodes the seedChakraborty et al. (2000) in all essential amino acids by 2.5- to 10-fold. specific protein, amaranth seed albumin (AmA1) from Amaranthus hypochondriacus, and the GBSS tuber-specific promoter gene. Plasmids were then inserted into potato shoots using Agrobacterium-mediated transformation Dancs et al. (2008) Increase in the content of free and bound methionine by Cotransferring cystathionine γ-synthase (CgSΔ90) and methionine2- to 6-fold. Also an increase in the amounts of soluble rich storage protein (15-kD β-zein) genes in potato (Desiree isoleucine and serine variety) using Agrobacterium strains Wheat Increase in the amount of protein, zinc, and iron in the grain Insertion of the gene, dubbed gpc-B1, from a wild emmer wheat by 10–15% into conventional wheat plants



Lysine

Uauy et al. (2006)

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deficiencies in these nutrients account for the majority of the problems in micronutrient-deficient individuals (WHO/WFP/UNICEF, 2001). Biofortification of staple foods with iodine is virtually nonexistent, as consumption of iodized salt is easily the best way to prevent iodine deficiency disorders. The following sections will discuss examples of biofortification with the micronutrients vitamin A, zinc, and iron, focusing primarily on rice. More information on rice biotechnology can be found in the recent review by Bajaj and Mohanty (2005). Other staple crops have also been the target of biofortification with micronutrients. HarvestPlus, a nongovernment organization committed to the development of biofortified crops for malnourished populations, is biofortifying seven key staple crops that will have the greatest impact in alleviating micronutrient malnutrition or hidden hunger in Asia and Africa. These crops are beans, cassava, maize, pearl millet, rice, sweet potato, and wheat. The countries that their current projects are targeting include D. R. Congo, Rwanda, Nigeria, Zambia, Uganda, Mozambique, India, Bangladesh, and Pakistan (HarvestPlus, 2009). 1.2.1.1 Vitamin A Although rice plants do possess carotenoids in their photosynthetic tissues they are absent in the endosperm, which is the edible part remaining after rice has been milled to remove the oil-rich aleurone layer that turns rancid during storage. Because the endosperm is lacking in carotenoids, vitamin A deficiency (VAD) tends to be a serious health issue in those parts of the world where rice is consumed as the staple food, namely Asia, Africa, and Latin America. In Asia, for example, more than 180 million children and women suffer from VAD (Chong, 2003). The development of golden rice (GR) was motivated by the need to alleviate VAD, which represents a major global health problem. GR is the generic name given to types of GM rice that produce β-carotene (a precursor of vitamin A) in the endosperm. It is so called because the yellow color of the grain, which can be ascribed to the high content of carotenoid, is easily discernible after milling and polishing. GR was initially produced by modifying the Japonica variety Taipei 309. β-Carotene is naturally synthesized in the vegetative tissues of rice rather than in the endosperm, which lacks two of the steps in the biosynthetic pathway. These two steps are controlled by two genes, namely phytoene synthase (psy) and phytoene desaturase (crt 1). GR technology is based on introducing the entire β-carotene biosynthetic pathway into the rice endosperm through the insertion of these two genes (psy and crt 1) using an Agrobacterium-mediated transformation (Ye et al., 2000). This technology was later also shown to work with other rice cultivars (Datta et al., 2003). The archetypal technology, however, needed improvement in order to increase the amount of carotenoid available per gram of rice, as it could only produce a maximum total carotenoid level of 1.6 μg per gram dry weight of rice (Al-Babili and Beyer, 2005; Al-Babili et al., 2006; Grusak, 2005), of which about half was in the form of β-carotene (Grusak, 2005). The children most at risk of VAD would therefore need to consume unrealistic amounts of GR in order to meet their recommended daily intakes of vitamin A equivalents. Further research led to the development of a new generation of GR (referred to as GR 2) using an enzyme from maize which increased grain carotenoid levels more than 23-fold. The use of this enzyme overcomes a bottleneck in β-carotene synthesis. GR 2 contains levels of total carotenoids of up to 37 μg per gram, of which a very high proportion is β-carotene. GR 2 development involved the replacement of the daffodil psy gene originally used with the equivalent gene from maize because psy, one of the two genes used in the development of GR, was found to be the limiting factor in β-carotene accumulation in the endosperm of rice. Psy from maize was selected after systematic testing of psy from various other plants in a model plant system because of its ability to substantially increase carotenoid accumulation (Paine et al., 2005). GR 2 has the potential to provide 50% of the Recommended Dietary Allowance (RDA) for vitamin A, though the overall bioavailability would depend on the presence of dietary oils and proteins (Anonymous, 2005). 1.2.1.2 Iron Unlike VAD, iron deficiency, which is the most widespread nutritional deficiency in the world, is a major problem both in developed and developing countries, though it is 3–4 times more prevalent in © 2010 Taylor and Francis Group, LLC


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developing countries (HarvestPlus, 2007). As with β-carotene, the removal of the outer layer (which includes the aleurone layer) of the rice seed during commercial milling decreases the iron content of rice (Doesthale et al., 1979), because most of the iron in rice accumulates in the aleurone layer. The average iron content of polished rice is 2 ppm, although some varieties naturally have high iron content (IRRI, 2006). Through the use of biotechnology, significant progress has been made to enhance the iron content of rice. Ferritin, an iron-storage protein found in both plants and animals, has been found to provide a good source of iron when orally administered to rats suffering from anemia (Beard et al., 1996). This finding suggests that increasing the ferritin content of cereals through genetic engineering may be a good way to solve iron deficiency problems. Based on this hypothesis, Goto and coworkers (1999), introduced the soybean ferritin gene into rice plants by Agrobacterium-mediated transformation. This was done under the control of a rice seed storage protein glutelin promoter, GluB-1 (−1302/ +18), to ensure that the iron accumulated specifically in the grain. Transgenic seeds that accumulated the soybean ferritin in the endosperm could store up to three times more iron than normal seeds. The International Rice Research Institute (IRRI) based in the Philippines has produced an iron dense rice, IR68144, through conventional breeding by crossing a high-yielding variety (IR72) with a tall traditional variety (Zawa Bonday) containing a relatively high iron content (4.1 ppm) in the grain (IRRI, 2006). The IRRI whole grain had an iron content of 21 ppm, which is about double the normal level in rice. Scientists have also found that after polishing for 15 min, IR68144 had approximately 80% more iron than a well known but low-iron commercial variety (ISIS, 2004). Human studies using women with low iron stores proved that the consumption of IR68144 increased body iron by 20% (IRRI, 2006), indicating that increased iron content does indeed translate into enhanced iron status in the consumer. IR68144 was also found to have a high zinc content (34 ppm), which is not surprising because research has shown that zinc and iron densities are positively correlated. It has also been reported that IR68144 combines a high vitamin A content and a high yield with good flavor, texture, and cooking qualities (ISIS, 2004). Vasconcelos and coworkers (2003) were able to transform IR68144 with the soybean ferritin gene driven by the glutelin promoter to obtain transgenic rice plants with an even higher content of iron and zinc, even after polishing. Transformation of IR68144 was achieved through a biolistic-mediated method using a construct of the plasmid pGPTV bar/Fer (which encodes for the soybean ferritin protein from Glycine max L.) and the endosperm-specific promoter Glu B-1. The amount of iron that is bioavailable depends both on iron intake and absorption, and hence increasing the iron content of foods will not necessarily translate into a proportional increase in absorbed iron. In the developing world, dietary iron mainly comes from nonheme sources such as grains and legumes, which are high in phytic acid, a recognized potent inhibitor of iron absorption (Hurrell et al., 1992). Thus, increasing iron intake through the provision of iron-enriched crop varieties will not solve iron deficiency problems unless the diet is also low in iron absorption inhibitors such as phytic acid or contains components that enhance the absorption and utilization of iron. Lucca and coworkers (2002) addressed this problem through the insertion of ferritin (pfe), metallothionein-like (rgMT), and phytase (phyA) genes into rice embryos through Agrobacterium-mediated transformations. Regenerated plants expressing these three genes showed a 2-fold increase in the iron content of seeds compared to negative controls. The plants also showed an increase in phytase activity of as much as 130-fold for some of the plant lines compared to controls. About 90% of phytase activity was retained after incubating the transgenic ground rice under stomach conditions, and the phytic acid content of the seed was greatly reduced. Other studies have shown that cysteine and cysteine-containing peptides obtained from meat have the capacity to enhance the absorption of nonheme iron by binding the iron through its thiol group (Layrisse et al. 1984; Taylor et al., 1986). Thus, by overexpressing a cysteine-rich protein, metallothionein, through the insertion of the rgMT gene, the cysteine content of the seed protein in the endosperm could be increased to levels that would further enhance iron bioavailability in the transgenic rice (Lucca et al., 2002). © 2010 Taylor and Francis Group, LLC

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Micronutrients are as essential for plant life as they are for humans, and abnormalities in plant growth and development occur when they are not available in optimum quantities. Globally, about 30% of the cultivated soils are considered calcareous, with low iron availability, because the iron present is only sparingly soluble in the soil solution. Crops grown on these soils are therefore also deficient in iron (Takahashi, 2003). Hence, another approach to tackling the iron deficiency problem is to produce crops that are tolerant to low iron availability, thus ensuring that crops grown on calcareous soils are not iron deficient. Plants have naturally developed mechanisms to cope with iron deficiency stress. One such mechanism adopted by graminaceous plants (which include rice) is to release phytosiderophores, which form a soluble complex with the inorganic iron in the soil by chelation which can then be absorbed by the roots (Curie et al., 2001). The level of tolerance exhibited by crops under iron deficiency stress is directly proportional to the amount of phytosiderophores produced and secreted into the soil (Takagi et al., 1984). Compared to other members of the graminaceous family (wheat, barley, rye, oats, sorghum, and maize), rice has the least tolerance in iron deficient situations because it produces only small amounts of phytosiderophores due to low activity of the nicotianamine aminotransferase (NAAT) gene responsible for the production and secretion of phytosiderophores. Barley has the highest tolerance among plants in this group (Römheld and Marschner, 1990). Takahashi (2003) produced GM rice with enhanced NAAT activity by purifying the NAAT protein from barley roots and inserting it into rice using the vector pBIGRZ1, which permits the expression of inserted genes under regulation by their own promoters. The transgenic rice plants harboring the NAAT gene from barley exhibited enhanced phytosiderophore release and consequently greater tolerance to soils with low iron availability. 1.2.1.3 Zinc Zinc deficiency occurs both in crops and humans, causing decreased crop yields and predisposing humans to diseases (Hotz and Brown, 2004; Welch and Graham, 2004). It is prevalent in regions whose soils are deficient in zinc, resulting in crop plants that are zinc deficient (Cakmak et al., 1999; Hotz and Brown, 2004). Fifty per cent of cereal growing areas suffer from soils with low plant availability of zinc (Cakmak, 2002). As with other micronutrients, most of the zinc present in seed is located in the embryo and aleurone layers, leaving the endosperm with a very low zinc concentration (Ozturk et al., 2006). Thus, milling to remove the embryo and aleurone layers further reduces the concentration of zinc in cereals. At present, polished rice has an average zinc content of 12 ppm (IRRI, 2006). Enrichment of cereal grains with zinc has become a high-priority area for research as part of the effort to minimize zinc deficiency and its associated health problems. Although originally developed to boost iron content, as discussed in the previous section, IR68144 (IRRI, 2006) and its improved transgenic variety (Vasconcelos et al., 2003) are also examples of GM zinc-enriched rice varieties. The IR68144 improved variety was found to have a zinc content of 34 ppm (ISIS, 2004) and the four lines obtained from the GM IR68144 have zinc contents ranging from 36.2 to 55.5 ppm, which compare favorably to the average rice zinc content of 20 ppm (Vasconcelos et al., 2003). Because iron and zinc concentrations are positively correlated, selecting for high-iron varieties will also tend to select for high-zinc varieties, as seen in the examples above.

1.2.2

BIOFORTIFICATION WITH PHYTOCHEMICALS

Phytochemicals, also referred to as phytonutrients, are a group of plant-based chemicals that have been identified as being active in disease prevention. Among their health benefits are maintaining bone and joint health (Cerhan et al., 2003; Wattanapenpaiboon et al., 2003), cancer prevention (Manson et al., 2007), and lowering serum cholesterol levels (Lerman et al., 2008). Phytochemicals are found notably in fruits and vegetables and fall under several categories, such as terpenoids or isoprenoids, which include: the carotenoids found in carrots and tomatoes, respectively; polyphenolics, which include the isoflavones and anthocyanins found in soybeans and green tea, respectively;



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and glucosinolates, which include the indoles and isothiocyanates found in broccoli and mustard, respectively. The amount of these phytochemicals varies greatly in plants, and levels in some plants may be either low or nonexistent. Much research has been done in this area to improve the phytochemical composition of certain food crops by either increasing their levels or preventing their degradation and/or conversion to nonbiologically active compounds. Efforts have also been made to ensure phytochemicals are available in widely consumed plants that do not naturally accumulate them. Biofortification is, therefore, not only used to increase levels of essential micronutrients in staple foods to address global malnutrition related deficiencies, but also to provide varieties that contain higher than normal amounts of phytochemicals to address particular ailments. Epidemiologic studies have demonstrated that the high consumption of foods derived from soybeans is linked to a low incidence of hormone-related cancers, menopausal symptoms, osteoporosis, and CVDs (Cornwell et al., 2004; Dixon and Ferreira, 2002; Watanabe et al., 2002). This protective action of soybean-derived food products is ascribed to the isoflavonoid content of soybean. This has led to an extensive research effort to biofortify more widely consumed foods such as vegetables, grains, and fruits with isoflavonoids in order to enhance the dietary intake of these compounds and thus their associated health benefits. Genistein, which is the backbone of all isoflavonoids, is produced from the dehydration of 2-hydroxyisoflavanone, which may occur spontaneously or through the catalytic action of dehydratase. Formation of 2-hydroxyisoflavanone, which represents the first step in the biosynthesis of isoflavonoids, is catalyzed by the enzyme 2-hydroxyisoflavanone synthase, commonly referred to as IFS. Cloning of genes encoding IFS from several leguminous plants, including red clover, licorice, and soybeans, has made it possible to synthesize isoflavones in plants that do not ordinarily accumulate such metabolites (Liu et al., 2007). The consumption of onions and related alliums such as garlic and leeks is associated with the lowering of serum cholesterol (Gorinstein et al., 2006; Vidyashankar et al., 2008) and is also known to have a protective effect on platelet aggregation (Chang et al., 2004; Hubbard et al., 2006), which translates into a reduced risk of CVDs. Garlic and onion extracts have also been reported to have antibiotic (Tsao and Yin, 2001; Yin and Tsao, 1999), antitumor (Li et al., 2002), antioxidant (Campos et al., 2003, Drobiova et al., 2009), hypoglycemic (El-Demerdash et al., 2005), and antithrombotic properties (Fukao et al., 2007; Yamada et al., 2004). The health-promoting properties of alliums have been linked to several organosulfur containing compounds. For example, the hypocholesterolemic effect of onion and garlic is attributed to S-methylcysteine sulfoxide (SMCS) (Augusti and Mathew, 1974; Sainani et al., 1979). When onion is broken through smashing or cutting, amino acid sulfoxides and a particular enzyme (lachrymatory factor synthase) are released. This enzyme catalyzes the conversion of the sulfoxides into a vapor form, which enters the eyes and causes the tears associated with the cutting of onions. The discovery of the gene responsible for triggering this tear production has led to the production of a GM onion that does not produce tears, by turning off the gene that expresses the enzyme. The GM onion was created by Dr. Colin Eady at the New Zealand Crop & Food Research Laboratory. Shutting down the gene was achieved using the process of RNA interference. By turning off the lachrymatory factor synthase gene, valuable sulfur compounds which otherwise would have been converted to the tear-causing agent and lost are instead made available for redirection into compounds that are known for their flavor and health properties (Environmental Graffiti, 2008). Individuals who avoid cooking with onions as a result of the tear factor now have an alternative that will enable them to reap the health benefits that onions have to offer.

1.2.3

MODIFICATION OF MACRONUTRIENTS

1.2.3.1 Oils CVD is the leading cause of death in the United States, with statistics indicating that CVD causes approximately 1.4 million deaths annually (Johnson et al., 2007). The fatty acid (FA) profile of the


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diet is the predisposing risk factor for CVD, with the main culprits being saturated FAs (SFAs), trans-FAs, and cholesterol. Unlike SFAs, monounsaturated FAs (MUFAs) and polyunsaturated FAs (PUFAs), especially omega-3 PUFAs, convey several health benefits. Consequently, there is a steadily increasing demand for unsaturated FAs (UFAs) to be incorporated in the diet and in addition to the search for alternative sources, the FA profiles of certain oils are being modified to increase their levels of unsaturated fats and decrease their saturated and transfat content in order to produce even healthier oils and products made from them. 1.2.3.1.1 Oils with Healthier FA Profile The essential FAs linoleic and α-linolenic acid, which are C18 PUFAs, may be metabolized into very long chain PUFAs (VLCPUFAs) (C20 and C22) in the body once consumed, although the conversion process is slow and inefficient compared to the direct consumption of these VLCPUFAs in the form of fish oils (Domergue et al., 2005). Nutritionally, the most important VLCPUFAs include arachidonic acid (AA, C20H32O2), eicosapentanoic acid (EPA, C20H30O2), and docosahexaenoic acid (DHA, C22H32O2). At present fish oil is the major source of EPA and DHA. The machinery and materials necessary for the biosynthesis of a wide range of FAs from conventional oilseed crops is already in place, but they lack several additional enzymes (certain fatty-acyl desaturases and elongases) that are necessary for the biosynthesis of VLCPUFAs (Abbadi et al., 2004). Various genes responsible for the biosynthesis of PUFAs have been cloned from organisms such as fungi, algae, mosses, higher plants, and mammals (Warude et al., 2006). The use of these isolated genes allows the manipulation of plants to enhance their PUFA profile. Because of the continuing decrease in marine resources as a result of overfishing, coupled with the environmental impact of fish farming, neither farmed nor wild fish represent a sustainable source of the VLCPUFAs necessary to ensure healthy nutrition for the ever growing world population (Abbadi et al., 2004). In addition, it has been recommended that the consumption of many types of fish be limited because of widespread contamination with pollutants such as heavy metals and dioxins (America.gov, 2004). Making VLCPUFAs available in nonfish sources (notably in the form of annual oilseed crops) would be a sustainable solution to this imminent depletion of our supply of VLCPUFAs from fish, and would also avoid exposure to toxic environmental contaminants such as dioxin. Abbadi and coworkers (2004) have produced linseed and tobacco plants that synthesize VLCPUFA in their seed by introducing genes for fatty-acyl desaturases and elongases obtained from a variety of organisms that produce VLCPUFAs. The best results were obtained using plant and algal gene sequences. The transgenic plants accumulated significant levels of the VLCPUFAs (5% of C20 VLCPUFA including AA and EPA) in their seed. The researchers were able to identify bottlenecks in the accumulation of desirable PUFAs, which paves the way for further research aimed at improving the levels of VLCPUFAs (Abbadi et al., 2004). This achievement holds promise for the production of healthier and more nutritious oils for human consumption. Also, the use of such oils in the production of animal feed could improve the PUFA content of animal products such as eggs, meat, and dairy food. In a further development, scientists at Rothamsted Research Institute in Hertfordshire, UK have genetically engineered plants to produce fish oils. Key genes were first isolated from a species of microscopic single-celled marine algae known as Thalassiosira pseudonana. The isolated genes were then inserted into crops such as linseed and oil seed rape (canola) and the transgenic plants were found to synthesize omega-3 PUFAs in their seed oils. The researchers are currently working to optimize and improve the levels of omega-3 PUFAs produced by these transgenic plants (BBC News, 2007). 1.2.3.1.2 Stearic Acid Rich Oils Although SFAs have been shown to raise serum total cholesterol and consequently increase the risk of CVD, recent studies have indicated that stearic acid (C18H36O2) has no effect on total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol (Yu et al., 1995), when



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compared to other SFAs such as palmitic (C16H32O2), lauric (C12H24O2), and myristic (C14H28O2) FAs (Muller et al., 2001). Bakery products usually require the use of solid fat to provide the desired functionality. To date, the choices of solid fats have been largely limited to animal fats such as lard and butter and tropical fats from palm kernel and coconut, both of which are hypercholesterolemic due to their high contents of palmitic, lauric, and myristc FAs. The other solid fat options, namely partially hydrogenated oils, are also harmful due to the transfats they contain. Food manufacturers are, therefore, faced with the challenge of finding healthier alternatives capable of replacing animal fats and tropical oils without compromising quality and functionality (Dirienzo et al., 2008). Stearic acid rich oils are one possible alternative being considered (Kris-Etherton et al., 2005). Facciotti and coworkers (1999) have reported the development of a type of canola oil that contains almost 40% stearic acid compared to the 1% of naturally occurring canola oil. Their success was based on earlier work by Hawkins and Kridl (1998), who isolated an enzyme that helps make stearic acid from the tropical plant mangosteen, whose seeds contain large amounts of stearic acid. They then inserted the gene for this enzyme, which belongs to a family of enzymes known as thioesterases, into the canola plant. The enzyme allows stearic acid to accrue in the plant by disrupting the biosynthesis of oleic acid, which accounts for most of the fat available in commercial canola oil. Both oleic and stearic acids have 18 carbon atoms, but the former contains a double bond whereas the latter has no double bond. In the course of the biosynthesis of these two FAs, an enzyme called desaturase creates the double bond to form oleic acid when the carbon chain reaches 18 atoms. The lengthening of the carbon chain is accomplished by various thioesterases; the thioesterase obtained from the mangosteen releases stearic acid before desaturase can convert it into oleic acid, resulting in the accumulation of stearic acid. However, although the amount of stearic acid produced increased, the content was still insufficient. Facciotti and coworkers (1999) created mutants of this gene (Garm FatA1) and tested them to determine which of their enzymes were most active in producing stearic acid. The best performing genes were then introduced into canola plants, resulting in oils that contain almost 40% of stearic acid. 1.2.3.1.3 Healthier and More Stable Cooking Oils Though PUFAs are nutritionally highly valuable, they are easily oxidized and readily break down (turn rancid) in storage and under extreme heat, rendering them unsuitable for cooking, especially in deep frying situations. To enhance their thermal stability, oils are partially hydrogenated to reduce the level of unsaturation. In the process, however, transfats are formed. Because of the deleterious health effect of transfats, the Food and Drug Administration (FDA) makes it mandatory for food manufacturers to list all transfat content on product labels (21 CFR Part 101) (FDA, 2003). There is, therefore, a trend toward producing transfat free but thermally stable cooking oils to increase their acceptability and utility. Until relatively recently, rapeseed was a fairly minor crop due to its natural content of erucic acid (C22H42O2) and glucosinolates. The bitter taste of erucic acid prevented its use in food and the toxic effect of the glucosinolates that remain after the pressing of rapeseed meal prevented its use in animal feed. However, modern plant breeding has led to improved rapeseed cultivars free of erucic acid and glucosinolates, and these new cultivars are referred to as “double zero.” “Double zero” rapeseed was developed in Canada and was renamed “canola” (Canadian oil, low acid) to distinguish it from nonedible rapeseed. As a result of its high-level content of MUFAs (60–70%) and PUFAs (about 22%), coupled with its low-level content of SFAs (about 7%) (Canola Council, 2008; GMOCompass, 2008), rapeseed oil has become significant as a healthy cooking oil. Canola oil is now seen to have the potential to help consumers achieve dietary recommendations and hence reduce the risk of CVD. Compared to the other oils commonly consumed in the United States, namely soybean, peanut, sunflower, corn, cottonseed, palm, and olive oils, canola oil has the lowest concentration of SFAs. In addition, canola oil is abundant in MUFAs and is also the richest source of the omega-3 essential PUFA, α-linolenic acid, of any of these oils (Johnson et al., 2007). © 2010 Taylor and Francis Group, LLC

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The oil extracted from traditional varieties of canola has only limited cooking applications because of its relatively high proportion of PUFAs. Through genetic engineering, canola oil with increased oleic acid (C18H34O2) content and/or reduced PUFA content has been developed. Oleic acid is a MUFA which is thought to confer lower heart disease and cancer risk benefits on olive oil. Pioneer High-Bred International, Inc. has developed two novel canola lines, 45A37 and 46A40, which have high oleic acid content and low linolenic acid content. These were produced by first inducing mutagenesis by exposing canola varieties to a solution 8 mM ethylnitrosourea in dimethylsulfoxide to achieve the high oleic acid trait, followed by traditional breeding with canola varieties such as Apollo and Stellar to achieve the low linolenic acid trait. Oils processed from these novel lines are referred to as P6 canola oil and 45A37 and 46A40 have 24% higher levels of oleic acid and 40% or 75% lower levels of linoleic and linolenic acid, respectively, compared to traditional canola oil (Health Canada, 2000). On June 6, 2004, at the BIO conference in San Francisco, Dow AgroSciences LLC showcased its improved canola oil, dubbed NatreonTM. This new canola oil variety was produced using the latest tools in plant breeding technology (Dow AgroSciences, 2004). Natreon canola oil contains 7% saturated fats, 75% oleic acid, 3% linolenic acid, and 15% linoleic acid, and is virtually transfat free. In addition to its nutritional and stability qualities, Natreon canola oil has a neutral flavor and therefore does not interfere with the natural flavor of food (Dow AgroSciences, 2004; NRCC, 2002). Monola oil, produced by Nutrihealth Pty Limited in Melbourne, Australia is another example of high oleic canola oil. This was obtained from normal canola modified by traditional plant breeding, with no genetic engineering involved. Monola oil contains 6% SFAs (1% less than traditional canola oil), 70% oleic acid, 20% linoleic acid, and 2.6% linolenic acid (GRDC, 2007). Lowering the levels of PUFAs through an increase in the oleic acid content eliminates the need for partial hydrogenation as the oil is less liquid and less prone to rancidity, and results in the production of few or no trans-FAs. In this way a healthier, and at the same time thermally stable, cooking oil is produced. At present, as a result of its superior stability, high oleic acid canola oil is being used by manufacturers instead of hydrogenated vegetable oils in products such as breads, cakes, and potato chips, which results in more healthful and better quality products (AFIC, 2004). Other vegetable cooking oils including cottonseed, sunflower, safflower, soybean, peanut, and palm oils have also been produced through biotechnology to improve thermal stability as well as to boost health-enhancing qualities. Detailed information on the compositions and applications of commercially available GM oils are available and can be found in the book by Richard D. O’Brian (2008). 1.2.3.2 Proteins Corn is used globally as a major cereal crop for human nutrition and as feed for livestock. In developing countries, animal protein is scarce and costly, making it unavailable to an appreciable number of people in the population. Corn accounts for about 15–65% of total daily calories in about 25 developing countries, mostly in Africa and Latin America (FAO Agrostat, 1992). Several million people in these areas depend on maize for the great majority of their protein and calorie requirements. However, corn proteins, as with all cereal proteins, have poor nutritional value for humans and monogastric animals because of their low content of the essential amino acids lysine, tryptophan, and methionine (Mertz et al., 1964). On average, cereal proteins have a lysine content of only 2%, which is less than one-half of the concentration recommended for human nutrition by the Food and Agriculture Organization (FAO) of the United Nations (UN) (FAO/WHO/UN, 1985). Enriching the protein content of corn would therefore have a tremendous impact on the millions of poor and malnourished people in such regions of the world. One high-protein corn variety known as quality maize protein (QMP) was developed at The International Maize and Wheat Improvement Center (CIMMYT) in Mexico. This protein-rich corn variety was developed using traditional breeding techniques to incorporate a series of special genes to offset the undesirable side effects of the opaque 2 (o2) mutation without jeopardizing the valueadded protein trait (CIMMYT, 2000). o2 is a previously discovered mutation that endowed grain



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proteins in the endosperm with twice the nutritional qualities of normal maize owing to a 2-fold increase in the levels of lysine and tryptophan (Mertz et al., 1964). Utilization of o2 in breeding programs in the past, however, was fraught with problems such as a soft endosperm that resulted in damaged kernels, increased susceptibility to pests and fungal diseases, lower yields, and inferior food processing qualities, none of which were easily overcome (Bjarnason and Vasal, 1992). QMP has a similar yield and agronomic performance to regular corn, but with 30% more lysine, 55% more tryptophan, and 38% less leucine. The higher tryptophan and lower leucine content leads to higher niacin availability. Only 34% of regular corn protein intake is utilized compared to 74% of the same amount of QMP. Comparing the nitrogen balance of QMP to skimmed milk indicates that the protein quality of QMP is 90% of that of milk (Graham et al., 1980). Other nutritional benefits of QMP include higher calcium and carbohydrate content and increased carotene utilization (Prasanna et al., 2001). In a related development, a maize breeder (Raman Babu) working at the Indian Council for Agricultural Research (ICAR) used a combination of biotechnology and conventional methods to further improve the quality of QMP. This was achieved by crossing lines of QMP with the parents of a popular normal hybrid known as Vivek Hybrid-9. Molecular markers were then used to quickly select the offspring that contained both the desirable parentage of the original hybrid and the quality protein trait. Using this procedure, they were able to develop the QMP hybrid in less than half the time it would have taken if only conventional selection methods had been employed. The qualities of this new hybrid, in addition to the high protein content it derives from the QMP line, are that it is early maturing compared to both QMP and Vivek Hybrid-9 and out-yields both parents. This is of particular importance, as years of research to develop a variety that could out-yield Vivek Hybrid-9 had previously been unsuccessful (cgiarNews, 2005). Other biotechnological means have also been employed to increase the protein content of maize. Programmed cell death is widely utilized during plant development to discard tissues or organs that are no longer needed, to adjust to environmental changes or to alter existing organs (Young et al., 2004). Every corn kernel results from a flower on an ear of a corn. During the developmental process the ear produces a pair of flowers for every kernel. However, through programmed cell death one of the pair of flowers is aborted, leaving a single flower for each group (ScienceDaily, 2005). By introducing the cytokinin synthesizing isopentenyl transferase (IPT) gene under the control of the Arabidopsis senescence-inducible promoter from the cysteine protease gene SAG (senescence associated gene) 12 in tobacco leaves, it has been shown that cytokinin plays a role in regulating entry into a senescence program (Gan and Amasino, 1995). Although programmed cell death can occur at both early and later stages of development whereas senescence occurs only at later stages of development and is thus developmentally separate, similar hormones may regulate entry into either program (Young et al., 2004). Based on this information, Young and coworkers (2004) set out to investigate whether cytokinin might affect the abortion of corn floral organs through the introduction of the SAG12-IPT construct into maize. The insertion of the construct essentially rescues the aborted flower, resulting in the fusion of two embryos in one kernel. This hinders the growth of the endosperm, resulting in kernels with an increased ratio of embryo to endosperm content. Because much of the oil and protein reserves available in the maize grain are stored in the embryo, this fusion event, generating kernels composed of two embryos with diminished endosperm content, increases the contribution of these storage reserves, thereby improving the nutritional value (i.e., high protein and low starch) of this corn variety. The reduced starch content that results from the decreased endosperm in this corn variety, if applied to sweet corn, would appeal to the large number of weight-watchers who are interested in low-carbohydrate diets and hence normally avoid corn in their diets. Despite the fusion the kernels are no bigger than normal and look just like ordinary corn, but are nutritionally superior (ScienceDaily, 2005). As noted above, the poor nutritional value of corn arises due to its low content of the essential amino acids lysine, tryptophan, and methionine. The poultry industry spends over $1 billion annually on synthetic methionine as a dietary supplement in feed (ScienceDaily, 2002). It is therefore imperative that the levels of methionine in corn be increased, not only to address global


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malnutrition problems in areas that are dependent on corn as a staple, but also to reduce the costs involved in breeding and raising livestock and poultry where maize is used as a major component of animal feed. Two researchers (Jinsheng Lai and Joachim Messing) at the Waksman Institute of Microbiology, at Rutgers University in New Jersey, found a way to increase the methionine content of corn. This was achieved without the addition of foreign DNA by first isolating the gene for methionine and then adjusting the genetic signals that control the synthesis of methionine. Through this manipulation the researchers were able to increase the plant’s ability to produce more of its own naturally occurring protein (Lai and Messing, 2002; ScienceDaily, 2002).

1.2.4

PRODUCTION OF HYPOALLERGENIC FOODS

Soy offers several health benefits such as the potential to reduce the incidence of coronary heart diseases (CHD), prostate cancer and breast cancer, as well as the ability to decrease and increase LDL and HDL cholesterol levels, respectively. The antioxidant properties of soy isoflavones have beneficial effects on the function of blood vessels and also protect LDL from oxidation (Chema et al., 2006). Thus, on 26 October 1999, the FDA published a final rule (64 FR 57700) which approves the health claim for soy protein as reducing the risk of CHD (FDA, 1999). Increased awareness of the health benefits of soy consumption, as well as its functional properties as a food ingredient, has led to widespread utilization of soybean products in a diverse range of food products. However, some individuals are allergic to soy proteins. The increasing use of soybean products in processed food products poses a potential threat to such allergic individuals. Therefore, recent research interests have been directed toward producing hypoallergenic soybeans and soy products. The development of hypoallergenic soybeans, and hence soy products, would not only protect sensitive individuals from allergic reactions, but would also enable allergic individuals to gain the aforementioned benefits of soy proteins, as at present avoidance of the food containing the allergenic moiety is the only treatment for individuals with allergy. The soybean proteins Gly m Bd 60K, Gly m Bd 30K, and Gly m Bd 28K are the main seed allergens in soybean-allergic individuals. Gly m Bd 30K (also referred to as P34), although accounting for only 1% of total seed protein, represents the major soy protein allergen. Biotechnology provides a way to eliminate undesirable proteins such as Gly m Bd 30K in order to enhance food safety and allows allergic individuals to avail themselves of the health benefits that such food items have to offer. The use of transgene-induced gene silencing to prevent the accumulation of Gly m Bd 30K in soybean seeds has been achieved. The resultant transgenic seeds do not accrue Gly m Bd 30K protein and the property and quality of the seed remain unchanged compared to controls despite the removal of this allergenic protein, indicating that it does not play a role in either seed protein processing or maturation (Herman et al., 2003). In a related development, Song and coworkers (2008) at the University of Illinois employed liquid and solid fermentation processes using benign microorganisms to produce hypoallergenic soy flour. They reported that allergenic protein production was lowered by as much as 97%, depending on the type of microorganism used. The bacterial species Lactobacillus plantarum, commonly found in sauerkraut, achieved the best hypoallergenic result. The fermentation process also had an added benefit of increasing the content of some essential amino acids, thereby improving the nutritional quality of the product. The product did, however, have a flavor typical of fermented oriental soy products such as soy sauce and miso soup. This may not pose a problem as Americans are fast becoming accustomed to these fermented soy sauce products already on the market. In other parts of the world, particularly Europe where GM foods are strongly opposed, removal of soy allergens without genetic engineering presents a viable alternative process (Song et al., 2008). Another area of research has been to reduce the allergenicity of other food varieties such as wheat and products derived from them, such as wheat flour. Protecting individuals who are allergic to wheat proteins is an enormous task, as many processed foods which are consumed daily contain wheat flour as an ingredient. The availability of hypoallergenic flour would thus greatly benefit



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these individuals. A great deal of research has therefore been devoted to this effort in order to meet consumer demand for hypoallergenic flour. Watanabe and coworkers (2000) have produced hypoallergenic flour by enzymatic fragmentation using the enzymes cellulase and actinase. Subsequent research proved that the allergy suppressive effect of the new allergenic wheat flour acted by hindering allergen absorption from the intestinal tract and inducing oral tolerance (Watanabe et al., 2004). Unfortunately this product has a consistency that makes processing by usual methods difficult and gelatinizing of the starch in the product and the addition of surfactants are necessary to make the hypoallergenic wheat flour suitable for food processing (Watanabe et al., 2000).

1.2.5

REDUCTION OF ANTINUTRIENTS

Antinutrients are plant compounds that decrease the nutritional value of plant food by making an essential nutrient unavailable or indigestible when consumed by humans or animals. They achieve this either by binding nutrients to make them unavailable or by inhibiting the enzymes needed for digestion. Phytic acid, the principal storage form of phosphorus in mature seeds or grains, is a major antinutritive factor in whole legume seeds and cereal grains. It is considered antinutritive because it limits the bioavailability of minerals such as zinc, iron, and calcium by forming indigestible chelates with these metals (Saha et al., 1994). It would therefore be useful to reduce the phytic acid content of foods in order to improve their nutritional value. In April 2002, the ARS–USDA and the Arkansas Agricultural Experiment Station released a low phytic acid mutant of rice known as KBNT lpa1-1. The mutation was induced by γ radiation of the Arkansas rice cultivar, Kaybonnet (KBNT) (Wells et al., 1995). The proportion of seed phosphorous tied up as phytic acid in KBNT lpa1-1 is reduced from 71% to 39% and the inorganic seed phosphorous is increased from 5% to 32%, with little effect on the total seed phosphorous (Larson et al., 2000). Rutger et al. (2004) produced the goldhull low phytic acid (GLPA) rice by hybridizing the low phytic acid mutant (KBNT lpa1-1) with the goldhull color cultivar “Bluebelle” (Bollich et al., 1968). This was followed by selection for recombinants that possess the recessive gene lpa1-1 (responsible for low phytic acid) from the first parent and the recessive gene gh (responsible for the goldhull color) from the second parent. Although the original low phytic acid mutant KBNT lpa1-1 is phenotypically the same as the original parent, GLPA is set apart by the goldhull color, allowing for identity preservation of the line. Considerable effort has gone into the enrichment of rice and other staples with metals like zinc and iron to address global malnutrition associated with their deficiencies, but these efforts will be futile if increasing the concentration of these metals in staple food crops is offset by the chelating effect of phytic acid. Low phytic acid foods that increase the bioavailability of these essential metals will serve as a necessary complementary measure to biofortification efforts. Attempts to reduce the phytic acid content have not been limited to staple crops but have also included products made from these crops. In recent times, brown rice has been prized as a healthy food ingredient for its high nutritive value. This is, however, limited by its relatively large amount of phytic acid. Akiko et al. (2005) used enzyme hydrolysis to reduce the phytic acid content of brown-rice bread. They added 0.2 and 1.0 g of phytase (Aspergillus niger phytase) to 521 g of dough material before mixing, fermenting, and baking. They reported that the addition of 0.2 g of the phytase to the dough material before baking reduced the phytic acid content of brown-rice bread with less negative effect on bread appearance compared to the addition of 1.0 g of the phytase. Starting with a raw material that is low in phytic acid and implementing this enzyme hydrolysis would lead to a final product with a very low phytic acid content and hence a more nutritious product.

1.3 BIOTECHNOLOGY FOR THE PRODUCTION OF ANIMAL-BASED FUNCTIONAL FOODS As with plant-based foods, biotechnology has also been used in the production of animal-based functional foods to provide specific nutritional and health benefits. Currently these biotechnological


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approaches have focused mainly on reducing the fat tissue and improving the FA profile of meat and dairy products with their naturally high saturated fat content. Although most consumers generally resist the idea of eating food derived from bioengineered animals, the FDA permits meat and milk from clones of adult cattle, pigs, goats, and their offspring and they are deemed to be as safe to eat as food from conventionally bred animals, according to three documents released by the FDA on 15 January 2008 (Osborne, 2009). However, little has been done with animal biotechnology compared to plant biotechnology because the animal genome is larger and more complex than that of plants and hence genetic modification of animals is more difficult and costly (Montaldo, 2006). This section will review some of the meat- and dairy product-based foods that have been developed with improved health values.

1.3.1

MEAT PRODUCTS

1.3.1.1 In Vitro Meat In vitro meat is effectively animal flesh that has never been part of a complete living animal. It is different from synthetic and artificial meat, both of which have the taste and texture of meat but do not consist of meat (Innovation Watch, 2007). The technology works by taking from a live animal stem cells, or myoblasts, that are preprogrammed to grow into muscle, placing them in a growth medium, and supplying the necessary nutrients for growth, such as glucose, minerals, and amino acids. The stem cells are then poured into a three-dimensional sponge-like scaffolding made of protein to which they can attach themselves; growth is stimulated by firing electrical impulses into the muscle cells, ultimately forming muscle fibers that can be harvested to produce a minced-meat product (The New York Times, 2005; The Times, 2008). It is predicted that 20 years from now it will be possible to use this technology to grow a whole beef or pork loin. The production of in vitro meat would circumvent many of the problems, especially pollution, associated with conventional farming. For example, in the United States livestock farming produces 1.4 billion tons of animal waste annually. Once a meat-cell culture has been produced it will be equivalent to regular yeast or yoghurt cultures; meat growers would not need to use a new animal for each set of starter cells and hence the meat industry would no longer be dependent on slaughtering animals (The New York Times, 2005). This technology holds great promise in the area of meat-based functional foods owing to the fact that the nutrients can be controlled. For example, most meats have a high content of omega-6 FAs, which can cause high cholesterol and other health problems. With in vitro meat, omega-6 FAs can be replaced by the healthier omega-3 FAs (Edelman et al., 2005). In vitro meat could also lead to easy control of the fat content of meat and reduce the incidence of food-borne diseases associated with the consumption of contaminated meat (The Times, 2008). 1.3.1.2 Meat with a Modified FA Profile It has been well documented that the consumption of omega-3 FAs offers some protection against CVD. However, meat from pigs, cows, and other food mammals typically has higher levels of omega-6 FAs as a result of an animal diet of grains that are rich in such FAs, as well as the inability to transform it into its healthier version of omega-3 FAs. For example, pork generally contains approximately 15% omega-6 FAs and 1% omega-3 FAs (Pig Progress.net, 2008). High levels of omega-6 FAs translate into a high omega-6 to omega-3 FA ratio associated with CVD, cancer, and inflammatory and autoimmune diseases. On the other hand, increased levels of omega-3 FAs, and hence a low omega-6 to omega-3 FA ratio, are known to exert a protective effect (Simopoulos, 2002). It has been demonstrated that the greatest risk factor for ischemic heart disease and arteriosclerosis is not high cholesterol intake but a high omega-6 to omega-3 FA ratio in the diet (Okuyama and Ikemoto, 1999). As such it has been recommended that the omega-6 to mega-3 FA ratio should not exceed 4 (Wood et al., 2004). Western diets typically have an omega-6 to omega-3 FA ratio of 15–20, well above the recommended range of 1–4 (Simopoulos, 2002). Consumption trends for © 2010 Taylor and Francis Group, LLC


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omega-3 FAs are either static or declining (Lee et al., 2006). Professional organizations and health agencies are therefore recommending an increase in the consumption of omega-3 FA rich foods in order to promote a reduction in the omega-6 to omega-3 FA ratio (Garg et al., 2006; Kolanowski and Laufenberg, 2006). There has therefore been a great deal of research devoted to producing meat products with a lower omega-6 to omega-3 FA ratio to improve meat quality and counteract the popular belief that meat products are inherently unhealthy. One such effort has resulted in the production of healthier meat from pork through an increase in the omega-3 FA content. This achievement was based on prior research where mice capable of transforming omega-6 FAs into omega-3 FAs were created by transplanting a gene from the roundworm Caenorhabditis elegans into normal mice (Kang et al., 2004). This raised the possibility of genetically engineering livestock with higher levels of omega-3 FAs, as livestock do not normally have the ability to convert omega-6 to omega-3 FAs because they lack an omega-3 FA desaturase gene (Lai et al., 2006). The roundworm gene (fat 1 gene) was first transferred into fetal pig cells, after which the cells were cloned and transferred into 14 mother pigs. Six of the 12 offspring produced tested positive for the gene and its ability to synthesize omega-3 FAs. The omega-3 FA content of the engineered pigs was 8% of the total muscle fat compared to 1% for their unmodified counterparts (Lai et al., 2006).

1.3.2

DAIRY FOODS

Milk is a high quality food source that is rich in protein, fat, carbohydrate, minerals, vitamins, and growth factors. The supply of human milk is not continuous and this has led to the use of livestock milk as a substitute. However, livestock milk is not a perfect substitute for human milk and is associated with problems such as lactose intolerance and allergy. Thus, in addition to engineering milk products to improve their FA profile as previously mentioned, biotechnology has been used to engineer livestock milk that is as similar as possible to human breast milk in order to address some of the problems associated with the consumption of livestock milk. 1.3.2.1 Milk for the Lactose-Intolerant Population About 70% of adults are denied the nutritional benefits of milk because they suffer from lactose intolerance, an intestinal disorder that arises due to the lack of the enzyme lactase. Lactose remains unabsorbed in the intestines of sufferers, causing severe intestinal discomfort characterized by abdominal pain and diarrhea (FoodReactions, 2005). In western societies, where milk represents a major component of the diet, lactose intolerance effectively restricts the use of this precious nutritional source for many people. Since milk can provide much of the calcium necessary to maintain bone structure, lactose intolerance has been associated with osteopenia in the later stages of life (Saltzman and Russell, 1998). The nutritional value of milk, coupled with its widespread utilization in many food formulations, means that low-lactose milk offers immense benefits to a large percentage of the adult population. Several biotechnological methods have been developed to produce low-lactose milk. The following paragraphs summarize the techniques utilized in the production of lactose-free or reduced lactose milk. Popular methods involve postharvest treatment of milk with lactose hydrolyzing enzymes obtained from microbiological sources to produce low-lactose milk. These techniques involve the use of enzymes (free or immobilized) from various sources to hydrolyze lactose into galactose and glucose. For example, the Organic Valley Family of Farms in LaFarge, Wisconsin, market a lactosefree milk produced by the hydrolysis of the lactose in milk using lactase enzyme derived from the dairy yeast, Kluyveromyces. The company first skims organic whole milk obtained from pasturefed cows to the desired fat content. The enzyme lactase is then added to the milk and the mixture stirred slowly for 24 h, the time required for the lactase enzyme to break down the lactose. The milk is then tested to ensure it is lactose free before it is pasteurized to inactivate the enzyme (Organic Valley, 2008). This treatment is, however, very expensive and the end products of the enzymatic


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hydrolysis (glucose and galactose) render the milk much sweeter, which appears unnatural to most consumers (Jelen and Tossavainen, 2003). Fermentation using various enzymes has also been employed to produce low-lactose milk. Lins and Leao (2002) used fermentation of skim milk to convert lactose into ethanol and carbon dioxide using the enzyme Kluyveromyces marxianus CBS 6164 (free or immobilized in Ca-alginate (2%) beads) obtained from yeast. According to the researchers, it is possible to remove all the lactose in skimmed milk in less than 4 h using 30 or 50 g cells per liter of milk. The carbon dioxide, ethanol, water, and other volatile products resulting from the fermentation are removed when the fermented milk without cells is subjected to spray drying. This procedure makes it possible to obtain a powdered skim milk free of any kind of sugar and without significant alterations in the lipid and protein profile that can be consumed not only by the lactose intolerant, but also by diabetics and obese individuals, as well as individuals suffering from galactosaemia. Genetic engineering to produce low-lactose milk offers a low cost alternative to these approaches. So far, research has tended to focus on the use of mice as convenient stepping stones for ultimate applications in dairy cattle. Jost et al. (1999) reported the development of transgenic mice that expressed intestinal lactase in the mammary gland and produced low-lactose milk. This was achieved by introducing a DNA construct into the mice that contained the rat intestinal lactasephlorizin hydrolase cDNA under the control of the mammary-specific α lactalbumin promoter. The transgenic mice expressed the foreign lactase construct during lactation, as evidenced by the presence of lactase in the milk secreted. Lactase synthesis led to a 50% and 85% reduction in milk lactose in milk collected at 0 and 8 h after milking, respectively. Both cases were associated with a corresponding increase in galactose and glucose content, thus demonstrating the enzymatic hydrolysis of lactose during storage in the mammary gland. Newborn mice suckling low-lactose milk from these transgenic mice displayed growth patterns akin to mice suckling milk from nontransgenic control mice, indicating that the nutritional quality of the milk from these transgenic mice was not significantly changed. 1.3.2.2 Milk with Enriched Antimicrobial Protein, Lysozyme Lysozyme is a widespread antimicrobial protein that is found in the saliva, tears, and milk of all mammals (Jolles and Jolles, 1984). It restricts the growth of bacteria that cause intestinal infections and diarrhea, but stimulates the growth of beneficial intestinal bacteria. Lysozyme is therefore considered to be one of the major components of human milk responsible for the health and well-being of breast-fed infants (Lonnerdal, 2003). Lactation, and hence the supply of these beneficial proteins from human milk, is not permanent, requiring milk from livestock to be substituted for human milk. Although milk from livestock can be easily and continuously obtained, its content of antimicrobial proteins such as lysozyme is much lower than the levels found in human milk. Enriching the milk of cows and goats with lysozyme is thus expected to be beneficial in protecting infants and children from diarrheal diseases which, according to the Institute for One World Health (iOWH), kill two million children worldwide annually (iOWH, 2006). Maga and coworkers (2006a) reported the generation of transgenic goats expressing human lysozyme (HLZ) in the mammary gland. Dairy goats were chosen as the experimental model because they are small ruminants with relatively short gestation periods and generation intervals compared to cattle, allowing results to be obtained faster and more economically. The transgenic goats were created by standard pronuclear microinjection with a DNA construct made up of 23 kb of the promoter and the 3′ regulatory elements of the bovine αS1-casein gene coupled to the 540-bp cDNA for HLZ. This gene was previously expressed in the milk of transgenic mice (Maga et al., 1995). Expression of HLZ in the transgenic goats was confirmed by Northern blot analysis of RNA isolated from sloughed somatic cells in milk. Expression of the transgene did not affect the gross composition of the milk. The transgenic goats exhibited a healthier udder based on fewer sloughed mammary epithelial cells and leukocytes, which are indicators of lower levels of intramammary bacterial infection. However the presence of HLZ in transgenic milk affected several of the processing



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characteristics of milk. For example, the rennet clotting time of HLZ milk was significantly lower than that of nontransgenic controls (Maga et al., 2006a). To test the efficacy of the HLZ milk, the researchers fed pasteurized HLZ milk to young goats (ruminants) and pigs (nonruminants). Because pigs have a digestive system similar to humans, they were chosen as a model to provide an idea of how the HLZ milk would impact the digestive tract of humans. In both animal models, the results of the study indicated that HLZ milk had an impact on the growth of bacteria in the gastrointestinal tract, though in opposite ways. Piglets fed HLZ milk had lower levels of coliform bacteria in the small intestine, including fewer Escherichia coli compared to the control group that were fed milk from nontransgenic goats. On the other hand, kid goats fed HLZ milk had higher levels of coliform bacteria and about the same level of E. coli compared to their control group. However, both sets of young animals were healthy and exhibited normal growth patterns. The researchers concluded that the differences observed in the two species were as a result of the fact that goats, being ruminants, have a different digestive system and as such a different collection of bacteria compared to pigs, which have a single stomach. Such transgenic HLZ dairy herds hold a great deal of promise in developing countries where intestinal diseases threaten the lives of infants and children. The researchers argued that the benefit of their research would be more pronounced if the technology was applied to dairy cattle rather than goats, because the amount of milk produced by cows is much greater than is possible with goats (Maga et al., 2006b). 1.3.2.3 Milk with an Improved FA Profile As already pointed out, high quantities of dietary fat, especially saturated fats, have been associated with an increase in blood cholesterol and consequently an increased risk for CHD and atherosclerosis. In contrast, unsaturated fats (poly- and monounsaturated) have a beneficial effect on the heart by reducing serum cholesterol. In the United States approximately 33% of saturated fats in the diet are obtained from the consumption of dairy products (Havel, 1997). Changing the FA content of milk through a reduction in its SFA content and an increase in its UFA content promises to be a worthwhile approach towards reducing the risk of CHD. Based on dietary recommendations, the nutritionally ideal milk should possess a FA composition of 10% PUFA, 8% SFA, and 82% MUFA. This differs markedly from the typical cows’ milk FA composition of 5% PUFA, 70% SFA, and 25% MUFA (Grummer, 1991). Researchers have sought to improve the FA profile of milk by feeding livestock diets such as linseed oil or fish oil rich in these heart-healthy UFAs, with the hope that milk from livestock raised on such diets will have higher levels of these UFAs. However, the presence of healthy FAs in the diet does not necessarily transfer into the milk produced by these animals (NUTRAingredients, 2005). Consumed UFAs are substantially biohydrogenated in the rumen before absorption in the small intestine, which results in the milk FAs becoming more saturated (Doreau et al., 1997). In ruminants, high quantities of long-chain SFAs absorbed in the intestine are not reflected in the milk FA composition. This is because the activity of the enzyme stearoyl-CoA desaturase (SCD) present in the epithelial cells of the mammary gland converts SFA to MUFA (Tocher et al., 1998). Increasing the SCD activity in the mammary gland would therefore not only increase the MUFA content but also decrease the SFA content of milk. Reh and coworkers (2004) tested this hypothesis by generating transgenic dairy goats using a DNA construct designed to express rat SCD cDNA in the mammary gland under the control of the bovine β-lactoglobulin promoter through a standard pronuclear microinjection procedure. The FA composition of milk from four female transgenic goats was analyzed on days 7, 14, and 30 of their first lactation. In two of the animals, the expression of the transgene altered the overall FA composition of the resulting milk such that at day 7 the transgenic milk had less saturated and more MUFA content compared to milk from nontransgenic controls. However, this effect diminished by day 30 of lactation. The percentages of protein and fat in milk from the four transgenic goats were within the same range as milk from nontransgenic goats, indicating that expression of the transgene had no effect on the gross composition of the milk.


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1.4


FINAL REMARKS

The use of biotechnology in the production of functional foods holds promise for efforts to address worldwide global malnutrition and reduce chronic diseases. The practice also addresses problems of pollution and the high production costs associated with conventional food production and processing. However, as a result of consumer attitudes toward foods derived from biotechnology coupled with current governmental regulations, most of the functional foods derived from biotechnology have not yet emerged from the laboratory to find commercial applications. Public interest groups, religious organizations, professional bodies, and environmental activists have all expressed their concern regarding the introduction of GM foods. The three major concerns most commonly raised are that they represent a potential environmental hazard, they may pose a health risk to humans in the longer term, and they will be very costly. However, given the right approach and information, consumers can be convinced to accept GM foods that provide specific health benefits. A study conducted by Purdue University revealed that consumers may be willing to pay a premium for particular GM foods if they are informed about the health benefits that they may receive from eating those foods (Lusk, 2003). This is a positive indication that the nutritional benefits of a GM food can outweigh the consumer’s perception of risk. Consumers’ attitudes toward GM foods vary across geographical locations and between individuals, particularly regarding the benefits that consumers stand to benefit from the consumption of GM food. In the United States there is general apathy toward GM foods, unlike in the Europe and Japan, where consumers are passionate about the issue. People generally have different attitudes toward food depending on the region of the world that they come from. Thus, food is not only considered for its nutritional value but also often has societal, historical, political, and religious importance. Therefore, persuading consumers to accept GM functional foods would require good communications with regard to risk assessment efforts and cost/benefit evaluations. A holistic evaluation of GM foods that considers not only its safety but also food security, social, economical, and ethical aspects, access and capacity building would contribute greatly to the future development of GM functional foods.

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Foods and 2 Functional Biotechnology in Japan Harukazu Fukami CONTENTS 2.1 2.2 2.3

Introduction ............................................................................................................................ 29 Bioactive Peptides Processed by Proteases or Microbes ........................................................ 29 Glycosidated Products ............................................................................................................ 33 2.3.1 Saccharides ................................................................................................................. 33 2.3.1.1 Oligosaccharides .......................................................................................... 33 2.3.1.2 Dietary Fibers .............................................................................................. 35 2.3.1.3 Others ........................................................................................................... 35 2.3.2 Glycosylated Products ................................................................................................ 36 2.3.2.1 Glucosylation of Flavonoids......................................................................... 37 2.3.2.2 l-Ascorbic Acid Glucosides .........................................................................40 2.3.2.3 Other Glycosylated Products ....................................................................... 41 2.4 Functional Lipids and Acylated Products ............................................................................... 42 2.4.1 Functional Lipids or Acylated Products Processed by Lipase ................................... 42 2.4.2 Functional Foods Fermented by Microbes ................................................................. 43 2.5 Perspective ..............................................................................................................................44 References ........................................................................................................................................44

2.1 INTRODUCTION Functional foods are being actively developed in Japan. The Food for Specified Health Uses (FOSHU) designation, which refers to foods containing ingredients with health functions, was officially approved for claims of physiological effects on the human body by the Ministry of Health, Labor and Welfare (MHLW) (Japanese Ministry of Health, Labor Welfare, n.d.). At present, eight categories of health claim are approved (Table 2.1), and FOSHU can issue these foods with a seal of approval (Figure 2.1). In order to sell a food as FOSHU, its safety and effectiveness on human health must be assessed, and the claim must be approved by the MHLW. There were 847 such items approved as of April 2009. In addition to traditional foods, many more functional foods processed by enzymes or microbes have been produced or are being researched and developed. These functional foods will be reviewed in this chapter.

2.2 BIOACTIVE PEPTIDES PROCESSED BY PROTEASES OR MICROBES Angiotensin I-converting enzyme (ACE) catalyzes the formation of the vasoconstrictive peptide angiotensin II (Skeggs et al., 1956). Its inhibitors have vasodilating action (Ondetti et al., 1977) and have been used as pharmaceuticals. ACE inhibitory peptides have been found in the enzymatic digests of many food proteins. More than 10 peptides were isolated from a gelatin digest using Clostridium histolyticum collagenase (Oshima et al., 1979). Among them, several peptides with Ala–Hyp (Hyp: hydroxyproline) at the 29 © 2010 Taylor and Francis Group, LLC

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TABLE 2.1 Health Claims and their Active Ingredients Approved as FOSHU Category

Specified Health Uses

1

Foods to modify gastrointestinal conditions

2 3

Foods related to blood cholesterol levels Foods related to blood sugar levels

4

Foods related to blood pressure

5 6 7

Foods related to dental hygiene Cholesterol plus gastrointestinal conditions; triacylglycerol plus cholesterol Foods related to mineral absorption

8 9

Foods related to osteogenesis Foods related to triacylglycerol

Principal Ingredients (Ingredients Exhibiting Health Functions) Oligosaccharides, lactose, bifidobacteria, lactic acid bacteria, dietary fiber 8 ingestible dextrin, polydextrol, guar gum, psyllium seed coat, etc. Chitosan, soybean protein, degraded sodium alginate Indigestible dextrin, wheat albumin, guava tea polyphenol, l-arabiose, etc. Lactotripeptide, casein dodecaneptide, tochu leaf glycoside (geniposidic acid), sardine peptide, etc. Paratinose, maltitiose, erythrytol, etc. Degraded sodium alginate, dietary fiber from psyllium seed husk, etc. Calcium citrated malate, CPP, hem iron, fructooligosaccharide, etc. Soybean isoflavone, MBP (Milk basic protein), etc. Middle-chain fatty acid, etc.

C-terminus served as substrates for ACE, and the dipeptide generated by ACE was a potent inhibitor of ACE. ACE inhibitory dodecapeptide (FFVAPFPQVFGK) and pentapeptide (FFVAP) were isolated from bovine casein hydrolysate using trypsin (Maruyama and Suzuki, 1982; Maruyama et al., 1985). The inhibitory activity of the dodecapeptide was weaker by 10-fold than that of the pentapeptide (see Table 2.2). Thermolysin treatment of α-zein yielded three tripeptides (LRP, LSP, and LQP), which were potent ACE inhibitors (Miyoshi et al., 1991). Recently, three active tripeptides (LVY, LQP, and LKY) were isolated from thermolysin hydrolysate of sesame protein (Nakano et al., 2006). ACE inhibitory peptides were also found in enzymatic hydrolysates of marine organisms (Fujita and Yoshikawa, 2008). It was reported that a thermolysin digest of dried bonito gave rise to eight active peptides (IKPLNY, IVGRPRHQG, IWHHT, ALPHA, FQP, LKPNM, IY, and DYGLYP) (Yokoyama et al., 1992). Among these, LKPNM and IWHHT were substrates for ACE, and LKP and IWH were active as ACE inhibitors. These peptides were of a prodrug type in the same way as the gelatin hydrolysate (Oshima et al., 1979). Sardine muscle hydrolysate generated using alkaline protease (Bacillus licheniformis protease) afforded nine active peptides (MF, RY, LY, YL, VF, KW, GRP, AKK, and GWAP), among which KY was the most potent (Matsufuji et al., 1994). Two active components (GIG and DW) were isolated from chum salmon head hydrolysate using Biopurase

FIGURE 2.1

The seal for FOSHU approval.


Functional Foods and Biotechnology in Japan

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TABLE 2.2 ACE-Inhibitory Activity of Peptides Peptide GPAGA(HyP) GPPGA(HyP) FFVAPFPQVFGK FFVAP LRP LSP LQP LVY LQP LKY IKPLNY IKP IVGRPRHQG IWHHT ALPHA FQP LKPNM LKP IY DYGLYP IY KW AKK DW IY MKY AKYSY LRY YNKL IVY YYSP IY PTHIKWGD VPP IPP

Origin

Enzyme

IC50

References

8.3 8.6 77 6.0 0.27 1.7 1.9 0.92 0.5 0.48 43 1.7 6.2 5.1 10 12 17 1.6 3.7 62 10.5 1.63 3.13 13 2.69 7.26 1.52 5.06 21

Oshima et al. (1979)

Matsui et al. (2006)

Gelatin

Collagenase

Casein Casein α-Zein

Trypsin Trypsin Thermolysin

Sesame protein

Thermolysin

Dried bonito

Thermolysin

Sardine muscle

Alkaline protease

Chum salmon heads Nori (Porphyra yezoensis)

Biopurase SP-10® Subtilisin

Wakame (Undaria pinnatifida) Royal jelly

Pepsin

Mycoleptodonoides aitchisonii Tuna Skim milk

Aqueous extract

0.5 1.3 3.7

Acid extract Lactobacillus helveticus (Fermentation)

2 9 5

Orientase ONS®

Maruyama and Suzuki (1982) Maruyama et al. (1985) Miyoshi et al. (1991)

Nakano et al. (2006)

Yokoyama et al. (1992)

Matsufuji et al. (1994)

Ohta et al. (1997) Suetsuna (1998)

Suetsuna and Nakano (2000)

Sakamoto et al., (2001), Cheung et al.(1980) Kohama et al. (1988) Nakamura et al. (1995)

SP-10® (Nagase Biochemical Co. Ltd., Kyoto) derived from Bacillus subtilis (Ohta et al., 1997). The IC50 values of GIG and DW were 730 and 13 μM, respectively. Although their activity was weak, oral administration of the hydrolysate to spontaneously hypertensive rats (SHR) decreased their blood pressure significantly. Nori (laver seaweed, Porphyra yezoensis) was digested by subtilisin to generate four active peptides (IY, LRY, MKY, and AKYSY) (Suetsuna, 1998). Wakame (Undaria pinnatifida) was also hydrolyzed using pepsin to give four tetrapeptides (AIYK, YKYY, KFYG, and YNKL) (Suetsuna and Nakano, 2000); YNKL was more potent than the others. Royal jelly proteins were hydrolyzed recently using Aspergillus oryzae derived protease (Orientase ONS®, HBI Enzyme Inc. Shiso, Hyogo) to produce several inhibitory peptides (LY, LW, IVY, YYSP, etc.)


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TABLE 2.3 Peptides Approved as FOSHU Name Casein dodecapeptide Sardine peptide Bonito oligopeptides Lactotripeptides Isoleucyltyrosine Wakame peptide Nori pentapeptide Sesame peptide Royal jelly peptide

Peptides

Supplier

References

FFVAPFPQVFGK VY LKPNM, IWHHT VPP, IPP IY YNKL AKYSY LVY VY, IY, IVY

Kracie Holdings Ltd. Senmi Ekisu Co. Ltd. Q’sai Co. Ltd. Calpis Co. Ltd. Kirin Holdings Co. Ltd. Riken Vitamin Co. Ltd. Shirako Co. Ltd. Suntory Ltd. Yamada Bee Farm

Maruyama and Suzuki (1982) Matsufuji et al. (1994) Yokoyama et al. (1992) Nakamura et al. (1995) Sakamoto et al. (2001) Suetsuna and Nakano (2000) Suetsuna (1998) Nakano et al. (2006) Matsui et al. (2006)

(Matsui et al., 2006). A dipeptide, IY, was isolated from an aqueous extract of the fruiting body of a mushroom, Mycoleptodonoides aitchisonii (Sakamoto et al., 2001). The ACE-inhibitor activity of IY had already been reported in 1980 (Cheung et al., 1980), and this peptide was also isolated from some of the protease hydrolysates mentioned above (Yokoyama et al., 1992; Suetsuna, 1998). Since oral administration of mushroom extract in which IY is an active ingredient showed an antihypertensive effect for both SHR and in humans, it was approved as FOSHU (Table 2.1, Category 4). Acid (1 M acetic acid–20 mM hydrochloric acid) extract of tuna (Neothunnus macropterus) muscle as a nonenzymatic treatment also afforded an inhibitory peptide (PTHIKWGD) (Kohama et al., 1988). Calpis sour milk (Calpis®), which is made from skim milk fermented by a starter culture containing Lactobacillus helveticus and Saccharomyces cerevisiae, is a famous Japanese soft drink. Two ACE inhibitory peptides (VPP and IPP) were isolated from Calpis (Nakamura et al., 1995). These peptides were produced by fermentation from milk protein, rather than by enzymatic digestion. The origins, methods of hydrolysis, and ACE inhibitory activity cited in references to these peptides are summarized in Table 2.2. Table 2.3 shows the antihypertensive peptides approved as FOSHU (Table 2.1, Category 4). Foods or beverages containing lactotripeptides, dried bonito oligopeptides, and sardine peptides as active ingredients were marketed for the first time in 1997. Since then, many food items containing the above peptides cited as active ingredients have been marketed in Japan. It was reported that opioid-related peptides from food proteins such as casein and gluten were obtained by enzymatic hydrolysis. Opioid antagonists YPSYGLN (casoxin A), YPYY (casoxin B), and YIPIGYVLSR (casoxin C) were isolated from a tryptic digest of κ-casein (Chiba et al., 1989). Casoxin C was active at 5 μM in the guinea pig ileum assay. Opioid peptide YPVEPF (neocasomorphin) was also found in trypsin or pepsin and chymotrypsin two-step digestions of β-casein (Jinsmaa and Yoshikawa, 1999). Gluten hydrolysate with pepsin-thermolysin or pepsin-trypsin-chymotrypsin gave several opioid peptides (GYYPT, GYYP, YGGWL, YGGW, and YPISL) (Fukudome and Yoshikawa, 1992, 1993). Soy protein is known to have a hypocholesterolemic effect in animals (Carroll, and Kurowska, 1995) and humans (Anderson et al., 1995). Peptic digest of soy protein has also been shown to exhibit a hypocholesterolemic effect (Yashiro et al., 1985). The effect of the peptic hydrolysate was suggested to be due to cholesterol absorption inhibition (Nagaoka et al., 1997). A mixture of the peptic hydrolysate with phospholipase A2-modified soy phospholipids (soy protein hydrolysate with bound phospholipids) was found to decrease cholesterol absorption markedly in rats (Nagaoka et al., 1999). A digest of soy protein using protease from B. subtilis (Hynute-D1) was developed by Fuji Oil Co. Ltd. The average molecular weight (MW) of the digested soy peptides (SPI-H) was 3000. In addition, SPI-H was effective against obesity in genetically obese male KK-Ay mice (Aoyama et al., 2000). Furthermore, SPI-H inhibited the absorption of dietary lipid, increased the absorption of



33

dietary carbohydrates, and promoted postprandial energy expenditure, which was accompanied by a postprandial increase in the oxidation of dietary carbohydrates in KK-Ay mice (Ishihara et al., 2003). Korean traditional soybean fermentation starter culture (Meju) produced soybean protein hydrolysate with proteases secreted from the starter. These proteases were neutral and alkaline protease derived from Bacillus amyloliquefaciens FSE-68 isolated from Meju. Some peptides isolated from the hydrolysate stimulated LDL-receptor transcription (Cho et al., 2007). This may be the mechanism of serum cholesterol lowering in soy protein. Globin peptide (VVYP) isolated from a globin digest with acidic protease exhibited a potent hypoglycemic activity in both mice and humans (Kagawa et al., 1996, 1998). Tryptic hydrolysate of β-lactoglobulin decreased serum and liver cholesterol levels in rats. A novel hypocholesterolemic peptide (IIAEK) was found in the tryptic hydrolysate. This peptide showed the same activity in vivo (Nagaoka et al., 2001). Fish protein from fish flesh remnants on salmon bone frames hydrolyzed by Protamex® (Bacillus protease complex) also showed hypocholesterolemic activity in obese Zucker rats. The report indicated that the mechanism was different from that of soy protein. Fish protein hydrolysate reduced the acyl-CoA:cholesterol acyltransferase activity, while soy protein elevated the excretion of bile acids in feces (Wergedahl et al., 2004). Among these, soy protein, soy protein hydrolysate with bound phospholipids, and globin peptide are approved as FOSHU (Table 2.1, Categories 2 and 3, respectively). Phosphorylated fragments of casein, casein phosphopeptides (CPPs), have a high calcium binding capacity to form a soluble complex with calcium ions, which increases calcium absorption (Tsuchita et al., 2001). Meiji Seika Kaisya Ltd. in Japan have developed CPPs produced by a tryptic hydrolysate of whole bovine casein (Hirayama et al., 2002). CPPs are known to be generated by the digestion of milk (Meisel and Frister, 1989), and to be contained in matured cheese (Addeo et al., 1994). CPPs are approved as FOSHU (Table 2.1, Category 7). Sardine muscle hydrolysate with alkaline protease showed α-glucosidase inhibitory activity (IC50: 48.7 mg/mL) (Matsui et al., 1996). Considering that green tea extract exhibited an IC50 of 11.1 mg/mL in the same experiment, the sardine muscle hydrolysate activity was moderate. Antioxidative peptides have been reported. Prawn (Penaeus japonicus) muscle hydrolysate digested using pepsin gave potent antioxidant activity. Three peptides (IKK, FKK, and FIKK) isolated from the hydrolysate exhibited antioxidative activity (Suetsuna, 2000). These synthetic peptides prolonged the induction period (days) of linoleic acid oxidation in ethanol at 60°C. The induction period of IKK (0.1 mM) was about 15 days, while that of α-tocopherol (0.5 mM) was 7 days (Suetsuna, 2000). A tryptic digest of β-casein inhibited enzymatic and nonenzymatic lipid peroxidation (Rival et al., 2001). These peptides have not yet been approved as FOSHU.

2.3 GLYCOSIDATED PRODUCTS 2.3.1

SACCHARIDES

2.3.1.1 Oligosaccharides Nondigestible oligosaccharides are known to stimulate the growth of beneficial bacteria such as Bifidobacterium species in the colon and to suppress the growth of harmful bacteria such as Bacteroides and Enterococcus species, thereby promoting health benefits. Thus, they are recognized as “prebiotics” (Gibson and Roberfroid, 1995). The following active ingredients have been developed: lactulose, raffinose, and soybean oligosaccharides, in which stachyose and raffinose are the main components, fructo-oligosaccharides, lactosucrose, galacto-oligosaccharides (GOS), isomalto-oligosaccharides (IMO), xylo-oligosaccharides, and manno-oligosaccharides derived from coffee mannan. Most of these have been developed by Japanese companies. Lactulose was synthesized for the first time in 1930 (Montgomery and Hudson, 1930). Subsequently, efficient synthetic methods and the reaction mechanism of lactulose formation were developed, and lactulose was formed by the alkaline isomerization of glucose in lactose into fructose (Aider and de Halleux, 2007). This industrial process was patented by Morinaga Milk Industry


34


Co. Ltd. (Tomita et al., 1993). Three hydrate crystals were found in 1992 (Jeffrey et al., 1992). The melting point of the crystal was lower (68°C) than the anhydrous material (169°C), and it dissolved endothermically in water (Mizota et al., 1994). Lactulose is also found in milk pasteurized by the ultra-high-temperature process (Elliott et al., 2005). Raffinose (α-d-galactosylsucrose) is found in beans, cabbage, Brussels sprouts, broccoli, asparagus, other vegetables, and whole grains; its supplier in Japan, Nippon Beet Sugar Manufacturing Co. Ltd. has been producing raffinose industrially from sugar beet molasses (Sayama et al., 1992). Soybean oligosaccharides composed of stachyose (α-d-galactosylraffinose) and raffinose are included in soybean at a proportion of 3–4% and can be isolated from soybean whey (Suzuki, 1995). Fructo-oligosaccharides are produced industrially using β-d-fructosyl transferase derived from Aspergillus niger by Meiji Seika Kaisha Ltd. (Hidaka et al., 1988). They are composed of 1-kestose (1-β-fructofuranosylsucrose, GF2), nystose (1F-β-d-fructofuranosyl-1-kestose, GF3), and 1F-β-dfructofuranosylnystose (GF4). The enzyme selectively transfers fructose to the 1-hydroxy group of the fructose moiety of saccharides. Lactosucrose (β-d-galactosylsucrose) is found in the fermentation of yogurt containing sucrose as a sweetener. It has been prepared from sucrose and lactose using levansucrase (Avigad, 1957); the industrial production of lactosucrose was established by Ensuiko Sugar Refining Co. Ltd. in Japan. A β-fructofuranosidase derived from Arthrobacter sp. catalyzes transfructosylation and predominantly transfers fructosyl residues to lactose with hydrolyzing sucrose to produce lactosucrose (Arakawa et al., 2002). In this reaction, an invertase-deficient yeast is added to assimilate the generated glucose, which inhibits the reaction. The high lactosucrose content syrup is called “Nyuka-oligo” and is available commercially as a low caloric sweetener and a prebiotic agent. GOS (β-d-galactosyllactose) were first prepared using β-galactosidase from lactose in 1951 (Wallenfels, 1951). Subsequently, it was shown that GOS exhibited prebiotic activity, and many studies have focused on the enzymatic synthesis of GOS. During these studies, the commercial production of GOS was established using β-galactosidase derived from A. oryzae (Matsumoto et al., 1989) and Cryptococcus leurentii OKN-4 (Otsuka et al., 1988). A variety of β-galactosidases- or β-galactosidase-like enzymes derived from bacteria and fungi are used in the preparation of GOS. An A. oryzae derived enzyme glycosylates the 6-hydroxyl or 3-hydroxyl group of the galactosyl moiety of lactose, while the enzymes derived from Cryptococcus leurentii OKN-4 and Steigmaltomyces elviae CBS 8119 (Onishi et al., 1995) give 4′-galactosyllactose (trisaccharide) as a major product. IMO are well known ingredients of traditional fermented foods such as Japanese “sake” (alcohol brewed from rice), “miso,” and soy sauce, and are produced industrially using three enzymes from corn starch (Yasuda et al., 1986). Corn starch was hydrolyzed by α-amylase and pullulanase to break the α-1,6-linkage. The solution from the hydrolysis was treated with α-glucosidase derived from A. niger, and then the resulting glucose was removed to give a commercial product (Isomalto900®). Isomalto-900 is composed of 37.2% isomaltose, 26.8% 6′-α-glucosylmaltose (panose), 21.4% 6″-α-glucosylmaltotriose, 10.5% maltose and maltotriose, and 4.1% glucose. IMO is digestible; however, its digestibility was lower than that of maltose (Kaneko et al., 1994). Xylo-oligosaccharides were prepared by xylanase derived from Trichoderma sp. from xylan (Fujikawa et al., 1990). The yield of xylobiose using Trichoderma sp. enzyme was higher than with enzymes derived from other microbes such as Humicola sp. and Bacillus pumilus. Coffee beans are known to contain hemicelluloses such as mannan and arabinogalactan. Coffee mannan is insoluble in water and remains present in spent coffee grounds. Coffee manno-oligosaccharides were produced by hydrolysis of spent coffee grounds using high-pressure steam at a high temperature (220°C) (Asano et al., 2003). The major components of manno-oligosaccharides were mannobiose, mannotriose, and mannotetraose. Coffee manno-oligosaccharides also exhibited a prebiotic activity. Gentio-oligosaccharides are oligosaccharides with β-1,6-glycosidic linkage containing gentiobiose. These oligosaccharides are prepared industrially using β-glucosidase derived from A. niger by Nihon Syokuhin Kako Co. Ltd. (Unno, 1995). The enzyme shows both condensation (dehydration) and transglucosylation reactions of glucose, so these oligosaccharides are composed from di-, tri-, and tetrasaccharide.


Functional Foods and Biotechnology in Japan Extraction

Beet Extraction Sucrose

Transglycosylation

Extraction Lactose Hydrolysis

Extraction

Isomerization

Fructo-oligosaccharides Lactosucrose Lactulose

Transglycosylation Galacto-oligosaccharides

Soluble starch

Soybean

Raffinose

Transglycosylation

Cow’s milk

Starch

35

Hydrolysis Transglycosylation

Soybean whey

Extraction Hydrolysis

Xylan Hydrolysis Spent coffee grounds Glucose

Transglycosylation

Isomalto-oligosaccharides

Soybean oligosaccharides Xylo-oligosaccharides Manno-oligosaccharides Gentio-oligosaccharides

FIGURE 2.2 A schematic presentation of production processes of nondigestible oligosaccharides. (Reprinted from Sako, T., Matsumoto, K., and Tanaka, R., 1999. Int. Dairy J. 9: 69–80. With permission. Copyright 1999 from Elsevier.)

Gentiobiose is known to have a bitter taste. As a result, the commercial product (Gentose®45) exhibited the bitter taste of gentiobiose and the sweet taste of glucose that are both contained in the product. The production processes of these oligosaccharides have been reviewed previously (Crittenden and Playne, 1996; Sako et al., 1999) and are shown schematically in Figure 2.2 (Sako et al., 1999). All of these are low caloric carbohydrates and exhibit prebiotic activity. These oligosaccharides improve the properties of the stool, alter lipid metabolism, and enhance calcium absorption (Mitsuoka, 2002). The oligosaccharides approved as FOSHU to modify gastrointestinal conditions along with their Japanese suppliers are summarized in Table 2.4. 2.3.1.2 Dietary Fibers Dietary fibers also improve the gastrointestinal condition. They are usually insoluble in water; in other cases they may assume the form of viscous liquids if solubilized in water. Guar gum, which is produced from the endosperm of guar seeds (Cyamopsis tetragonolobus), is a viscous liquid in water. Guar gum partially hydrolyzed by endo-β-mannanase with MW in the range of 1000–100,000 also exhibited dietary fiber activities and low viscosity (McCleary and Matheson, 1983; Yamatoya, 1994). Guar gum was partially hydrolyzed by β-endogalacto-mannase from A. niger to give polysaccharides of 20,000 MW (Yamamoto et al., 1990d). β-mannanase derived from Penicillium oxalicum produced oligosaccharides with 2–7 degrees of polymerization (DP) (Kurakake et al., 2006). This partially hydrolyzed guar gum is on the market as FOSHU (Table 2.1, Category 1). 2.3.1.3 Others Phosphorylated oligosaccharides (POs) are prepared from potato starch by reaction with α-amylase, glucoamylase, and pullulanase followed by separation of neutral sugars using an anion-exchange resin (Kamasaka et al., 1995). POs are composed of monophosphoryl malto-oligosaccharides (triose, tetraose, and pentaose as the main components) and diphosphoryl malto-oligosaccharides (pentose and hexose as the main components). The phosphate group is attached to the 6- or 3-hydroxy group (Kamasaka et al., 1997). POs increase calcium solubility to enhance calcium absorption in the


36


TABLE 2.4 Oligosaccharides Approved as FOSHU Name

Main Component

Lactulose

Gal(β-1,4)Fru

Raffinose

Gal(α-1,4)Glu(α-1,4-)Fru

Soybean oligosaccharides (stachyose and raffinose)

Gal(α-1,4)Gal(α-1,4) Glu(α-1,4-)Fru (stachyose), Gal(α-1,4)Glu(α-1,4-)Fru Glu (α-1,2) Fru(β-6,2) Fru (1-kestose) Gal(β-1,4)Glu(α-1,2)Fru

Fructo-oligosaccharides (1-kestose, etc.) Lactosucrose (Nyukaoligo) GOS

IMO

Xylo-oligosaccarides Coffee mannooligosaccharides

Gal(β-1,4) Gal(β-1,4)Glu

Glu(α-1,6) Glu (isomaltose) Glu(α-1,6)Glu(α-1,4)Glu (panose) Xyl(β-1,4)Xyl (xylobiose) Man(β-1,4)Man (mannobiose)

Japanese Supplier

References

Morinaga Milk Industry Co. Ltd. Nippon Beet Sugar Manufacturing Co. Ltd. Calpis Co. Ltd.

Tomita et al. (1993)

Meiji Seika Kaisha Ltd.

Hidaka et al. (1988)

Ensuiko Sugar Refining Co. Ltd. Yaklut Honsya Co. Ltd. Nissin Sugar Manufacturing Co. Ltd. Showa Sangyo

Arakawa et al. (2002)

Suntory Ltd. Aginomoto Co. Inc.

Fujikawa et al. (1990) Asano et al. (2003)

Sayama et al. (1992) Suzuki (1995)

Matsumoto et al. (1989) Otsuka et al. (1988) Yasuda et al. (1986)

intestine like CPP. They also promote recalcification of both enamel and dentin lesions (Kamasaka et al., 2004). Thus, a sugar-free chewing gum (POSCAM®) containing a calcium salt of POs as an active ingredient has been sold as a food related to dental hygiene as FOSHU (Table 2.1, Category 5). Ezaki Glico Co. Ltd. marketed a highly branched cyclic dextrin (HBCD, Cluster Dextrin®). HBCD was prepared from amylopectin using a thermo-stable branching enzyme [1,4-α-dglucan:1,4-α-d-glucan 6-α-d-(1,4-α-d-glucano)-transferase] derived from Bacillus stearothermophilus (Takata et al., 1996). This branching enzyme catalyzes transglycosylation to form an α-1,6-glucosidic linkage of amylopectin or glycogen (Figure 2.3). Although HBCD is a high MW molecule (MW: 400,000), it shows high water solubility. The solution exhibits good stability, low osmotic pressure, low viscosity, and so on. (Takata, 2004). These properties show that HBCD is able to supply energy for sporting activities. HBCD enhanced swimming endurance in mice (Takii1 et al., 1999). A beverage containing HBCD is sold as an athletic drink.

2.3.2

GLYCOSYLATED PRODUCTS

Glycosylation is a useful method for the structural and functional modification of bioactive compounds contained in food. It enhances their solubility, physicochemical stability, bioactivity, and intestinal absorption and also improves their taste qualities. With these advantages in mind, many studies have investigated the glycosylation of natural bioactive compounds. Enzymatic transglycosylation is catalyzed by glycosyltransferase, which uses a nucleotide-activated sugar as a glycosyl donor. This reaction can also be catalyzed by glycoside hydrolases such as α-amylase and α-glucosidase which use a polysaccharide or oligosaccharide as donors. Some glucoside hydrolases, such as cyclodextrin glucanotransferase (CGTase) (Funayama et al., 1993), α-glucosidase (Nakagawa et al., 1996), α-amylase (Nishimura et al., 1994), and sucrose phosphorylase (Kitao et al., 1993), are used to transfer monoglucosyl or oligoglucosyl groups to the hydroxyl group of alcohols and phenols. The research and development into the glucosylation of flavonoids, ascorbic acid, and others are cited in this section.



37

DP 27–28 Amylopectin

BE

Highly-branched cyclic dextrin (Cyclic Cluster Dextrin®)

FIGURE 2.3 A model of the action of a branching enzyme (BE) on amylopectin. Solid line, α-1,4-glucan chain; arrow, α-1,6-glucosidic linkage; open triangle, α-1,4-glucosidic linkage to be cleaved by BE; closed triangle, glucosyl residue to be used as an acceptor. (From Takata, H., 2004. J. Appl. Glycosci. 51: 55–61. With permission from the Japanese Society of Applied Glycoscience.)

2.3.2.1 Glucosylation of Flavonoids Flavonoids (Figure 2.4) such as catechins, anthocyanidins, and quercetin are known to exhibit a variety of biological activities which involve benefits to human health (Tanaka and Suzuki, 2004; Vita, 2005; Lakhanpal, 2007). The glucosylation of flavonoids usually increases their water solubility and stability against light or oxidation and improves their pharmacological properties compared with the original substrate. OH 3’ 4’ OH 2’ 1 1’ B 8 HO 7 5’ O A C 2 6’ 6 4 3 OH 5 OH O Quercetin

8 HO 7

1 O

6

4

5 OH

OH 3’ 4’ OMe 1 1’ 5’ O 2 6’ 4 3

8

HO 7 6

OH 3’ 4’ OH 2’ 1’ 5’ 2 6’ OH

5

O

2” 3” OH 1” 4” 6” 5” OH OH

The chemical structures of flavonoids.


6 5 OH

Hesperetin

Epigallocatechin gallate (EGCG)

FIGURE 2.4

8 HO 7

OH O

3 O

OH 3’ 4’ OH 1 1’ 5’ O 2 6’ 4 3 OH 2’

2’

(+)–Catechin

8 HO 7 6 5

OH 3’ 4’ OH 2’ 1 1’ 5’ O 2 6’ OH 4 3 OH

OH Epicatechin

38


Rutin and isoquercitrin glucosides are sold as enzymatically modified rutin (extract) and enzymatically modified isoquercitrin, respectively. These products are included in the “List of Existing Food Additives” [Notification No. 120 (April 16, 1996), Ministry of Health and Welfare, Japan] as food additives. They are produced from rutin [3-O-(6-O-α-l-rhamnopyranosyl)-β-d-glucopyranosyl) quercetin] and isoquercitrin (3-O-β-d-glucopyranosylquercetin) by the actions of B. stearothermophilus CGTase, using dextrin as a gulcosyl donor, and Rhizopus sp. glucoamylase (Suzuki and Suzuki, 1991). This enzyme glycosylates the 4-hydroxyl group of the glucose moiety of both rutin and isoquercitrin. Rutin glucosides are composed of rutin, rutin monoglucoside, rutin malto-oligosaccharides, isoquercitrin, and isoquercitrin monoglucoside as major components, while isoquercitrin glucosides are composed of isoquercitrin, isoquercitrin monoglucoside, and isoquercitrin maltoside (Akiyama et al., 2000). Rutin glucosides are 3000 times more soluble than rutin. Hesperidin [(6-O-α-l-rhamnopyranosyl)-β-d-glucopyranosylhesperetin] is contained in the peel of Citrus sp. It is called “vitamin P,” and it decreases capillary permeability and fragility in the same manner as rutin and quercetin (Middleton and Kandaswami, 1993). Hesperidin glucosides are prepared by CGTase from an alkalophilic Bacillus sp. with soluble starch as a glucosyl donor (Kometani et al., 1994). The enzyme transglycosylates the 4-hydroxyl group of glucose moiety of hesperidin in pH 9.0 solution which renders hesperidin highly soluble. Hesperidin glucosides consist of hesperidin, hesperidin monoglucoside, hesperidin maltoside, and hesperidin maltotrioside as the major components, and these compounds show 10,000 times higher solubility than the parent compound. This product is also included in the List of Existing Food Additives as a food additive. The transglycosylation of catechins was also reported. CGTase derived from Bacillus macerans gave 3′-O-α-d-glucopyranosyl-(+)-catechin and unidentified glucosylated products using (+)-catechin and soluble starch as a glucosyl donor (Funayama et al., 1993). The unidentified products were hypothesized to be malto-oligosaccharides of catechin. Sucrose phosphorylase from Leuconostoc mesenteriodes produced catechin glucosides and epigallocatechin gallate (EGCG) glucosides in the presence of sucrose as a glucosyl donor (Kitao et al., 1993, 1995). The enzyme preferentially glycosylates the 3′-hydroxyl group (B ring) of (+)-catechin, while it glycosylates the 4′-hydroxyl (B ring) and both of 4′- and 4″-hydroxyl groups (gallate moiety) of EGCG. These compounds exhibited increased water solubility and increased stability against light or oxidation. (+)-Catechin was glycosylated by an enzyme derived from Xanthomonas campestris sp. in the presence of maltose as a glucosyl donor to give 4′-O-α-d-glucopyranosyl-(+)-catechin (Sato et al., 2000). Mutans streptococci, including Streptococcus mutans and Streptococcus sorbirinus, induce dental caries by secreting a glucosyltransferase that generates glucan from sucrose. Glucosyltransferases catalyzed the glucosylation of (+)-catechin to produce 4′-O-α-d-glucopyanosyl- and 4′,7-di-O-α-d-glucopyanosyl-(+)-catechin (Nakahara et al., 1995; Meulenbeld et al., 1999). A lactic bacterium, Leuconostoc mesenteroides, which produces glucan or dextran from sucrose, also generated a glucosyltransferase to give 4′-O-, 7-O-α-d-glucopyanosyl and 4′,7-di-O-α-d-glucopyanosyl-(−)-epigallocatechin gallate (Moon et al., 2006). The Arthrobacter sp. β-fructofuranosidase responsible for synthesizing lactosucrose (Arakawa et al., 2002) also catalyzed transfructosylation of epicatechin and EGCG in the presence of sucrose as a glucosyl donor (Nakano et al., 2002). 3-β-Fructofuranosylepicatechin was obtained from epicatechin, while the chemical structure of three β-fructofuranosylated products obtained from EGCG were not assigned. An α-glucosylation enzyme was also found in Trichoderma viride (Noguchi et al., 2008), and this was the first identification of commercial cellulase products such as Cellulase Onozuka RS and Pancelase BR (Yakult Pharmaceutical Industry Co. Ltd. in Tokyo) derived from Trichoderma sp. glycosylate (+)-catechin in the presence of dextrin. Cellulase Onozuka RS exhibited transglycosylation activity in the presence of soluble starch, dextrin, γ-cyclodextrin, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose as glucosyl donors, but not in the presence of maltose, cellobiose, isomaltose, carboxymethyl cellulose, α-cyclodextrin, or dextran. Thus, the enzyme was suggested to be an α-amylase-like enzyme, not a cellulase or β-glucosidase. An α-amylase family enzyme gene was cloned and expressed in Saccharomyces cerevisiae EH1315. © 2010 Taylor and Francis Group, LLC


39

The enzyme also showed α-glucosylation activity with some flavonoids, including (+)-catechin, EGCG, and daizein. The (+)-catechin and EGCG glucosylated products obtained using these enzymes were identified as follows, 5-O-α- and 7-O-α-d-glucopyranosyl-(+)-catechin, 5-O-α- and 7-O-α-d-glucopyranosyl, 7-O-α-d-maltosyl, 7-O-α-d-maltotriosyl, 7-O-α-d-maltotetraosyl, and 7-O-α-d-maltopentaosylepigallocatechin gallate from commercial cellulase products, while 3′-Oα-, 4′-O-α-, and 5-O-α-d-glucopyranosyl-, 3′-O-α-, 4′-O-α-, and 5-O-α-d-maltosyl-(+)-catechin, 3′-O-α-d-glucopyranosyl, 3′-O-α-d-maltosyl, 3′-O-α-d-maltotriosylepigallocatechin gallate, and unidentified EGCG glucosides were obtained using the recombinant enzyme. The commercial and recombinant enzymes had rather different specificity for glucosylating positions. The water solubility of 5-O-α-d-glucopyranosylepigallocatechin gallate increased more than 5 times, as seen for EGCG. The results of taste analyses of the aqueous solution using a multichannel taste sensor (Taste Sensing System SA402B; Intelligent Sensor Technology, Kanagawa, Japan) are shown in Figure 2.5. The astringency and astringent stimulation of the compound were markedly lower than those of EGCG. Glucosylation of (+)-catechin using plant cultured cells has also been performed (Otani et al., 2004. (+)-Catechin was added to the Eucalyptus perriniana cultured cells and incubated for 3 days to obtain three catechin glucosides; 7-O-β-d-, 5-O-β-d-, and 3′-O-β-d-glucopyranosyl-(+)-catechin with yields of 44%, 13%, and 33%, respectively. The reaction gave monoglucosides with only β-glycosidic linkages, and the conversion ratio from (+)-catechin was high. Bioconversion using cultured cells may be a useful production method. The flavonoid glycosides are summarized in Table 2.5.

Sourness 20.00 10.00 0.00 –10.00 –20.00

Umami

Relative taste sensitivity

Bitterness

Saltiness

–30.00

Astringency

Sweetness

Kokumi

Astringent stimulation

FIGURE 2.5 The taste sensitivities of EGCG (closed square) and 5-O-α-d-glucopyranosylepigallocatechin gallate (open triangle). Relative taste sensitivities for these flavonoids are shown (the value for EGCG = 0) as the average values of four independent determinations. The 20% difference in concentration between standard and sample solutions is converted to a graduation of relative taste sensitivity; in other words, relative taste sensitivity equals log 1.2 (the difference in concentration between standard and sample solutions). “Kokumi” is a taste-enhancing quality and has been described variously as continuity, mouthfulness, mouthfeel, and thickness. (Reprinted from Noguchi et al., 2008. J. Agric. Food Chem. 56: 12016–12024. With permission. Copyright 2008 from American Chemical Society.)


40


TABLE 2.5 The Enzymes (Origins) Generate Flavonoid Glycosides Name Rutin glucoside Rutin glucosides Isoquercitrin glucosides Hesperidin glucosides (+)-Catechin glucoside (+)-Catechin glucoside EGCG glucosides

Compound Quercetin-3-O-βG(6-O-αRhm-4-O-α-G) Quercetin-3-O-βG[6-O-αRhm-4-O-α-G(α-1,4-G)n] Quercetin-3-O-βG[4-O-αG(α-1,4-G)n] Hesperetin-7-O-βG[6-O-αRhm-4-O-α-G(α-1,4-G)n] (+)-Catechin-3′-O-α-G and unidentified (+)-Catechin-3′-O-α-G

(+)-Catechin glucoside

EGCG-4′-O-α-G, -4′, 4″-di-O-α-G (+)-Catechin-3′-O-α-G

(+)-Catechin glucoside

(+)-Catechin-4′-O-α-G

(+)-Catechin glucosides

(+)-Catechin-4′-O-α-G, -4′,7-di-O-α-G EGCG-7-O-α-G, -4′-O-α-G, -4′, 7-di-O-α-G Epicatechin-3-O-α-G and unidentified (+)-Catechin-5-O-α-G, -7-O-α-G EGCG-7-O-α-G, -5-O-α-G, -7-O-α-G(α-1,4-G)n (+)-Catechin-5-O-α-G, -4′-O-α-G, -3′-O-α-G, -5-O-α-G(α-1,4-G), -4′-O-α-G(α-1,4-G), -3′-O-α-G(α-1,4-G) EGCG-3′-O-α-G, -3′-O-α-G(α-1,4-G)n, and unidentified (+)-Catechin-7-O-β-G, -5-O-β-G, -3′-O-β-G

EGCG glucosides Epicatechin (+)-Catechin glucosides EGCG glucosides (+)-Catechin glucosides

EGCG glucosides

(+)-Catechin glucosides

Enzyme (Origin)

References

CGTase (B. stearothermophilus) glucoamylase (Rhizopus sp.) CGTase (B. stearothermophilus)

Suzuki and Suzuki (1991)

CGTase (B. stearothermophilus)

Akiyama et al. (2000)

CGTase (Bacillus sp.)

Kometani et al. (1994)

CGTase (B. macerans)

Funayama et al. (1993)

Sucrose phosphorylase (Leuconostoc mesenteriodes) Sucrose phosphorylase (Leuconostoc mesenteriodes) Glucosyl transferase (X. campestris) Glucosyl transferase (Streptococcus sobrinus) Glucosyl transferase (Streptococcus mutans) CGTase (Leuconostoc mesenteriodes)

Kitao et al. (1993)

β-fructofuranosidase (Arthrobacter sp.) Glucosyl transferase ? (Trichoderma viride) Glucosyl transferase ? (Trichoderma viride) Glucosyl transferase ? (recombinant Trichoderma viride)

Glucosyl transferase ? (recombinant Trichoderma viride) E. perriniana cultured cells

Akiyama et al. (2000)

Kitao et al. (1995) Sato et al. (2000) Nakahara et al. (1995) Meulenbeld et al. (1999) Moon et al. (2006) Nakano et al. (2002) Noguchi et al. (2008) Noguchi et al. (2008) Noguchi et al. (2008)

Noguchi et al. (2008)

Otani et al. (2004)

2.3.2.2 L-Ascorbic Acid Glucosides l-Ascorbic acid (vitamin C) is extremely unstable upon exposure to light, heat, oxygen, or metal ions. Various attempts to modify the structure have been made in order to improve its stability. Some attempts to introduce substituents at the hydroxyl groups of the 2,3-enediol, which is the unstable and antioxidizing moiety of l-ascorbic acid, were performed to develop a more stable ascorbic acid derivative, known as “provitamin C” which can be easily cleaved to generate active ascorbic acid in the body. 2-O-Sulfate (Mead and Finamore, 1969) and 2-O-phoshate (Nomura et al., 1969) have greatly enhanced stability and these products are known to be converted to ascorbic



41

acid in vivo and by intracellular hydrolysis through the action of sulfatases or phosphatases. These are compounds produced chemically for use in cosmetics and quasi-drugs. Enzymatic glycosylations of l-ascorbic acid have been performed. 2-O-α-d-Glucopyranosyl-lascorbic acid (AA2-αG) was prepared in both rat intestinal and rice seed α-glucosidase in the presence of maltose as a glucosyl donor (Muto et al., 1990; Yamamoto et al., 1990b). AA2-αG was also stable in aqueous solution (Yamamoto et al., 1990a) and exhibited “provitamin C” activity (Yamamoto et al., 1990c). AA2-αG has been produced industrially using CGTase derived from B. stearothermophilus in the presence of α-cyclodextrin as a glucosyl donor, by the successive treatment of glucoamylase to hydrolyze l-ascorbic acid 2-malto-oligosaccarides (Aga et al., 1991). AA2-αG is included in the List of Existing Food Additives as a food additive and is now broadly used as an active ingredient in cosmetics and food. 5-O- and 6-O-α-d-Glucopyranosyl-l-ascorbic acid were obtained as by-products of AA2-αG production (Mandai et al., 1993), but only 6-O-α-dglucopyranosyl-l-ascorbic acid was detected using a reaction with A. niger α-glucosidase (Muto et al., 1990). These products showed antioxidative activity similar to l-ascorbic acid (Mandai et al., 1993). 2-O-β-d-Galactopyranosyl-l-ascorbic acid was prepared by the reaction of 5,6-Oisopropylidene-l-ascorbic acid and lactose with A. oryzae β-galactosidase and successive acid hydrolysis of the isopropylidene group (Shimono et al., 1994). 2-O-β-d-Glucopyranosyl-l-ascorbic acid (AA2-βG) was isolated from Lycium (Lycium barbarum) fruit (Toyoda-Ono et al., 2004). The content in dried fruit was about 0.5%. AA2-βG is only present in the fruit, not the leaf or root, and is not detected in other plants of the Solanaceae family such as green pepper or tomato, nor other fruits such as Barbados cherry, lemon, camu-camu, sea buckthorn, or grapefruit, which contain abundant l-ascorbic acid. AA2-βG was orally absorbable in rats (Toyoda-Ono et al., 2004) and showed “provitamin C” activity in ODS (osteogenic disorder Shionogi) rats, which have a hereditary ascorbic acid biosynthesis defect (Toyoda-Ono et al., 2005). Although AA2-βG was formed by Trichoderma sp.-derived cellulase in the presence of cellobiose as a glucosyl donor, it was a minor product, and 6-O-β-d-glucopyranosyl-l-ascorbic acid was obtained as a major product (Toyoda-Ono et al., 2005). 2.3.2.3 Other Glycosylated Products Menthyl α-d-glucopyranoside (α-MenG) was prepared using yeast α-glucosidase with maltose as a glucosyl donor (Nakagawa et al., 1998). Menthol is volatile and insoluble in water. Glycosylation of menthol was attempted because a water-soluble menthol derivative might be useful as a food additive. α-MenG tastes slightly sweet at first and in a few minutes a refreshing flavor spreads in the mouth. However, the β-MenG contained in fresh leaves of peppermint is rather bitter. Stevioside (Figure 2.6), which is a major sweet glycoside contained in the leaves of Stevia rebaudiana Bertoni, is 150 times sweeter than sucrose. However, it has a slightly bitter taste. Stevioside was modified by enzymatic glucosylation, and B. macerans CGTase gave α-d-glucopyranosyl derivatives of stevioside in the presence of soluble starch as a glucosyl donor (Fukunaga et al., 1989). OR2 13

24

OR1

HO H

OH

H

3 R1O

CO2R2 19 R1 Steviol Stevioside

FIGURE 2.6

H β-G

R2 H β-G(2-O-β-G)

R1

The chemical structures of terpenoid sweeteners.


R2

Mogrol H H Mogroside V β-G(6-O-β-G) β-G(2-O-β-G, 6-O-β-G)

42


Stevioside was glucosylated at the 4-hydroxyl group of the β-d-glucopyanosyl moieties. Among them, mono-α-glucosylated products, in which α-d-glucose is attached at the 4′-hydroxyl of the sophorose moiety (position 13 of steviol) and the 4-hydroxyl of the β-glucosyl ester moiety (position 19 of steviol), showed higher sweetness and a delicious taste. β-Glucosyl steviosides were obtained by an extracellular enzyme system of Streptomyces sp. using cardlan (1,3-β-glucan) as a glucosyl donor (Kusama et al., 1986). In this method, stevioside was β-glucosylated at the 3-hydroxyl group of β-d-glucopyanosyl moieties. The three major components were 13-O-β-sophorosyl-19-βlaminaribiosyl, 13-O-β-(3′-O-β-glucosyl)sophorosyl-19-O-β-glucosyl, and 13-O-β-sophorosyl-19β-laminaritriosyl steviol. The sample mixture gave a better quality of taste than the individual components. Rubusoside (13-O-β-glucosyl-19-O-β-glucosyl steviol) contained in the leaves of Rubus suavissimus S. Lee is 100 times sweeter than sucrose. Rubusoside was also α-glucosylated by CGTase using starch to generate malto-oligosyl (1,4-α-glucosyl units) products (Ohtani et al., 1991). 13-O-β-Maltotriosyl-9-O-β-glucosyl steviol was 278 times sweeter than sucrose. α-Glucosylation of the 13-O-β-glucosyl moiety enhanced sweetness. Mogroside V (Figure 2.6) in the fruits of Luo-han-guo (Siraitia grosvenori Swingle) is a powerful sweetening agent, 378 times sweeter than sucrose. Mogroside V was α-glucosylated by CGTase using starch (Yoshikawa et al., 2005). Between one and three glucosylated products with α-1,4-linkages were obtained and resulted in improved taste quality. Arbutin (hydroquinone-O-β-d-glucopyanoside), found in the plant Uvae ursi, exhibits a whitening effect on the skin by the inhibition of tyrosinase, which is a key enzyme of melanogenesis and is used in cosmetics. α-Arbutin (hydroquinone-O-α-d-glucopyanoside) was prepared by several researchers from hydroquinone using B. subtilis α-amylase (Nishimura et al., 1994), Leuconostoc mesenteroides sucrose phosphorylase (Kitao and Sekine, 1994), and X. campestris α-glucosidase (Kurosu et al., 2002), as mentioned in the section on the glucosylation of flavonoids. These enzymes only produced monoglucoside. α-Arbutin also showed the same activity as arbutin and is used in cosmetics.

2.4 FUNCTIONAL LIPIDS AND ACYLATED PRODUCTS 2.4.1

FUNCTIONAL LIPIDS OR ACYLATED PRODUCTS PROCESSED BY LIPASE

Diacylglycerol is approved as FOSHU for foods related to triacylglycerol (Table 2.1, Category 9). The product was prepared using immobilized 1,3-specific lipase (Lipozyme RM IM®, Rhizomucor meihei lipase, produced by Novozyme Ind., Denmark) from a hydrolysate (fatty acid mixture) of soybean oil and glycerol (Watanabe et al., 2003). The reaction was performed at 50°C under high vacuum (1 mm Hg) to remove the water generated in the reaction. The product, composed of 1,3- and 1,2-diacylglycerol (7:3), suppressed excess body fat accumulation compared with triacylglycerol in a double-blind controlled test for 4 weeks in healthy men (Nagao et al., 2000). Medium- and long-chain triacylglycerols (MLCT) containing caprylic acid as the fatty acid (FA) component are produced by lipase (Lipase QL® derived from Alcaligenes sp., Meito Sangyo Co. Ltd, Japan) by catalyzing the transesterification of soybean oil and tricaprylin (Negishi et al., 2003). The product also suppressed the accumulation of body fat in a double-blind randomized test of 82 healthy humans for 12 weeks (Kasai et al., 2003) and is approved as FOSHU for foods related to triacylglycerol. Palm oil mid fraction is interesterified with ethyl stearate using 1,3-specific lipase from Rhizopus delemer (Hashimoto, 1993). The obtained product was composed of POP (1,3-di-O-palmitoyl-2-Ooleoylglycerol), POS (the mixture of 2-O-oleoyl-1-O-palmitoyl-3-O-stearoylglycerol and 2-O-oleoyl3-O-palmitoyl-1-O-stearoylglycerol), and SOS (1,3-di-O-stearoyl-2-O-oleoylglycerol). Although cocoa butter has the same components, their ratio is different. Cocoa butter contains a lower proportion of POP than the interesterified product which gives cocoa butter a higher melting point. The product is of better quality than cocoa butter for chocolate, which is called chocolate equivalent (CBE), and is used as chocolate butter.


Functional Foods and Biotechnology in Japan (b) 200 a

150 100

a,b

50 0

ASTAX 900H® Monoester Diester

b 0

5

a a,b b 10

15

20

24

Liver astaxanthin concentration (ng/g tissue)

Plasma astaxanthin concentration (ng/mL)

(a)

43

Time (h)

500 400

a

300

a a,b

200 100 0

ASTAX 900H® Monoester Diester

b

b b 0

5

10

15

20

24

Time (h)

FIGURE 2.7 Astaxanthin concentrations in plasma and liver following the oral administration of astaxanthin n-octanoic acid esters. Astaxanthin n-octanoic acid monoester, astaxanthin n-octanoic acid diester and Astax 9000H® (extract from Haematococcus algae) diluted with olive oil to obtain the amount corresponding to 100 mg/kg of free astaxanthin were administered orally to 5-week-old male Wistar rats. The plasma (a) and liver (b) astaxanthin concentrations after 3, 5, 7, and 10 h were determined by high performance liquid chromatography (HPLC). (From Fukami et al., 2006. J. Oleo Sci. 55: 653–656. With permission from Japan Oil Chemists’ Society.)

Structured lipids, that is, triacylglycerols with a specified chemical structure not in a mixture, are synthesized by 1,3-specific lipase to improve their nutritional and pharmaceutical properties. Mammalian pancreatic lipases preferentially hydrolyze the 1,3-positions of triacylglycerol to generate 2-monoacylglycerol, which is easily absorbed through the intestinal mucosa. To improve the absorption of functional polyunsaturated FA (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid (ARA) cited in the next section, the high oil content of DHA or ARA was derived from 1,3-specific lipases originating from Rhizopus delemer and Rhizomucor meihei in tricaprylin to obtain 1,3-di-O-capryloyl triacylglycerols (Iwasaki et al., 1999; Nagao et al., 2003). The capryloyl ester moiety is hydrolyzed efficiently by gastrointestinal lipases to improve the intestinal absorption of PUFA (Ikeda et al., 1991). DHA is well known to be abundant in fish oil (about 25% in tuna oil). If DHA is used for pharmaceutical or other materials, it needs to be purified. Attempts have been made to raise the DHA purity of tuna oil using lipase hydrolysis. The use of nonspecific Candida cylindrcea lipase exhibited lower hydrolysis potential for DHA than other FA in tuna oil. Therefore, 65% hydrolysis of tuna oil and the successive removal of free FA gave a 53% DHA content glycerol mixture including triacyl-, diacyl-, and monoacylglycerols (Tanaka et al., 1992). Furthermore, ARA rich oil (25% ARA content) obtained by microbial fermentation, referred to in the next section, was conducted using the same lipase. The oil obtained, in which triacylglycerol was the major component, contained 60% ARA, and the recovery of AA was 70% (Shimada et al., 1995). Astaxanthins are widely found in seafood such as shrimps and flesh and eggs of redfish or trout, and are usually present as long-chain FA esters. It was found that astaxanthin monocaprylate showed better absorbability than the corresponding long-chain FA ester mixture extracted from Haematococcus algae (Figure 2.7) (Fukami et al., 2006). Thus, the enzymatic synthesis of astaxanthin caprylic acid esters was attempted by immobilized Candida cylindrcea lipase in tricaprylin (Nakao et al., 2008). However, the yield was only 36.4% and the reaction was not practical.

2.4.2

FUNCTIONAL FOODS FERMENTED BY MICROBES

ARA and DHA are important constituents of phospholipids forming the cell membrane. Both DHA and ARA are known to be necessary in human infants for growth and neural development (Wright et al., 2006). These compounds are increasingly being added to baby formula. DHA is abundant in


44


fish oil. However, ARA is not especially abundant in foods even though it is present in meat, egg, and seafood. These PUFAs were produced by fermentation (Ward and Singh, 2005). Oil with a high ARA content was produced industrially using fungi, Mortierella alpina 1S-4 (Higashiyama et al., 1998). The oil was obtained at 24.2 g/L and contained 45% ARA as a FA composition. DHA was also produced by fermentation using microalga Crypthecodinium cohnii (Behrens and Kyle, 1996; Swaaf et al., 2003) and Schizochytrium sp. (Yaguchi et al., 1997). The DHA content was approximately 40% by weight in the oil. Dihomo-γ-linolenic acid (DGLA), a precursor of ARA biosynthesis, was produced using a Δ5 desaturase-defective mutant of Mortierella alpina that is not able to synthesize ARA (Kawashima et al., 2000). The obtained oil contained 45% DGLA by weight. These ARA- and DGLA-rich oils advanced the understanding of the physiological functions of ARA and DGLA, respectively (Kawashima, 2005).

2.5

PERSPECTIVE

Following all this progress in genetic and protein engineering, it has become possible to obtain useful enzymes without the traditional screening of microbes. Enzyme-encoding DNA sequences can be modified by genetic engineering using methods such as error-prone PCR and gene shuffling. A library composed of numerous proteins expressed from the obtained modified genes is screened by advanced high-throughput small-scale screening systems. It was reported that a protein library expressed on the surface of yeast was screened in a microwell array (well size: picoliter scale) (Fukuda et al., 2005). Active yeast was picked up from the well and proliferated. This yeast, called “arming yeast,” enables its use as a bioreactor (Seong et al., 2006). Transcriptome, proteome, and metabolome technologies in functional food and food components (known as nutrigenomics), have become popular (Kato, 2008). These “omics” provide a wealth of information on the physiological functions, mechanism of action, biomarkers, and functional food components in foods and functional foods. For example, the mode of function of sesamin was clarified by transcriptome analysis in rat liver (Tsuruoka et al., 2005). The gene cloning of key enzymes for the biosynthesis of useful metabolites has become easy. The obtained gene is transferred to a plant and the transgenic plant is bred to obtain the metabolite. A transgenic land plant producing DHA-containing oil is under investigation (Robert, 2006). In the near future, new functional foods or functional food components will be created to promote human health.

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Yamamoto, I., Muto, N., Murakami, K., Suga, S., and Yamaguchi, H., 1990a. l-Ascorbic acid alpha-glucoside formed by regioselective transglucosylation with rat intestinal and rice seed alpha-glucosidases: Its improved stability and structure determination. Chem. Pharm. Bull. (Tokyo). 38: 3020–3023. Yamamoto, I., Muto, N., Nagata, E., Nakamura, T., and Suzuki, Y. 1990b. Formation of a stable l-ascorbic acid alpha-glucoside by mammalian alpha-glucosidase-catalyzed transglucosylation. Biochim. Biophys. Acta (general subjects). 1035: 44–50. Yamamoto, I., Suga, S., Mitoh, Y., Tanaka, M., and Muto, M., 1990c. Antiscorbutic activity of l-ascorbic acid 2-glucoside and its availability as a vitamin C supplement in normal rats and guinea pigs. J. PharmacobioDynam. 13: 688–695. Yamamoto, T., Yamamoto, S., Miyahara, I., Matsumura, Y., Hirata, A., and Kim, M., 1990d. Isolation or a β-mannan hydorlyzing enzyme and hydrolysis of guar gum by the enzyme isolated. Denpun. Kagaku. 37: 99–105. Yamatoya, K., 1994. Hydrolyzed guar gum. A new generation water-soluble dietary fiber. Int. Food Ingredients 4: 15–19. Yashiro, A., Oda, S., and Sugano, M., 1985. Hypocholesterolemic effect of soybean protein in rats and mice after peptic digestion. J. Nutr. 115: 1325–1336. Yasuda, E., Takaku, H., and Matsumoto, H., 1986. Japan Patent 61-212296. Yokoyama, K., Chiba, H., and Yoshikawa, M., 1992. Peptide inhibitors for angiotensin I-converting enzyme from thermolysin digest of dried bonito. Biosci. Biotechnol. Biochem. 56: 1541–1545. Yoshikawa, S., Murata, Y., Sugiura, M., Kiso, T., Shizuma, M., Kitahara, S., and Nakao, H., 2005. Transglycosylation of mogroside V, a triterpene glycoside in Siraitia grosvenori, by cyclodextrin glucanotransferase and improvement of the qualities of sweetness. J. Appl. Glycosci. 52: 247–252.


and Clinical Studies 3 Basic on Active Hexose Correlated Compound Takehito Miura, Kentaro Kitadate, Hiroshi Nishioka, and Koji Wakame CONTENTS 3.1 Introduction ............................................................................................................................ 51 3.2 Manufacturing Process ........................................................................................................... 51 3.3 Ingredient Composition and Structure ................................................................................... 52 3.4 Safety Assessment and Drug Interaction ................................................................................ 52 3.5 Previous Remarkable Research Results.................................................................................. 53 3.6 Alleviating Effect on Chemotherapeutic Drug-Induced Side Effects .................................... 53 3.7 Immunomodulating Action..................................................................................................... 54 3.8 Protective Action Against Infections ...................................................................................... 55 3.9 Anti-Inflammatory Effect ....................................................................................................... 57 3.10 Summary ................................................................................................................................ 58 References ........................................................................................................................................ 58

3.1 INTRODUCTION Active hexose correlated compound (AHCC) is a collective term for the botanical polysaccharides extracted from a liquid culture of the mycelia of the basidiomycete, shiitake (Lentinula edodes). In a survey carried out by the Ministry of Health, Labor and Welfare research group, AHCC was listed as a commonly used health food among Japanese cancer patients, second only to the fungus, agaricus (Hyodo et al., 2005). Recent studies have demonstrated AHCC’s efficacy in the treatment of infectious and inflammatory diseases as well as cancer. This article summarizes recent experimental results where AHCC has been used in the treatment of various diseases including cancer and hepatitis as a complementary and alternative medicine (CAM) in significant medical institutions.

3.2

MANUFACTURING PROCESS

A number of basidiomycetes form the sexual organ or carpophore (fruit body), which produces basidiospores under certain conditions (light, temperature, humidity, change of nutritional status, etc.) subject to sufficient growth of the mycelia. However, if the basidiomycetes are cultured in a liquid medium, they proliferate and form globular fungal bodies rather than carpophores (Furukawa, 1992). It is thought that these properties of the mycelia of basidiomycetes produce AHCC which contains medium components modified by the diverse mycelia-produced enzymes. In the actual AHCC manufacturing process, the mycelia of edible shiitake are subjected to the liquid medium and finally cultured in large 15-ton tanks. Saccharolytic enzymes such as cellulase and glucosidase, and proteolytic enzymes such as protease are produced during this culture. After 51 © 2010 Taylor and Francis Group, LLC

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fermentation, AHCC itself is produced through manufacturing processes including separation, concentration, sterilization, and freeze drying (Hosokawa, 2003).

3.3 INGREDIENT COMPOSITION AND STRUCTURE Nutritional information with regard to AHCC and mushrooms is shown in Table 3.1 (Hosokawa, 2003). AHCC contains abundant carbohydrates as compared to agaricus (Agaricus blazei Murill) and dry shiitake (Lentinus edodes). It is thought that these carbohydrate elements are mainly polysaccharides. In basidiomycete (mushroom)-derived substances, β-glucan is known as a physiologically active ingredient (Furukawa, 1992). However, AHCC substantially differs from other mushrooms and mushroom-derived food products in that it contains only 2% β-glucan but abundant α-glucan. In particular, there is reportedly α-1,4-glucan present, in which the hydroxyl group of C-2 and/or C-3 position is partially acylated, and which is considered to be one of the active ingredients. It is deduced that this partially acylated α-glucan is not obtained by simple extraction from basidiomycete culture broth but is generated by the enzymatic modification of normal α-glucan in the unique patented manufacturing process of AHCC (Hosokawa, 2003).

3.4 SAFETY ASSESSMENT AND DRUG INTERACTION AHCC is considered to be safe since the raw material of AHCC is a basidiomycete derived from edible shiitake, which has been consumed over a long period. The various preclinical safety assessments of AHCC were carried out according to Good Laboratory Practice (GLP) standards. In a single-dose oral toxicity test using rats, the LD50 value (50% lethal dose) exceeds 12,500 mg/kg, which is the maximum dose that can be administered as AHCC. Moreover, when 2% or 5% AHCC mixed with powder diet was given to rats in a 4-month repeated dose oral toxicity test, there were no physiological and biochemical changes, demonstrating a high level of safety of AHCC (Hosokawa, 2003). Spierings and coworkers executed the clinical trial corresponding to a Phase I study to evaluate the safety of AHCC in healthy volunteers. Twenty-six male and female healthy volunteers aged 18–61 years received 9 g of AHCC daily for 14 days, which was three times the recommended dose. No adverse events were admitted and it was therefore concluded that AHCC is safe as a food product in clinical practice (Spierings et al., 2007). Matsui et al. (2002) also reported that there were no adverse events in 113 postoperative patients with hepatocellular cancer who had been given AHCC for 9 years. In CAM, dietary supplements are widely used by cancer patients and these foods are taken alongside chemotherapeutic agents in cancer treatments. In this particular case, there is some concern

TABLE 3.1 General Nutritional Ingredient Analysis and β-Glucan Content Ratio AHCC Freeze-Dried Powder (%) Protein Fats Carbohydrates Dietary fiber Ash contents β-Glucan

13.1 2.2 71.2 2.1 8.9 0.2

Agaricus Agaricus blazei Murill (%)

Shiitake Lentinula edodes (%)

Analysis Method

40–45 3–4 38–45 6–8 5–7 11.4

19.3 3.7 59.2 10.0 3.9 3.5

Kjeldahl method Acid decomposition method As per the balance Enzymatic–gravimetric method Direct ashing method Enzymatic method, ELISA method

Source: From Hosokawa, M. (Supervisor), Yamasaki, M., Kamiyama, Y. (Editors), 2003. Basic and Clinical Situation of Active Hexose Correlated Compound, 1st Edition, pp. 7–15. Tokyo: Lifescience Co., Ltd. With permission.


Basic and Clinical Studies on Active Hexose Correlated Compound

53

TABLE 3.2 CYP 450 Metabolism Profile of AHCC CYP 450 Isoenzyme 3A4 2C8 2C9 2D6

Substrate

Inhibitor

Inducer

− − − +

− − − −

− − − +

Source: Mach, C. et al., 2008. J. Soc. Integrative Oncol. 6(3): 105–109. With permission.

about drug interaction between dietary supplements and anticancer drugs. Mach et al. (2008). examined the mutual interaction of AHCC on CYP450 (Table 3.2). Results showed that AHCC does not have an effect on drug metabolism except for possibly promoting the metabolism of this drug, which is a substrate of the 2D6 pathway, indicating that AHCC has no interaction with most of the chemotherapeutic agents in liver metabolism (Mach et al., 2008). This finding suggested that AHCC should not have an effect on cancer chemotherapy and could be used safely.

3.5 PREVIOUS REMARKABLE RESEARCH RESULTS It was reported that, as an adjunctive therapy after hepatectomy in patients of hepatocellular carcinoma, AHCC contributes to the prevention of cancer recurrence, liver function improvement, and prolongation of postoperative survival rate (Matsui et al., 2002). Moreover, its safety was confirmed by the various safety assessments mentioned above (Hosokawa, 2003; Spierings et al., 2007), and there is no interaction with chemotherapeutic agents, resulting in a safe supplement causing no concern in combination with conventional chemotherapies (Mach et al., 2008). Thus, AHCC can be used widely as a dietary supplement in patients not only with hepatocellular carcinoma but also with other types of cancer. However, since there has only been this retrospective clinical study on hepatocellular carcinoma so far, clinical reports and double-blind trials on other cancers will be required.

3.6

ALLEVIATING EFFECT ON CHEMOTHERAPEUTIC DRUG-INDUCED SIDE EFFECTS

When these dietary supplements are used in clinical practice, they are commonly taken in combination with conventional treatments. In particular, they are ingested along with chemotherapeutic agents in cancer treatment. AHCC is mainly used to reduce the side effects of anticancer drugs. Although its mechanism of action is not clear, it appears to attenuate hair loss, anorexia, nausea, and bone marrow suppression. These findings were proved in several animal studies, and it was reported that AHCC ameliorates the renal dysfunction and bone marrow suppression associated with cisplatin (Hirose et al., 2007). In a colon cancer-inoculated mouse model, administration of AHCC prevented the elevation of blood urea nitrogen and creatinine concentrations resulting from cisplatin-induced renal dysfunction (Table 3.3) and alleviated the reduction of bone marrow cells. At the same time, importantly, AHCC did not inhibit the antitumor action of cisplatin (Figure 3.1). Hair loss is one of the serious side effects that lower the quality of life (QOL). It is known that the chemotherapeutic agent cytarabine sometimes causes hair loss. However, the hair loss in rats treated with cytarabine (30 mg/kg, ip) for seven successive days was reduced by supplementation with 500 mg/kg of AHCC 1 h before the treatment of cytarabine (Kitadate, 2008). In addition to anticancer drug monotherapy, the alleviating effects of AHCC against various side effects induced by multidrug chemotherapy were investigated in normal mice treated with multiple


54


TABLE 3.3 Effect of AHCC on Cisplatin-Induced Renal Damage Treatment Group Control Cisplatin Cisplatin + AHCC

Urea Nitrogen

Creatinine

20.2 ± 1.5 34.0 ± 5.0* 26.0 ± 3.3**

0.88 ± 0.05 1.09 ± 0.20* 0.98 ± 0.04

Source: From Hirose, A. et al., 2007. Toxicol. Appl. Pharm. 222: 152–158. With permission. Unit: mg/dL; *p < 0.01 versus control; **p < 0.01 versus cisplatin.

anticancer agents such as paclitaxel/cisplatin, 5-flurouracil (5-FU)/irinotecan, and so on. The results revealed that AHCC attenuated bone marrow suppression, and hepatic and renal dysfunction related to multidrug treatments (Shigama et al., 2009). Thus, the alleviating outcome of AHCC on anticancer drug-induced side effects was demonstrated in several animal studies. This leads to the expectation that AHCC should be useful in maintaining and improving the QOL of cancer patients, and in the completion of their chemotherapy. In a clinical trial, 44 patients with unresectable progressive liver cancer were divided into two groups: 34 subjects in the AHCC group who received 6 g/day of AHCC, and 10 in the placebo group. When the survival rate and QOL were compared in both groups, the AHCC group showed a significant improvement in survival rate and QOL including mental stability, general health status, and normal activity (Cowawintaweewat et al., 2006).

3.7

IMMUNOMODULATING ACTION

Many botanical polysaccharides derived from mushrooms are being trialed as immune enhancers. Matsushita et al. (1998) reported that AHCC improved the reduction of natural killer (NK) activity and mRNA expressions of IL-1β and TNF-α in UFT-treated (uracil and tegafur in a 4:1 molar concentration) SST-3 breast cancer-transplanted rats. It was also reported that supplementation with (b)

(a)

10

8000

Cisplatin

6000

Tumor weight (g)

Tumor size (mm3)

Control

Cisplatin + AHCC

4000

5

*

* , * **

2000

*, **

0

0 0

10 20 Days after tumor transplantation

30

Control

Cisplatin

Cisplatin + AHCC

FIGURE 3.1 Antitumor effect of cisplatin alone and cotreatment with cisplatin and AHCC. (a) Growth curves of colon-26 tumor cells and (b) weight of colon-26 solid tumor at day 28. *p < 0.01 versus control; **p < 0.05 versus cisplatin. (From Hirose, A. et al., 2007. Toxicol. Appl. Pharm. 222: 152–158. With permission.)


Basic and Clinical Studies on Active Hexose Correlated Compound (a)

(b)

(c)

p = 0.025

30

p = 0.038

25 20 15 10 5 0

p = 0.119

25 20 15 10 5 0

Pre Post AHCC

Pre Post Control

12 Number of DC2 (×103)

p = 0.038

Number of DC1 (×103)

Number of total DC (×103)

35

55

p = 0.314

p = 0.021

10 8 6 4 2 0

Pre Post AHCC

Pre Post Control

Pre Post AHCC

Pre Post Control

FIGURE 3.2 Comparison of DC numbers in the AHCC and control groups. (a) Total DC; (b) DC1; (c) DC2. (From Terakawa, N. 2008, Nutr. Cancer 60(5): 643–651. With permission.)

3 g/day of AHCC for 2 weeks enhanced NK activity in cancer patients previously showing low NK activity (Ghoneum et al., 1995). Terakawa et al. (2008) conducted a double-blind randomized clinical trial to evaluate the number of dendritic cells (DC) in pheripheral blood. Twenty-one healthy volunteers were divided into two groups: 10 and 11 subjects in the AHCC and placebo groups, respectively. In the AHCC group, 3 g/day of AHCC was administered for 4 weeks. Blood was collected before and after administration. The quantity of total dendritic cells (total DC), myeloid dendritic cells (DC1), and lymphoid dendritic cells (DC2) was measured, and an increase in the number of total DC and DC1 was shown in the AHCC group (Figure 3.2). DC is in the upstream of the immune cascade and DC1 play an important role in antitumor action via näive T lymphocytes, suggesting that AHCC might improve the immune capacity of healthy subjects. On the other hand, in neither group was there was a significant difference in NK activity and production of cytokines such as interferon and interleukin. When AHCC was orally administered into C57BL/6 mice inoculated with B16 melanoma cells or EL4 lymphoma cells, a significant delay in the tumor growth was observed. Furthermore, AHCC promoted the proliferation and activation of antigen-specific CD4+ and CD8+ T cells, enhanced production of IFN-γ, and increased the number of NK and γδT cells (Gao et al., 2005). These results suggest that AHCC not only strengthens immunity surveillance against tumor cells and shows protective action against cancer growth, but might also be effective in microbial infections.

3.8 PROTECTIVE ACTION AGAINST INFECTIONS As mentioned above, oral administration of AHCC reinforces immunity surveillance and modulates both natural and acquired immunities, anticipating protection against external microbial infections as well as tumor cells. There have been some reports on the effects of AHCC in experimental infectious models (Ishihashi et al., 2000; Ikeda et al., 2003; Aviles et al., 2003, 2004, 2006, 2008; Ritz et al., 2006; Fujii et al., 2007; Ritz, 2008; Nogusa et al., 2009). In cyclophosphamide (CY)-induced neutropenia models, AHCC exhibited defensive effects against Candida albicans, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA) (Ishihashi et al., 2000). In an experimental granulocytopenia mouse model treated with CY, 5-FU, doxorubicine, or prednisolene, a protective effect was seen against C. albicans infection (Ikeda et al., 2003). These results indicate that ingestion of AHCC provides a defensive function against opportunistic infection in the immunosuppressive state related to anticancer drugs. Aviles


56


TABLE 3.4 NK Cell Activity (% Cytotoxicity) in the Spleen of Influenza-Infected Mice Control AHCC

Day 0

Day 1

Day 2

Day 3

5.6 ± 1.0 5.3 ± 0.7a

14.6 ± 3.2 13.6 ± 2.5a,b

10.4 ± 1.6 20.4 ± 3.6b,**

6.3 ± 1.2 6.7 ± 0.5a

Source: From Ritz, B. et al., 2006. J. Nutr. 136: 2868–2873. With permission. Mean value ± SEM (n = 3); a, b: significant difference between signs of a and b (p < 0.05); **p < 0.01 versus control.

and coworkers reported the protective effect of AHCC against Klebsiella pneumoniae infection in the hindlimb-unloading murine model of space flight conditions (to evoke an immunosuppressive state). AHCC prolonged the survival rate of infected mice (Aviles et al., 2003), increased spleen cell proliferation and cytokine production, and elevated cytokine production in peritoneal exudate cells (Aviles et al., 2004). Furthermore, Aviles et al. (2006, 2008) reported that AHCC enhanced resistance to K. pneumoniae infection in a surgical wound infection model in mice, resulting in an extension of the survival rate. It is possible that supplementation with AHCC improves immunosuppression due to trauma, infection, and food deprivation, and other adverse effects in living organisms. It has also been reported that AHCC modulates natural and acquired immunities and is effective against viral as well as fungal and bacterial infections (Ritz et al., 2006; Fujii et al., 2007; Aviles et al., 2008; Ritz, 2008; Nogusa et al., 2009). In the case of influenza infection, treatment with AHCC increased NK activity 2 days after the infection (Table 3.4), and resulted in lower reduction and earlier recovery of body weight following the infection (Figure 3.3). This defensive effect against viral infections was also seen with a low dose of AHCC (Nogusa et al., 2009). The other experiment was conducted to evaluate the effect of AHCC for H5N1-type bird influenza virus, and the survival rate improved in mice infected with a lethal dose of the virus (Fujii et al., 2007). Recently, a report on protection against the West Nile virus (WNV) demonstrated that AHCC up-regulated production of the IgG antibody against the WNV and affected the γδT cell subset, resulting in an alleviation of the severity of the infection through modulating host immunity (Wang et al., 2009). Clinical studies of infectious diseases have been eagerly anticipated. 28 27

Weight (g)

26 25 24 23 22 AHCC Control

21 20 19

0

1

2

3 4 5 6 7 8 Time after infection (days)

9

10

FIGURE 3.3 Body weights of control and AHCC-supplemented young mice following infection with 100 HAU influenza A/PR8 through d 10 postinfection. Values are means + SEM, n = 20. ***Different from AHCC. p < 0.001. (From Ritz, B. et al., 2006. J. Nutr. 136: 2868–2873. With permission.)



57

TABLE 3.5 Effect of AHCC Administration for Microorganisms in Faces of TNBS Colitis Model Rat Microorganisms

Control

TNBS

Sulfasalazine

AHCC100

AHCC500

Aerobic bacteria Anaerobic bacteria Lactobacillus Bifidobacteria Clostridium

7.83 ± 0.21a 8.34 ± 0.18 7.46 ± 0.28a 5.99 ± 0.37a 3.06 ± 0.26b

6.36 ± 0.16b 8.76 ± 0.21 5.90 ± 0.35b 5.39 ± 0.12b 4.52 ± 0.19a

6.79 ± 0.20b 8.16 ± 0.13 5.50 ± 0.27b 8.21 ± 0.22a 2.90 ± 0.65b

8.03 ± 0.21a 9.17 ± 0.23 7.58 ± 0.37a 6.73 ± 0.48a 3.46 ± 0.14b

7.58 ± 0.31a 8.58 ± 0.26 7.00 ± 0.40a 6.84 ± 0.45a 2.95 ± 0.14b

Source: Daddaoua, A. et al., 2007. J. Nutr. 137: 1222–1228. With permission. The values are mean value ± SEM (n = 6), and represent colony forming units (CFU). There is a significant difference between signs of a and b ( p < 0.05).

3.9

ANTI-INFLAMMATORY EFFECT

NO production (nmol/106 cells)

The impact of AHCC on inflammatory diseases has been supported by several recent research projects. Daddaoua and coworkers reported that in a trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model, the damage score of large intestines, cytokine production of IL-1β, IL-1 receptor antagonist, MCP-1 and TNF-α, and intestinal flora were improved by administering 100 and 500 mg/kg/day of AHCC. Although treatment with TNBS induced a reduction in lactobacillus and bifidobacteria and an increase of clostridia, administering AHCC increased lactobacillus and bifidobacteria and decreased clostridia. The efficacy of AHCC was as same as that of sulfasalazine (200 mg/kg) used as a remedy (Table 3.5) (Daddaoua et al., 2007). Nitric oxide (NO) production exerts bactericidal and antiviral action in bacterial and viral infections. In hepatic inflammation, injury and cancer, inducible NO synthase (iNOS) is induced and a large amount of NO is generated, leading to an aggravation of the symptoms. Matsui et al. (2007) found that treatment with AHCC reduces iNOS-induced NO production in rat hepatocytes stimulated by IL-1β (Figure 3.4), and this reduction is attributed to the degradation of iNOS mRNA by AHCC treatment. This experiment also provoked a new finding that not only transcribed mRNA of the sense chain of the iNOS gene but also the antisense transcript of iNOS are simultaneously synthesized in the cultured hepatocytes, and the antisense transcript contributes to the stability of 15

IL-1β IL-1β + AHCC

10

5

0 0

2 4 6 8 10 Time after IL-1β stimulation (h)

12

FIGURE 3.4 Suppression of NO synthesis by AHCC. Amount of NO (NO2−) in the medium of primary cultured rat hepatic cells was measured by the Griess method. Square and Circle represent the NO amount in the medium supplemented with IL-1β (1 nM) and IL-1β plus AHCC (8 mg/mL), respectively. (From Matsui, K. et al., 2007. J. Parenter. Enteral. Nutr. 31: 373–381. With permission.)


58


iNOS mRNA (Matsui et al., 2008). The mechanism of regulating post-transcription via the antisense transcript is a new finding about the functions of noncoding RNA, and is expected to provide a seed for new nucleic acid medicine. AHCC is closely associated with the antisense transcript, suggesting that it might slow the anti-inflammatory effect by modulating iNOS mRNA and result in protection for the liver.

3.10 SUMMARY Although there are many points of uncertainty about the active ingredients of AHCC and the mechanism of its action so far, the research results discussed in this chapter should contribute towards identifying the active ingredients and explaining the partial mechanism of action. In addition to the immunostimulatory activity that many mushroom-derived supplements possess, AHCC can normalize suppressed or overstimulated immunity. Thus, it is possible to help a living body respond to various extracorporeal stimuli such as infections. Moreover, since AHCC controls the proliferation of cancer by reinforcing surveillance and shows no drug interaction with most chemotherapeutic agents, further applications are hereafter expected as a safe supplement or as a food ingredient in combination with conventional therapies.

REFERENCES Aviles, H., Belay, T., Fountain, K., et al., 2003. Active hexose correlated compound enhances resistance to Klebsiella pneumoniae infection in mice in the hindlimb-unloading model of spaceflight conditions. J. Appl. Physiol. 95: 491–496. Aviles, H., Belay, T., Vance, M., et al., 2004. Active hexose correlated compound enhances the immune function of mice in the hindlimb-unloading model of spaceflight conditions. J. Appl. Physiol. 97: 1437–1444. Aviles, H., O’Donnell, P., Orshal, J., et al., 2008. Active hexose correlated compound activates immune function to decrease bacterial load in a murine model of intramuscular infection. Am. J. Surg. 195: 537–545. Aviles, H., O’Donnell, Sun, B., et al., 2006. Active hexose correlated compound (AHCC) enhances resistance to infection in a model of surgical wound infection. Surg. Infect. 7(6): 527–535. Cowawintaweewat, S., Manoromana, S., Sriplung, H., et al., 2006. Prognostic improvement of patients with advanced liver cancer after active hexose correlated compound (AHCC) treatment. Asian Pac. J. Allergy Immunol. 24: 33–45. Daddaoua, A., Martinez-Plata, E., Lopez-Posadas, R., et al., 2007. Active hexose correlated compound acts as a prebiotic and is antiinflammatory in rats with hapten-induced colitis. J. Nutr. 137: 1222–1228. Fujii, H., Nishioka, H., Wakame, K., et al., 2007. Nutritional food active hexose correlated compound (AHCC) enhances resistance against bird flu. JCAM 4(1): 37–40. Furukawa, H., 1992. Science of Mushrooms, 1st edition, p. 71. Tokyo: Kyoritsu publications. Gao, Y., Zhang, D., Sun, B., et al., 2005. Active hexose correlated compound adaptive immune responses. Cancer Immunol. Immun. 55(10): 1258–1266. Ghoneum, M., Wimbley, M., Salem, F., et al., 1995. Immunomodulatory and anticancer effects of active hemicellulose compound (AHCC). Int. J. Immunother. XI(1): 23–28. Hirose, A., Sato, E., Fujii, H., et al., 2007. The influence of active hexose correlated compound (AHCC) on cisplatin-evoked chemotherapeutic and side effects in tumor-bearing mice. Toxicol. Appl. Pharm. 222: 152–158. Hyodo, I., Amano, N., Eguchi, K., et al., 2005. Nationwide survey on complementary and alternative medicine in cancer patients in Japan. J. Clin. 23(12): 1–10. Hosokawa, M. (Supervisor), Yamasaki, M., Kamiyama, Y. (Editors), 2003. Basic and Clinical Situation of Active Hexose Correlated Compound, 1st Edition, pp. 7–15. Tokyo: Lifescience Co., Ltd. Ikeda, T., Ishihashi, H., Tansei, S., et al., 2003. Preventing action of mushroom product AHCC against Candida albicans infection in an experimental granulocytopenia infection mouse model. Jpn. J. Med. Mycol. 44: 127–131. Ishihashi, H., Ikeda, T., Tansei, S., et al., 2000. Preventing action of mushroom product AHCC in an opportunistic infection mouse model. Yakugaku Zasshi 120(8): 715–719. Kitadate, K., 2008. AHCC enhances anticancer activity and alleviates side effects. Food Style 12(5): 69–72.



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Mach, C., Fujii, H., Wakame, K., et al., 2008. Evaluation of active hexose correlated compound hepatic metabolism and potential for drug interactions with chemotherapy agents. J. Soc. Integrative Oncol. 6(3): 105–109. Matsui, K., Kawaguchi, Y., Ozaki, T., et al., 2007. Effect of active hexose correlated compound on the production of nitric oxide in hepatocytes. J. Parenter. Enteral. Nutr. 31: 373–381. Matsui, K., Kawaguchi, Y., Ozaki, T., et al., 2008. Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes. Hepatology 47(2): 686–697. Matsui, Y., Uhara, J., Satoi, S., et al., 2002. Improved prognosis of postoperative hepatocellular carcinoma patients when treated with functional foods: A prospective cohort study. J. Hepatol. 37: 78–86. Matsushita, K., Kuramitsu, Y., Ohiro, Y., et al., 1998. Combination therapy of active hexose correlated compound plus UFT significantly reduces the metastasis of rat mammary adenocarcinoma. Anticancer Drugs 9: 343–350. Nogusa, S., Gerbion, J. and Ritz, B., 2009. Low-dose supplementation with active hexose correlated compound (AHCC) improves the immune response to acute influenza infection in C57BL/6 mice. Nutr. Res. 29: 139–143. Ritz, B., 2008. Supplementation with active hexose correlated compound increases survival following infectious challenge in mice. Nutrition 66(9): 526–531. Ritz, B., Nogusa, S., Ackerman, A., et al., 2006. Supplementation with active hexose correlated compound increases the innate immune response of young mice to primary influenza infection. J. Nutr. 136: 2868–2873. Shigama, K., Nakaya, A., Wakame, K., et al., 2009. Alleviating effect of active hexose correlated compound (AHCC) for anticancer drug-induced side effects. J. Exp. Ther. Oncl. 8: 43–51. Spierings, E., Fujii, H., Sun, B., et al., 2007. A phase I study of the safety of the nutritional supplement, active hexose correlated compound, AHCC, in healthy volunteers. J. Nutr. Sci. Vitaminol. 53: 536–539. Terakawa, N., Matsui, Y., Satoi, S., et al., 2008. Immunological effect of active hexose correlated compound (AHCC) in healthy volunteers: A double-blind, placebo-controlled trial. Nutr. Cancer 60(5): 643–651. Wang, S., Walshe, T., Fang, H., et al., 2009. Oral administration of active hexose correlated compound enhances host resistance to west Nile Encephalitis in mice. J. Nutr. 139: 598–602.



and Breeding 4 Biotechnology for Enhancing the Nutritional Value of Berry Fruit Jessica Scalzo and Bruno Mezzetti CONTENTS 4.1 4.2 4.3

Introduction ............................................................................................................................ 61 Nutritional Values and Phytochemical Composition of Berry Fruits..................................... 63 Breeding Berry Fruit for Improved Nutritional Values ..........................................................64 4.3.1 Case Study: Blueberry ................................................................................................64 4.3.2 Case Study: Strawberry .............................................................................................. 68 4.4 The Use of Biotechnology for Improving Fruit Quality and Nutrition: A Short Review....... 71 4.4.1 The Application of Biotechnology in Improving Strawberry Nutritional Values ...... 74 4.5 Conclusion .............................................................................................................................. 75 Acknowledgments............................................................................................................................ 75 References ........................................................................................................................................ 75

4.1 INTRODUCTION The reasons for consuming particular fruits or vegetables, the quantities consumed and the preferred quality are related to social and cultural life conditions. Overall fruit consumption is related to several factors, including fruit price, availability, and consumer preference and quality acceptance. Recently, consumers have become increasingly interested in the health benefits and nutritional values (vitamins content, minerals, phytochemicals, antioxidants, etc.) of fruits and vegetables, and research has produced important information about these properties. Increasing consumption of fruits and vegetables has been associated with general health benefits (Ames et al., 1993), and the benefits of fruit consumption have been associated with their antioxidant activity. Studies of a diet rich in flavonoids and anthocyanins have associated their antioxidant activities with low rates of mortality from coronary heart disease (Hertog et al., 1993, 1997; Knekt et al., 1996) and stroke (Keli et al., 1996), vasoprotective effects (Shin et al., 2006), anti-inflammatory activities (Wang et al., 1999), benefits to vision (Matsumoto et al., 2003) and protection against H2O2-induced cell toxicity effects and DNA damage (Ghosh et al., 2006). The health effects of other phytochemical compounds such as vitamin C and carotenoids have also been shown to be linked to their antioxidant activity (Cumming et al., 2000). A general theory is emerging that “bioactive components” in plants induce metabolic effects such as antioxidant functions. Bioactive components are generally defined as compounds in foods that deliver a health benefit beyond basic nutrition (International Life Science Institute, 1999). Fruits and vegetables are rich in phytochemical compounds with potential benefits to health. Although the recommended minimum daily adult intake is 400 g (WHO, 2003), it is not known for certain which of the compounds are the major contributors to good health. 61 © 2010 Taylor and Francis Group, LLC

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TABLE 4.1 Antioxidant Activity of a Range of Fruit Assessed With Different Methods Fruit Wild strawberry Blackberry Red raspberry Strawberry Cherry Blueberry Plum Kiwifruit Orange Pineapple Apple Pear Banana Peach Apricot

FRAP (mmol TE/100 g)a

ORAC (mmol TE/100 g)b

TEAC (mmol TE/100 g)c

— 4.023 2.334 2.159 2.009 1.854 1.826 1.017 0.901 0.600 0.311 0.226 0.163 0.147 —

— 5.35 4.93 3.58 3.36 2.43 7.34 0.92 1.81 0.79 3.25 1.77 0.88 1.86 —

3.306 2.731 2.594 1.503 0.756 2.252 — — — — 0.160 — — 0.525 0.39

Fruit have been ordered according to their FRAP content. a Halvorsen et al. (2006). b Wu et al. (2006). c Scalzo et al. (2005a).

Phytochemical composition and antioxidant properties vary considerably among fruits and vegetables (Wang et al., 1996; Cao and Prior, 1998; Prior and Cao, 2000; Agius et al., 2003; Brat et al., 2006; Mattila et al., 2006). Studies have demonstrated that the antioxidant activity may depend on the food matrix (Scalzo et al., 2005a; Wu et al., 2006; Halvorsen et al., 2006) as summarized in Table 4.1. There is a clear variation in antioxidant activity among different fruit types, with berry fruits being high in antioxidants. The dietary intake of antioxidants can be raised by increasing the quantity of fruits and vegetables consumed or by consuming a higher proportion of food with a high antioxidant activity such as berry fruits. Enhancement of the nutritional value in fruit through breeding and/or application of emerging biotechnology is an important option in order to support, for example, a higher antioxidant intake even when the consumption of fruit is low. If nutritional components are combined with high sensorial fruit quality, prospective consumer health may be further improved by increased consumption. The breeding approach can succeed if the variability and heritability of the nutritional trait indicate the possibility of achieving a breeding progress. The biotechnological approach is now an integrated option to extend this improvement. However, the success of both breeding and biotechnological approaches is related to knowledge of the genetic diversity to be used in genetic and genomic studies. In this chapter we discuss whether the nutritional values of berry fruits, in particular the phytochemical composition and antioxidant activity, can be improved with breeding and biotechnology. We will review the results of two breeding programs (blueberry and strawberry) as case studies and we will report results obtained by studying the nutritional quality of fruit from genetically modified strawberries.


Biotechnology and Breeding for Enhancing the Nutritional Value of Berry Fruit

4.2

63

NUTRITIONAL VALUES AND PHYTOCHEMICAL COMPOSITION OF BERRY FRUITS

Berry fruits are well known for their important nutritional values and as a good source of flavonoids (USDA, 2005, 2007) and have been widely studied for their health benefits due to antioxidant activity. As with other fruits and vegetables, berries are an important dietary source of fibres, essential vitamins, and minerals (Beattie et al., 2005). When compared to other fruits, berry fruits are rich in anthocyanins which are responsible for their colors (McGhie and Walton, 2007), and single servings of some berry fruit can provide 10–100 mg of anthocyanins. Anthocyanin composition varies among berry fruit (Beattie et al., 2005; Wu et al., 2006; Scalzo et al., 2008). Their high antioxidant activity in vitro has been extensively studied in the Genus Vaccinium L., Fragaria, Ribes, and Rubus (Wang et al., 1997; Prior et al., 1998; Kalt et al., 1999, 2001; Deighton et al., 2000; Ehlenfeldt and Prior, 2001; Connor et al., 2002a, 2002b, 2002c, 2005; McGhie et al., 2002; Moyer et al., 2002; Proteggente et al., 2002; Wada and Ou, 2002; Agius et al., 2003; Scalzo et al., 2004, 2005a, 2005b; Currie et al., 2006) and the role of the genotype on blueberry and strawberry phytochemical composition is well known (Clark et al., 2002; Connor et al., 2002c; Wang et al., 2002; Azodanlou et al., 2003; Scalzo et al., 2005a, 2005b; Capocasa et al., 2008). The anthocyanins present in blueberry are galactosides, glucosides, and arabinosides of the aglycones delphinidin, cyanidin, petunidin, peonidin, and malvidin. Additionally, these glycosides may also be acetylated (Wu and Prior, 2005). According to Wu et al. (2006), among the fruits and vegetables they analysed, blueberries were found to be rich in malvidins and petunidins, while other authors (Kader et al., 1996; Goiffon et al., 1999) have reported that blueberries are rich in delphinidin-3-galactoside and petunidin-3-glucoside. In strawberries the major representatives are pelargonidin and cyanidin glucosides or acylated forms with a range of aliphatic acids (Goiffon et al., 1999; Lopes-da-Silva et al., 2002) and the presence of the main derivates seems to be constant in all varieties, although qualitative and quantitative variation has been observed among cultivars (Maatta-Riihinen et al., 2004). Anthocyanin concentrations may differ widely within the same variety, dependent on the degree of ripeness, on climatic factors, and on postharvest storage (Lopes-da-Silva et al., 2007). Other compounds such as proanthocyanidins have also been surveyed for their health benefits (Beecher, 2004), in particular those of the genera Vaccinium (Maatta-Riihinen et al., 2005). For example, the protective effect of cranberry toward urinary tract infection is probably due to specific proanthocyanidins (Howell, 2002). Different Vaccinium spp. seem also to be rich in resveratrol (Rimando et al., 2004) which has been shown to possess cancer chemopreventive activities (Jang et al., 1999). Berry fruits also contain ellagic acid (Amakura et al., 2000; Maatta-Riihinen et al., 2004; Koponen et al., 2007) which, according to Hakkinen et al. (1999), is responsible for more than 50% of total phenolics quantified for strawberries and raspberries. Together with other berries, the strawberry is also a relevant source of folate (Bailey and Gregory, 1999) and vitamin C, which is responsible for more than 20% of the total antioxidant activity of the fruit extracts (Carr and Frei, 1999). Breeding to increase one or more beneficial phytochemicals in fruit is likely to be achievable as many different fruit traits have been successfully modified through breeding strategies. Classical genetic, as well as transgenic, approaches are being used to increase the content of specific bioactive compounds of plants (Davuluri et al., 2005) and there is an increasing awareness that multiple genetic and environmental factors affect production and accumulation of bioactive compounds (Davik et al., 2006), although these factors are seldom taken into consideration when a functional food-fruit is marketed. Breeding and biotechnological programs aimed at increasing fruit nutritional values need to be undertaken with knowledge of the decision process used for determining the efficacy of a bioactive compound and/or group of compounds.


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In the next section we will describe the breeding approaches used to improve the nutritional values of berry fruit, in particular the phytochemical composition and antioxidant activity, and we will discuss the results of two breeding programs aimed towards obtaining blueberry and strawberry cultivars with improved nutritional qualities.

4.3 BREEDING BERRY FRUIT FOR IMPROVED NUTRITIONAL VALUES Berry fruit represent a good source of bioactive compounds and the increase of consumption of berry fruit is seen as an appropriate strategy for improving antioxidant activity intake. Another option to support a high intake of “healthy compounds” even when the consumption of fruit is low is by increasing their level in fruit through breeding. Breeding for increased nutritional value of fruit can be a long-term procedure and a good knowledge of the target phytochemical(s) is necessary. This breeding approach can only succeed if the genetic variation, heritability, and impact of environmental parameters on the expression indicate that progress through breeding is feasible. Whether the breeding target is a specific phytochemical compound or a fruit trait, its variation within the plant population needs to be known. The inheritance, correlation to other traits, environmental effects on the trait, and population size are important aspects of the breeding process that will allow the development of the most effective strategy. Berry fruits are highly perishable—often sold immediately after harvest—and expensive, especially when hand picked (fresh fruits are almost exclusively hand picked). Offering good berry fruit of a consistent quality would be the way to increase interest from consumers and to satisfy their expectations. The quality of fruit is considered an extremely complex matter because it is difficult to describe objectively and it changes during the fruit maturation. Consumer acceptance is related to specific perceived aspects (external quality) such as fruit appearance, lack of visible defects, shape, and color. However these are not sufficient to define the fruit quality as fruit attributes such as texture, sweetness, and acidity, combined with aroma and flavor are also important. Nowadays, the fruit quality traits associated with long storage and high nutritional value are major topics of several breeding programs, even when these traits are controlled by a complex genetic background and are frequently associated with negative agronomic characters. There is a high genetic variability of the traits previously described as well as environmental influences. Thus, the challenge for the breeder who wants to obtain a berry fruit with high nutritional values while maintaining an outstanding fruit quality is not only the knowledge of a single trait, but also what is affecting the variation and how different traits are correlated together. We focus the following discussion on the progress achieved in breeding to increase the nutritional values (phytochemical composition and antioxidant activity) of blueberry and strawberry while maintaining an outstanding fruit quality.

4.3.1

CASE STUDY: BLUEBERRY

A recent survey (Scalzo et al., 2008, 2009) on blueberry cultivars and breeding selections of northern highbush (NHB), southern highbush (SHB), and rabbiteye (RE) types maintained at HortResearch showed variation among genotypes for most fruit traits, including the phytochemicals. Figure 4.1 demonstrated the results of phytochemical composition as total antioxidant activity assessed by the ferric reducing antioxidant power (FRAP) assays, while the total anthocyanin content (total ACY) was measured by a reversed-phase high performance liquid chromatography (HPLC). Both traits were assessed following the protocols described by Connor et al. (2005). Genotypes considered in this study are planted in observation plots at the Ruakura Research Centre, New Zealand (37°48′S 175°17′E), and fruits were harvested on three occasions over each of two growing seasons (2005– 2006 and 2006–2007). For FRAP and total ACY, the variance attributable to genotype was up to an


Biotechnology and Breeding for Enhancing the Nutritional Value of Berry Fruit FRAP

65

Total ACY

“Hortblue petite” “Northland” “Duke” 18 22 19 “Nui” “Hortblue poppins” 17 21 16 “Bluecrop” “Reka” 20 26 “Misty” “Marimba” 25 “O Neal” 28 29 27 23 24 30 “Maru” “Centurion” 34 “Centra blue” 31 33 32 “Powderblue” “Rahi” 10

20

30

40

1000

2000

3000

4000

FIGURE 4.1 Phytochemical composition (i.e., total antioxidant activity assayed by FRAP and total ACY) of a collection of blueberry cultivars HortResearch advanced selections 16–34 are maintained at the Ruakura Research Centre of the Horticulture and Food Research Institute of New Zealand Ltd. (HortResearch) (37°48′S 175°17′E). For each genotype the symbol (● NHB, ○ SHB, and □ RE) indicates the average of 2 years and the solid lines indicate the range (min–max, both years). FRAP was measured as μmol Trolox equivalent/g fruit weight, and total ACY as μg cyanidin 3-O-glucoside chloride equivalents/g fruit weight. Genotypes have been ordered according to FRAP.

order of magnitude greater than the residual variance (Scalzo et al., 2008, 2009) and this is reflected in the mean genotype values and their ranges (Figure 4.1). The highest average values of FRAP and total ACY were obtained from fruit of a RE advanced selection (Selection 30). FRAP average values were also high in the fruit of other RE blueberry genotypes (“Maru,” “Centurion,” Selection 34, “Centra Blue,” and Selections 31 and 33) and in the fruit of two NHB blueberry cultivars, “Hortblue Petite” and “Northland.” Fruit of these genotypes also had a high total ACY. In the literature there are numerous studies of the variation of phytochemical compounds in absolute and relative terms within Genus Vaccinium (Ehlenfeldt and Prior, 2001; Kalt et al., 1999, 2001; Prior et al., 2001; Sellappan et al., 2002; Cho et al., 2004, 2005; Maatta-Riihinen et al., 2005; Scalzo et al., 2008). The antioxidant activity and the phenolic content of blueberry are not only affected by genotype, but can also show a year-to-year variability (Connor et al., 2002a), a variation during fruit maturation and ripening (Castrejon et al., 2008), during postharvest storage conditions (Kalt et al., 1999; Connor et al., 2002d), and with different cultural conditions (Wang et al., 2008). The strong and positive correlation between total anthocyanins, total phenolics, and antioxidant activity was also noted (Prior et al., 1998; Ehlenfeldt and Prior, 2001; Connor et al., 2002c; Moyer et al., 2002; Sellappan et al., 2002).


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TABLE 4.2 Mean Content of Individual Anthocyanins Over All 34 Blueberry Genotypes and Range (Min–Max) Individual Anthocyanin Malvidin galactoside Cyanidin galactoside + delphinidin arabinoside Delphinidin galactoside Malvidin arabinoside Malvidin glucoside Acylated anthocyanin Petunidin galactoside Petunidin arabinoside Minor anthocyanins Delphinidin glucoside Petunidin glucoside Cyanidin arabinoside Cyanidin glucoside

Content (μg/g) 310 (67–882) 229 (24–686) 205 (8–674) 183 (40–388) 141 (2–560) 131 (0.5–809) 124 (14–369) 91 (21–304) 73 (5–256) 69 (1–364) 52 (1–232) 43 (1–273) 28 (1–155)

Source: Scalzo, J. et al., 2009. Acta Hort. (ISHS) 810: 823–830.

Blueberry contains anthocyanins of the five phenolic aglycones (delphinidin, cyanidin, petunidin, peonidin, and malvidin) conjugated with galactose, glucose, or arabinose, and acyl derivates of anthocyanins are also present (Scalzo et al., 2008). In this survey Scalzo et al. (2008, 2009) identified individual anthocyanins (Table 4.2) and the chromatogram traces recorded at 530 nm show the variation of anthocyanin components among three NHB blueberry genotypes (Figure 4.2). A high variability in anthocyanin content in absolute and relative terms was also found among and within five different blueberry populations of Vaccinium corymbosum. Results of the total Blueberry #1

Blueberry #2

Blueberry #3

FIGURE 4.2 RP-HPLC chromatogram traces (530 nm) of anthocyanins present in three genotypes of NHB blueberry.



67

5000 4500 4000

Total ACY

3500 3000 2500 2000 1500

“Hortblue petite” “Duke”

Selection 18

“O’Neal”

“O’Neal”

“O’Neal”

Selection B

Selection 28 Selection 23

1000 Selection A

500 0 1

2

3

4

5

FIGURE 4.3 Total ACY as cyanidin 3-O-glucoside chloride equivalents (μg/g fruit weight) of five blueberry populations. For each family the symbol ● indicates the mean, the symbol ▲ indicates the parent mean, and the solid lines indicate the range of values (min–max). The number of offspring for each family varied between 50 and 100.

ACY, average values per family, and variation within family are shown in Figure 4.3. The five crosscombinations were chosen after having being screened as potential parent candidates for their anthocyanin and antioxidant properties. Four cross-combinations had fruit of the parental plants with similar anthocyanin content (Family 1, Family 3, Family 4, and Family 5), while Family 2 arose from a cross-combination between the lowest (maternal—Selection A) and the highest (paternal— “Hortblue Petite”) anthocyanin content in fruit. Interesting results were also found in Family 5, where it was possible to identify six albino fruittype seedlings from a cross-combination of fully blue fruit-type parents. The anthocyanin content of the albino fruit was low for each of these seedlings. The potential exists to breed blueberries with high nutritional values, which could be facilitated by estimating the heritabilities and genetic correlations among phytochemicals and other traits of commercial importance. In breeding for a trait such as antioxidant activity, which in blueberry and other berries appears to be due to numerous phenolic compounds including anthocyanins, phenolic acids, and other flavonoids, many genes would be expected to influence that expression. In our study, the heritability estimates for FRAP and total ACY were high (0.75 and 0.74, respectively) excluding the albino fruit-type blueberry (Table 4.3). Connor et al. (2002c) reported a moderate narrow-sense heritability (0.43) for antioxidant activity in populations derived from crosses representing NHB (V. corymbosum L.), lowbush (V. angustifolium Ait.), and half-high (V. corymbosum × V. angustifolium derivatives) blueberry. A moderate to high (0.46–0.80) narrow-sense heritability estimates the antioxidant activity and anthocyanin composition found in blackcurrant populations (Currie et al., 2006). The authors also found a high genetic correlation among the main antioxidants. A similar study in red raspberry (Connor et al., 2005) found very high narrow-sense heritability for total anthocyanin and a high phenotypic correlation between total phenols and antioxidant activity, suggesting that phenolics, including anthocyanins, were the most important compound with antioxidant activity and that, with a high heritability of the trait, selection based on phenotype would be successful. According to our study, the genotypic and phenotypic correlations between FRAP and total ACY were also positively high (Table 4.3).


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TABLE 4.3 Heritability Estimates (Diagonal, Bold), Genotypic Correlations (Upper Triangle), and Phenotypic Correlations (Lower Triangle) for Fruit Weight (Weight), Scar Diameter (Scar), Fruit Firmness (Firmness), and the log10 Transformations of Phytochemical Variates: FRAP and Total ACY (With Standard Error Estimatesa in Brackets) Weight Scar Firmness FRAP Total ACY a

Weight

Scar

Firmness

FRAP

Total ACY

0.91 (0.024) 0.19 (0.085) 0.53 (0.085)

0.38 (0.192) 0.49 (0.079) 0.09 (0.098)

0.65 (0.110) 0.29 (0.183) 0.87 (0.059)

−0.27 (0.084) −0.05 (0.087)

−0.19 (0.084) −0.16 (0.084)

−0.15 (0.097) −0.06 (0.098)

−0.40 (0.195) −0.15 (0.220) −0.30 (0.216) 0.75 (0.064) 0.73 (0.058)

−0.11 (0.212) −0.09 (0.212) −0.07 (0.281) 0.81 (0.092) 0.74 (0.057)

Standard errors for genotypic and phenotypic correlations were estimated using the jackknife procedure.

Obtaining blueberries with the phytochemical traits previously described while still maintaining fruit quality traits such as big and uniform size, high berry firmness, and small pedicel scar is currently an important target for the breeding program at HortResearch. These traits were used for advancing breeding selections of NHB, SHB, and RE blueberry and the results obtained from a breeding population were correlated (Table 4.3). Fruit weight correlated positively (genotypically and phenotypically) with fruit firmness and pedicel scar, which reflects the selection pressure on both these traits. The high correlation between FRAP and total ACY suggests that either assay could be used for selection and result in genetic progress for both traits. The phytochemical traits were inversely correlated with the quality traits, in particular fruit weight with FRAP, and similar results were found in strawberry (Capocasa et al., 2008). Narrow-sense heritability estimation for fruit weight, pedicel scar, and fruit firmness were medium to high (Table 4.3). Exploring new genetic sources for increasing phytochemical composition while maintaining a good fruit quality will be explored in breeding blueberries as it has been successfully approached in strawberries, where the use of Fragaria virginiana glauca has improved the fruit of the breeding populations in antioxidant and soluble solid content (Capocasa et al., 2008).

4.3.2

CASE STUDY: STRAWBERRY

The effect of cultivars in controlling the phytochemical composition of strawberries is well known (Wang et al., 2002; Azodanlou et al., 2003; Meyers et al., 2003), but few genotypes are well characterized for these important features. Furthermore, limited knowledge is available about the possibility of improving strawberry nutritional traits by breeding. The evaluation of the phytochemical composition and important fruit traits of strawberries is a significant task which is necessary in order to exploit new cultivars for commercial release. Bearing in mind the complexity of strawberry phytochemical composition, about 8 years ago we started a breeding program at the Marche Polytechnic University, focused on the improvement of these traits while maintaining outstanding fruit quality. The phytochemical traits considered in our study were the total antioxidant activity assessed with two different methods, the FRAP and the Trolox equivalent antioxidant capacity (TEAC), total polyphenols (TPH), anthocyanin content (total ACY) both in absolute and relative terms, vitamin C, and folates (Tulipani et al., 2008). The abovementioned traits are used for screening strawberry genotypes and selecting new genetic material derived from breeding programs, and the results are shown in Tables 4.4 and 4.5. © 2010 Taylor and Francis Group, LLC


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TABLE 4.4 Phytochemical Composition of Fruit From a Collection of 20 Strawberry Genotypes Maintained at the UNIVPM Genotype

FRAP (μmol TE/g)

“Maya” “Sveva” AN94.414.52 AN93.371.53 “Cilady” “Cifrance” “Patty” “Don” “Camarosa” “Paros” “Roxana” “Alba” “Madeleine” “Queen Elisa” “Onda” AN94.268.51 91.143.5 “Idea” “Irma” “Adria”

17.0 a 15.0 b 14.8 b 13.7 bc 13.4 bcd 13.3 bc 12.1 cdef 12.0 cdef 12.0 cdef 11.9 cdef 11.9 cdef 11.8 cdef 11.8 cdef 11.6 cdef 11.2 def 11.1 fg 10.9 fg 10.0 fg 9.7 g 9.5 g

TPH (mg GAE/g)

TEAC (μmol TE/g) 15.5 bcd 18.4 a 17.3 ab 12.4 ef 16.6 bc 16.5 bc 12.8 ef 11.8 ef 14.5 cd 15.0 cd 12.6 ef 14.2 d 14.8 cd 12.6 ef 13.5 de 15.6 bcd 15.1 cd 12.7 ef 11.2 f 12.9 def

2.2 fg 2.8 bc 3.0 ab 2.9 b 2.6 cd 3.2 a 2.6 cd 2.0 gh 2.6 cd 2.6 cd 2.0 gh 2.0 fgh 2.2 efg 2.0 gh 2.0 gh 2.3 efg 2.4 de 1.9 h 1.8 h 1.8 h

Source: Capocasa, F. et al., 2008. Food Chem. 111: 872–878. All the results are a mean value of two consecutive production years (2003 and 2004 northern hemisphere) and fruit sampled in the three main harvests for each genotype. Total antioxidant activity was assayed by FRAP and TEAC and measured as μmol Trolox equivalent/g fruit weight; the TPH was measured as mg of gallic acid equivalent/g fruit weight. Values in the same column that are followed by different letters are significantly different (p ≤ 0.01) using the Student Newman Keuls (SNK). Genotypes have been ordered according to FRAP.

TABLE 4.5 Quantification of Total ACY and Single Anthocyanins Based on HPLC Data Genotype AN00.239.55 AN94.414.52 AN99.78.51 Adria AN03.338.51 Alba Patty Sveva Irma

Total ACY

Cyanidin Glucoside

Pelargonidin Glucoside

296.23 250.33 209.22 176.52 167.40 162.97 155.39 137.08 99.00

5.28 11.15

282.34 227.94 172.46 160.41 164.74 128.82 125.76 132.95 95.80

2.31 2.67 6.60 1.06 20.3 1.61

Pelargonidin Rutinoside 6.31 7.46 9.96 13.80 7.26 4.94 0.87 0.71

Source: Tulipani, S. et al., 2008. J. Agric. Food Chem. 56(3): 696–704. Results are expressed as μg per gram of fruit weight and genotypes have been ordered according to total ACY.


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24 22

FRAP

20 18 16 AN93.371.53

“Sveva”

AN 94.414.52

“Queen Elisa”

91.143.5

14 12

“Paros” “Queen Elisa”

“Onda”

AN 94.414.52

“Sveva” “Patty”

“Onda”

10 8 1

2

3

4

5

6

Family

FIGURE 4.4 Total antioxidant activity as FRAP (μmol Trolox equivalent/g fruit weight) of six strawberry populations. For each family the symbol ● indicates the mean, the symbol ▲ indicates the parent mean, and the solid lines indicate the range of values (min–max). The number of offspring for each family was 10.

Important phytochemical traits such as antioxidant activity and the total polyphenolic content were used to characterize different breeding populations (Scalzo et al., 2005a), where the crosscombinations and parental plants were selected for the highest composition and where the use of wild germplasm has improved the phytochemical composition of strawberries (Capocasa et al., 2008). The results of the total antioxidant activity of six different strawberry populations assessed with FRAP are reported in Figure 4.4. Families with the highest FRAP average values were Families 4 and 6. Seedlings of these families arose from cross-combinations in which one of the parental plants (AN94.414.52) was a hybrid between two Fragaria species (Fragaria × ananassa and F. virginiana glauca). We found a negative correlation between fruit size and most of the nutritional quality parameters. In particular, strawberry fruit size negatively correlated with the soluble solid content (SS) and the titratable acidity (TA) and with phytochemical parameters such as the total antioxidant activity assessed with both TEAC and FRAP, and TPH (Capocasa et al., 2008). Since the most recent breeding programs consider phytochemical traits as being of low priority, the genotypes rarely associate production efficiency and sensorial quality with these traits. “Cifrance” and “Cilady” are the only cultivars showing a sufficient combination of standard quality and nutritional parameters (quite high SS, TA, and antioxidant features), but both lack fruit firmness. New Italian varieties as “Queen Elisa,” “Adria,” “Alba,” and “Sveva” and the selection AN94.268.51 had the required firmness, but only in “Sveva” was this combined with high nutritional parameters (Capocasa et al., 2008). Furthermore, the cultivar/genotype studies showed a high correlation of antioxidant activity versus TPH (Heinonen et al., 1998; Proteggente et al., 2002; Meyers et al., 2003). We have found a negative correlation between fruit color (both L* and Chroma Index) and phytochemical traits (Table 4.6). A pale shiny strawberry fruit such as “Idea” resulted in lower antioxidant activity, while a dark dull fruit (e.g., fruit of AN94.414.52 and “Sveva”) has the highest antioxidant values. The results of a study carried out by Capocasa et al. (2008) showed for the first time the importance of F. virginiana spp. glauca genetic base in improving the nutritional quality of commercial strawberries, as already observed for other characteristics such as male fertility, fruit size, and disease resistance (Hancock et al., 2002). In fact, the plant materials providing the best fruit nutritional quality were selected from Families 4 and 6 (Capocasa et al., 2008), reflecting the maternal parent (AN94.414.52) behavior. Among the other cross-combinations only families having “Sveva” as a



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TABLE 4.6 Pearson’s Correlation and Significance Based on Genotype Means for Standard Quality and Nutritional Parameters Variable TA SS F L* Chroma index TPH FRAP

SS

F

L*

Chroma Index

TPH

FRAP

TEAC

0.43** – – – – – –

−0.19* −0.07ns – – – – –

−0.12 ns −0.18*s 0.17*

−0.02 ns −0.02 ns 0.10 ns 0.77**

0.43** 0.38**

0.25** 0.20*

0.38** 0.23**

−0.23** −0.39** −0.28** − −

−0.08 ns −0.26** −0.24** 0.51**

−0.14* −0.21* −0.13 ns 0.52** 0.43**

− − − −

− − −

−

Source: Capocasa, F. et al., 2008. Food Chem. 111: 872–878. Titratable acidity (TA), soluble solids content (SS), firmness (F), brightness (L*), chroma index, total polyphenol content (TPH), and total antioxidant activity assessed by FRAP and TEAC. ns. *. ** Nonsignificant or significant at P ≤ 0.05 or 0.001, respectively.

parent contained offspring with an increased value of phytochemical parameters, particularly TPH, but combined with lower standard quality parameters. These results demonstrate that: (i) the loss of these attributes occurred during domestication; (ii) the nutritional value of fruit can be considered an inheritable trait; and (iii) the genetic background available in F. × ananassa cultivars can be improved by using wild strawberry species such as F. virginiana spp. glauca.

4.4

THE USE OF BIOTECHNOLOGY FOR IMPROVING FRUIT QUALITY AND NUTRITION: A SHORT REVIEW

Conventional breeding is one means of achieving the development of berries rich in nutritional compounds; the genetic diversity available within sexual compatible species of any given crop will limit the extent of improvement. Transgenic approaches can provide an alternative, although there is currently public concern about their use in contemporary agriculture, particularly when genes derived from organisms other then plants are used. Up to now, transgenic approaches have been used successfully to increase the nutritional value of several crops with worldwide importance, such as rice (Paine et al., 2005) and tomato (Davuluri et al., 2005). However, these approaches have not increased both carotenoids and flavonoids simultaneously, except in cases associated with several plant developmental defects (Gilberto et al., 2005). All major breeding and biotechnology programs on berries have as their major priority the improvement of fruit quality. In general, a clear definition of quality is quite difficult and varies according to the eventual destination of the fruit. However, for berries the following components may be considered of major importance: flavor (a complex combination of sweetness, acidity, and aroma), firmness, and shelf-life. All these aspects are controlled by a complex developmental process related to fruit ripening, involving specific changes in gene expression and cellular metabolism (Manning, 1994). In climacteric fruits these events are coordinated by the gaseous hormone ethylene, which is synthesized autocatalytically in the early stages of ripening. Nonclimacteric fruits do not synthesize or respond to ethylene in this manner, yet undergo many of the same physiological and biochemical changes associated with the production of a ripe fruit. Wild strawberry is an attractive model system for studying ripening in nonclimacteric fruit, because of its small diploid genome, its short reproductive cycle, and its capacity for transformation.


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Eight ripening-induced cDNAs were isolated from this species after differential screening of a cDNA library (Nam-YoungWoo et al., 1999). The predicted polypeptides of seven clones exhibit similarity to database protein sequences, including acyl carrier protein, caffeoyl-CoA 3-O-methyltransferase, sesquiterpene cyclase, major latex protein, cystathionine γ-synthase, dehydrin, and an auxin-induced gene. None of these proteins appears to be directly related to events generally associated with ripening such as cell wall metabolism or the accumulation of sugars and pigments; rather, their putative functions are indicative of the wide range of processes regulated during fruit ripening. mRNA populations in ripening strawberry (F. × ananassa) fruit were examined using polymerase chain reaction (PCR) differential display (Wilkinson et al., 1995). Five mRNAs with ripening-enhanced expression were identified using this approach. Three of the mRNAs appeared to be fruit-specific, with little or no expression detected in vegetative tissues. Sequence analysis of the cDNA clones revealed positive identities for three of the five mRNAs based on homology to known proteins. These results indicate that the differential display technique can be a useful tool for studying fruit ripening and other developmental processes in plants at the RNA level. Tissue softening accompanies the ripening of many fruits and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. Expansin gene expression was examined in the strawberry (F. × ananassa) (Civello et al., 1999). The strawberry differs significantly from the tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberries are nonclimacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripeningregulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, the results suggest that expansins are a common component of ripening and that nonclimacteric signals other than auxin may coordinate the onset of ripening in strawberries. A novel E-type endo-β-1,4-glucanase (EGases) with a putative cellulose-binding domain is highly expressed in ripening strawberry fruits of the octoploid cultivar Chandler (Trainotti et al., 1999). Two full-length cDNA clones (faEG1 and faEG3, respectively) have been isolated by screening a cDNA library representing transcripts from red fruits. Southern blot analysis of genomic DNA suggests that the strawberry EGases are encoded by a multigene family and are predominantly expressed during the ripening process. In agreement with other ripening-related genes in strawberry, the expression of faEG1 and faEG3 is also down-regulated by treatment with the auxin analogue 1-naphthaleneacetic acid (NAA). Differences in temporal expression of the two EGase genes in fruits are not accompanied by differences in spatial expression. The pattern of expression and the sequence characteristics of the two polypeptides suggest that the two strawberry EGases operate in a synergistic and coordinated manner. Antisense technology resulted an useful tool to prevent strawberry fruit from softening by suppressing particular genes involved in fruit softening without altering fruit quality (Woolley et al., 2001; Palomer et al., 2006; Sesmero et al., 2007). Llop Tous et al. (1999) isolated two cDNA clones (Cel1 and Cel2) from a cDNA library obtained from ripe strawberry (F. × ananassa) fruit encoding divergent EGases (cellulases). The EGases differ in their secondary and tertiary structures and in the presence of potential N-glycosylation sites. By in vitro translation it was shown that Cel1 and Cel2 bear a functional signal peptide, the cleavage of which yields mature proteins of 52 and 60 kDa, respectively. The Cel2 EGase was expressed in green fruit, accumulating as the fruit turned from green to white and remaining at an elevated, constant level throughout fruit ripening. In contrast, the Cel1 transcript was not detected in



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green fruit and only a low level of expression was observed in white fruit. The level of Cel1 mRNA increased gradually during ripening, reaching a maximum in fully ripe fruit. The high levels of Cel1 and Cel2 mRNA in ripe fruit and their overlapping patterns of expression suggest that these EGases play an important role in softening during ripening. Carbohydrate content and balance are important in determining the flavor and processing quality of the strawberry. As the fruit ripens, the sugar balance favors the hexoses glucose and fructose rather than sucrose. This has consequences for the osmotic potential of the fruit cells and may result in an overall adjustment of water import, fruit growth, and potential for processing. In many fruit species the enzyme invertase (β-fructofuranosidase) is responsible for catalyzing the breakdown of sucrose. An invertase that is located in the cell wall may regulate the unloading of sucrose from the phloem and controls assimilate accumulation, whereas an invertase located in the vacuole may regulate sucrose and hexose storage. Invertase genes cloned from potato, encoding cell wall and vacuolar forms, were integrated into two strawberry cultivars, Symphony and Senga Sengana, via A. tumefaciens-mediated transformation (Bachelier et al., 1997). Transgenic plants were assessed for modified growth, sugar balance, and flavor and processing quality (Graham et al., 1997). Recently, Park et al. (2006) generated transgenic plants which incorporated an antisense cDNA of ADP-glucose pyrophosphorylase (AGPase) small subunit (FagpS) driven by the strawberry fruitdominant ascorbate peroxidase (APX) promoter, to evaluate the effects on carbohydrate contents during fruit development. The results showed that the levels of AGPase mRNA were drastically reduced in the red stage of fruits in all the transgenic plants. The suppression of the AGPase small subunit in transgenic plants resulted in a 16–37% increment of total soluble sugar content and a 27–47% decrease of the starch content in mature fruit without significantly affecting other fruit characteristics such as color, weight, and hardness. Results from previous studies suggested that, through biotechnological alternation, the AGPase gene might be used for improving soluble sugar content and decreasing starch content in strawberry fruits. Fruit flavor is a result of a complex mix of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. DNA microarrays and appropriate statistical analyses were used to identify a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit (Aharoni et al., 2000). Volatile esters are quantitatively and qualitatively the most important compounds in providing fruity odors. Biochemical evidence for the involvement of the SAAT gene in the formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s). MADS-box genes encode putative transcription factors which are highly conserved among eukaryotes. The name arises from the four original members of this family, which are MCM1 in yeast, AG in Arabidopsis, DEFA from Antirrhinum, and SRF in humans (Schwarz-Sommer et al., 1992). Genetic analyses have shown that plant MADS-box genes are homeotic and control the spatial and temporal locations of specific organs. AGAMOUS, from Arabidopsis, is a MADS-box gene responsible for stamen and carpel identity. It is also involved in maintaining the floral meristem and preventing it from reverting from a determinate state to an indeterminate state (Mizukami and Ma, 1995). Homologs of this gene have been found in diverse plant species including tobacco (Kempin et al., 1993), tomato (Pnueli et al., 1994), and recently also in strawberry (Aharoni et al., 2000). This molecular research into the factors that affect strawberry fruit quality should open up interesting future opportunities to improve our knowledge of the mechanisms controlling such complex but important agronomic characteristics.


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4.4.1 THE APPLICATION OF BIOTECHNOLOGY IN IMPROVING STRAWBERRY NUTRITIONAL VALUES An early interesting result in berry research was the demonstration of the biosynthesis of l-ascorbic acid in ripe strawberry fruit which can occur through d-galacturonic acid. Furthermore, it was demonstrated that vitamin C levels can be increased in A. thaliana plants by overexpressing the strawberry gene GaiUR, encoding a d-galacturonic acid reductase (Agius et al., 2003). The role of DefH9-iaaM and rolC genes in improving fruit production and nutritional quality was studied for the first time in transgenic strawberries (Mezzetti et al., 2004a, 2004b). The aim of this study was to better identify the potential use of both genes in improving strawberry agronomic performances and the antioxidant attributes of control and transgenic lines of two strawberry (F. × ananassa) genotypes (breeding selection AN93.231.53 and “Calypso”). In similar studies, differences were found in phytochemical traits like the antioxidant activity by using TEAC and the total phenolic content (TPH) by Folin–Ciocalteu. The increased productivity led by DefH9-iaaM did not alter the antioxidant activity while the pleiotropic changes induced by rolC improved fruit antioxidant activity. The increase in strawberry plant productivity caused by the DefH9-iaaM gene is likely to be due, either directly or indirectly, to transgene expression, and consequently may increase auxin synthesis (indoleacetic acid—IAA) in the flower buds (Mezzetti et al., 2004b). The increased IAA content in flowers induced by the expression of the DefH9-iaaM gene strongly affected plant morphogenic development, but without changing the fruit components controlling quality and antioxidant activity (Scalzo et al., 2005a). The increased plant cytokinin metabolism induced by the expression of rolC gene had an effect on plant development (increased vigour and adaptability), but also led to an improvement in the fruit nutritional quality (mainly sugar content and total antioxidant activity). The highest yield of rolC lines was mainly related to a larger fruit number with a reduced fruit weight but was also associated with increased fruit total sugar content and antioxidant activity (Scalzo et al., 2005a). This study represents the first evidence of rolC effect on fruit quality and antioxidant capacity. Further agronomical studies for the genetically modified strawberry, as well as the appropriate risk assessment, are now required. Recently a great stride has been taken in identifying and characterizing, through biochemical and molecular means, the major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development (Almeida et al., 2007). The cloning and biochemical characterization of a glucosyltransferase involved in anthocyanin biosynthesis in strawberry fruit was reported by Griesser et al. (2008). Data were reported on the ripening-related and auxin-controlled expressions of this gene. By using other techniques to verify the function of glycosyltransferases, the RNA interference (RNAi)-mediated down-regulation of an anthocyanidin-3-O-glycosyltransferase gene in a commercially important fruit crop was also reported, thus confirming its function in the plant. While an ongoing work has produced AmDFR and MiANS transgenic lines of Calypso and Sveva and preliminary transcriptomic studies on Sveva, DFR evidenced a deep perturbation of the whole pathway. These new transgenic lines represent unique new material for molecular and biochemical studies to elucidate the regulation of flavonoid pathways and to improve the nutritional properties of the strawberry (Montironi et al., 2009). As strawberries are a niche product, these findings will mainly be relevant for smaller farms. Under increasing global warming conditions, the importance of growing strawberries may actually increase, but there may also be a need for new high-quality varieties adapted to different growing conditions and/or cultivation systems. Furthermore, strawberries are a charismatic crop, consumed directly without being processed, hence the population will react sensibly to a potential offer of transgenic strawberries. This will require a particularly sound ecological risk assessment for the commercial cultivation of transgenic strawberries. In addition, strawberries may serve as a model for other cultivated Rosaceae such as fruit trees, transgenic varieties of which have already been developed (e.g., plum and cherry trees) or are under



75

development (e.g., apple or peach trees). Strawberries have the same basic flower structure and the same main pollen vectors (honey bees), but much shorter life cycles, and hence are more amenable to experimentation.

4.5 CONCLUSION In this discussion we have tried to delineate the breeding and biotechnology opportunities available for increasing the nutritional value of berry fruit. The enhancement of the level of phytochemicals in fruit through breeding and/or biotechnology is an important option to support a higher antioxidant intake even when the consumption of fruit is low. The success of both breeding and biotechnology approaches is related to the deep knowledge of the genetic diversity to be used in genetic and genomic studies. However, the manipulation of plant metabolism is still not easy to address. There is an increasing awareness that multiple genetic and environmental factors affect production and accumulation of bioactive compounds, but these factors are rarely taken into account when fruit is marketed. Phytochemical composition varies among fruits and berry fruits are particularly rich. However, there is a high variability in phytochemicals among and within berry fruit. We have shown that for two berry fruit crops, blueberry and strawberry, the phytochemical composition varies among genotypes and that the nutritional value can be improved with breeding. Combining the nutritional components with a high standard sensorial fruit quality is a challenge that breeders are now required to undertake and establishing success in this is likely to require a few breeding generations. Inheritance, correlation of the traits, environmental effects on the trait, and population size are important aspects of the breeding process that will allow the development of the most effective strategy. Finally, biotechnological applications can be used as an integrated option to breeding. In our discussion we have reviewed the role of two different genes (DefH9-iaaM and rolC) in modifying the nutritional quality of strawberry fruit.

ACKNOWLEDGMENTS We wish to acknowledge people involved in the blueberry and strawberry research: Dr. Tony McGhie, Shirley Miller, Carolyn Edwards, John Meekings, Judith Rees, and Dr. Franco Capocasa. The valuable help of Dr. Ron Beatson and Dr. Peter Alspach in editing the manuscript is gratefully acknowledged.

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the Bioavailability 5 Improving of Polyphenols Tetsuya Konishi and M. Mamunur Rahman CONTENTS 5.1 5.2

Introduction ............................................................................................................................ 81 Bioavailability of Flavonoids .................................................................................................. 82 5.2.1 Factors Affecting Bioavailability................................................................................ 82 5.2.2 Anthocyanin Bioavailability .......................................................................................84 5.3 Strategy to Improve the Bioavailability of Flavonoids ........................................................... 85 5.4 Conclusion .............................................................................................................................. 86 References ........................................................................................................................................ 87

5.1

INTRODUCTION

Polyphenols are a large family of compounds having more than two phenolic OH groups in their structure, although simple phenolic acids and alcohols are also included in the group and are widely distributed in nature, especially in plants (Halvorsen et al., 2002; Shahidi and Ho, 2005). Epidemiologically, it is suggested that the consumption of polyphenol-rich foods or beverages is associated with the prevention of diseases, cancer, and aging. Now people are living longer, issues of cardiovascular protection, anticancer effects, and neurodegenerative disease prevention are attracting particular attention (Chen et al., 2008; Ness and Powles, 1997; Visioll and Hagen, 2007). Indeed, various reports on polyphenols have been published, discussing their essential antioxidant properties (Lopez-Velez et al., 2003), cardiovascular protection (Tuoie et al., 2007; Yung et al., 2008), anticancer effects (Steinmetz and Potter, 1996), antidiabetic properties (Venables et al., 2008), reduction in cataract development (Chethan et al., 2008), anti-inflammatory effect (Rahman, 2006), and so on. These are described in several review articles (D’Archivio et al., 2007; Prasain and Barnes, 2007; Scalbert and Willamson, 2000; Singh et al., 2008; Willamson and Manach, 2005; Yang et al., 2008). Fruits, vegetables, spices, and herbs are major sources of dietary polyphenols, and are therefore attracting attention as major foods which play a critical role in human health promotion and disease prevention. Among the polyphenols, flavonoids are important because they are the most abundant in the daily diet (Grotewold, 2006). Flavonoids are characterized as a group of compounds having a threering structure with a C6 –C3–C6 skeleton. They include the anthoxanthins, which have six structurally related family members: flavones, flavonols, flavanols, flavanones, isoflavones, and isoflavanes. The types and amounts of flavonoids in the diet fluctuate widely, depending on habits related to culture, ethnic group, and geological location. For example, Asian people consume high amounts of isoflavones and other flavonoids derived from tea (Fletcher, 2003). Anthocyanins are another group of flavonoids found ubiquitously as plant pigments in nature. These are also attracting attention as active dietary ingredients with a potential role in preventing cardiovascular disease, brain degenerative disease, and in chemoprevention (Konishi and Ichiyanagi, 2008). Their unique color is attributed to 81 © 2010 Taylor and Francis Group, LLC

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characteristic structural changes occurring at different pHs, such as flavylium cation at an acidic pH and quinoidal forms when alkaline. Although a wide range of health benefits has been postulated for flavonoids, the precise mechanism of their action and their actual physiological role in diet are still not fully understood. One reason lies in their low bioavailability, which makes it difficult to extrapolate the positive biological functions observed in vitro, into in vivo studies (Scalbert and Willamson, 2000). There is generally a large discrepancy between the plasma concentration of flavonoids that appear after oral administration and the active in vitro concentration, and thus the bioavailability of polyphenols is still unclear.

5.2

BIOAVAILABILITY OF FLAVONOIDS

Several reviews have been published on the bioavailability of bioflavonoids and its implication for their physiological functioning (D’Archivio et al., 2007; Prasain and Barnes, 2007; Scalbert and Willamson, 2000; Singh et al., 2008; Willamson and Manach, 2005; Yang et al., 2008). The term bioavailability indicates the extent of utilization of orally taken food ingredients or drugs in the body (Stahl et al., 2002), but the values are dealt with differently among researchers in different fields. In the nutritional research field, bioavailability has simply been evaluated from the recovered amount of ingested molecules in the urine. In the pharmacokinetic research field, however, the term is defined as the rate of pharmacological utilization of a dose of drugs in the body after administration. Orally administered drugs usually enter the body through several paths, known as ADME (absorption from gastrointestinal tract, distribution into plasma and tissues, phase I and phase II metabolism in tissues, and excretion into urine or feces). To evaluate the efficacy of utilization of an administered dose of any particular drug, bioavailability is evaluated by a comparison of the area under the plasma concentration curves (AUC) obtained for intravenous and orally administered drugs, respectively (Ichiyanagi et al., 2006a). A drug which is administered intravenously skips gastrointestinal absorption and metabolism processes, but follows the same liver metabolism and tissue distribution processes as an orally administered drug. Thus, it might be more useful to calculate the overall utilization profile in the body after oral ingestion in order to find the bioavailability value. Prasain and Barnes (2007) discussed the definition and significance of bioavailability more extensively in their review.

5.2.1

FACTORS AFFECTING BIOAVAILABILITY

As described above, bioavailability means the rate of utilization of an administered dose of a certain target molecule and thus is independent of the quantity of the respective molecule contained in the food. However, this value is useful in order to estimate a reasonable dose required for the expected functioning of either medicines or food factors. The absorption of food ingredients including flavonoids usually occurs in the gut tract, and thus the determining factor for absorption is primarily the amount of the target compound reaching the enterocyte in a form suitable for absorption (Scholz and Willamson, 2007). In addition to an inherent instability due to their chemical nature, biotransformation by intestinal microflora and degradation is a critical factor affecting the bioavailability of orally ingested flavonoids (Scalbert and Willamson, 2000; Willamson and Manach, 2005). Flavonoids exist mainly in sugar-conjugated form in dietary plants. They undergo intestinal hydrolysis by microfloral glucosidase or hydrolase to release the respective aglycone, or in host cells when they are taken orally and then transported across the gut membrane into plasma. They are also subjected to metabolic transformation, such as methylation of the free phenolic group mediated by cathecol O-methyl transferase (COMT) (Day et al., 2000; Hur et al., 2000). Such biotransformation might reduce the apparent bioavailability of the original parent molecules and, when the metabolites are physiologically active, a large discrepancy might be expected as is shown in bioflavonoids between the apparent bioavailability and the physiological effects observed


Improving the Bioavailability of Polyphenols

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by the intake of original food factors. Such an example is isoflavone. It is well known that an isoflavone such as daidzein is transformed by intestinal microflora into the active metabolite, (S)-equol, in which estrogen-like activity is much higher than in the original parent molecule (Setchell et al., 2002; Setchell et al., 2005). Moreover, during intestinal uptake, flavonoids, either intestinally transformed or in intact form, undergo glucuronate or sulfate conjugation reactions in enterocytes, and are also subjected to further metabolism after absorption into plasma, mainly in the liver. Indeed, the quercetin glycoside level is quite low in blood plasma after oral administration; the metabolites and their secondary modified glucuronate or sulfate conjugates appear as the major component, and the plasma level of the original ingested form is quite low (Corona et al., 2006). Therefore, bioavailability must be evaluated not only in terms of the original parent molecule but also of the metabolites generated. Recently, highly sensitive tandem mass spectrometry (MS) studies have addressed this issue and have been reviewed by many authors (Prasain and Barnes, 2007). Mennen et al. (2008) have recently challenged our whole understanding of the beneficial health effects of dietary flavonoids, by targeting several polyphenols and their metabolites in a spot of urine as the biomarker of polyphenol intake. The intestinal mucosa, on the other hand, are not a simple barrier against xenobiotics but work for selective absorption, biotransformation, and excretion back to the lumen (Sergent et al., 2008). The presence of ATP-binding cassette (ABC)/multiple drug resistance (MDR) family translocators on the lumen-side membrane of enterocytes is also a factor which negatively modulates the net intestinal uptake of flavonoids (O’Leary et al., 2003; Zjhang et al., 2004). MDR is a membrane transporter protein which acts to prevent the disadvantageous uptake of xenobiotics and thus confers multidrug tolerance in cancer chemotherapy (Benet et al., 1999). Flavonoids as micronutrients are also xenobiotic substances and thus a significant efflux of tea isoflavones from the basal to the apical side was shown in the Caco-2 monolayer model; the efflux rate was greater in epicatechin (EC) than in epigallocatechin (EGC) and epigallocatechin gallate (EGCG) (Chan et al. 2007). Basically, absorption efficacy depends on the amount or concentration of respective flavonoids to be absorbed in the gut. When intestinal absorption is not mediated by specific membrane transporters, the absorption of molecules is generally regulated by the physicochemical properties of a molecule such as its hydrogen bonding character, molecular weight, and log P value (P being the partition coefficient between oil and water), known as Lipinski’s rule (Zjhang et al., 2004). Thus, molecules having a large molecular size, high polarity, and large apparent size (hydration) are poorly absorbed so long as any specific transporters are not available (Vaidyanathan and Wall, 2001). This rule is also adapted to polyphenols including flavonoids, although the P value here is usually less than 5. Since aglycones and metabolites are more hydrophobic and have a small molecular size, they are absorbed more easily in diets than the parent flavonoids with conjugated sugar (Hong et al., 2002). In the same way, the uptake efficiency of EGCG was lower than that of EC and ECG when compared among tea catechin aglycones (Hong et al., 2003). As discussed above, sugar conjugation decreases the lipophilicity of molecules, which is a critical requirement for the intestinal absorption of dietary ingredients, but on the other hand, it improves their stability. Moreover, it is known that the type of sugar affects the absorption efficiency in several flavonoids such as quercetin, where glucoside was much more easily absorbed than rutinoside (Erlund et al., 2000). This was because quercetin glucoside is better hydrolyzed by brush-border glucosidase in the small intestine in order to release the aglycone, whereas rhamnoside is hydrolyzed by microfloral enzymes in the colon. Thus, intestinal stability is the factor controlling bioavailability. Flavonoids in nature are acylated in addition to glycosidation. The extent and type of the acylated group also affect the bioavailability of flavonoids. Henning and Heber compared the bioavailability of gallated and nongallated flavan-3-ols in tea and found acylation resulted in decreased absorption. This rule is also adapted to anthocyanins from the purple sweet potato (Suda et al., 2002) and cooked black carrot (Kurilich et al., 2005), where it was shown that the plasma level of diacylated anthocyanin (peonidin-3-caffeoyl sophoroside-5-glucoside) in purple potato was six times lower


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than in nonacylated anthocyanin (aglycon) after oral administration. It was, however, noticed that p-coumaroyl delphinidin from eggplant showed better absorption than nonacylated delphinidin. Although the mechanism of enhanced absorption is unclear, the type of acylation may alter the absorption efficiency, probably due to improved stability (Ichiyanagi et al., 2006b). In addition to the factors modulating ADME mentioned above, the physicochemical properties of molecules, including stability, protein binding, and (coexisting) matrix effect, are also factors which affect bioavailability. For example, polyphenols bind to salivary proteins, particularly proline-rich proteins and basic residues of histatins, and thus are prevented from interacting with gut enzyme proteins such as α amylase, α glucosidase, and protease (Bennick, 2002; Griffiths, 1986; McDougall and Stewart, 2005). Recent studies have indicated a significant binding of flavonoids to blood plasma protein. This protein binding usually restricts the tissue uptake of flavonoids, that is, restricts the molecules from reaching the target site of action. Moreover, the profile of bound flavonoids is different from that of free flavonoids in the plasma, indicating that protein binding alters the functional role of the diet from that which would have been expected from the composition of existing active ingredients (Manach et al., 1995). In relation to this, the effect of milk protein on the bioavailability of tea polyphenols has been extensively studied. Primarily, it was reported that milk lowers the bioavailability of catechin when tea was added with milk (Reddy et al., 2005) but others reported that milk did not alter the plasma level of catechin (Van het Hof et al., 1998; Serafini et al., 1996; Richelle et al., 2001). A similar discussion has taken place about the effect of milk on cocoa polyphenols, but recent studies including double blind cross-over studies on healthy human subjects rather concluded that the effect of milk on cocoa polyphenols was marginal and does not give rise to significant physiological effects (Keogh et al., 2007; Roura et al., 2007). The effect of the addition of milk to black tea was also assessed for its ability to modulate oxidative stress and antioxidant status in adult male volunteers using catechin as a marker. Milk addition may not obviate the ability of black tea to modulate the antioxidant status of subjects and the consumption of black tea with/without milk prevents oxidative damage in vivo. This rule was typically observed when the tea catechin family was studied for their intestinal absorption and back flux behavior, mediated by MDR using the Caco-2 cell monolayer system (Reddy et al., 2005).

5.2.2

ANTHOCYANIN BIOAVAILABILITY

Anthocyanin has a unique property which makes it different from other flavonoids in several respects; for example, the chemical structure is variable dependent on pH. In an acidic pH below 4, the major form is flavylium cation, but in an alkaline pH above 8 it is in the quinoidal form. Both are rather stable. However, at a neutral pH covering physiological pH, it turns to a colorless open ring structure, chalcone, and eventually decomposes (Rahman, 2008). In particular, aglycones are more instable than their glycosides. Moreover, it is known that anthocyanins are absorbed in their intact form in a different way from other flavonoids such as quercetine (Erlund et al., 2000). These characteristics of anthocyanin defined their bioavailability as quite low. Nevertheless, many human and animal experiments have indicated a high potentiality in the prevention of diseases, particularly cardiovascular and neurodegenerated diseases (Konishi and Ichiyanagi, 2008). Ichiyanagi et al. (2006a) closely studied the bioavailability of 15 anthocyanins in bilberry by the pharmacokinetic approach and showed that the values varied among the anthocyanins in bilberry from 0.028% to 0.6%, still quite low compared to other flavonoids. When the plasma uptake among anthocyanins was compared with the same aglycone but a different type of conjugated sugar in rats, galactosyl anthocyanin showed a higher plasma concentration than glucosyl or arabinosyl anthocyanins after oral administration (Ichiyanagi et al., 2006a; Konishi and Ichiyanagi, 2008; Rahman, 2008). Sugar conjugation stabilizes anthocyanin aglycone but it is not yet clear whether the stability of galactosyl conjugate is superior to other sugar conjugates, and involvement of an unknown transporter preferentially mediates galactosyl anthocyanin. Wu and coworkers (2005) studied the effect of conjugated



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sugar on intestinal absorption and found that cyanidin-3-O-rutinoside is better absorbed than cyanidin-3-O-glucoside after oral administration of blackcurrant extract. They indicated that the higher plasma level of rutinoside is due to its stability in the small intestine compared to glucoside (Wu et al., 2005). Since the natural content of anthocyanin in daily diets is quite large as a micronutrient—for example, one serving of blueberry supplies 10–100 mg of anthocyanins (Grotewold, 2006)—improved bioavailability will be of practical importance to exploit the beneficial health effects of flavonoids anthocyanins in daily life.

5.3

STRATEGY TO IMPROVE THE BIOAVAILABILITY OF FLAVONOIDS

Currently, many trials have been carried out in order to improve the absorption of low bioavailable food factors, since bioavailability is a limiting factor for active ingredients to really play their important beneficial role in preventing diseases and aging in daily life. This is especially important in the case of flavonoids which have a wide range of physiological functions but quite low bioavailability. As discussed above, there might be several targets which could be manipulated to improve bioavailability and these are summarized in Table 5.1. Chemical modification of hydrophilic phenolic OH makes molecules more hydrophobic and increases their metabolic stability in the gut; many approaches to this have been reported. As is shown for flavones, O-methylation of phenolic OH occurring in intestinal tract improves the metabolic and chemical stability of aglycone and also increases the hydrophobicity of the molecule so that intestinal absorption is improved (Walle, 2007a). Walle (2007b) precisely discussed the advantageous use of methoxylated flavones in cancer chemoprevention in his review. Henning et al. (2008) also observed the stabilization of EGC by methylation. However, Landis-Piwowar et al. (2008) studied the effect of the methylation of plant polyphenols including green tea catechins and other flavonoids on their proteasome inhibitory and apoptosis-inducing abilities in human leukemia HL60 cells and concluded that methylation reduced the potent cytotoxic effect of nonmethylated flavonoids toward cancer cells and would be likely to have limited ability as a chemopreventive agent. Acetylation of EGCG to the peracetylated derivative increased the AUC of plasma EGCG 2.4 times compared to unmethylated EGCG and also enhanced biological activities both in vitro and in vivo (Lambert et al., 2006; Landis-Piwowar et al., 2007). Esterification is another approach to improve the absorption of flavonoids such as quercetin. The esters are hydrolyzed by esterase to release parent molecules when incorporated in the cells and thus function as a prodrug. Biasutto and coworkers (2007) synthesized esterified quercetin and studied transepiterial transport across the cell monolayers of three different epithelial cell lines, MDCK-1 and -2, and human Caco-2. Although quercetin was subjected to phase II modification by all three cells, no phase II conjugation reaction was observed in the esterified quercetin during the transport process. They suggested that ester

TABLE 5.1 Factors Affecting the Bioavailability of Ingested Food Factors 1. ADME related factors • Physicochemical property (chemical and metabolic stability, solubility, molecular weight, charge, etc.) • Microflora • Metabolic modification (phase I and phase II metabolism, COMT) • ABC/MDR transporters • Protein binding • Excretion 2. Matrix or coadministration effects


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derivatization might be a useful method to increase systemic aglycone concentrations (Biasutto et al., 2007). Matrix or coadministered substances often give rise to a marked increase of intestinal absorption and bioavailability. The first example of enhancing the absorption of anthocyanin by a coexisting factor is reported by Matsumoto et al. (2007). They administered blackcurrant anthocyanins together with 2.5% phytic acid to rats and humans, and the plasma uptake was measured. The resulting absorption was remarkably enhanced approximately ten times. Since residual anthocyanin in the gastrointestinal tract was significantly different in the presence and absence of phytic acid, it was suggested that the enhancing effect was due to gastrointestinal motility (Matsumoto et al., 2007). However, phytic acid is a strong metal ion chelator and thus might inhibit metal absorption instead. Gibson and coworkers suggested possible control of the bioavailability of flavonoids with a simple compound (Gibson et al., 2006). On the other hand, in the villus tip enterocytes of the small intestine, phase I drug metabolizing enzymes and the multidrug efflux pump, MDR, or P-glycoprotein (P-gp), are present at high levels. Since they demonstrate a broad overlap in substrate and inhibitor specificities, it is suggested that they act as a concerted barrier to xenobiotic absorption, including micronutrients (Benet et al., 1999). Therefore, the substances that can alter the activity of these enzymes will contribute to the bioavailability of polyphenols. Such an example is piperine, an alkaloid from black pepper (Piper nigrum). Piperine is a strong inhibitor of hepatic and intestinal aryl hydrocarbon hydroxylase and uridine 5′-diphosphate (UDP)-glucuronyl transferase and has been documented to enhance the bioavailability of a number of therapeutic drugs as well as phytochemicals (Srinivasan, 2007), such as tea catechins (Lambert et al., 2004) and curcumin (Anand et al., 2007). Since the peak plasma time of EGCG appeared slower but the AUC was larger with piperine coadministration rather than with EGCG alone, piperine appears to improve the bioavailability not only by inhibiting enzymes but by slowing down gastrointestinal morbidity. MDR might be another target of molecules in manipulating the bioavailability of flavonoids. Certain flavonoids such as EGCG and curcumin are substrates of MDR but, at the same time, behave as inhibitors. Accordingly, the bioavailability of both is increased when they are coadministrated (Hong et al., 2003). Although the mechanism is not yet clear, such synergistic enhancement of bioavailability was observed when bilberry anthocyanins were administered to rats as a mixture. Their bioavailability was markedly improved compared to the value obtained for the respective anthocyanins when administered independently, even though the value was several times lower than the values (1–10%) reported for other flavonoids (Konishi and Ichiyanagi, 2008; Rahman, 2008; Zafra-Stone et al., 2007). Several other factors such as alcohol, sugar, and fat have been reported on, but their effects were not significantly evaluated compared to the above examples (Andlauer et al., 2003).

5.4

CONCLUSION

As discussed above, the factors affecting the bioavailability of polyphenols, especially of flavonoids, are diverse and variable dependent on the type of flavonoids. It appears that a single factor affects different type of flavonoids in different ways. However, the basic strategy for improving the absorption of flavonoids would be to improve the stability and solubility in the intestinal tract; those are possibly modulated by the chemical modification of hydrophilic phenolic OH, matrix effects, and the inhibition of phase I drug metabolizing enzymes and the multidrug efflux pump, MDR, or P-gp. The choice of an appropriate matrix or the proper combination with other food factors, in addition to the chemical modification approach, would be a promising strategy for enhancing the dietary absorption of flavonoids. Food processing technology could be valuable here. For example, it has been observed that flavonoids in processed juice are better absorbed than in a pure form; nanoemulsion technique was applied to the flavonoid solution to facilitate absorption (Parada and Aguilera,



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2007). Henning et al. (2008) reported an approach to increasing the bioavailability of tea catechins, which included the administration of tea in combination with fruit juices, coadministration with piperine, and peracetylation of EGCG. The same approach has been adapted for curcumin (Anand et al., 2007). By controlling the processing steps, the flavonoid profile of cocoa was manipulated to improve its bioavailability (Tomas-Barberan et al., 2007). Since fermentation and the drying process reduce flavonoid content (Shahidi and Naczk, 2004), in addition to a high temperature and longer roasting time (Wollgast and Anklam, 2000), an unfermented, nonroasted, and branch-treated cocoa powder was prepared that contained four times more procyanidins, and eight times more epicatechin and procyanidin B2 than conventional cocoa powder. Intake of a milk drink prepared from this flavonoid-enriched cocoa powder increased plasma flavonoid levels five times as assessed by plasma epicatechin glucuronide, and urinary excretion was also markedly enhanced when evaluated by the main metabolites, particularly methyl epicatechin sulfate. It is also reported that flavanols, notably tea catechins, are affected by such factors of epimerization reaction occurring in the processing stages (Scholz and Willamson, 2007). Thus, processing is critical to change the bioavailability of flavonoid-rich resources. Currently, the third function of foods—associated with pharmacological or physiological functions of food ingredients—has attracted much attention in developing a functional strategy for health promotion and complementary therapy, especially in cancer treatment. It is well recognized that foods can have significant pharmacological or physiological functions; this is the third function of foods, after the nutritional and sensory functions which are defined as the primary and secondary functions, respectively. Therefore, many studies have focused on the functional ingredients of food resources and there has been discussion about their dietary role in preventing diseases such as vascular aging and cancer. There has been a focus on flavonoids as a group of active substances with beneficial health functions. As described above, their bioavailability is usually quite low, and a large proportion of an oral dose goes missing. It is almost certain that flavonoids have various beneficial roles in health promotion and disease prevention, but it is still unclear whether the functional principals are the original absorbed flavonoids or their metabolites, in addition to their degradation products. For a deeper understanding of the beneficial role of polyphenols including flavonoids in health, further studies on ADME are required to elucidate the real bioavailability of these dietary functional factors.

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Function of the 6 The Next Generation Polyphenol, “Oligonol” Takehito Miura, Kentaro Kitadate, and Hajime Fujii CONTENTS 6.1 6.2 6.3

Introduction ............................................................................................................................ 91 Conversion to Low-Molecular Weight of Proanthocyanidin .................................................. 91 Lychee Polyphenols ................................................................................................................92 6.3.1 Ingredients and Structure ...........................................................................................92 6.3.2 Absorption in the Human Body.................................................................................. 95 6.3.3 Safety Assessments ..................................................................................................... 95 6.4 Functions of Oligonol .............................................................................................................96 6.4.1 Blood Flow Improvement ...........................................................................................96 6.4.2 Antifatigue Effect .......................................................................................................96 6.4.3 Visceral Fat Reducing Action ..................................................................................... 98 6.4.4 Beauty Effects.............................................................................................................99 6.5 Summary .............................................................................................................................. 100 References ...................................................................................................................................... 101

6.1

INTRODUCTION

Polyphenols are ingredients contained in various kinds of plants. They have diverse functions including antioxidant activity and are raw materials commonly used in the food industry. In particular, proanthocyanidin is a pigment component substantially contained in cacao, grapes, persimmons, and bananas, which is well known to have an astringent and/or bitter taste. Proanthocyanidins are structurally characterized as polymers of catechin and have a high molecular weight, showing high antioxidant activity in vitro. However, their absorption in the body is low when administered orally, so that their in vivo antioxidant activity is not as high as expected. Moreover, proanthocyanidins with a high molecular weight are practically insoluble in water and have an astringent taste, binding to saliva proteins and mucous membranes in the mouth (Haslam, 1998), and making them difficult to use in the food industry. In collaborative research with Faculty of Pharmacy, Nagasaki University, we have been successful in converting high molecular weight proanthocyanidin into low molecular proanthocyanidin, which is utilizable in the food industry. Thus, we have developed “Oligonol,” which shows an excellent in vivo antioxidant activity. The manufacturing method, structure, and functions of Oligonol are reviewed in this chapter.

6.2 CONVERSION TO LOW-MOLECULAR WEIGHT OF PROANTHOCYANIDIN For a long time, a thiolysis method has been used for the structural analysis of proanthocyanidin. In this method, based on nucleophilic reaction, a compound possessing a thiol group binds to the end 91 © 2010 Taylor and Francis Group, LLC

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Biotechnology in Functional Foods and Nutraceuticals OH

R1

OH HO

O

OH O-R

OH HO

OH HO OH

O

H+

OH OH O-R

OH HO

O

OH

O

(Acidic condition)

OH

R1 OH

OH HO

O-R2

+

O O-R2

Carbocation

OH HO

OH R1 OH

O O-R2

OH OH O-R

OH HO

R1

OH

O

OH

HO

O

O-R OH

Soluble and insoluble polymers in fruits

OH

δ–

OH

Soluble oligomers

O-R2 OH

Tea catechins

FIGURE 6.1 A conceptual diagram showing the convertion of proanthocyanidin to a low molecular weight. (From Tanaka, T., Yoshitake, N., Zhao, P., Matsuo, Y., Kouno, I., and Nonaka, G., 2008. J. Jpn. Soc. Food Chem. With permission.)

unit of proanthocyanidin fragmented under acidic conditions, and accordingly the low-molecular proanthocyanidin is stably obtained. However, as most compounds with a thiol group are not suitable for consumption, their application to food products is limited. In collaboration with the Faculty of Pharmacy, Nagasaki University we have developed a technique to convert high-molecular proanthocyanidins to low-molecular proanthocyanidins without using thiol compounds, and have been successful in making the technology practicable. This method is to bind a compound possessing a phloroglucinol ring structure (such as catechin) as a nucleophilic compound to proanthocyanidin fragmented under acidic conditions (Tanaka et al., 2007). As shown in Figure 6.1, catechin monomers are substituted at the C-4 position of fragmented proanthocyanidins with a high molecular weight and, as a consequence, low-molecular proanthocyanidins are stably generated. While it is possible to use proanthocyanidin polymers derived from any plant origin for this method, we have selected lychee fruit as a raw material to develop Oligonol.

6.3

LYCHEE POLYPHENOLS

The lychee (Litchi chinensis, Sapindaceae) has been consumed since ancient times in China and the southern area of Southeast Asia. Legends state that the lychee was the secret behind the beauty of Youkihi (Yang Guifei) who was considered to be one of the four most beautiful ladies in the world, because she was a great eater of lychees. The lychee is rich in polyphenols; Brat et al. (2006) reported that its polyphenol content per edible part is second only to strawberries. A particular feature of lychee polyphenols is that they contain a comparatively large number of A-type procyanidin units (Sarni-Manchado et al., 2000) (Figure 6.2).

6.3.1

INGREDIENTS AND STRUCTURE

The low-molecular polyphenol, Oligonol, is produced by using proanthocyanidins derived from lychee fruit and tea extract as a nucleophilic reagent. It has been found that the low-molecular proanthocyanidin content of Oligonol is markedly higher than that of lychee fruit polyphenol used as the raw material, and grape seed and pine bark polyphenols that are similarly rich in proanthocyanidins (Figure 6.3). The proportion of monomers (flavanols) and proanthocyanidin dimers and trimers in grape seed and pine bark polyphenols is around 10% and it is less than 20% even in lychee fruit polyphenol which has a comparatively higher content of low-molecular proanthocyanidins, © 2010 Taylor and Francis Group, LLC

The Function of the Next Generation Polyphenol, “Oligonol”

93 OH

OH OH

OH

O

HO

O

HO OH O

OH

OH OH

O

OH O OH

O

HO

HO OH

HO

OH

HO

Procyanidin A1

Procyanidin A2 OH OH

HO

O OH OH

n

OH OH HO

O

OH OH Procyanidin polymer

FIGURE 6.2

Lychee polyphenols.

100 Others

Polyphenol composition (%)

40 Trimer

30 Dimer

20

10

Monomer

0 Oligonal

Lychee fruit

Pine burk

Grape seed

FIGURE 6.3 The polyphenol composition of each substance. The contents of monomers to trimers in Oligonol, lychee fruit polyphenol (used as the raw material), and polyphenols available in the market (pine bark and grape seed) was analyzed and compared by the high performance liquid chromatography (HPLC) method at UV 280 nm using a normal phase column.


94

Biotechnology in Functional Foods and Nutraceuticals OH

OH

OH

OH O

HO

O

HO

OH

OH OH

OH

(+)- catechin

(–)- epicatechin (EC)

OH

OH OH

OH

O

HO

OH OH

OH

OC O

OH

O

HO

OH

OH

OH

(+)- epicatechingallate (ECG)

OH

OC O

OH

(–)- epigallocatechingallate (EGCG)

OH

OH OH

OH

O

HO

O

HO OH O

OH

OH O

OH OH

O

OH

O

HO

HO OH

HO

OH

HO

Procyanidin A1

Procyanidin A2

OH

OH OH

OH

O

HO

O

HO OH

OH HO

OH OH

OH

OH

OH OH HO

O

O OH

OH

OH

OH Procyanidin B1

Procyanidin B2 OH OH O

HO OH

OH

OH O

HO

OH

HO

OH

OH O

OH O C O

OH

OH

HO

OH

HO

OH

(–)-epicatechin-(4β→8, 2β→O→7) epicatechin-(4β→8)-epicatechin (A2-EC)

Flavanols and proanthocyanidins in Oligonol.


OH O

HO

OH

(–)-epicatechin-(–)-epigallocatechingallate (EC-EGCG)

FIGURE 6.4

OH OH

O

OH OH HO

OH O


95

whereas the content reaches around 40% in Oligonol (Figure 6.4). Sakurai et al. (2008) reported that Oligonol contains 15.7% of monomers such as catechin and epicatechin, and 13.3% of dimers including procyanidins A1, A2, B1, and B2. The existence of trimers has also been confirmed.

6.3.2

ABSORPTION IN THE HUMAN BODY

A study was conducted in healthy volunteers to clarify the biological absorption of polyphenols after the oral intake of Oligonol. The polyphenol concentration in the blood after ingesting 100 mg of Oligonol or lychee fruit polyphenol was measured using the previously reported method (Fujii et al., 2007). In both cases, blood polyphenol concentrations were highest 2 h after the intake and decreased gradually thereafter. However, the highest blood concentration (Cmax) of polyphenol in the Oligonol group was around three times greater than that of the lychee fruit polyphenol group, and a high blood concentration was maintained in the Oligonol group even after 4 and 6 h (Figure 6.5). This result demonstrated that the bioavailability of polyphenols contained in Oligonol is superior to lychee fruit polyphenol (Wakame, 2007).

6.3.3

SAFETY ASSESSMENTS

Since the principal ingredients of Oligonol containing flavanols and proanthocyanidin oligomers are polyphenols derived from lychee fruit and tea extract, there can be no doubt about its safety, based raw materials commonly used as food. In addition, the safety of Oligonol has also been evaluated in great detail in safety studies according to Good Laboratory Practice (GLP) standards. In a single dose oral toxicity test in rats, the 50% lethal dose (LD50) of Ologonol was over 2000 mg/kg. There was no toxicity in a 90-day repeated dose oral toxicity test, where rats were given Oligonol at a dose of 1000 mg/kg. Besides this, both a reverse mutation test and a micronucleus test in mice came out negative (Fujii et al., 2008). In a phase I safety study, 600 mg/day of Oligonol was administered into 29 healthy volunteers aged 18–60 years (13 males and 16 females) for 14 days, and no 10

Polyphenol concentration (μg/mL)

Oligonol 100 mg/day Lychee fruit polyphenol 100 mg/day 8

*#

* versus 0 h # versus Lychee fruit polyphenol (2 h)

6

*

4

*

2

0 0

2

4

6

Time (h)

FIGURE 6.5 The blood polyphenol concentration after Oligonol intake. Thirty-seven healthy volunteers were divided into two groups: the Oligonol group with 18 subjects (14 males and 4 females) and the lychee polyphenol group with 19 subjects (14 males and 5 females). Both groups were given a 100 mg single dose of the respective sample. Blood samples were collected before the intake, and 2, 4, and 6 h after the intake. The amount of polyphenol in serum was measured using the Prussian Blue method. (From Wakame, K., 2007. Food Style 11(10): 65. With permission.)


96


abnormalities were observed in blood pressure and general blood chemistry examinations, indicating that Oligonol is a safe food (in preparation for submission). Furthermore, Oligonol is certified as NDI (new dietary ingredient) by the U.S. Food and Drug Administraion (FDA).

6.4 FUNCTIONS OF OLIGONOL In a questionnaire given to subjects taking part in an exploratory 3-month intake study carried out as a preliminary evaluation of Oligonol, the subjects reported improvements in shoulder stiffness, eye strain, quality of sleep (falling asleep easily and waking up easily), fatigue and recovery from fatigue, and intelligence as subjective symptoms during the intake period. So far, this effectiveness has been verified by the following tests.

6.4.1 BLOOD FLOW IMPROVEMENT It is known that when vascular endothelial cells are damaged due to hyperglycemia and oxidative stress, the production of nitric oxide (NO) decreases and a loss in smooth microcirculation occurs, resulting in the occurrence of vasoconstriction, inflammation, and thrombus. The effect of Oligonol on NO production was investigated in vascular endothelial cells stimulated by bradykinin under hyperglycemic conditions. Although a high concentration of glucoseinduced reduction of NO production in 30 μM bradykinin-stimulated vascular endothelial cells was found, an addition of Oligonol recovered the reduction (Figure 6.6). Body surface temperature was measured by thermography in clinical trials to confirm the blood flow improvement effected by Oligonol. A thermograph taken 90 min after a single dose of 600 mg Oligonol is shown in Figure 6.7. An elevation of the body surface temperature in the upper half of the body and the fingers was observed in the thermograph taken 90 min after intake, suggesting an enhancement of peripheral blood flow. A similar change in the body surface temperature was also confirmed after an administration of 50 mg Oligonol (data not shown).

6.4.2 ANTIFATIGUE EFFECT

[NO2–] + [NO3–](nmol/105 cells)

Questionnaires from the exploratory 3-month intake study suggested that Oligonol might show antifatigue effects. Consequently, in a forced swimming mouse model, it was investigated whether 3.0 2.5 2.0 1.5 1.0 0.5

0.0 Glucose (mM) Oligonol (μg/mL) Bradykinin (30 μM)

5.6 0 +

22.4 0 +

22.4 100 +

FIGURE 6.6 The effect of Oligonol on decreased NO production. The effect of Oligonol on NO production was examined in porcine aortic vascular endothelial cells stimulated by bradykinin. Although there was a significant decrease in the NO production ability when glucose concentration was altered from physiological concentration (5.6 mM) to high concentration (22.4 mM), the decrease was significantly improved by the addition of Oligonol. (*p < 0.01 versus bradykinin + normal glucose, bradykinin + high glucose + Oligonol.)



Before intake

97

90 min after intake

FIGURE 6.7 The change in the body surface temperature after Oligonol intake in a thermograph (43-yearold male). The subject was asked to lie quietly in bed in a temperature controlled room (room temperature 26 ± 1°C, humidity 50%) for 30 min prior to the sample intake and the body surface temperature was analyzed immediately before and every 30 min after the sample intake. JTG-4310S (Japan Electron Optics Laboratory, Japan) was used to measure the temperature.

Oligonol has an antifatigue action (Ohno et al., 2007). Oligonol was mixed with a commercialized powder diet (CE-2, CLEA Japan) and administered into ddY mice at a dose of 50 mg/kg. Mice were forced to swim in a 23°C water bath once a day (for 10 min) from day 10 to 14 after commencement of the supplementation for a total of 5 days. Their spontaneous locomotor activity was measured by the Open Field method before forced swimming at days 12 and 13. A 10-min break was given after the forced swimming and then the riding time was evaluated using a Rota-Rod treadmill. The mice that were not subjected to fatigue (forced swimming) were considered as a normal group. When the spontaneous locomotor activity and treadmill riding time were represented as a ratio to those of the normal group (% of normal), the spontaneous locomotor activity of the Oligonol group was greater than that of the control group and the riding time was also longer. These results demonstrated that Oligonol attenuated the fatigue caused by forced swimming (Figure 6.8). Moreover, when the values of TEAC (Trolox equivalent antioxidant capacity) and LPO (lipid peroxide) in blood were measured at the end of the experiment (day 15), supplementation with Oligonol increased and decreased the blood TEAC and LPO values, respectively, suggesting that Oligonol might decrease the level of fatigue through suppressing the oxidative stress increased by forced swimming. Ohno et al. (2008) of Kyorin University studied the antifatigue effect of Oligonol in athletes of the track team of the university. Forty-seven athletes (25 males and 22 females) were divided into two groups. The study was a single (subject)-blind placebo-controlled crossover trial where

Open field

Rota-rod treadmill 60

120 % of normal

*

*

80

40

40

20

0

Control

Oligonol

0

Control

Oligonol

*p < 0.05 versus control

FIGURE 6.8 The effect of Oligonol on fatigue induced by forced swimming. Water flow was created by agitating (75 rpm) at 23 ± 1°C and mice were forced to swim in it for 10 min. The results at day 13 are shown in the graph. In Open Field, spontaneous locomotor activity was evaluated for 3 min, and riding time was measured using a Rota-Rod treadmill (Model 7650, UGO BASILE Company, USA) with a rotating speed of 20 rpm. (From Ohno, H., 2007. Jpn. J. Mt. Med. 27: 43–60. With permission.)


98

Biotechnology in Functional Foods and Nutraceuticals Fatigue by exercise training 5

Recovery from fatigue by exercise training 5

Group A Group B

4 *

*

***

Score

Score

4 3 **

2 1

Group A Group B

Group A Group B

**

2

Day 0 Day 26 Day 52

***

3

1

Day 0 Day 26 Day 52

Placebo Oligonol Oligonol Placebo

Day 0 Day 26 Day 52

FIGURE 6.9 The antifatigue effect of Oligonol. The level of fatigue was scored after training using a subjective questionnaire. Group A had 24 subjects (13 males and 11 females) and Group B had 23 subjects (12 males and 11 females).*p < 0.05 versus day 0; **p < 0.05 versus day 26 (Group B); ***p < 0.05 versus day 26 (Group A). (From Ohno, H., Sakurai, T., Hisajima, T., et al., 2008. Adv. Exerc. Sports Physiol. 13(4): 93–99. With permission.)

Oligonol and a placebo were alternately administered to the athletes for 26 days each. Fatigue and recovery from fatigue were assessed by scores in the questionnaire. One group took 200 mg/day of Oligonol in the first half and another group received 200 mg/day of Oligonol in the second half after a 9-day washout period. One capsule of 100 mg Oligonol was respectively given in the morning and evening. An improvement of fatigue and recovery from fatigue was observed during the Oligonol intake period in both groups (Figure 6.9). In addition, there was a significant decrease of RPE (ratings of perceived exertion) in the Oligonol-taking group, and scores related to body pain also improved. Taken together, although the fatigue recovery mechanism of humans is not clear, supplementation with Oligonol improved subjective symptoms such as fatigue and recovery from fatigue in the athletes undergoing daily training. The result of the animal study using the forced swimming model suggested that Oligonol might be involved in suppressing the oxidative stress caused by exercise.

6.4.3 VISCERAL FAT REDUCING ACTION Recently, it has been found that visceral fat-type obesity increases the risk of life-threatening diseases such as cardiac infarction. Hence, it is important to prevent excessive accumulation of visceral fat, and improvement of dietary habits and moderate exercise are being promoted. A dysregulation in the production of adipokines including adiponectin and TNF-α is associated with the accumulation of visceral fats (Mohamed-Ali et al., 1998). It has been reported that Oligonol modulates adipokine production (Sakurai et al., 2008). Sakurai et al. (2008) of Kyourin University fed a high-fat diet to C57BL/6J mice treated with Oligonol (100 mg/kg body weight) for 5 weeks and afterwards measured the mRNA of adipokines in the adipose tissue. They found that the expression of adiponectin mRNA decreased due to a highfat diet was recovered by supplementation with Oligonol and that the increased mRNA expressions of TNF-α, PAI-1, and MCP-1 were conversely attenuated (Figure 6.10). To verify the effects of Oligonol in a clinical trial, 18 male and female adult volunteers with over 85 cm of abdominal circumference or with more than one item of abnormal value in blood lipid parameters were randomly divided into two groups and were given a capsule with 100 mg of Oligonol or a placebo twice a day. Physical examination, blood test, and abdominal CT scan were


The Function of the Next Generation Polyphenol, “Oligonol” 2

1

3

1

a

β-actin

1.2 1.0 0.8 0.6 0.4 0.2 0.0

a

Oligonol LEP 20 mg/mL 20 mg/mL

Control

1

2

a


3

a

a


Relative density (control = 1)


3

β-actin

a

2

a


3

Leptin

β-actin

Control

1.2 1.0 0.8 0.6 0.4 0.2 0.0

Control

1

Adiponectin 1.2 1.0 0.8 0.6 0.4 0.2 0.0

2

PAI-1


a



β-actin

Control

3

MCP-1

TNF-α 1.2 1.0 0.8 0.6 0.4 0.2 0.0

2

1

99

1.2 1.0 0.8 0.6 0.4 0.2 0.0

β-actin a

Control

a


FIGURE 6.10 The regulation of adipokine production in adipose tissues by Oligonol. Total RNA was extracted from completely differentiated HW cells treated with 20 μg/mL of Oligonol or lychee fruit polyphenol (LFP) for 24 h and was subjected to RT (reverse transcription)-PCR. Lane 1: Control; Lane 2: Oligonol 20 μg/mL; Lane 3: LFP 20 μg/mL. Each expression amount was normalized by the amount of β-actin expression. The bar graphs are shown as the relative density to control. The values represent average ± SE (n = 3). (From Sakurai, T., Nishioka, H., Fujii, H., et al., 2008. Biosci. Biotechnol. Biochem. 72(2): 463–476. With permission.)

done before the test, and 5 and 10 weeks after the test. A reduction of subcutaneous and visceral fat areas in the Oligonol group was shown in the abdominal CT scans (in preparation for submission). As mentioned above, Oligonol demonstrated an inhibitory effect against visceral fat accumulation through regulating the production of adipokines, suggesting that it might be effective in improving metabolic syndromes.

6.4.4 BEAUTY EFFECTS The oxidative stress enhanced by ultraviolet ray (UV)-induced reactive oxygen species is involved in various adverse effects such as skin aging and skin cancer (Cerutti et al., 1994), and administration of antioxidative substances is considered to be very useful in the field of dermatology. The application of Oligonol was also evaluated in this field. Kundu et al. (2009) investigated the expression of COX-2 induced by UV-B irradiation in the skin of hairless mice. They reported that topical administration of Oligonol lowered the expression of COX-2 in the mice (Figure 6.11). Surh et al. (2008) have also found that Oligonol inhibits phorbol ester-induced chemical carcinogenesis in mouse skin. The effect of an oral intake of Oligonol was examined in an open-label clinical trial with 17 women aged 26–60 years. One capsule with 100 mg of Oligonol was respectively taken in the morning and evening (200 mg/day) for 12 weeks. Number of pigmentations, pigmentation area, and length and total area of wrinkles at the outer corners of the eyes were estimated using Robo Skin Analyzer CS50 (Inforward Company, Japan) before and 4 and 12 weeks after intake. As shown in Table 6.1, the oral intake of Oligonol indicated a decreasing tendency in all evaluated items in subjects over 40 years old.


100

Biotechnology in Functional Foods and Nutraceuticals Oligonal

–

–

+

UV-B

–

+

+

COX-2

Actin

Relative band intensity (% of COX-2/actin)

80

*

60

**

40

20

0

FIGURE 6.11 The protective effect of Oligonol against UV exposure in mouse skin. HR-1 hairless mice underwent UV-B (312 nm) irradiation (BIO-LINK BLX-312, Vilber Lourmat), and the expression of COX-2 protein was evaluated by the Western blot method. Oligonol (0.25 mg/head) was topically applied 30 min prior to UV-B irradiation. *p < 0.001 (Control versus UV-B); **p < 0.001 (UV-B versus Oligonol). (From Kundu, J.-K., Choi, K.-S., Fujii, H., et al., 2009. J. Functional Food 1: 98–108. With permission.)

TABLE 6.1 Skin Improvement by Oligonol in Subjects Aged Over 40 years Evaluation Parameter Number of pigmentations Pigmentation area (mm2) Length of wrinkles at the tail of eyes (mm) Total area of wrinkles at the tail of eyes (mm2)

0 Weeks

4 Weeks

12 Weeks

61 210 45 53

53 194 38 42

54 199 35 37

Oligonol attenuates various types of skin damage caused by UV-induced reactive oxygen species, and is expected to play a preventative role related to skin aging.

6.5

SUMMARY

Oligonol shows predominant bioavailability compared to traditional proanthocyanidins, which enables it to produce an improvement in blood flow, antifatigue, visceral fat reduction, and in the field of beauty treatment. However, some issues, including the mechanisms of the action and pharmacokinetics of each proanthocyanidin molecular species, still need to be elucidated, and further research is desired.



101

REFERENCES Brat, P., George, S., Bellamy, A., et al., 2006. Daily polyphenol intake in France from fruit and vegetables. J. Nutr. 136: 2368–2373. Cerutti, P., Ghosh, R., Oya, Y., et al., 1994. The role of the cellular antioxidant defense in oxidant carcinogenesis. Environ. Health Perspect. 10: 123–129. Fujii, H., Nishioka, H., Wakame, K., et al., 2008. Acute, subchronic and genotoxicity studies conducted with Oligonol, an oligomerized polyphenol formulated from lychee and green tea extract. Food Chem. Toxicol. 46: 3553–3562. Fujii, H., Sun, B., Nishioka, H., et al., 2007. Evaluation of the safety and toxicity of the oligomerized polyphenol Oligonol. Food Chem. Toxicol. 45: 378–387. Haslam, E., 1998. Practical Polyphenolics, From Structure to Molecular Recognition and Physiological Action. Cambridge, Cambridge University Press (ISBN 0-521-46513-3). Kundu, J.-K., Choi, K.-S., Fujii, H., et al., 2009. Oligonol, a lychee fruit-derived low molecular weight polyphenol formulation, inhibits UVB-induced cyclooxygenase-2 expression, and induces NAD(P)H: Quinone oxidoreductase-1 expression in hairless mouse skin. J. Functional Food 1: 98–108. Kundu, J.-K., Hwang, D.-M., Lee, J.-C., et al., 2009. Inhibitory effects of Oligonol on phorbol ester-induced tumor promotion and COX-2 expression in mouse skin: NF-κB and C/EBP as potential targets. Cancer Lett. 273: 86–97. Mohamed-Ali, V., Pinkney, J.H. and Coppack, S.W., 1998. Adipose tissue as an endocrine and paracrine organ. Int. J. Obes. 22: 1145–1158. Ohno, H., Sakurai, T., Hisajima, T., et al., 2008. The supplementation of Oligonol, the new lychee fruit-derived polyphenol converting into a low-molecular form, has a positive effect on fatigue during regular trackand-field training in young athletes. Adv. Exerc. Sports Physiol. 13(4): 93–99. Ohno, H., Sakurai, T., Kizaki, T., et al., 2007. Significance of intake of antioxidative supplements in high altitude taking into consideration symposium high altitude and nutrition—focus on Oligonol. Jpn. J. Mt. Med. 27: 58–60. Sakurai, T., Nishioka, H., Fujii, H., et al., 2008. Antioxidative effects of a new lychee fruit-derived polyphenol mixure, oligonol, converted into a low-molecular from in adipocytes. Biosci. Biotechnol. Biochem. 72(2): 463–476. Sarni-Manchado, P., Le Roux, E., Le Guerneve, C., et al., 2000. Phenolic composition of lychee fruit pericarp. J. Agric. Food Chem. 48: 5995–6002. Tanaka, T., Yoshitake, N., Sho, H., et al., 2007. Method of manufacturing proanthocyanidin Oligomer by polymer fragmentation. J. Jpn. Soc. Food Chem. 14(3): 134–139. Wakame, K., 2007. The function of lychee fruits-derived low molecular polyphenol. Food Style 11(10): 65.



of Biotechnology 7 Application in the Development of a Healthy Oil Capable of Suppressing Fat Accumulation in the Body Hiroyuki Takeuchi CONTENTS 7.1 7.2

Introduction .......................................................................................................................... 104 Edible Oil and Biotechnology .............................................................................................. 104 7.2.1 Gene Recombination ................................................................................................ 104 7.2.1.1 Genetically Modified Crops ....................................................................... 104 7.2.1.2 Herbicide-Resistant Soybean ..................................................................... 104 7.2.1.3 Harmful Insect-Resistant Corn .................................................................. 105 7.2.1.4 Crops with Modified Fatty Acid Composition ........................................... 105 7.2.2 Transesterification..................................................................................................... 105 7.2.2.1 Transesterification Reaction....................................................................... 105 7.2.2.2 Examples of the Application of Transesterification................................... 105 7.2.3 Others........................................................................................................................ 106 7.2.3.1 Oil Production by Microorganisms ........................................................... 106 7.2.3.2 Biomass Fuels ............................................................................................ 106 7.3 Healthy Cooking Oils which Suppress Fat Accumulation in the Body ............................... 106 7.3.1 Introduction .............................................................................................................. 106 7.3.2 Medium-Chain Fatty Acids ...................................................................................... 106 7.3.3 Digestion and Absorption of Medium-Chain Triacylglycerol .................................. 107 7.3.4 Metabolism of Medium-Chain Fatty Acids.............................................................. 107 7.3.5 Less Body Fat Accumulation of MCT ...................................................................... 108 7.3.6 Application of Medium-Chain Fatty Acids in Foods ............................................... 109 7.3.7 Application as an Edible Oil ..................................................................................... 109 7.3.8 Less Body Fat Accumulation of MLCT ................................................................... 109 7.3.9 Summary .................................................................................................................. 112 7.4 Conclusion ............................................................................................................................ 112 References ...................................................................................................................................... 112

103 © 2010 Taylor and Francis Group, LLC

104

7.1


INTRODUCTION

Various types of biotechnology are already being utilized in the field of edible oils, with the main examples shown in Table 7.1. This chapter outlines these biotechnologies as applied to the oil and fat industry. In the second half of the chapter, the development of a healthy cooking oil which is less likely to cause body fat accumulation and which utilizes the physiological action of medium-chain fatty acids and transesterification is described as an example. Countermeasures against obesity are significant for the prevention of lifestyle related diseases. Animal experiments have shown that medium-chain fatty acids are readily utilized as energy compared to long-chain fatty acids, and are less likely to accumulate as body fat. However, there are problems, such as fuming and bubbling in deep-frying. Applying transesterification, a functional medium- and long-chain triacylglycerol (MLCT) oil suitable for cooking and less likely to cause body fat accumulation was developed.

7.2 EDIBLE OIL AND BIOTECHNOLOGY 7.2.1

GENE RECOMBINATION

7.2.1.1 Genetically Modified Crops In Japan, the Ministry of Health, Labor and Welfare completed the safety examination of six genetically modified crops including those for oil production, such as soybean, corn, rapeseed, and cotton, and approved their import. The first generation of genetically modified crops was developed aiming at simplifying cultivation and reducing the production cost for agricultural workers by increasing the resistance to herbicides and harmful insects. In the second generation, oil crops with resistance to environmental conditions, such as cold- and salt-resistance, and seeds of oil crops with a modified fatty acid composition to increase their dietary value are being developed. Typical genetically modified crops are reviewed below. 7.2.1.2 Herbicide-Resistant Soybean The genetically modified crop most widely cultivated worldwide is soybean, with the largest yield among the edible oilseeds. Herbicide-resistant soybean was developed by the Monsanto company, which developed the herbicide Roundup®. Roundup is a nonselective herbicide which kills plants by blocking their amino acid-synthesizing pathways. The main ingredient of Roundup is degraded when it comes into contact with soil, leaving little residue, inhibiting the emergence of resistant weeds. However, this agent is not applicable during the raising of crops because it kills all plants. Monsanto identified a resistance gene against this agent in soil bacteria, and developed a soybean recombined with this gene. The area planted with this recombinant soybean has been

TABLE 7.1 Examples of the Application of Biotechnology in the Oil and Fat Industry Classification

Main Objective

Gene recombination

Improvement of oilseed productivity (reduced labor costs) Modification of oil

Transesterification

Improvement in physical properties Improvement in cooking suitability Improvement in nutritional function Polyunsaturated fatty acid production Reduction of CO2 emission

Culture of microorganisms Biofuel


Example Herbicide-resistant rapeseed Pest-resistant corn Lauric acid-rich rapeseed oil Oleic acid-rich soybean oil Cacao butter substitute MLCT Structured oil γ-linolenic acid, arachidonic acid, DHA Biodiesel fuel made from palm oil

Development of a Healthy Oil Capable of Suppressing Fat Accumulation in the Body

105

rapidly increasingly because of the high-level productivity. In 1997, about 8% of all soybeans cultivated for the commercial market in the United States were genetically modified. In 2006, the figure skyrocketed to 89%. 7.2.1.3 Harmful Insect-Resistant Corn The second most widely planted recombinant crop after soybean is corn. In farm work, not only weeds but also harmful insects are troublesome. Northrop King Co. (now named Novartis Seed) prepared Ostrinia nubilali-resistant corn by introducing the gene of Bt protein, utilized for 30 years as a biological pesticide. O. nubilalis is a species of moth, and its extermination is not possible after it enters corn. Bt protein reacts with the digestive juice (alkaline) of O. nubilali and converts it to a peptide. This peptide reacts with receptors on intestinal epithelial cells and creates a hole in the digestive tract, which impairs digestion and leads to the death of the insect. The peptide is nontoxic to other biologics because they have no receptor binding to this peptide. 7.2.1.4 Crops with Modified Fatty Acid Composition Edible oilseed varieties with fatty acid compositions modified by gene recombination have been developed. One example is lauric acid-rich rapeseed. The normal variety of rapeseed does not contain lauric acid (12-carbon saturated fatty acid), but lauric acid-rich rapeseed oil contains about 40% lauric acid. It has been approved as a food product by the U.S. Food and Drug Administration (FDA), and is added to coffee cream, ice cream, and sherbet. Soybean with a higher oleic acid content than that in the normal variety has also been developed. Normal soybean oil contains about 20% oleic acid and about 50% linoleic acid, but the oleic acid content was increased to 85%, while the linoleic acid content of the modified soybean oil was reduced by several percentage points by gene recombination. Oleic acid-rich oil is superior to linoleic acid-rich oil with regard to oxidative stability.

7.2.2

TRANSESTERIFICATION

7.2.2.1 Transesterification Reaction In the transesterification of oils and fats, the positions of the fatty acid groups of triacylglycerol are changed within or between molecules to produce new triacylglycerols. The reaction causes the exchange of ester groups between oils, between oil and fatty acid (acidolysis), and between oil and alcohol (alcoholysis). The history of the transesterification of oil is long; it was in practical use to improve the physical properties of lard in the 1940s in the United States. Transesterification is generally performed chemically using sodium methoxide and sodium hydroxide, but an enzyme (lipase) has recently been applied for the industrial transesterification of oil. There are two types of lipase: one randomly acts without position specificity, while the other has position specificity (positions 1 and 3). Utilizing the specific reaction of position-specific lipase, structural lipids with specific fatty acids bound at specific positions of glycerol are synthesized. 7.2.2.2 Examples of the Application of Transesterification The most well-known example of enzymatic transesterification is in the production of cacao butter substitute. Cacao butter is an expensive fat which creates the characteristics of chocolate, hard at room temperature but rapidly melting in the mouth. The main constituents of this fat are saturated fatty acids bound at positions 1 and 3 (palmitic acid and stearic acid) and an unsaturated fatty acid (oleic acid) bound at position 1 of triacylglycerol. A fat with physical properties similar to those of cacao butter is prepared by transesterification between fractionated palm oil (medium melting point fraction) and stearic acid using position-specific lipase. Palmitic acid accounts for about one-fourth of fatty acids in breast milk, and about 70% of these are bound at position 2. The digestion and absorption of palmitic acid are better when bound at


106


position 2, and no inhibition of calcium absorption is caused. Fat enriched with palmitic acid bound at position 2 by enzymatic transesterification has been developed for babies overseas. Transesterified oil containing medium-chain fatty acids are detailed in the second half of this report. Other examples of its application include the development of structural lipids containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and margarine containing no trans fatty acids.

7.2.3

OTHERS

7.2.3.1 Oil Production by Microorganisms Oil production by microorganisms has been investigated since the early twentieth century because the production rate is very high, oils with high-level additional values not present in oil plants can be obtained, and constant production is possible regardless of the weather. The production of oil composed of γ-linolenic acid was initially industrialized by culturing lipid-accumulating filamentous fungi. γ-Linolenic acid exhibits hypotensive, anti-inflammatory, and cholesterol-reducing effects, and γ-linolenic acid produced by microorganisms is utilized as a health food material. Stomach mucosa- and liver-protective actions and a cytotoxic effect on cancer cells of arachidonic acid have been reported. The importance of arachidonic acid in infancy has also been clarified. Since plant oils do not contain arachidonic acid, its production by filamentous fungi isolated from the soil has been investigated and industrialized. The production of dihomo-γ-linolenic acid, EPA, and DHA is being investigated. 7.2.3.2 Biomass Fuels Biomass fuels are prepared from biomass, such as plants. The Kyoto Protocol led to the introduction of biomass fuels. Since organic compounds originally converted from carbon dioxide in the air by photosynthesis are burned, they are viewed as “carbon neutral,” and so emissions are regarded as zero in the Kyoto Protocol. The methylesterification of oil and its utilization as a fuel for diesel engines (biodiesel fuel) are being investigated. In Japan, the use of palm oil, dietary oil waste, and rapeseed oil produced in Japan has been proposed, but it is still limited. The production of soybean oil-based biodiesel fuel has been increased to 80,000 tons in the United States, and that of rapeseed oil-based fuel to 1,600,000 tons in Europe. This background has led to support for biomass fuels from countries and states and to its promotion by agricultural organizations.

7.3 7.3.1

HEALTHY COOKING OILS WHICH SUPPRESS FAT ACCUMULATION IN THE BODY INTRODUCTION

Medium-chain fatty acids have been shown to be readily converted into energy and less likely to accumulate as body fat in animal studies. We attempted to develop a dietary oil with these nutritional characteristics, and to overcome previous problems concerning its cooking suitability, by using transesterification, producing of a dietary oil suitable for cooking and less likely to accumulate as body fat. The cost performance of its industrial production has been increased by utilizing a powdered enzyme for transesterification. After confirmation of the low accumulation of body fat following ingestion of this oil and its safety, the oil was certified as a food specified for health use in 2002, and marketed as a dietary oil “less likely to cause body fat accumulation” (product name: Healthy Resetta) by Nisshin Oillio, Japan.

7.3.2

MEDIUM-CHAIN FATTY ACIDS

Triacylglycerol is composed of many types of fatty acids with different numbers of double bonds and carbons (Table 7.2). Medium-chain fatty acids, which are saturated fatty acids composed of © 2010 Taylor and Francis Group, LLC

Development of a Healthy Oil Capable of Suppressing Fat Accumulation in the Body

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TABLE 7.2 Classification of Fatty Acid and Representative Fatty Acid

Short-chain fatty acid (C2–6) Medium-chain fatty acid (C8–10) Long-chain fatty acid (C12 or more)

Saturated Fatty Acid (Double Bond = 0)

Monounsaturated Fatty Acid (Double Bond = 1)

Acetic acid, butyric acid (vinegar, butter) Caprylic acid, capric acid (breast milk, palm oil) Palmitic acid, stearic acid (beef tallow, lard)

—a

—

—

—

Oleic acid (olive oil)

Polyunsaturated Fatty Acid (Double Bond = 2 or More)

Linoleic acid (corn oil), linolenic acid (flaxseed oil), EPA and DHA (fish oil)

Note: Examples of food containing abundant fatty acids are shown in parentheses. a Not present or rarely present in nature.

8–10 carbons, have unique nutritional characteristics different from those of long-chain fatty acids (fatty acids composed of 6–12 carbons may be regarded as medium-chain fatty acids in some cases). The following characteristics were already reported in the 1950–1960s: medium-chain fatty acids are (1) readily digested and absorbed, (2) transported to the liver and easily utilized as energy, and (3) less likely to accumulate as body fat. Medium-chain fatty acids are present as triacylglycerol in general foods, although the content is low. For example, 100 g of butter or fresh cream contain about 3 or 2 g of medium-chain fatty acids, respectively. They are also contained in cow and breast milk at about 1–3%. The general daily intake of medium-chain fatty acids is estimated to be about 200 mg in Japan. Coconut and palm kernel oils contain about 14% and 7%, respectively, serving as the main sources of medium-chain fatty acids used for foods.

7.3.3

DIGESTION AND ABSORPTION OF MEDIUM-CHAIN TRIACYLGLYCEROL

Fats and oils composed of medium-chain fatty acids, that is, medium-chain triacylglycerol (MCT), are more readily digested and absorbed than the fat and oil contained in general meals (long-chain triacylglycerol, LCT). LCT is hydrolyzed at the 1,3-ester bonds of glycerol to 2-monoacylglycerol by pancreatic lipase in the small intestine. The resulting 2-monoacylglycerol is dissolved in bile acid micelles and absorbed by the small intestinal mucosa cells. In contrast, MCT is completely hydrolyzed to free fatty acids and glycerol by pancreatic lipase, and rapidly absorbed. MCT is absorbed relatively well even when bile and pancreatic secretions are reduced. When labeled MCT or LCT was injected into the small intestinal loop in rats, and the digestion/absorption was compared, 92% of MCT was degraded to fatty acids within 15 min, but only 29% of LCT was degraded to fatty acids. Long-chain fatty acids absorbed from the small intestine are resynthesized to triacylglycerol in the small intestinal mucosa cells, form chylomicrons, flow into the systemic circulation via lymph vessels, and reach peripheral tissues (adipose tissue and muscles). In contrast, medium-chain fatty acids are not readily resynthesized to triacylglycerol. Those absorbed by the small intestinal mucosa are mostly bound by albumin as free fatty acids, forming no chylomicrons, and transferred to the portal vein (Table 7.3).

7.3.4

METABOLISM OF MEDIUM-CHAIN FATTY ACIDS

For the biosynthesis of triacylglycerol from fatty acids, it is necessary that it binds with CoA to become the activated form (acyl-CoA). The enzyme that activates fatty acids by binding them to


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TABLE 7.3 Nutritional Characteristics of MCT and LCT Intestinal hydrolysis Intestinal absorption Resynthesis of TG Route of absorption Metabolic process Fat accumulation Energy density

MCT

LCT

Rapid Rapid Unnecessary Mainly by portal vein Mostly oxidized to acetyl-CoA or CO2 6000 >1200

50–1000

100

500

100 100

600 2000

10–50

100

3000

3–15

Source: Based on Lee (2001); Spolaore et al. (2006); Shimamatsu (2004); Carlsson et al. (2007); Bimbo (2007); Doesum (2006). a Based on an average β-carotene content in Dunaliella of 5% DW.


Microalgal Biotechnology in the Production of Nutraceuticals

283

monospecific cultures of microalgae are difficult to establish in open systems for longer periods of time and the productions are not free of contaminants. For the production of nutraceuticals, production hygiene must be taken into account, and with the development of cost-competitive, closed photobioreactor technology, it must be expected that regulatory mechanisms will favor closed photobioreactor technology for the production of nutraceuticals in future.

17.1.2

PRODUCTION COSTS OF MICROALGAL BIOMASS

It is difficult to obtain exact information about the cost of producing microalgal biomass in largescale production plants because such information is invariably confidential. Instead, production costs for various microalgal products have been estimated by researchers. On the basis of information from producers, Lee (2001) estimated microalgal production costs with raceways to range from US$8 to US$15 per kg. Potential production costs for unspecified biomass in plants of a capacity of 100 ton dry weight (DW)/year were estimated (Chisti, 2007) as US$2.95/3.80 per kg DW for photobioreactors and raceways, respectively, with considerable economy of scale benefits from increasing production to 10,000 ton/year (US$0.47/0.60 per kg). However, the estimate concerned biomass production for biodiesel, and did not take for instance the above-mentioned quality control costs into account. There are not many examples in the literature of production cost calculations for algae in photobioreactors (Carvalho et al., 2006) and the ones that are available are for systems that can be considered as industrial prototypes. Consequently, the estimated production costs are high. For the production of Phaeodactylum in Spain in an acrylic tubular photobioreactor, a biomass production cost of US$32 per kg was calculated (Grima et al., 2003). For the production of Nannochloropsis in Spain in a flat-plate reactor, a cost of US$90 per kg DW was estimated (Cheng-Wu et al., 2001). The challenge is to build photobioreactor systems with simultaneous optimization of energy costs for mixing and gas transfer and material costs for construction (Wijffels, 2008). Such systems are under way, with developments, for example, in Italy (Fotosintetica & Microbiologica S.r.l, http://www. femonline.it/), Belgium (Proviron http://www.proviron.com/Proviron_GB/bio/algae_alg.php), and United States (Solix, http://www.solixbiofuels.com/). These biomass production cost figures may also be related to the bulk price levels of pond-cultured Spirulina, which range from US$3 to 15 per kg, the lower end typically representing Indian or Chinese products and the higher end American products. Chinese-produced Chlorella costs about US$10 per kg and Spirulina in 20 ton shipments (delivered in the United States) costs about US$5 per kg (Carlsson et al., 2007). With the laborintensive open pond systems, it should be considered that the low cost of Indian and Chinese labor will not last long. Photobioreactor-produced algal products are today small specialty products for aquaculture and human nutrition purposes. They currently fetch prices up to US$90 per kg and are delivered as chilled products. Preservation of the product constitutes a significant part of the cost. High-purity Chlorella from the tubular photobioreactor plant in Germany is reported sold at about € 50 per kg (Carlsson et al., 2007). For comparison, the price level of fermenter-produced algae, for example, dry Schizochytrium biomass, is about US$25 per kg (Harel et al., 2002). For food supplements, biomass production costs constitute only a minor fraction of the retail price of the nutraceutical product. As an example, retail sales prices per gram astaxanthin sold as human food supplements are typically about 20–30 times higher than production costs at dry biomass level; hence the direct influence of reduction of costs is minor. However, for microalgal nutraceutical products sold as bulk, in competition with other sources of product, production costs are critical.

17.1.3

MICROBIOLOGICAL FOOD QUALITY ASSURANCE

Microalgae are consumed either as whole-cell food supplements or as extracts of varying purity, and, in general, without any heat treatment or other procedure to eliminate foreign microorganisms.


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As food supplements, they enter into a market, characterized as a “regulatory vacuum” (Miller and Longtin, 2000). WHO has currently not approved of microalgae as foods based on the lack of evidence of historical consumption safety (Gantar and Svircev, 2008). Microbiological product quality standards for microalgal food supplements in different EU countries are still not completely harmonized (Belay, 2007). In some EU countries, microbial standards for dried plant materials are applied to microalgal products. In addition, various trade organizations have developed their own quality standards for plant-based foodstuffs that a microalgal producer may have to subscribe to, and criteria for aerobic bacterial count may, for example, vary by several orders of magnitude (Belay, 1997, 2007). According to Grobbelaar (2003), so far India is the only country that has introduced quality requirements specifically for pond-cultured microalgae. As a rule, both in Europe and in the United States, food supplements can be placed on the market without preapproval. Since 1994, in the United States, the DSHEA (Dietary Supplement Health and Education Act) requires FDA (Food and Drug Administration) to prove the detrimental effect of a product to order it off the market. Product labeling must be non-misleading and advertising should be regulated. Basic FDA quality standards apply to pond-cultured Spirulina: it should contain no foreign algae, no contaminants, insect fragments less than 30 pieces per 10 g, and rodent hairs less than 1.5 pieces per 150 g (Shimamatsu, 2004). The insect fragment and rodent hair criteria are standards derived from grain and cereal products and are difficult to manage with the strongly colored and finely ground microalgal products (Belay et al., 1997). However, if the product is a new dietary ingredient, which means that it was not sold in the United States before October 15, 1994, preapproval is required. When putting a new dietary ingredient on the market, a producer is required, 90 days prior to the intended market placement time, to notify the FDA on which grounds the product is assumed to be safe, by either documenting a history of safe use or submitting reasonable evidence that the product is safe (Gantar and Svircev, 2008). In the United States, the GRAS (generally recognized as safe) list serves as a public blueprint for food supplements. A product may obtain GRAS status (“approved as GRAS”) if the regulatory authority (FDA) does not object to the documentation supporting the GRAS claim put forward by the company. Spirulina and Dunaliella have GRAS status (FDA, 2003; Cognis, 2009). In Europe, microalgal products, like other plant materials, are regulated under the general foods law (178/2002/EC) that places the responsibility of food safety assurance on the manufacturer. In addition, there are food manufacturing hygiene regulations (EC No. 2073/2005) on microbiological standards for food, which are important for manufacturers of microalgal food supplements. EFSA recently compiled an assessment of food safety aspects of a range of botanicals, but so far it has no jurisdictional implications. New food ingredients that do not have a sales history (to a substantial extent) with the European market before May 15, 1997, must be preapproved according to the novel foods regulation (EC/258/97) (European Union, 1997). Normally, food supplements qualify as ingredients. Vitamins, colorants, and technical adjuvants require preapproval as food additives (commonly identified as substances “with an E-number”). In some cases, it is not clear from the character of the substance whether it should be regulated as a food ingredient or as a food additive, like for instance β-carotene that may as well be a food ingredient (antioxidant) as an additive (food colorant) or some fatty acids that may be marketed both as antioxidants/ immune system regulators and as vitamins. In both cases, approval requires submission of a dossier that includes a description of product and processing, ample toxicology studies, descriptions of intended use, analysis of ingestion patterns, health effects with the intended use, and so on. Depending on the “novelty” of the product, this can be a very important process. In both cases, the advantage from the point of view of the manufacturer is permission to market the product over the entire EU market. For novel food, a simplified procedure exists (substantial equivalence procedure) if it can be justified that the product is essentially similar to an existing and legally marketed product, and this procedure has been granted with, for example, different strains of the same algal species (e.g., Haematococcus). A somewhat similar legislative situation for novel food approval applies in Australia.



17.1.4

285

HEALTH CONCERNS WITH MICROALGAL PRODUCTS

Few health concerns with microalgal and cyanobacterial health foods have been reported so far. Heavy metal accumulation, however, is a concern with microalgae as many algae accumulate large quantities of heavy metals if they are present in cultivation media. Sources of heavy metal contamination of media are either atmospheric fallout or impurities in fertilizers. There are no natural or normal levels of heavy metal concentrations in microalgae (Becker, 2004). Producers are setting their own quality standards for heavy metal contents, and for high-quality products, they are so low that a consumption of several kilograms of algal DW per day is required to exceed the WHO maximum recommended heavy metal intake. This should be compared with a typical maximum recommended intake level of a few grams of algal DW per day. Occasionally, imports of microalgae with elevated levels of heavy metals are found. Natural microalgal toxins are another concern, which is well known from shellfish poisoning, but it should be noted that it is related to certain algal species. In 2000, a large number of cyanobacterial strains were verified to be able to produce the neurotoxic amino acid b-N-methylamino-l-alanine (BMAA), suggested to be a potential causative agent in the development of amyotrophic lateral sclerosis (ALS) (Gantar and Svircev, 2008). The substance has not been found in Spirulina. A neurotoxin, microcystin, formed in the cyanobacteria Microcystis aeruginosa (Gilroy et al., 2000) remains a concern as a pollutant in natural stocks of Aphanizomenon flos-aquae. Microcystin also does not occur in pure Spirulina products. A Canadian investigation documented microcystin traces in all nonpure Spirulina batches examined (Grobbelaar, 2003). A standard is now introduced setting the maximum level of microcystin to 1 μg/g of product, which is comparable to the WHO standard for microcystin in drinking water (1 μg/L) (Gantar and Svircev, 2008). Processed under poor quality control, microalgal chlorophyll (similar to chlorophyll from other plants sources) can degrade to form phaeophytin, which has strong allergic properties. The enzyme responsible for the degradation, that is, phytase, can be inhibited or eliminated, for instance, by heat treatment. A number of common bacterial pathogens (zoonoses) can also occur with microalgal products. Depending on national food legislation, microalgae may have to comply with food safety standards, for example, for vegetable products. Special food safety standards have been proposed for microalgal products, for example (Jassby, 1988). Adsorption and absorption of heavy metals during production is probably the most pertinent health issue with microalgal products. Heavy metals may enter the products through the use of lowquality fertilizers.

17.1.5

QUALITY BY SELF-REGULATION

The management quality assurance standards ISO 9001–2000 have now been adopted by a large number of microalgal food supplement suppliers. The essence of the procedure is that all elements of the manufacturing process follow defined quality requirements and that methods to assure compliance with these internal standards are in place. ISO standards can be purchased and adopted internally, but many companies find it useful to also seek certifi cation—that is, to take an audit, carried out by a certification body, that documents that the company’s management of quality assurance is in compliance with ISO standards. The certification organization may itself be accredited: registered by IAF (International Accreditation Forum and the ISO organization) as competent to carry out certification according to the given standards. One of the steps in the process is the HACCP (“hazzap”) analysis for the production process. HACCP means hazard analysis and critical control points and has been mandatory for EU food supplements producers since 2006 (EHPM, 2007). It is, in principle, a very straightforward and intuitively basic process. It implies that all potential risks to public health from the manufacturing process and means (control points) to prevent these hazards are identified. In addition, critical limits for each control point and principles for documentation that these limits are met and actions required otherwise, are established.


286

17.2 17.2.1


LIPID PRODUCTION WITH MICROALGAE LIPID CONTENTS

The lipid content of microalgal biomass can be very high: more than 75% of the DW (see Table 17.2). The basic composition, of current and potential microalgal sources of lipids for nutraceutical use, is also indicated in Table 17.2. The present interest in nutraceuticals production with microalgae is focused on the polyunsaturated fatty acids DHA, C22:6 (ω3), EPA, C20:5 (ω3), ARA (arachidonic acid), C20:4 (ω6), and linolenic acid, C18:3 (ω3). A number of microalgal groups with high amounts of long-chain polyunsaturated fatty acids (PUFAs) have been identified, including diatoms, chrysophyceans, cryptophyceans, and dinoflagellates (Apt and Behrens, 1999). The dinophycean Crypthecodinium can be produced with a lipid content of 56% DW, DHA constituting 19% of the fatty acids (De Swaaf et al., 2003). DHA fractions as high as 56% of the fatty acids have been reported (Behrens and Kyle, 1996). Schizochytrium is also a producer of DHA; it is a so-called thraustochythrid, a colorless, simple microorganism, belonging to the stramenopila that also contain diatoms, yellow algae, and brown algae (seaweeds) (Honda et al., 1999). Another genus in this group is Ulkenia, also known as a DHA producer. These organisms are typically found among decaying organic matter at seashores. Schizochytrium can be produced with fatty acids constituting 70% DW and DHA constituting 35% of the fatty acid. With obtainable biomass densities of 50–70 g DW/L, it clearly has a strong potential as a DHA producer. The organisms are of marine origin and one of the patents of Martek describes the substitution of NaCl in the medium with other salts to make production in normal fermenters possible.

TABLE 17.2 Composition (% % DW) of Microalgae with Potential Nutraceutical Applications Oil Content % DW Anabaena cylindrica Botryococcus braunii Chlorella sp. Chlorella vulgaris Chlamydomonas reinhardtii Crypthecodinium cohnii Cylindrotheca sp. Dunaliella primolecta Dunaliella salina Isochrysis sp. Nannochloris sp. Nannochloropsis sp. Neochloris oleoabundans Nitzschia sp. Nitzschia alba Phaeodactylum tricornutum Scenedesmus obliquus Porphyridium cruentum Schizochytrium sp. Spirulina maxima Synechococcus sp. Tetraselmis sueica


Protein

Carbohydrate

Lipid

% DW

% DW

% DW

43–56

25–30

4–7

25–75 28–32 51–58

12–17

21 20

14–22 53

16–37 23 57

32

6

25–33 20–35 31–68 35–54 45–47 50 20–30 50–56 28–39

10–17 40–57

12–14 9–14

60–71 63

13–16 15

6–7 11

50–77

15–23

Source Chisti (2007) Chisti (2007) Becker (2004) Becker (2004) Chisti (2007), De Swaaf et al. (2003) Chisti (2007) Chisti (2007) Becker (2004) Chisti (2007) Chisti (2007) Chisti (2007) Chisti (2007) Chisti (2007) Barclay et al. (1994) Chisti (2007) Becker (2004) Becker (2004) Chisti (2007) Becker (2004) Becker (2004) Chisti (2007)


287

For EPA, Barclay et al. (1994) refer to heterotrophic Nitzschia alba strains with 50% DW lipids that could be produced in densities of 45–48 g DW/L in 64 h in fermentors. EPA would constitute about 5% of fatty acids, resulting in volumetric fermenter productivities of 250 mg EPA/L/day. This value is probably the highest published EPA productivity. The authors also refer to Nitzschia alba strains with EPA constituting 10–29% of total fatty acids, but with total lipids constituting only 3.7–6% DW. It would appear that there is development potential in simultaneously optimizing EPA percentage and lipid content in Nitzschia alba.

17.2.2 LIPID CLASSES For downstream processing, it is important to establish whether fatty acids are present as triglycerids in the cytoplasm (preferable) or as phospholipids in membranes (difficult to extract). In Nitzschia species, Pythium and Crypthecodinium, PUFAs are mainly triglycerides, while PUFAs in Phaeodactylum tricornutum and Porphyridium cruentum are mainly galactolipids (Ward and Singh, 2005).

17.2.3

MODIFICATION OF LIPID CONTENTS BY GROWTH CONDITIONS

In Pavlova viridis, lipid class composition and fatty acid composition are strongly altered through growth phases, with the stationary phase exhibiting higher total lipid (233 mg/g) than the late exponential phase (166 mg/g). EPA decreased through the growth cycle while DHA remained constant. The hydroxylated sterols (Pavlova sterols) increased from 2.4 to 4.3 mg/g while stigmasterol and sistosterol decreased through the growth cycle (Xu et al., 2008). Carbon dioxide also influences fatty acid composition in some microalgae, with the degree of unsaturation being higher in Chlorella vulgaris cultivated at low CO2 whereas the lipid classes were not affected in Chlamydomonas reinhardtii, and Dunaliella reacted in the same manner. CO2 did not have that effect on Euglena gracilis, Porphyridium cruentum, and Anabaena variabilis (Tsuzuki et al., 1990).

17.2.4

YIELD OF LIPIDS IN AUTOTROPHIC MICROALGAL PRODUCTIONS

Outdoor phototrophic production of EPA with Phaeodactylum resulted in productivities of 56 mg EPA/L/day (Fernandez Sevilla et al., 2004), which is most likely the best EPA autotrophic productivity data published so far. It is possible that on a direct EPA base, phototrophic production of EPA with Phaeodactylum is competitive to heterotrophic Nitzschia alba as a result of lower production costs associated with outdoor tubular reactors compared to fermenter productions. However, the processing costs (see Section 17.2.2) and production GMP conditions should also be considered.

17.3

CAROTENOID PRODUCTION WITH MICROALGAE

Dietary carotenoids are important in human antioxidative protection in a number of tissues, including retina (Lutein and Zeaxanthin) and liver (β-carotene) (refs). Carotenoids can protect skin against photo-damage and counteract aging of skin (refs), and substantial in vitro and cell model evidence indicates a role in cancer prevention (refs) and prevention of cardiovascular diseases (refs). Fucoxanthin is emerging as an active ingredient in slimming products. Uses of carotenoids also include animal feed such as fish and poultry feed. The global carotenoid market for all carotenoids was in 2004, according to a report from the company BCC research, cited in Del Campo et al. (2007), US$887 million and projected to increase to US$1 billion in 2009. The total production of microalgae-derived carotenoids is still quite limited (see Table 17.1). The largest contribution comes from β-carotene from Dunaliella. The scale of global Dunaliella biomass production is indicated to be 1200 ton (Del Campo et al., 2007; Pulz and Gross, 2004). Assuming


288


an average β-carotene content of about 5% DW, the produced β-carotene thus corresponds to about 50 ton pure substance, which would indicate a market share at about 25%; however, it should be kept in mind that this figure covers Dunaliella marketed both as dry biomass and as extracted β-carotene product. The volume of microalgal astaxanthin is estimated to be 0.5–1 ton (pure substance) and thus constitutes less than 1% of the market. At present, there is no available information about substantial microalgal production of other carotenoids. There has been considerable academic interest in the microalgal production of lutein, which is the only one of the major nutraceutical carotenoids that is not synthesized. Potential microalgal production is both autotrophic and heterotrophic. The dominant source of lutein is produced by Cognis from Marigold petal leaves.

17.3.1

CHEMICAL NATURE AND BIOLOGICAL FUNCTION OF CAROTENOIDS

Carotenoids are C40 terpenoids synthesized from isopentenyl diphospate (IPP) as repetitive C5 isoprene units (equivalent to 2-methyl, 1,3-butadiene). More than 600 naturally occurring carotenoids have been identified. In all organisms, the synthesis proceeds via IPP, but two different pathways for the formation of IPP have been identified. In the cytoplasm of plant cells (and mammals), synthesis of IPP proceeds via the mevalonate pathway whereas IPP for the production of carotenoids in the chloroplast is believed to be formed only via the deoxyxylulose 5-phosphate (DXP) pathway (Lange and Croteau, 1999; Grunewald et al., 2000). Carotenoids may chemically be classified into two groups: hydrocarbon carotenoids (or simply carotenes, including β-carotene, α-carotene, and lycopene) or oxygen-containing xanthophylls (including lutein, astaxanthin, β-cryptoxanthin, and zeaxanthin). Functional oxygen groups include hydroxyl, carbonyl, epoxy, and oxo groups. A carotenoid may be either acyclic or cyclic—which implies that there is a cyclic group at one or both ends of the isoprenoid molecule. The cyclic group is typically an ionone: a cyclohexane ring with one or more endocyclic double bonds. These ionone groups, as breakdown products from carotenoids, are important constituents of flower fragrances. The structure of carotenoids and position in membranes may reflect in their tissue location and antioxidative functions; lycopene and β-carotene are thus effective against radicals generated in the inner face of the membranes, whereas the less hydrophobic lutein and zeaxanthin act in the hydrophilic side of the membranes (Slattery et al., 2000). Phytoene, the simplest carotenoid, is formed from two molecules of geranyl-geranyldiphosphate by the enzyme phytoenesynthase, which is a key enzyme in the metabolic engineering of carotenoid synthesis as it is believed to be limiting for carotenoid synthesis rate in many microalgae (Yan et al., 2005). The configuration of a number of nutraceutical carotenoids is shown in Figure 17.1. Of these, currently only β-carotene and astaxanthin are industrially produced by microalgae. The possibility of enhancing the carotenoid productivity of microalgal cultures depends on a detailed understanding of their function and synthesis regulation. Carotenoids may be either primary or secondary: primary carotenoids are normal constituents of the photosynthetic apparatus in chloroplasts together with chlorophyll a and b (in green algae) and proteins (Barber and Nield, 2002). Their quantity reflects light acclimation status of the cells and they do not therefore normally accumulate to a significant extent. Carotenoids (β-carotene) have important functions in the photosynthetic apparatus. They absorb blue–green light and assist in transferring energy to the reaction centers. They quench triplet state chlorophylls in the antenna molecules, thereby preventing them from transferring the exitation energy to oxygen—which would result in detrimental and toxic singlet state oxygen molecules. In the reaction centers, β-carotenes do not quench triplet state chlorophylls, but they can react directly with singlet oxygen (1O2), during which they decompose into nonreactive products (Telfer, 2002). In chloroplasts, carotenoids function both as accessory pigments and as antioxidants. All carotenoids directly associated with photosynthesis are referred to as primary carotenoids, but they may serve different functions. Primary carotenoids are present as a relatively constant proportion



289

All-trans phytoene

β-carotene

Lycopene OH

Lutein HO

OH

Zeaxanthin O

HO

Canthaxanthin O

O OH

Astaxanthin HO O

FIGURE 17.1 Configuration formula of nutraceutical carotenoids from microalgae.

to chlorophylls in concentrations generally under 1% of the biomass (Lamers et al., 2008). Their concentrations thus reflect the general photo-acclimation state of the algae, which means that very high concentrations of these substances in the biomass cannot be achieved by simple manipulations of growth conditions. Secondary carotenoids are carotenoids that are not found in the thylakoid membranes and are not directly involved in photosynthesis (Grünewald et al., 2001), but may accumulate either in or outside the chloroplast. They typically do not accumulate under conditions of fast growth, but if cell division is impeded as a result of lack of nutrients, massive accumulation of secondary carotenoids may take place. β-Carotene may serve both as a primary carotenoid in the chlorophyll–protein complexes of photosystems and as a secondary pigment accumulating under conditions of retarded growth, while astaxanthin is an example of an exclusively secondary pigment with no primary photosynthetic functions. At the present time, only two carotenoids are produced industrially at significant scale (β-carotene and astaxanthin) and both of them are secondary carotenoids. Industrial production of these carotenoids is based on enhanced accumulation under conditions of reduced cell division.


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17.3.2


BIOLOGICAL FUNCTION OF CAROTENOIDS

Light absorption in chloroplasts takes place in chlorophyll–protein complexes called antenna. The chlorophylls absorb light in two regions: 350–460 and 700–960 nm. Carotenoids function as accessory pigments, absorbing light in the blue and green region from 350–550 nm, where chlorophyll light absorption is low. These carotenoids are physically closely associated with the antenna chlorophylls to which excitation energy is transferred. Several different carotenoids are found in the photosynthetic antennas (Telfer et al., 2008). However, the efficiency of excitation energy transfer to chlorophylls is not very high: 20–30% is referred to in Telfer (2002) whereas bacterial photosynthetic excitation energy transfer efficiency may approach 100%. But carotenoids perform a second function, which has been exploited by many organisms that do not produce the carotenoids themselves but depend on dietary intakes of plant carotenoids, including humans. In photosystem II (PSII), excess light (at which photosynthetic reactions are saturated) will result in the formation of triplet state chlorophyll radicals that are relatively long-lived and may transfer their excitation energy to molecular oxygen, whereby reactive oxygen radicals are formed. In the antenna, carotenoids can prevent singlet oxygen (1O2) by rapidly quenching the chlorophyll triplet state, and should singlet oxygen stages nevertheless be formed, carotenoids can furthermore scavenge them (Telfer et al., 2008). In the antenna of algae and vascular plants, lutein is the main carotenoid, but β-carotene is more efficient in 1O2 scavenging and the presence of β-carotene may have been developed mostly for that purpose. In the reaction centers, on the other hand, β-carotenes do not appear to be able to quench triplet state chlorophylls (Telfer, 2002). Carotenoids also quench excess excitation energy in PSII (nonphotochemical quenching), which diminishes the formation of superoxide and hydroxyl radicals (Telfer et al., 2008). β-Carotene is found in PSII reaction centers, and zeaxanthin and lutein (vascular plants and green algae), diatoxanthin and peridinin (dinophyceans), and fucoxanthin (phaeophyceans and diatoms and chrysophyceans) are found in the antenna (Grossman et al., 1995). The xanthophyll cycle is a series of reversible carotenoid transformations carried out in response to variations in light intensities. The xanthophyll cycle serves to stabilize chlorophyll–protein complexes and dissipate excess excitation energy from the chlorophyll (also referred to as nonphotochemical quenching) (Young and Frank, 1996; Young, 1991). In green vascular plants and green algae, the xanthophyll cycle denotes the violaxanthin → antheraxanthin → zeaxanthin transformation that takes place over a timescale of hours during exposure to excess irradiation and contributes to nonphotochemical quenching. For some green microalgae, including Haematococcus, the xanthophyll cycle has been found to contribute less to nonphotochemical quenching than in higher plants (MasojÌdek et al., 2004). In diatoms, a similar chain of transformations involves diatoxanthin and diadinoxanthin. In diatoms, diadinoxanthin in antenna complexes is converted to diatoxanthin under high light conditions in a xanthophyll cycle (Telfer et al., 2008), with a function similar to that in green algae.

17.3.3 SECONDARY CAROTENOIDS Secondary carotenoids are carotenoids that are not integral parts of membranes, but accumulate in large quantities in lipid vesicles. There are not many examples of algae with described accumulation of secondary metabolites. Examples include Dunaliella (β-carotene), a number of snow algae such as Chlamydomonas nivalis and Chloromonas (astaxanthin), Euglena sanguinea (astaxanthin), and Haematococcus sp. (astaxanthin). The aerial green alga Coelastrella striolata var. multistriata accumulates cantaxanthin in high quantities, 4.75% DW (Abe et al., 2007). They may accumulate inside the chloroplast (β-carotene in Dunaliella) or outside the chloroplast (astaxanthin in Haematococcus). Secondary carotenoids may accumulate in significant quantities, typically where photosynthesis remains significant while cell division is reduced. β-Carotene may reach concentrations of 12–14% of the dry biomass in Dunaliella (Ben-Amotz and Avron, 1990; Spolaore et al., 2006) while astaxanthin content may reach up to 8% of the DW of Haematococcus; however, typically the content in most industrial productions does not exceed 3% (Lorenz and Cysewski, 2000; Olaizola, 2000).



17.3.4

291

β-CAROTENE

Dunaliella salina has the unique ability of accumulating β-carotene in large quantities. β-Carotene is a primary carotenoid present in chloroplasts in the reaction center of photosystem II of all photosynthetic organisms known (Telfer et al., 2008). In Dunaliella, it may also be formed as a secondary carotenoid and may accumulate in significant quantities. An excess of 12% of DW was reported by Ben-Amotz and Avron (1990). The reason for this accumulation is not completely understood, but a frequently referred hypothesis suggests that β-carotene accumulating in the interthylakoid regions as small globules in the chloroplast serves to protect the algae against detrimental effects of strong illumination simply by acting as a filter. The large β-carotene accumulation prevents photoinhibition in Dunaliella, particularly the photoinhibitory effects of blue light (Ben-Amotz et al., 1989). The lack of effect toward red light and the specific effect on blue light seem consistent with that hypothesis. However, the formation of reactive oxygen species (ROS) may also be involved in triggering the formation of secondary β-carotene, because enhancing ROS formation in the cells and inhibiting catalase and superoxide dismutase enhance β-carotene accumulation (Shaish et al., 1993). For a recent review of mechanisms of β-carotene accumulation and signaling in Dunaliella, see Lamers et al. (2008). Compared to the astaxanthin accumulation process in Haematococcus, much less is known about the β-carotene accumulation process in Dunaliella, perhaps because the industrial carotenoid formation process is much simpler. The degree of accumulation is related to the amount of illumination received over the cell cycle (Lamers et al., 2008). The effect of other environmental conditions that enhance β-carotene accumulation may act indirectly by reducing the growth rate, but the exact function so far remains a matter of speculation. However, it has important advantages in industrial exploitation because the molecule is accumulating in lipid globules in the chloroplast and is not bound in membranes. The Chlorophycean strains Dunaliella tertiolecta and Dunaliella bardawill are used for β-carotene production. Harvesting is generally carried out by flotation and filtration (Dufossé et al., 2005). Dunaliella possesses a few peculiar traits that contribute to making the production economically interesting. Dunaliella lacks a rigid cell wall and is able to adapt to a wide range of salinities from freshwater to concentrated saline conditions that prevail in salt lakes. As an osmolarity regulating agent, Dunaliella produces glycerol. Three products contribute to the industrial value of the species: β-carotene, food protein, and glycerol (Dufossé et al., 2005). Furthermore, Dunaliella has a suitable composition of carotenoids that gives it a high dietary value compared with other microbial sources, including lutein, neoxanthin, zeaxanthin, violaxanthin, cryptoxanthin, and α-carotene. In total, these carotenoids constitute about 15% of carotene concentration (Dufossé et al., 2005).

17.3.5

INDUCTION OF β-CAROTENE FORMATION

Since carotenogenesis requires high light intensities, Dunaliella is grown in raceway ponds at low biomass densities, as light would be completely absorbed in only 5 cm of water column at biomass levels of 0.2–0.6 g DW/L−1 (Ben-Amotz and Avron, 1990). The minimum practical culture depth in ponds is about 15 cm—otherwise, it would be preferable to produce β-carotene with Dunaliella at lower depths.

17.3.6

ASTAXANTHIN

Astaxanthin may be produced biologically with a number of different organisms, including the fungi Dendrophyllomyces dendrorhus (Phaffia rhodozyma), the flower plant Adonis aestivalis, the microalgae Haematococcus pluvialis, Chlorella zofingiensis, Chlamydomonas nivalis, Euglena gracilis, Chlorella sorokiniana, and a few other species. Furthermore, astaxanthin is produced from


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shrimp and shellfish waste in small scale. The only industrial microalgal production today depends on Haematococcus because of the very high pigment concentrations obtainable (up to 8% DW). The current product standard in, for example, dried biomass product from BioReal (Sweden) is 5% (BioReal, 2009). The identity of the red pigment astaxanthin in Haematococcus pluvialis has been known since at least 1944 and the involvement of light and nitrogen deprivation in carotenogenesis enhancement has been known since 1909 (Droop, 1954). Probably the first attempts to exploit microalgal astaxanthin formation industrially were Norwegian attempts to grow Chlamydomonas nivalis for astaxanthin production for salmon feed (Kvalheim and Knutsen, 1985). After 1985 the development of large-scale cultivation of Haematococcus started in Japan, Israel, Sweden, France, and Hawaii, and in 2009 some 50 companies are well established in the commercial production of Haematococcus. Haematococcus is a Chlorophycean with a number of morphologically different growth stages. Normally five different cell types are recognized: flagellate, palmelloid, aplanospore, cyst, and microzoid. The flagellate stage is also called a macrozoid. Little is known about the genetic constitution of the different cell types of Haematococcus. Despite the many cell types, production is quite stable and reproducible once appropriate technology has been established. The dominant technology is a two-stage process. Biomass is produced under conditions that favor the green flagellate stage. Flagellate production can be autotrophic or heterotrophic, typically continuous, but requires closed, photobioreactor technology as the flagellate cultures are susceptible to contamination by other algae or protozoans. Product formation is carried out in a separate stage under some kind of induction of carotenogenesis, typically a combination of nutrient deprivation and high light intensity per biomass unit. As the biomass at the end of the cycle, which usually lasts 4–7 days, is entirely harvested and the cysts, which is the resulting growth stage, are resistant to bacteria and most protozoan infections, this stage is conveniently carried out outdoors, as light is required in high amounts for autotrophic carotenoid induction. The astaxanthin content in Haematococcus cysts may reach about 8% of DW. 7.7% was obtained under experimental conditions in acetate- and high CO2-supplemented light induction by Kang et al. (2005) in most productions; however, the astaxanthin concentration in the biomass for industrial production usually is taken to about 3%. Astaxanthin is used extensively in aquaculture; about 100 ton is currently used in feed to give salmonids and shrimp the pink flesh coloration. Products from Haematococcus are predominantly sold as human food supplements.

17.3.7

INDUCTION OF ASTAXANTHIN FORMATION

Theories about the biological purpose of astaxanthin formation include “sunshade” function, shielding the chloroplast against excess irradiation or damaging blue light (Bidigare et al., 1993), antioxidant defense as demonstrated from in vitro experiments (Hagen et al., 1993) or induced by excess photosynthetic reducing equivalents (NADPH) (Yamane et al., 1997), energy storage (Droop, 1954), singlet oxygen scavengers (Kobayashi and Sakamoto, 1999), or lipid peroxidation chain breakers (Miki, 1991). The close correlation between lipid formation and astaxanthin formation has been suggested to be important in understanding the biological function of astaxanthin accumulation (Zhekisheva et al., 2002). Even interpretation of the physiological status of the algae during astaxanthin formation is ambiguous. Cessation of cell division as induced by phosphate, sulfur depletion, or salt stress may lead to carotenoid accumulation. The maximum carotenoid synthesis rate, on the other hand, occurs just after the introduction of light stress or nitrate limitation, where growth rate and chlorophyll a synthesis are at the maximum (Boussiba et al., 1992). In 1991, Kobayashi et al. (1991) published the first quantitative description of enhancement of astaxanthin formation using high light intensity in combination with acetate. The inclusion of salt of 0.8% under optimum light conditions stimulated astaxanthin accumulation (Boussiba



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et al., 1992; Sarada et al., 2002). Above 1%, total astaxanthin formation decreased. Controlling nitrate depletion, light and temperature, and salinity or inclusion of 450 μmol/L FeSO 4 was found to enhance the accumulation of astaxanthin (Kobayashi et al., 1991; Harker et al., 1995; Hagen et al., 2001). With EMS (ethylmethanesulfonate) or NTG (1-methyl 3-nitro 1-nitrosoguanidine), algal strains with 2–3 times increased expression of lycopene cyclase and early-stage enhanced astaxanthin formation were obtained (Sandesh Kamath et al., 2008; Tripathi et al., 2001). The mutants exhibited increased astaxanthin formation under conditions of stress (high light or salinity), whereas the astaxanthin formation rate under less environmental stress conditions was similar to the wild strains.

17.3.8

BIOMASS PRODUCTIVITY IN NATURAL DAYLIGHT

Autotrophic production can yield high volumetric astaxanthin concentrations (175 mg/L) and high specific productivities (4–5 mg/L/day) (Hata et al., 2001; Kang et al., 2005). In outdoor, pilot-scale photobioreactors in Almeria, Spain, López et al. (2006) investigated biomass productivity and astaxanthin formation (a tubular photobioreactor and a bubble column). The maximum volumetric biomass productivity (0.55 g DW/L/day) was obtained at an average light intensity of 130 μmol/ m2/s; high biomass concentrations (8 g DW/L) were used and fluorescence measurements showed that the algae were not light inhibited even at noon. Biomass yield on light was 0.61 g/mol in the tubular reactor. In terms of both volumetric productivity and yield on light, these data presumably represent the maximum productivity that can be achieved with an autotrophic culture under daylight conditions.

17.3.9

ASTAXANTHIN FORMATION AND IRRADIATION

It is possible to relate astaxanthin formation rate directly to light absorption in Haematococcus. With white light-induced astaxanthin formation in cultures with biomass density kept constant, it could be established that astaxanthin formation is directly proportional to light absorption, the astaxanthin yield on light corresponding to approximately 10 mg/mol (Norsker, unpublished data). As the cost of delivering 1 mol of photons to the culture with artificial illumination is approximately 0.6–0.7 kWh, it can easily be verified that purely light-driven carotenogenesis is not feasible with artificial illumination for the production of astaxanthin for the feed market (US$1900–2200 per kg). On supplying acetate also during the carotenogenesis, the yield could be doubled. High relative light intensities may be applied by keeping the biomass concentration low in outdoor ponds or reactors or by increasing light intensity. In a tubular photobioreactor with light concentration in fresnel lenses, where peak irradiances could reach 6000 μmol/m 2/s1, it was possible to enhance the astaxanthin formation rate significantly. In control tubes, irradiated with ambient light conditions, the astaxanthin formation rate was 25% lower than in the light-concentrated tubes (Masojidek et al., 2008). Astaxanthin biomass concentration reached 3% DW in only four days. Fluorescence measurements indicated strong nonphotochemical quenching with high irradiance, with photochemical efficiency values (ΔF/F′ m) near 0. In contrast to the use of high-level irradiation in the above-mentioned examples, it was found that using low-intensity (2–12 μmol/m2/s) flashing blue LED lights (1 kHz, 17–67% light duty cycle) for product formation enhancement in a one-stage heterotrophic process, the irradiation requirements were reduced by one-third compared to continuous irradiation. Maximum volumetric yields of astaxanthin were about 28 mg/L and productivities about 2.5 mg/L/day (Hata et al., 2001). Given the substantial industrial interest in the product and considering that the basic characteristics of the astaxanthin formation process in Haematococcus have been known for almost 100 years, it may surprise one that the present knowledge about the mechanisms and control of astaxanthin formation is still so fragmented. However, the complex growth stage pattern and the presumed large genetic


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variability between strains complicate the research. Hopefully, molecular biological research can contribute by elucidating the regulation of astaxanthin formation and help to optimize the process.

17.3.10

INDUSTRIAL HAEMATOCOCCUS PRODUCTION

Industrial cultivation of Haematococcus has been applied since 1996. The Swedish company Astacarotene (now BioReal) was the first to market astaxanthin produced from Haematococcus, followed by the Hawaiian companies Cyanotech and Mera Pharmaceuticals, Fuji Chemical Company, Indian Parry Pharmaceuticals, and Portuguese Necton. Today, a large number of companies are establishing productions. The applied methods in industrial Haematococcus production are very different. At the Hawaiian companies Cyanotech and Mera Pharmaceuticals, biomass production takes place in closed photobioreactors to a variable degree while the product formation step takes place in outdoor open raceways of 500 m3 volume. Mera Pharmaceuticals undertakes biomass production in 41 cm diameter, disposable plastic sleeve, tubular photobioreactors with a high degree of process control and astaxanthin formation takes place in open raceway ponds (Olaizola, 2000; Lorenz and Cysewski, 2000). BioReal operates two production plants: one in Hawaii, where both steps are carried out in dome-shaped, acrylic photobioreactors, and one in Sweden, where both biomass production and astaxanthin formation take place in artificially illuminated photobioreactors.

17.4 17.4.1

CARBOHYDRATES CHLORELLA GROWTH FACTOR

A hot-water extract from various Chlorella strains named CGF, or Chlorella growth factor, has been marketed in Japan as a general health promoting product since the 1960s. CGF is a hot-water extract from Chlorella pyrenoidosa, the main active component thought to be a β-glucan (a glucoprotein rich in d-galactose) with a molecular weight of 63 kDa (Liang et al., 2004). In China, microalgal liquid extracts are beginning to enter the market, the ease of consumption being the main incentive. They are hot-water extracts from Chlorella and Spirulina with various flavor additives, including extracts from traditional Chinese medicine. CGF is considered an important active ingredient. Health and cosmeceutical products are being developed. A range of foodstuffs produced include noodles, bread, green tea, and beer. Companies active in the field include Guangdong Maoyuan Imp. & Exp. Corporation and Guangdong Guanghua Pharmaceutical Factory and Tianjin Meilin Health Products Co., Ltd. (Liang et al., 2004). A number of polysaccharides from Spirulina are being developed as nutraceuticals or drugs, including Ca-Spirulan (Ca-SP) and Immolina. Ca-SP is a sulfated polysaccharide that consists of two types of repeating disaccharide units: acofriose (1,2 linked O-rhamnosyl-3-O-methylrhamnose with sulfate at C-4) and aldobiuronic acid (1,3 linked O-hexuronosyl-rhamnose with sulfate at C-2 and C-4), the proportion of which is 3:5 (Kaji et al., 2004). The Spirulan molecule typically has a molecular weight about 210,000. Immolina is another polysaccharide from Spirulina. It is produced as a crude ethanol extract (Balachandran et al., 2006; Pugh et al., 2001) and is being marketed in a number of countries as an immunostimulant, which in many countries is compatible with marketing as a food supplement.

17.5 MICROALGAL PRODUCTION METHODS: AUTOTROPHIC, HETEROTROPHIC, MIXOTROPHIC In autotrophic nutrition cell carbon and energy are derived from photosynthesis, whereas in heterotrophic nutrition both carbon and energy are derived from reduced organic substances.



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The production of the largest part of the biomass for nutraceuticals production is autotrophic, but heterotrophic production of Chlorella was established in the 1990s using conventional fermentation technology. In 1996, heterotrophically produced DHA-rich oils produced with Crypthecodinium (Vazhappilly and Chen, 1998) reached the market and from 2002 also with Schizochytrium. In 1999, half the production of Chlorella in Japan was based on fermentation (H. Endo, personal communication) and new production capacity was entirely based on heterotrophic production. The advantage of heterotrophic production, from a nutraceutical product quality point of view, is that the production of biomass takes place under axenic conditions, whereas the presence of bacteria in photobioreactors cannot be completely eliminated. It is expected that it will be easier to have new foods approved according to various national novel food regulations if they take place under axenic conditions. Few algal species are obligate autotrophic—that is, only able to grow photosynthetically. Obligate photoautotrophy can in some cases be explained by a partially blocked tricarboxylic acid (TCA) cycle (Chen and Chen, 2006). On the other hand, few species are capable of true heterotrophic growth, that is, able to grow in the dark on organic carbon sources. A large number of reduced carbon sources can support growth in microalgae, including glucose, ethanol, acetate, amino acids, glycolate, citrate, pyruvate, and glyceraldehyde. In most species that are capable of growing both heterotrophically and autotrophically, the growth rate on glucose or acetate is similar to, or lower than, autotrophic growth. However, the advantages from a production point of view are that considerably higher biomass concentrations may be obtained by heterotrophic or mixotrophic cultivation than by autotrophic cultivation, with a proportional increase in volumetric productivity rate. Some microalgae can use several reduced carbon sources, but many are restricted to either acetate or glucose. Ukeles and Rose (1976) investigated 13 marine microalgal species with abilities to assimilate organic substances; only three could grow in the dark (heterotrophic growth). Fifteen sugar types, 19 acids, and 16 alcohols could be assimilated. Chlorella, Cyclotella, Tetraselmis, and Nitzschia are examples of true heterotrophic species (Apt and Behrens, 1999). Different strains of the same species may have different abilities to grow heterotrophically. The third growth mode, mixotrophic, in principle concurrent autotrophic and heterotrophic growth, is more difficult to define, as in some algae that are capable of growing both in the dark and autotrophically, photosynthesis can to some extent suppress the assimilation of organic substances and vice versa. For some algae, such as Chlorella vulgaris and Haematococcus pluvialis, the maximum specific growth rates appear to be the sum of heterotrophic and autotrophic growth rates (Kobayashi et al., 1992; Takahira and Shuichi, 1981) as long as the cultures are growing under light limiting conditions. Mixotrophic production is not currently applied to large scale because of the technical difficulties involved when using reduced carbon sources. Maintenance of axenic conditions requires the use of stainless steel tanks and steam sterilization. One area where mixotrophy is readily applicable is the formation of astaxanthin in Haematococcus. As the product formation step lasts only 3–6 days, acetate as a mixotrophic carbon source may be applied in addition to light-driven carotenogenesis, without resulting in any significant increase in bacterial load.

17.5.1

AUTOTROPHIC GROWTH

Photosynthesis consists of two major processes: light reactions, in which light is converted to the energy carrier ATP and the reductant NADPH, and dark reactions, in which ATP and NADPH are used to fix or convert carbon dioxide to carbohydrates. During light reactions, O2 is liberated when two oxygen atoms from a water molecule pass on their electrons to NADP+. Light reactions take place in two different protein–chlorophyll complexes, photosystem I (or P700) and photosystem II (or P680). The photosystems consist of light harvesting antenna complexes that contain several light harvesting compounds (chlorophyll a and b, carotenoids, and other accessory pigments, including phycocyanin, phycoerythrin, and allophycocyanin) and a core with


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the reaction centers to which the excitation energy of an intercepted photon is channeled. The photosystems are embedded in the thylakoid membranes in the chloroplasts. Thylakoids are flattened membrane sacs. The space inside the thylakoids is called the lumen. When the chlorophyll molecule in a photosystem II reaction center is excited by the capture of a photon, charge separation takes place and strong electropositive and electronegative radicals are formed. The positive radical is so strong that it can oxidize oxygen in water by attracting an electron, whereas the negative radical passes its electron on through a series of reactions, called the electron transport chain via photosystem I, where it is used to generate NADPH from NADP+ (two electrons required). As these processes involve powerful electrochemical reactions that constantly create harmful oxygen radicals, antioxidant enzymes and antioxidant carotenoids are provided for the containment. Some of these compounds, for example, β-carotene, zeaxanthin, and lutein, are retained in human tissues (liver and retina), where they presumably function as antioxidants. Several of the redox steps in the electron transfer chain result in the formation of protons that are released to the lumen (inner) side of the thylakoid membranes. As a result, a pH gradient (or proton gradient) of several pH units is formed, which serves to drive the formation of ATP. The Calvin cycle is frequently referred to as the dark reaction, in which CO2 is fixed using ATP and NADPH. Actually, the term “dark reaction” is misleading—it does not take place in the dark, but merely means that light is not involved in the process. Ribulose-diphosphate is the compound that reacts with carbon dioxide in the Calvin cycle, mediated by the enzyme ribulose-diphosphatecarboxylase (also known as Rubisco). The immediate product of the photosynthesis is glyceraldehyde3-phosphate. This or other intermediates from the cycle may be exported from the chloroplasts. Several amino acids are synthesized directly from glyceraldehyde-phosphate. Energy and reducing equivalents may also be transported from the chloroplasts into the cytoplasm in the form of ATP and NADPH through various shuttle mechanisms. It is important to note that while carbon and energy production may be entirely autotrophic, algae may still exhibit respiration. Two different types of respiration occur in microalgae: photorespiration and dark respiration. Photorespiration is the oxygenation of ribulose-diphosphate. This occurs as a result of the relatively high affinity of the initial CO2 fixing enzyme Rubisco for O2. As a consequence of the oxygenation, phosphoglycolate and 3-phosphoglycerate are formed. 3-Phosphoglycerate is a normal intermediate in the Calvin cycle, but phosphoglycolate must be re-entered through a series of energy requiring reactions and thus represents a loss of energy to the system. Photorespiration occurs to a variable extent in different species (Jordan and Ogren, 1981). It is favored by high temperatures, high oxygen concentration, and low CO2 levels near the enzyme and may be a significant factor limiting the photosynthetic efficiency of algae. There may be genetic engineering solutions under way to alleviate the energy losses through photorespiration and the potential advantages would be substantial (Leegood, 2007). Dark respiration, on the other hand, takes place in the cytoplasm or mitochondria. It is equivalent to respiration in animal cells and denotes the oxidation of photosynthetically formed carbohydrates or lipids, in which electrons are transferred to oxygen. This process is similar to respiration in heterotrophic cells. Significant proportions of the “day production” of fixed carbon can be lost during night—up to 46% of daytime production under conditions of suboptimal biomass concentrations (Masojídek et al., 2003). If biomass was increased, the losses in the example were reduced to around 5%. Dark respiration constitutes, at least partially, the requirement for energy or carbon structures for specific anabolic purposes. However, it has been demonstrated that it is possible to increase net productivity considerably by cooling the algal culture down by night and reheating it at daybreak (Grobbelaar and Soeder, 1985). Whereas little can be accomplished with pond cultures, in photobioreactors the reduction of dark respiratory losses by temperature management of the algal culture is possible.



17.5.2

297

EFFICIENCY OF THE PHOTOSYNTHESIS

The energy conversion of primary light capture processes is very high, with red light being the most effective. For daylight between 400 and 700 nm (PAR), the maximum energy efficiency is up to 21%. Considering the full daylight spectrum (that is, global radiation that includes UV and IR), the theoretical maximum efficiency is thus reduced to 9% (Kruse et al., 2005). With large-scale, outdoor cultures, current practice results in 1–4% photosynthetic efficiency (Janssen et al., 2003). Photosynthetic efficiency calculation is based on energy: the total energy in the absorbed radiation is related to the enthalpy change of the given process. For biomass production, this may be taken as the combustion energy (enthalpy) of the produced biomass. The combustion energy varies with the lipid content of the algal biomass, from about 20 to 28 kJ/g. Some typical values are Phaeodactylum 20.15 kJ/g, Spirulina 21.56 kJ/g (Acién Fernández et al., 2003; Tredici and Zittelli, 1998), and Chlorella protothecoides 23 kJ/g (autotrophic) and 27 kJ/g (heterotrophic) (Miao and Wu, 2004). Another frequently used measure of the efficiency of autotrophic growth represents the quantum requirements for biomass or carbon fixation as biomass production and light absorption in photons are measured comparatively easy. For the fixation of 1 mol of carbon, theoretically 8 mols of photons are required (the process is CO2 + H2O → CH2O + O2). The maximum theoretical efficiency of 9% global solar irradiation corresponds to an average annual yield of 60 g DW/m2/day or 218 ton DW/ ha/year for an optimum site with an average annual daily (24 h) irradiation of 200 W, such as southern Europe; hence despite the somewhat disappointing low efficiencies of autotrophic growth, there is still value to be gained from microalgal photosynthesis. Attaining this high efficiency furthermore requires relatively low light intensity as the reaction centers otherwise may saturate (being in a reduced state). Applying higher intensities than the photosystems can handle, result in energy dissipation by heat or fluorescence. One of the process engineering strategies for improving photosynthetic efficiency is to reduce the average light intensity at which the algae are growing, in practice by applying higher biomass levels per surface area. This can be accomplished in two different ways, either with densely spaced vertical photobioreactors with relatively low biomass densities or by applying high biomass densities in well-mixed horizontal or inclined systems. With the first option, the challenge is to construct functional, large-area photobioreactors at low cost; with the second strategy, it is a challenge to get the correct combination of biomass concentration and mixing.

17.5.3

GROWTH RATE AND LIGHT INTENSITY

In algal cultures, growth rates depend on light intensity. At low light intensities, the relationship is approximately linear as a result of an approximately linear photosynthetic efficiency. At higher light intensities, the photosynthetic efficiency is increasingly reduced (Figure 17.2). At very high light intensities, photosynthesis and growth rate may directly drop as a result of photoinhibition; the effect may be reversible or irreversible, in which case permanent photodamage has taken place. Chlorophyll fluorescence measurements may be used to characterize the state of the photosynthetic apparatus, including estimating photosynthetic efficiency and photoinhibition. The combination of high light intensities (relative to biomass concentration) and low temperature can result in photoinhibition, which can markedly reduce productivity (Torzillo et al., 1996). An advantage of vertical systems is that they are less prone to exhibit photoinhibition at high light intensities at noon because they absorb less light and conversely absorb more light in the morning and in the evening. To measure growth rate directly as a function of light intensity, it is necessary to apply very dilute cultures, as light is attenuated by algal absorption and the light intensity, measured at the surface of the culture, only applies to the surface layer. It must be emphasized that the applied microalgal literature is quite rich in examples of measurements of growth rates or productivities as a function of surface irradiance in dense cultures in vessels of various shapes and without describing the hydraulic regime, which means that the relations between irradiation and growth is little informative.


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FIGURE 17.2 The growth rate, μ, protein yield per absorbed photon Y, and light absorption coefficient of Chlamydomonas reinhardtii, as a function of light intensity. (Reprinted from Janssen, M., et al., 2002. Biotechnol. Bioeng. 81: 193–210. 2002 © Wiley Periodicals Inc. With permission.)

With biomass concentrations from fractions of a gram DW per liter, significant light gradients over just a few centimeters occur, and mixing and the ability of the algal culture to undertake light integration and hence the hydraulic regime become important. The light integration is a consequence of the functional relationship between light intensity and photosynthetic efficiency and its kinetics. With high biomass densities of algae and good mixing, it is possible to use high light intensities with a relatively high efficiency (Qiang and Richmond, 1996), which is also evident from Figure 17.3. In this example, a flat panel reactor with a 10 mm light path and biomass densities of 20–50 g DW/L was illuminated from both sides with artificial light of an intensity equaling twice that of full sunlight. In this way, a photon flux density (which more correctly should be termed photon fluence rate)

16 14

15 12 10

10

PE (%)

Areal output rate (g m–2 h–1)

20

8 5 6 0

0

1000

2000

3000

4000

5000

6000

7000

8000

4

PFD (μmol m–2 s–1)

FIGURE 17.3 Productivity of Spirulina in an artificially illuminated flat-panel reactor with a short light path. PFD: photon flux density; the flux of photons through the reactor area; a maximum total flux of 2 × 4000 μmol/s is applied to the photobioreactor. (Reprinted from Richmond, A., 2000. J. Appl. Phycol. 12(3): 441–451. Springer Copyright Clearance Center. With permission.)



299

of 8000 μmol photons/m2/s—four times that of direct full sunlight—was obtained. More remarkably, up to an irradiation level corresponding to full direct sunlight (2000 μmol/m2/s), the photosynthetic efficiency remains high and constant (15% PAR). This setup yielded maximum volumetric productivities of up to 1.2 g/L/h, which is probably the highest published volumetric productivity figure for a microalgal photobioreactor. In conclusion, it may be inferred that productivity is highly sensitive to a number of factors and that high productivities may be reached with careful adjustment of the physical and physiological conditions in photobioreactors.

17.5.4

HETEROTROPHIC MICROALGAL PRODUCTIVITY

A number of works have demonstrated that it is possible to obtain microalgal biomass densities in fermenters that are compatible to what may be obtained with bacteria or fungi (up to about 120 g DW/L) Still, growth rates exhibited by microalgae during heterotrophic cultivation are typically an order of magnitude smaller than those obtained by bacteria or fungi in industrial fermentations. Table 17.3 gives the biomass concentration, biomass productivity, and product productivity for a number of heterotrophic microalgal processes.

17.5.5

BIOMASS PRODUCTION SYSTEMS

The biomass for microalgal nutraceuticals production today is produced in open ponds, tubular photobioreactors, and fermenters. Emerging technologies also include flat-plate photobioreactors and bubble columns. The characteristics of these systems are briefly discussed in relation to the development and quality of nutraceuticals production.

TABLE 17.3 Productivity (Biomass or Lipid) and Biomass Density of Some Heterotrophic Microalgae Species Chlorella protothecoides Galdieria sulphuraria (red microalga) Crypthecodinium cohniia Crypthecodinium cohniib Chlorella pyrenoidosa Phaeodactylum tricornutumc Chlorella protothecoidesd Nitzschia laevise Schizochytrium Schizochytrium SR-21 Nitzschia alba a b c

d e

Product

Biomass Density (g DW/L)

Lutein Phycocyanin

45.8 80–110

DHA DHA

109 83.0 118 25 12.8–15

EPA (3%) EPA DHA DHA EPA

13 48.1 45–48

Biomass or Lipid Productivity

Reference

Shi et al. (1997) Scmidt et al. (2005), Graverholt and Eriksen (2007) DHA: 48 mg/L/h DeSwaaf et al. (2003) DHA: 53 mg/L/h Sijtsma and De Swaaf (2004) 1.02 g DW/L/h Wu and Shi (2007) EPA: 56 mg/L/day Fernandez Sevilla et al. (2004) Li et al. (2007), Xu et al. (2006) EPA: 73 mg/L/day Wen and Chen (2002) DHA 53 mg/L/h Fan et al. (2001) DHA:138 mg DHA/L/h Yaguchi (1997) EPA: 250 mg/L/day Barclay et al. (1994)

On acetate. On ethanol. Mixotrophic: outdoor bubble columns, glycerol as substrate. Mixotrophic cultures produced 3.0% EPA as opposed to 1.9% in autotrophic cultures, where EPA productivity was 18 mg/L/day. Large-scale fermentors, 3–11 m3. μ = 0.5–0.6 per day, gluc. = 20 g/L.


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17.5.6


OPEN PONDS

Spirulina, Dunaliella, and part of the Chlorella production is carried out in open pond technology. The largest nutraceutical product of microalgal origin is Spirulina. It is produced in open raceway ponds (Figure 17.4), where it is grown at high alkalinities. This gives Spirulina a competitive advantage, as very few other algae can grow under these conditions. Raceways are shallow (15–30 cm) circular channel systems through which the culture is recirculated by means of a large paddle wheel at velocities of 15–30 cm/s. The sizes of raceway ponds may reach 5000 m 2 and biomass levels may reach about 0.5 g DW/L (Ben-Amotz and Avron, 1990; Shimamatsu, 2004). Spirulina is comparatively easy to harvest because of the filamentous growth form, using, for example, a vibrating sieve. This results in low harvest costs, despite the relatively low biomass densities obtained in the ponds. The function of the paddle wheel is primarily to keep the algae suspended in the water column and to a lesser extent to provide vertical mixing. Hence, comparatively little energy is used for mixing: 20 mg/100 mL.


354

Biotechnology in Functional Foods and Nutraceuticals (b) 30

30

25

25 GABA (mg/100 g)

l-Glutamic acid (mg/100 g)

(a)

20 15 10

15 10 5

5 0

20

0 0

1 2 Culture time (day)

3

0

1 2 Culture time (day)

3

FIGURE 20.2 The time course of l-glutamic acid concentrations (a) and GABA concentrations (b) during the cultivation of Lc. lactis YIT 2027 and Lb. casei strain Shirota. ●, Lc. lactis YIT 2027; ▲, Lb. casei strain Shirota; and ■, Lc. lactis YIT 2027 + Lb. casei strain Shirota. Culture conditions: 20% skim milk solution (3 L) at 30°C.

GABA by the GAD of Lc. lactis YIT 2027 (Hayakawa et al., 2004). GABA productivity by coculturing was increased to nearly 2.5 times the GABA productivity of Lc. lactis YIT 2027 cultured alone (Figure 20.2). In this way, a novel fermented milk product containing GABA was produced (Figure 20.3).

20.5

ANTIHYPERTENSIVE EFFECT OF FERMENTED MILK CONTAINING GABA

The antihypertensive effects of the fermented milk product containing GABA (which we have called FMG—fermented milk containing GABA) was investigated via low-dose oral administration to SHR and normotensive Wistar–Kyoto (WKY/Izm) rats (Hayakawa et al., 2004). A single oral dose of FMG significantly decreased the blood pressure of SHR but not that of WKY rats (Figure 20.4). The hypotensive activity of GABA was dose-dependent in SHR (Figure 20.5). During long-term administration of various experimental diets to SHR rats, a significantly slower increase in blood pressure with respect to the control group was observed at 1 or 2 weeks after the start of feeding with the FMG diet, and this difference was maintained throughout the period of feeding (Figure 20.6). A placebo-controlled trial was performed on humans with mild hypertension. The difference between the products ingested by the experimental and control groups was the presence or

Milk protein & peptides Lb. casei strain Shirota l-Glutamic acid Lc. lactis YIT 2027 GABA

FIGURE 20.3 A flow chart of GABA production by coculture of two strains of LAB.


Synthesis of Antihypertensive GABA-Enriched Dairy Products (b) 200

130

195

125

190

120

SBP (mm Hg)

SBP (mm Hg)

(a)

355

185 180 175

115 110 105

170 0

15 20 5 10 Time after administration (h)

25

100

0

5 10 15 20 Time after administration (h)

25

FIGURE 20.4 The effect of a single oral administration of FMG or GABA on SBP in SHR/Izm rats (a) and WKY/Izm rats (b). The doses of saline (●), skim milk solution (6%, w/w) (△), FMG (■), and GABA solution (0.1 mg/mL) (◇) were 5 mL/kg of BW. Values are means ± SEM for six animals per group. *Significant difference versus control (saline) value at the same time point (P < 0.05: Dunnett’s test). 200

SBP (mm Hg)

195 190 185 180 175 170 0.01 Control

0.1 GABA (mg/kg)

1

10

FIGURE 20.5 The dose response of the antihypertensive effect of GABA in SHR/Izm rats. SBP measured 4 h after administration of a single oral dose (5 mL/kg of body weight). Values are means ± SEM for six animals per group. *Significant difference versus control (saline) value (P < 0.05: Dunnett’s test). 210

SBP (mm Hg)

200 190 180 170 Test diets

160 150 6

7

8 Age (weeks)

9

10

FIGURE 20.6 The effect of long-term administration of FMG or GABA on SBP in young SHR/Izm rats. ●, Control; ■, FMG; ◇, GABA. Values are means ± SEM for 10 animals per group. *Significant difference versus control value at the same time point (P < 0.05: Dunnett’s test).


150

Intake period

145 140 135 130 –4

0

4 8 Weeks

12

16

Diastolic blood pressure (mm Hg)

Biotechnology in Functional Foods and Nutraceuticals Systolic blood pressure (mm Hg)

356

95

FMG Placebo

90 85 80 75 –4

Intake period 0

4 8 Weeks

12

16

FIGURE 20.7 The time course of blood pressure after everyday intake of FMG or placebo. Values are means (n = 22–23) ± SEM, #P < 0.05 and ##P < 0.01 versus control (student t-test), *P < 0.05 and **P < 0.01 versus initial (Dunnett’s test).

absence of GABA (Inoue et al., 2003; Kajimoto et al., 2003) (Figure 20.7). Subjects took 100 mL of FMG or placebo once a day for 12 weeks. The change of systolic blood pressures (SBPs) from the start to 12 weeks after ingestion declined in the test group, but not in the placebo group (Figure 20.8). Blood tests and urinalysis revealed no abnormal changes over the intake period. In addition, medical examination and subjective symptoms revealed no adverse reactions to the test food. In an additional study, we investigated the safety of FMG in healthy adults with normal blood pressure (Kimura et al., 2002). We observed no remarkable changes in blood pressure, hematological, or biochemical indices. It is important that FMG had no effect on normal blood pressure in humans, because it is undesirable to reduce blood pressure further than necessary.

20.6 MECHANISM OF THE ANTIHYPERTENSIVE EFFECT OF GABA The mechanism of the hypotensive action of systemically administered GABA has not yet been fully elucidated; however, several hypotheses have been postulated. Because GABA has not (Kuriyama

Change of blood pressure (mm Hg)

FMG

Placebo

20

20

10

10

0

0

–10

–10

–20

–20

–30

–30 r = –0.56

–40 120

130 140 150 160 Initial blood pressure (mm Hg)

r = –0.03 –40 120

130 140 150 160 Initial blood pressure (mm Hg)

FIGURE 20.8 The change of blood pressure in various subjects after taking FMG compared to that in subjects taking placebo.


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260 Denervated–control Denervated–GABA Sham–control Sham–GABA

250

SBP (mm Hg)

240 230

Denervation

220 210

Test diet

200 190

7

8

9 10 Weeks of age

11

12

FIGURE 20.9 The effect of GABA administration on the development of hypertension after bilateral renal denervation or sham operation of SHR/Izm rats. Each point represents the mean ± SEM for 10 animals. SBP, systolic blood pressure. *P < 0.05 and **P < 0.01 versus GABA-free, sham-operated group (“sham-control”) at the same time point (Dunnett’s test).

and Sze, 1971) or rarely (Kuriyama and Sze, 1971) passed the blood–brain barrier, it is reasonable to consider that, at low doses, GABA cannot act centrally and acts only peripherally. In the mesenteric arterial bed, perivascular nerve stimulation-induced increases in perfusion pressure and noradrenaline release are inhibited by GABA in SHR, but not in normotensive rats; this inhibition of noradrenaline release by GABA is attenuated by the selective GABA B receptor agonist baclofen, GABA

Blood vessel

Kidney Sympathetic nerve

Sympathetic nerve Noradrenaline Noradrenaline

Juxtaglomerular cell

Renin

Vascular dilation

Natriuresis

Fall in blood pressure

FIGURE 20.10

The mechanism of the hypotensive effect of GABA.


358


but not by the selective GABAA receptor agonist muscimol. The same inhibition is completely antagonized by the selective GABAB receptor antagonist saclofen, but not by the selective GABAA receptor antagonist bicuculline. These results suggest that, in SHR, the antihypertensive effect of GABA is a result of the inhibition of noradrenaline release from sympathetic nerves in the mesenteric arterial bed via presynaptic GABAB receptors (Hayakawa et al., 2002). Long-term dietary administration of GABA significantly decreases blood pressure and plasma renin activity in sham-operated SHR rats but not in renal-sympathetic-denervated SHR rats (Hayakawa et al., 2005) (Figure 20.9). Water intake, urine volume, and urinary sodium were slightly higher in the GABA fed group than in the GABA-free control group (data not shown). These results suggest that the GABA-induced hypotensive effect is mediated by a reduction in the effects of renal sympathetic nerve activity in SHR (Figure 20.10).

20.7 CONCLUSIONS A novel fermented milk product containing GABA was produced by coculture of Lc. lactis YIT 2027 and Lb. casei strain Shirota. In 2004, this product was approved in Japan as a FOSHU (Food for Specified Health Use) for mild hypertensives, based on the evidence of its efficacy, quality and safety aspects. We hope that this fermented milk product may help to maintain the vascular health of people with mild hypertension.

REFERENCES Ackley, S., Barrett, C.E., and Suarez, L., 1983. Dairy products, calcium, and blood pressure. Am. J. Clin. Nutr. 38: 457–461. Appel, L.J., Moore, T.J., Obarzanek, E., Vollmer, W.M., Svetkey, L.P., Sacks, F.M., Bray, G.A., et al. 1997. A clinical trial of the effects of dietary patterns on blood pressure. New Engl. J. Med. 336: 1117–1124. DASH-Sodium Trial Collaborative Research Group, 2003. Effects of the dietary approaches to stop hypertension (DASH) diet on the pressure-natriuresis relationship. Hypertension 42: 8–13. DeFeudis, F.V., 1982. Muscimol and GABA-receptors: Basic studies and therapeutic implications. Rev. Pure Appl. Pharmacol. Sci. 3: 319–379. DeFeudis, F.V., Ossola, L., Sarliève, L.L., Schmitt, G., Rebel, G., Varga, V., and Mandel, P., 1981. GABA and muscimol binding processes in CNS tissue culture preparations. Adv. Biochem. Psychopharmacol. 29: 405–410. De Roos, N. and Katan, M., 2000. Effects of probiotics bacteria on diarrhea, lipid metabolism, and carcinogenesis: A review of papers published between 1988–1998. Am. J. Clin. Nutr. 71: 405–411. Elliott, K.A.C. and Hobbiger, F., 1959. Gamma aminobutyric acid: Circulatory and respiratory effects in different species; re-investigation of the anti-strychnine action in mice. J. Physiol. 146: 70–84. European Society of Hypertension and European Society of Cardiology, 2007. Guidelines for the management of arterial hypertension: The task force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). J. Hypertens. 25: 1105–1187. Fujimura, S., Shimakage, H., Tanioka, H., Yoshida, M., Suzuki-Kusaba, M., Hisa, H., and Satoh, S., 1999. Effects of GABA on noradrenaline release and vasoconstriction induced by renal nerve stimulation in isolated perfused rat kidney. Br. J. Pharmacol. 127: 109–114. Gelder van, M.N. and Elliott, K.A.C., 1958. Disposition of γ-aminobutyric acid administered to mammals. J. Neurochem. 3: 139–143. Gillis, R.A., DiMicco, J.A., Williford, D.J., Hamilton, B.L., and Gale, K.N., 1980. Importance of CNS GABAergic mechanisms in the regulation of cardiovascular function. Brain Res. Bull. 5(Suppl. 2): 303–315. Hata, Y., Yamamoto, M., Ohni, M., Nakajima, K., Nakamura, Y., and Takano, T., 1996. A placebo-controlled study of the effect of sour milk on blood pressure in hypertensive subjects. Am. J. Clin. Nutr. 64: 767–771. Hayakawa, K., Kimura, M., and Kamata, K., 2002. Mechanism underlying γ-aminobutyric acid-induced antihypertensive effect in spontaneously hypertensive rats. Eur. J. Pharmacol. 438: 107–113.


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Hayakawa, K., Kimura, M., Kasaha, K., Matsumoto, K., Sansawa, H., and Yamori, Y., 2004. Effect of a γ-aminobutyric acid-enriched dairy product on the blood pressure of spontaneously hypertensive and normotensive Wistar-Kyoto rats. Brit. J. Nutr. 92: 411–417. Hayakawa, K., Kimura, M., and Yamori, Y., 2005. Role of the renal nerves in gamma-aminobutyric acidinduced antihypertensive effect in spontaneously hypertensive rats. Eur. J. Pharmacol. 524: 120–125. Inoue, K., Shirai, T., Ochiai, H., Kasao, M., Hayakawa, K., Kimura, M., and Sansawa, H., 2003. Blood-pressurelowering effect of a novel fermented milk containing γ-aminobutyric acid (GABA) in mild hypertensives. Eur. J. Clin. Nutr. 57: 490–495. Kajimoto, O., Hirata, H., and Nishimura, A., 2003. Hypotensive action of novel fermented milk containing γ-aminobutyric acid (GABA) in subjects with mild hypertension. J. Nutr. Food 6: 51–64 (in Japanese). Kimura, M., Chounan, O., Takahashi, R., Ohashi, A., Arai, Y., Hayakawa, K., Kasaha, K., and Ishihara, C., 2002. Effect of fermented milk containing γ-aminobutyric acid on normal adult subjects. Jpn. J. Food Chem. 9: 1–6 (in Japanese). Kuriyama, K. and Sze, Y., 1971. Blood-brain barrier to H3-γ-aminobutyric acid in normal and amino oxyacetic acid-treated animals. Neuropharmacol. 10: 103–108. Manzini, S., Maggi, C.A., and Meli, A., 1985. Inhibitory effect of GABA on sympathetic neurotransmission in rabbit ear artery. Arch. Int. Pharmacodyn. Ther. 273: 100–109. Metchnikoff, E., 1908. The Prolongation of Life. New York: Putnam’s Sons . Miller, G.D., DiRienzo, D.D., Reusser, M.E., and McCarron, D.A., 2000. Benefits of dairy product consumption on blood pressure in humans: A summary of the biomedical literature. J. Am. Coll. Nutr. 19: 147S–164S. Monasterolo, L.A., Trumper, L., and Elias, M.M., 1996. Effects of γ-aminobutyric acid agonists on the isolated perfused rat kidney. J. Pharmacol. Exp. Ther. 279: 602–607. Moore, T.J., Conlin, P.R., Ard, J., and Svetkey, L.P., 2001. DASH (Dietary Approaches to Stop Hypertension) diet is effective treatment for stage 1 isolated systolic hypertension. Hypertension 38: 155–158. National High Blood Pressure Education Program Coordinating Committee, 2002. Primary prevention of hypertension: Clinical and public health advisory from The National High Blood Pressure Education Program. JAMA 288: 1882–1888. Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S., and Takano, T., 1995. Purification and characterization of angiotensin I-converting enzyme inhibitor from sour milk. J. Dairy Sci. 78: 777–783. Nakamura, Y., Yamamoto, N., Sakai, K., and Takano, T., 1995. Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme. J. Dairy Sci. 78: 1253–1257. Roberts, E., Lowe, P.I., Guth, L., and Jelinek, B., 1958. Distribution of γ-aminobutyric acid and other amino acids in nervous tissue of various species. J. Exp. Zool. 138: 313–328. Saito, T., Nakamura, T., Kitazawa, H., Kawai, Y., and Itoh, T., 2000. Isolation and structural analysis of antihypertensive peptides that exist naturally in gouda cheese. J. Dairy Sci. 83: 1434–1440. Salminen, S., Bouley, M.C., Boutron-Ruault, M.C., Cummings, J., Franck, A., Gibson, G., Isolauri, E., Moreau, M.C., Roberfroid, M., and Rowland, I., 1998. Functional food science and gastrointestinal physiology and function. Brit. J. Nutr. 80: S147–S171. Seppo, L., Jauhiainen, T., Poussa, T., and Korpela, R., 2003. A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects. Am. J. Clin. Nutr. 77: 326–330. Shortt, C., Shaw, D., and Mazza, G., 2004. Overview of opportunities for health-enhancing functional dairy products. In: C. Shortt and J. O’Brien (Eds), Handbook of Functional Dairy Products, Florida, CRC Press. Stanton, H.C., 1963. Mode of action of gamma amino butyric acid on the cardiovascular system. Arch. Int. Pharmacodyn. 143: 195–204. Stanton, H.C. and Woodhouse, F.H. The effect of gamma-amino-n-butyric acid and some related compounds on the cardiovascular system of anesthetized dogs. J. Pharmacol. Exp. Ther. 128: 233–242. Suter, P.M., Sierro, C., and Vetter, W., 2002. Nutritional factors in the control of blood pressure and hypertension. Nutr. Clin. Care. 5: 9–19. Takahashi, H., Tiba, M., Iino, M., and Takayasu, T., 1955. The effect of γ-aminobutyric acid on blood pressure. Jpn. J. Physiol. 5: 334–341. Takahashi, H., Tiba, M., Yamazaki, T., and Noguchi, F., 1958. On the site of action of gamma-aminobutyric acid on blood pressure. Jpn. J. Physiol. 8: 378–390. Tsukada, Y., Hirano, S., Nagata, Y., and Matsutani, T., 1960 Metabolic studies of gamma-aminobutyric acid in mammalian tissues. In: E. Roberts (Ed.), Inhibition in the Nervous System and Gamma-Aminobutyric Acid, 163–168. New York: Pergamon.


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World Health Organization, International Society of Hypertension Writing Group, 2003. World Health Organization (WHO)/International Society of Hypertension (ISH) statement on management of hypertension. J. Hypertens. 21: 1983–1992. Yamamoto, N., Maeno, M., and Takano, T., 1999. Purification and characterization of an antihypertensive peptide from a yoghurt-like product fermented by Lactobacillus helveticus CPN4. J. Dairy Sci. 82: 1388–1393.


of High-Quality 21 Production Probiotics Using Novel Fermentation and Stabilization Technologies Franck Grattepanche and Christophe Lacroix CONTENTS 21.1 Introduction .......................................................................................................................... 362 21.2 Selection Criteria of Probiotic Strains .................................................................................. 363 21.2.1 Safety Criteria ........................................................................................................... 363 21.2.2 Functional Properties ................................................................................................ 363 21.2.3 Technological Criteria ..............................................................................................364 21.3 Stresses Encountered by Probiotic Bacteria from Production to the Gastrointestinal Tract Following Ingestion .....................................................................................................364 21.3.1 Oxidative Stress ........................................................................................................ 365 21.3.2 Acid Stress ................................................................................................................ 366 21.3.3 Bile Stress ................................................................................................................. 367 21.3.4 Heat Stress/Cold Stress ............................................................................................. 367 21.3.5 Osmotic Stress .......................................................................................................... 369 21.4 Technologies to Improve Intrinsic Technological Properties of Probiotics.......................... 370 21.4.1 Exploitation of Cellular Stress Response.................................................................. 370 21.4.1.1 Genetic Manipulation of Probiotic Strains ................................................ 370 21.4.1.2 Application of Sublethal Stresses .............................................................. 371 21.4.1.3 High-Throughput Screening Method ......................................................... 373 21.4.2 Continuous Fermentation with ICT to Efficiently Produce Probiotics ..................... 374 21.4.2.1 Cell Entrapment within Polymeric Networks ............................................ 374 21.4.2.2 Advantages of ICT Over Free Cell Systems for Efficient Production of Probiotics ............................................................................................... 375 21.4.2.3 Changes in Cell Physiology Induced by ICT ............................................. 376 21.5 Encapsulation: An Efficient Stabilization Technology to Protect Probiotic Cells During Storage and in the Gastrointestinal Tract Following Ingestion................................ 377 21.5.1 Gel Particles .............................................................................................................. 377 21.5.2 Spray-Coating ........................................................................................................... 378 21.5.3 Production of Water-Insoluble Food-Grade Microcapsules by Emulsion and Spray-Drying ................................................................................ 379 21.6 Concluding Remarks ............................................................................................................ 380 References ...................................................................................................................................... 380

361 © 2010 Taylor and Francis Group, LLC

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21.1


INTRODUCTION

Since the beginning of their placement on the market, in the mid 1990s, functional foods have undergone a very important expansion driven by consumer demands and a heightened awareness of the link between health, nutrition, and diet. Functional foods are foods that claim to promote human health beyond nutritional effects. The global market of functional foods was estimated at nearly 61 billion $US in 2004 (Benkouider, 2004). In Europe this market has increased from 4–8 billion $US in 2000, depending on which definition of functional foods is considered, to about 15 billion $US in 2006 (Kotilainen et al., 2006; Menrad, 2003). Functional foods containing probiotics constitute a large segment of this market representing more than 1.4 billion euros in Western Europe (Saxelin, 2008). Probiotics are defined as “live microorganisms which when consumed in adequate numbers confer a health benefit on the host” (FAO/WHO, 2001). The success of probiotic foods is closely related to the growing number of scientific studies supporting the beneficial effects of specific probiotic microorganisms in sustaining gut health, alleviation of allergic disease, and lactose intolerance as well as for treatments of some diarrhea (Gueimonde and Salminen, 2005). In current use, most probiotic microorganisms are lactic acid bacteria (LAB), in particular lactobacilli, or bifidobacteria which have been isolated from the intestinal microflora of healthy human subjects. Although not exclusively so, for example Saccharomyces boulardii (nom. inval.), is a probiotic yeast first isolated from litchi fruit. In fact, only a very limited number of microbial strains exhibiting relevant probiotic properties are currently used in food products or as supplements. This can be primarily explained by the fact that beneficial health effects of a probiotic strain must be clearly demonstrated from in vitro tests to well-designed human studies. These experiments are very expensive and must be carried out for each strain tested because health benefits are strain specific and effects shown for particular strain cannot be extrapolated to other strains even within the same species (Pineiro and Stanton, 2007). In addition, for most of the health-promoting effects probiotic cells must be viable, metabolically active, and in sufficient number at the host target site for full efficacy. Probiotics encounter challenging conditions which can greatly affect their viability during production, downstream processing, storage, incorporation into foods, and throughout the entire shelf life of the food products. Furthermore, developments in probiotic production have mainly focused on achieving high viable cell yields while keeping costs low. On the other hand, the technology used for probiotic production directly impacts cell physiology, with possible effects on functional properties. Although effective doses are not well documented and might vary as function of the strain and the targeted health effect, a minimal somewhat arbitrary level of more than 10 6 viable probiotic bacteria per milliliter or gram of food product is generally recommended (Lacroix and Yildirim, 2007). However, probiotic bacteria often show poor technological properties, as many strains, being isolated from human or animal intestine, are difficult to propagate outside their natural environment (Ross et al., 2005). Moreover, in order to guarantee high survival rate in gastrointestinal tract following ingestion, probiotic bacteria must exhibit suitable properties in regard to their resistance against the acidic environment of the stomach and bile secreted in the duodenum. New probiotic strains with improved technological and functional properties and/or development of fermentation and stabilization technologies, to control cell physiology and to protect cells during downstream processing, storage, and in gastrointestinal tract following ingestion are therefore clearly needed. The present chapter addresses the latest criteria for selection of probiotic strains; the effects of the different stresses encountered by probiotic cells and, in regard to this knowledge, new technological approaches to improve the intrinsic tolerance of probiotic cells to stresses. A special focus will be placed on production of probiotic cells with improved technological and functional properties using immobilized cell technology (ICT) and continuous culture. Furthermore, encapsulation technologies conferring a physical protection of cells will be examined.


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21.2 SELECTION CRITERIA OF PROBIOTIC STRAINS Beside the fact that probiotics must be safe, as is the case for all microorganisms used in food or therapeutic applications, their beneficial health effects have to be clearly demonstrated before any health-related product claims could be approved. In addition, technological performance of probiotics has to be tested and must be suitable for large-scale applications. Safety and efficacy of probiotics in human health are discussed in greater detail in Chapter 19 of this book.

21.2.1

SAFETY CRITERIA

Safety criteria for probiotics have evolved largely over the last 10 years. The extended history of safe consumption of some bacteria, mainly LAB, was originally considered a sufficient criterion for safety. However, some newly isolated and characterized probiotic strains cannot claim such a long historical use and therefore their safety should be assessed. A FAO/WHO working group has recently proposed a list of criteria to assess safety of probiotics, even for those belonging to microbial groups that are generally recognized as safe (Pineiro and Stanton, 2007). These guidelines include safety concerns related to antibiotic resistance gene transfer, excessive metabolic activities (mainly d-lactate production and bile salt deconjugation), toxin production, hemolytic activity, and infectivity, particularly with regard to immunocompromised persons. Epidemiological surveillance has also been suggested as an additional safety measure for new probiotic strains (Pineiro and Stanton, 2007; Salminen and Ouwehand, 2002). Prevalence of food-related allergies related to food consumption has drastically increased in the last years. Since milk and some of its constituents are among the major allergenic foods and widely used for production of probiotics, it was then recommended to minimize or eliminate the use of these potential allergenic substances or at least to indicate the possible cross-contamination and/or deliberate use of potential allergenic ingredients during probiotic preparation (Del Piano et al., 2006).

21.2.2 FUNCTIONAL PROPERTIES For most of the health-promoting effects associated with probiotics, cells must be viable, metabolically active, and in sufficient number at the target site of the host. Probiotic cells are exposed to various adverse conditions which can affect their viability in the gastrointestinal tract following ingestion (see following sections for greater detail). Probiotics are therefore first assessed in regard to their tolerance to low pH, gastric, bile salts, and pancreatic juices. Such tests can easily be performed in vitro. Survival of probiotics through digestive steps is required for delivery of functional active probiotics in the gut. Probiotic strains must also exhibit at least one of the following functional characteristics: adherence to intestinal mucosa and mucus, production of antimicrobial substances, antagonism against pathogens and cariogens, competition for adhesion sites (competitive exclusion), interaction with gut-associated lymphoid tissue (immune modulation), inactivation of harmful components within the intestinal contents (binding to toxins and regulation of the metabolic activity of the intestinal microflora), a trophic effect on the intestinal mucosa (e.g., through the production of butyrate), and overall normalization of the intestinal microflora composition and activity (Salminen and Ouwehand, 2002). Some of these properties can be assessed using classic in vitro tests (e.g., agar diffusion assay to test production of antimicrobial compounds) or more elaborated systems such as colonic fermentation model to study, for example, interactions between the intestinal microflora, probiotics, and/or pathogens (Cinquin et al., 2006; Le Blay et al., 2009) prior to expensive and ethically regulated animal or human in vivo studies. Prevention and treatment of antibiotic-associated diarrhea diseases is one of the clinical interests in probiotic applications. Antibiotic tolerance is therefore a desired trait of probiotic cells. Antibiotic resistance however should not be conferred to other bacteria through gene transfer. Acquired


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antibiotic resistance can be detected by comparing resistance patterns of several bacteria belonging to the same species as well as by new molecular screening tools based on microarray hybridization allowing rapid screening of antibiotic resistance genes (Grattepanche et al., 2008).

21.2.3

TECHNOLOGICAL CRITERIA

Stability of probiotics during production, downstream processing, and storage in frozen or dried form and in food products following their incorporation is an important selection criterion (Table 21.1). Indeed, probiotics exhibit generally low survival rates with common methods used to prepare microbial food adjuncts such as freeze- and spray-drying, and are very sensitive to many environmental stresses such as oxygen exposure and low pH encountered during production, storage, and as found in some food products (Doleyres and Lacroix, 2005). Most probiotics are also nutritionally fastidious microorganisms, requiring expensive media and addition of growth-promoting factors for propagation (Ibrahim and Bezkorovainy, 1994). Probiotics are mainly marketed through fermented dairy products in which they can be incorporated after fermentation or involved in the fermentation process. In this regard, their ability to propagate in milk as well as to compete with starter culture should be considered (Table 21.1). Finally, incorporation of probiotics in foods must not negatively affect organoleptic properties of the products. Technological criteria often limit large-scale development of probiotic strains even for those exhibiting relevant functional health properties.

21.3

STRESSES ENCOUNTERED BY PROBIOTIC BACTERIA FROM PRODUCTION TO THE GASTROINTESTINAL TRACT FOLLOWING INGESTION

Probiotic bacteria encounter several stressing conditions from production to the gastrointestinal tract following ingestion that may affect their viability (Figure 21.1). Nevertheless, probiotics, like other bacteria, are able to quickly develop defense mechanisms overcoming, to a certain extent, adverse conditions. The following section deals with the cellular mechanisms of probiotics, with a special focus on lactobacilli and bifidobacteria, to cope with the main environmental stresses.

TABLE 21.1 Technological Criteria for Selection of Probiotics Process Step Biomass production

Downstream process Storage Incorporation into food


Criteria Inexpensive cultivation Ease of concentration to high cell densities Tolerance to shear forces encountered in the bioreactor Bacteriophage resistant Ability to grow in milk Reproducibility of the fermentation process Stability during freezing, freeze-, and spray-drying Tolerance to oxygen, low pH Stability during storage Absence of antagonistic activity when used in mixed culture Do not modify original organoleptic properties of the food products Stability over shelf life of the food products


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Downstream processes Production

Ingestion Storage

Freeze-drying –Composition of the growth medium –Toxic by-products –Dissolved oxygen –Final cell mass

–Powder form –In food matrix –Frozen liquid concentrate

Spray-drying

–Mechanical stress –Composition of freezing and drying media –Extreme temperatures (freezing, spray- and freeze-drying) –Oxygen stress –Cell dehydration (osmotic stress)

–Acidity of carrier food –Oxygen stress –Competition with other organisms in the product –Temperature –Moisture content

–Acidic concentration in stomach –Competition with other gut microorganisms –Composition of the environment (e.g., nutrient availability) –Bile salts in the small intestine

FIGURE 21.1 Stressing conditions encountered by probiotic bacteria from production to ingestion. (Adapted from Lacroix, C. and Yildirim, S., 2007, Curr. Opin. Biotechnol. 18: 176–183.)

21.3.1

OXIDATIVE STRESS

At industrial scale, strict anaerobic conditions cannot be easily controlled during production, downstream processing, and storage of probiotic bacteria which are then exposed to different levels of oxygen (Figure 21.1). Probiotic cultures, generally isolated from human gut, can be classified according to their tolerance to oxygen as microaerophilic (e.g., Lactobacillus acidophilus) and strictly anaerobic (most of the Bifidobacterium spp.). Oxygen sensitivity is however species and strain dependent (Talwalkar and Kailasapathy, 2004). The toxicity to oxygen of sensitive bacteria results from the accumulation of reactive oxygen species (ROS) such as O2− (superoxide radical anion), OH• (hydroxyl radical), and H2O2 (hydrogen peroxide), that react with proteins, lipids, and nucleic acids to cause lethal damages (Talwalkar and Kailasapathy, 2004; van de Guchte et al., 2002). These ROS are produced during NADH oxidizing reactions catalyzed by oxidases to regenerate NAD+. Some strains of Lactococcus lactis and Bifidobacterium longum possess a superoxide dismutase (SOD) that catalyzes the conversion of O2− into the less-damaging H2O2 and O2 (Sanders et al., 1995; Shimamura et al., 1992). It has also been reported that the extremely high manganese content of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus fermentum, and Leuconostoc mesenteroides can act as an efficient scavenger of O2− even in the absence of SOD activity (Archibald and Fridovich, 1981). In contrast to O2−, OH• cannot be enzymatically degraded, however, cell free extracts of LAB and bifidobacteria exhibit scavenging activity toward this highly ROS (Lin and Yen, 1999; van de Guchte et al., 2002). NADH peroxidase, present in some LAB and bifidobacteria, can reduce H2O2 into H2O (Miyoshi et al., 2003; Shimamura et al., 1992). This enzymatic activity is however generally low (10–30 times lower than that of NADH oxidases which generate H2O2) (Miyoshi et al., 2003). In many lactobacilli, H2O2 can also be eliminated by catalase (active in heme containing medium) or pseudocatalase (nonheme catalase) (De Angelis and Gobbetti, 2004; Engesser and Hammes, 1994). Reduction of the intracellular redox potential, repair of oxidative damages by overexpression of chaperones, and protection of potential targets (e.g., proteins) from oxidation have also been


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suggested as mechanisms to explain tolerance of LAB to oxygen toxicity (van de Guchte et al., 2002). The development of molecular tools and the increasing number of available genome sequences permitted identification of proteins or genes involved in these mechanisms in many bifidobacteria but the role for some of them remain unclear (Dubbs and Mongkolsuk, 2007; Klijn et al., 2005; O’Connell-Motherway et al., 2000; Schell et al., 2002).

21.3.2

ACID STRESS

From production to ingestion, probiotic cultures are exposed to several acidic environments which may differentially affect cell viability (Figure 21.1). Indeed, probiotic cultures at the end of fermentation first experience mild acidic environment that is nonlethal and less deleterious than the harsh conditions encountered in the human stomach with a pH of about 2. Probiotic cultures also exhibit poor resistance under prolonged acidic conditions such as those encountered in yoghurts, which are often used as probiotic carrier (Shah et al., 1995). Indeed, traditional yoghurt has an initial pH of about 4.1–4.4 that decreases to 3.8–4.2 during refrigerated storage due to lactic acid production by Lactobacillus delbrueckii subsp. bulgaricus. The pH of yoghurt or fermented milk containing probiotics is then usually set at a high value of 4.6–5.0 to enhance probiotic cell survival in the product. Resistance to acidic conditions of probiotic cultures is genera, species and strain dependent with lactobacilli being less affected than bifidobacteria (Champagne et al., 2005). The mechanism by which acids cause damages to bacterial cell is well established (Presser et al., 1997). Acids enter the cell by passive diffusion and then dissociate into proton and charged derivatives. Accumulation of protons in the cytoplasm leads to a reduction of the intracellular pH (pHi) that consequently affects the transmembrane potential (ΔpH), and the proton motive force (pmf) involved in many transmembrane transport processes. In addition, reduction of the pHi has deleterious effect on the activity of acid-sensitive enzymes, damages DNA and proteins, and favors concentration of oxidizing agents (Blankenhorn et al., 1999; van de Guchte et al., 2002). Some responses to acid stress are similar for both lactobacilli and bifidobacteria. In acidic condition, protons can be expulsed out of cells through the membrane-associated F0 –F1 ATPase (Corcoran et al., 2008). The proton extrusion function of this enzyme is however ATP dependent. It has been reported that the carbon balance of the glycolytic pathway in an acid-adapted strain of B. longum cultured under acid pH was higher than that of the wild-type strain, suggesting an optimization of the bifid shunt, probably directed toward an increase in the ATP production that can be used by the F0 –F1 ATPase (Sanchez et al., 2007a). In contrast to bifidobacteria, LAB produce an extra energy source from the arginine deiminase pathway. This pathway, composed of three enzymes, arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase, and an antiporter allowing the exchange of ornithine and arginine without energy consumption, catalyzes the conversion of arginine into ornithine, ammonia (two per arginine) with the concomitant production of 1 mol of ATP (Cunin et al., 1986). The ammonia formed reacts with proton leading to an alkalization of the extracellular environment. In response to acid pH, bifidobacteria can produced ammonia from glutamine deamination (Sanchez et al., 2008). Another reaction leading to the formation of ammonia is the hydrolysis of urea by urease that is found in many bacteria including LAB and bifidobacteria (Corcoran et al., 2008). Decarboxylation of carboxylic acidic compounds, such as malic acid or some amino acids (ornithine, glutamate, and histidine) is another way to increase alkalinity of the cytoplasm (Cotter and Hill, 2003). In this reaction, catalyzed by specific decarboxylases, an intracellular proton is consumed and the alkaline product is pumped out of the cell, increasing extracellular pH. In some cases, the alkaline products are transported in the extracellular environment via an electrogenic transporter leading to the formation of ATP. This system has already been reported for lactococci and lactobacilli but not yet in bifidobacterial cells. General and heat shock chaperones involved in repair of damaged proteins are always overexpressed in LAB under acidic conditions. The identity and the induction level of these chaperones vary however from one species to another (Champomier-Vergès et al., 2002; Lim et al., 2000; Streit



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et al., 2008). Surprisingly, proteome analysis of B. longum exposed to acidic pH did not reveal any increase in expression of general stress proteins (Sanchez et al., 2007a). These authors suggest that the increase of certain chaperones can only be detected after prolonged acid adaptation.

21.3.3 BILE STRESS Bile, mainly composed of bile salts, is a digestive secretion of the hepatic system that plays a major role in the dispersion and absorption of fats. Bile stress is obviously encountered by probiotics in the gastrointestinal tract following ingestion. Bile exhibits strong antimicrobial activity caused by protein misfolding, damages to DNA and membrane structure and secondary structure formation in RNA (Begley et al., 2005a). It may also induce oxidative stress through the generation of oxygen free radicals. It is not surprising that response of bacteria to the pleiotropic effects of bile salt shares common characteristics with other stress such as acid and oxidative stresses. In response to bile stress, lipidic composition of Bifidobacterium animalis subsp. lactis membrane changes toward an increase level of the branched-chain fatty acids, the unusual 10-hydroxyoctanodecanoic acid, and a decrease of cyclic fatty acids (Ruiz et al., 2007). These observations are consistent with transcriptomic and proteomic studies showing a down-regulation of a whole cluster of genes coding for enzymes involved in fatty acid biosynthesis and a decrease in expression of long-chain fatty acid CoA ligase, respectively, in B. animalis cells exposed to bile salts (Garrigues et al., 2005; Sanchez et al., 2007b). Chou and Weimer (1999) reported that the cell membrane of a bile tolerant Lb. acidophilus contained C14:1 at 0.9%, while this fatty acid was not detected in the parent strain. Bile stress in bifidobacteria also induces expression of chaperones that correct folding of proteins (Sanchez et al., 2005, 2007b). These authors also reported a lower number of overexpressed chaperones in B. longum NCIMB 8809 than in the more bile tolerant B. animalis. Several enzymes directly or indirectly involved in redox reaction are also overexpressed in B. animalis in order to cope with oxidative stress associated with bile salts (Sanchez et al., 2008). A proteomic approach based on two-dimensional gel electrophoresis revealed modifications in expression level of enzymes involved in carbohydrate metabolism toward an increase production of energy in B. longum and B. animalis subsp. lactis cells grown in presence of bile salts (Sanchez et al., 2008). This extra ATP production could be used by the F0 –F1 ATPase to cope with cytoplasm acidification (see Section 21.3.2 acid stress) caused by the membrane permeabilization effect of bile salt (Amor et al., 2002; Sanchez et al., 2006). Finally, several studies strongly support the evidence that bile salts hydrolases (BSH) play a role in tolerance of bacteria to bile salts (Begley et al., 2005b; De Smet et al., 1995; Dussurget et al., 2002; Grill et al., 2000; Noriega et al., 2006). BSH is a common enzyme of many bacteria, including lactobacilli and bifidobacteria, isolated from the gastrointestinal tract. Although its precise role in tolerance of bacteria to bile salts is still under debate, several physiological functions have been attributed to BSH: (i) nutritional role through the liberation, during hydrolysis of amino acid bile salt conjugates of taurine or glycine, that can be further metabolized, (ii) alteration of membrane characteristics by facilitating incorporation of bile or cholesterol, and (iii) bile detoxification as a result of bile salt deconjugation (see Begley et al., 2005a, 2006).

21.3.4

HEAT STRESS/COLD STRESS

Probiotic cultures encounter adverse temperature conditions during downstream processes such as freezing, freeze- or spray-drying, and frozen or refrigerated storage that can severely affect their viability. Effects of heat and cold stress on probiotic bacteria and general cellular responses are summarized in Table 21.2. Responses to temperature shifts are mainly related to the induction of chaperones and proteases but not limited these two classes of proteins. Indeed, Rezzonico et al. (2007) reported that 46% of B. longum NCC2705 genes, among which many with unknown function were


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TABLE 21.2 Effects of Heat and Cold Shock and Cellular Stress Responses in Probiotic Bacteria Heat Stress Reported effects

Stress responses of lactobacilli and bifidobacteria

Cold Stress

Denaturation and subsequently Alteration of cell membrane aggregation of proteins fluidity Destabilization of Stabilization of secondary macromolecules (e.g., structures of RNA and DNA ribosomes and RNA) Alteration of cell membrane Reduction of efficiency of fluidity transcription, translation and DNA replication Disturbance of transmembrane Reduction of enzyme activity potential Common responses Induction/overexpression of chaperones and proteases which participate in protein folding and degradation of misfolded proteins, respectively

Reference De Angelis and Gobbetti (2004), van de Guchte et al. (2002)

Beaufils et al. (2007), De Angelis and Gobbetti (2004), Kim et al. (1998), Rezzonico et al. (2007), Sanchez et al. (2008), Serror et al. (2003), Ventura et al. (2004, 2005a)

Specific responses Induction/overexpression of Induction/overexpression of • Heat shock proteins required • Cold shock proteins involved for normal growth, DNA and in RNA stabilization, reduction RNA stability, and prevention of negative DNA supercoiling, of inclusion body formation and acting as transcriptional • Proteins involved in other enhancer stresses (e.g., β chain of the • Glycolytic enzymes F0–F1 ATPase) • Changes in fatty acid • Glycolytic enzymes composition of membrane • RecA involved in DNA repair system

differentially expressed during a moderate heat treatment. In addition, overlapping between the cold and heat shock responses can occur. For example, cold shock enhanced the thermotolerance of Lc. lactis IL1403 (Panoff et al., 1995) and induction of heat shock response can confer a cross protective effect to freezing for Lc. lactis, Lc. lactis subsp. cremoris, and Lactobacillus johnsonii (Broadbent and Lin, 1999; Walker et al., 1999). In contrast, the survival capacity of Lactococcus cremoris MG1363 to freezing is not improved by heat shock (Wouters et al., 1999) suggesting that cross protection is strain dependent. More recently, induction of cold shock protein has been observed in B. longum NCC2705 cells subjected to a heat shock (Rezzonico et al., 2007). The induction of cold shock proteins in bifidobacteria in response to temperature downshift has not yet been investigated, nevertheless genes coding for these proteins were identified in different bifidobacterial strains (Kim et al., 1998; Rezzonico et al., 2007). An acid stress pretreatment can also improve tolerance of Lactobacillus bulgaricus cells to freezing and frozen storage (Streit et al., 2007). This cross protection phenomenon can be explained by induction of general and heat shock chaperones after exposure of cells to acidic condition as reported for Lb. bulgaricus and other LAB (see Section 21.3.2). In addition, an acid stress can modify the lipidic composition of cell membrane that plays an important role in maintaining membrane fluidity and consequently in resistance of LAB cells to freezing and frozen storage (Beal et al., 2001; Lebeer et al., 2008; Streit et al., 2008).



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Beside the formation of ice crystals that cause severe membrane damages, freezing also induces osmotic stress by cryoconcentration of solutes (Fonseca et al., 2006). Panoff et al. (2000) reported that pretreatment with osmotica such as lactose, sucrose, and trehalose improves the cryotolerance of Lb. bulgaricus to freeze-thaw stress.

21.3.5

OSMOTIC STRESS

Freezing, freeze-drying, and, to a lesser extent, spray-drying are frequently used for storage and distribution of probiotic cultures. During these treatments, osmolality of the environment increases as a result of cryoconcentration of solutes or dehydration leading to an excessive passage of water from the cell to the extracellular environment that compromises essential cell functions (Poolman et al., 2002). In contrast to bifidobacteria, the response of LAB, especially lactobacilli, to hyperosmotic stress has been well documented. Hyperosmotic stress caused by salts (e.g., KCl or NaCl) is more detrimental for Lb. plantarum than that imposed by nonelectrolyte compounds such as lactose or sucrose (Glaasker et al., 1998a). In contrast to other bacteria, Lb. plantarum cannot accumulate K+ or Na+ in sufficient concentration to restore turgor pressure under salt stress. On the other hand, sugars impose only a transient osmotic stress because external and internal sugar concentrations equilibrate rapidly by facilitated diffusion via systems with very low substrate affinity (Glaasker et al., 1998a). Lactobacilli retain water by accumulating intracellularly specific solutes (so-called compatible solutes) such as glycine-betaine or carnitine to maintain turgor pressure under salt stress. LAB are not able to synthesize these compatible solutes, or synthesize them at very low levels, which are then taken up from the medium by specific transporters (Glaasker et al., 1996; Poolman and Glaasker, 1998). In the absence of glycine-betaine in the medium, growth of Lb. plantarum is severely inhibited by salt stress (Glaasker et al., 1996, 1998a). The protective role of this compatible solute against osmotic stress has also been reported for Lb. acidophilus and Lc. lactis cells (Hutkins et al., 1987; Obis et al., 1999; van der Heide et al., 2001). Activation of the glycine-betaine transport system, QacT (quaternary ammonium compound transport) in Lb. plantarum is regulated by turgor parameter (Glaasker et al., 1998b) while glycine-betaine uptake in Lc. lactis is controlled at both the gene expression level and transport activity. Expression of genes coding for the glycine-betaine transporter, BusA (Obis et al., 1999) or OpuA (Bouvier et al., 2000), in Lc. lactis are regulated at the transcriptional level by BusR (Romeo et al., 2003). Beside their role in osmoregulation, OpuA or BusA may also sense changes in cytoplasmic ionic strength via alterations in membrane properties such as protein–lipid interactions (Poolman et al., 2002). Therefore, it seems likely that stress factors targeting cell membrane (e.g., acidic pH, heat, or cold shock) could modulate activity of this transporter and consequently induce an osmotic-like response. Inversely, changes in lipidic composition of cell membrane occurred following growth of Lb. casei under hyperosmotic conditions (Machado et al., 2004). In addition to their role in osmotic balance, compatible solutes may also stabilize enzymes and other macromolecules (Glaasker et al., 1998a) providing a cross protective effect against high temperature, freeze-thawing, and drying (van de Guchte et al., 2002). Koch et al. (2007) reported that salt stress pretreatment improved tolerance of Lb. delbrueckii subsp. lactis to lyophilization. Proteomic studies also revealed that chaperone proteins such GroES, GroEL, DnaK, and GrpE were overexpressed in Lc. lactis, Lactobacillus rhamnosus, and Bifidobacterium breve following an osmotic stress (Kilstrup et al., 1997; Prasad et al., 2003; Ventura et al., 2005b). Transcriptomic analyses showed similar response to heat and osmotic stresses in regard to expression of chaperone coding genes in B. breve suggesting an overlapping regulatory network controlling both osmotic and severe heat-induced genes (De Dea Lindner et al., 2007). Knowledge about mechanisms involved in cellular response of probiotic cells to stresses has considerably increased this last decade. The understanding of the basis of cellular stress responses might be crucial to screen and develop probiotic cultures with improved technological functionalities. Identification of the different stresses encountered by probiotic cultures from production to consumption is also very useful for the development and optimization of technologies for preserving cell viability.


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21.4 21.4.1


TECHNOLOGIES TO IMPROVE INTRINSIC TECHNOLOGICAL PROPERTIES OF PROBIOTICS EXPLOITATION OF CELLULAR STRESS RESPONSE

Probiotic cultures are able to withstand adverse conditions using different mechanisms. However, time to develop these cellular responses may be insufficient to cope with a quick shift from optimum to adverse conditions as, for example, the rapid increase in temperature encountered by bacteria during spray-drying. In addition, the tolerance level to a certain stress differs among bacteria even from the same species indicating that cellular responses exhibit different efficiencies. The following sections deal with two different approaches exploiting cellular stress responses to improve viability of probiotic cells. 21.4.1.1 Genetic Manipulation of Probiotic Strains Chaperone proteins such as GroES, GroEL, DnaK, DnaJ, and GrpE are involved in several stress responses [see Sugimoto et al. (2008) for a detailed review on chaperones and their roles in LAB and bifidobacteria]. The GroESL operon was homologously overexpressed in Lactobacillus paracasei NFBC 338 (Corcoran et al., 2006). Spray- and freeze-dried cultures overproducing GroESL exhibited 10- and twofold better survival, respectively, than the untransformed cells (Corcoran et al., 2006). Heterologous and homologous overexpression of GroESL in Lc. lactis NZ9800 and Lb. paracasei NFBC 338, respectively, also leads to a 10-fold better survival after a heat shock at 54°C (lactococci) and 60°C (lactobacilli) for 30 min compared to parent strains (Desmond et al., 2004). In addition, the tolerance of both GroESL overproducing strains to salt and to butanol stress for Lc. lactis was enhanced (Desmond et al., 2004). Almost all organisms from prokaryotes to eukaryotes possess small heat shock proteins (sHsp) (Narberhaus, 2002). They confer thermotolerance to cell cultures during prolonged incubations at elevated temperatures by preventing uncontrolled protein aggregation in concert with chaperone heat shock proteins (Hsp) (Han et al., 2008). They can also be constitutively expressed or induced under various conditions such as oxidative and osmotic stress. sHsp possess unique properties compared to Hsp. They function as ATP-independent chaperones, require the flexible assembly and reassembly of oligomeric complex structures for their activation and exhibit a wide range of substrate-binding capacities (Han et al., 2008). In contrast to the highly conserved chaperone proteins, sHsp show great variations in sequence, size of single polypeptide, and oligomer subunit number (Sugimoto et al., 2008). In addition, genes encoding sHps varies in number between LAB species from one in Lb. acidophilus, Lb. bulgaricus, Lactobacillus brevis, Lactobacillus gasseri, Lactobacillus reuteri, and Lactobacillus sakei up to three in Lb. plantarum (Han et al., 2008). The effect of homologous overproduction of each of the three sHsp of Lb. plantarum WCFS1 has been studied in regard to stress tolerance of transformed cells (Fiocco et al., 2007). A shift from the optimum temperature (i.e., 28°C) to 12°C, 37°C, and 40°C leads to a reduction in cell viability ranging between 2.0 and 4.0 log CFU/mL for the untransformed cells while the three sHsp-overproducing strains were not affected (Fiocco et al., 2007). The same authors also reported that sHsp overproduction in Lb. plantarum WCFS1 enhances butanol and ethanol tolerance. Heterologous expression in Lb. casei of the nonheme, manganese-dependent catalase (MnKat) of Lb. plantarum ATCC 14431, one of the very few LAB strains able to degrade H2O2, increased survival rate after exposure to H2O2 as well as during long-term aerated cultures (Rochat et al., 2006). Heterologous expression of MnKat in Lc. lactis and Lb. bulgaricus did not however, show any improvement in oxidative stress tolerance. This observation can be explained by the low intracellular manganese contents for both strains compared to Lb. plantarum and Lb. casei (Rochat et al., 2005, 2006). In contrast, the heterologous expression of KatE, a heme catalase produced by Bacillus subtilis, by Lc. lactis conferred an 800-fold increase in survival after 1 h exposure to 4 mM H2O2 and protected DNA from degradation during long-term aerated storage (Rochat et al., 2005). © 2010 Taylor and Francis Group, LLC


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Noonpakdee et al. (2004) reported that a recombinant strain of Lb. plantarum expressing KatA, a heme catalase of Lb. sakei, grew better than the parent strain under oxidative stress condition. The presence of SOD can also protect bacterial cells against hydrogen peroxide (see Section 21.3.1 oxidative stress). The gene sodA, encoding a manganese SOD of Streptococcus thermophilus, was successfully expressed in Lb. johnsonii, Lb. reuteri, Lb. acidophilus, and Lb. gasseri, and provided protection against H2O2 (Bruno-Barcena et al., 2004). Lactobacillus salivarius UCC118 exhibits relevant probiotic properties but poor robustness during spray-drying (Sheehan et al., 2006). As mentioned in Section 3.5, accumulation of compatible solutes is an efficient way for bacterial cells to withstand osmotic stress. Heterologous expression in Lb. salivarius of betL, a gene encoding a glycine betaine uptake transporter in Listeria monocytogenes, increased resistance to several stresses including elevated osmo-, cryo-, baro-, and chilltolerance as well as spray- and freeze-drying (Sheehan et al., 2006). The same experiment was conducted with B. breve UCC2003 as recipient strain. The recombinant B. breve UCC2003 strain exhibited improved tolerance to gastric juice and conditions of elevated osmolarity mimicking the gut environment (Sheehan et al., 2007). Genetic manipulation seems to be an efficient approach to improve stress tolerance of probiotic cultures as illustrated above. However, negative perception of genetically modified organisms by consumers as well as technical limitations, for example, bifidobacteria cannot be easily transformed, could hinder its development. Furthermore, genetic modifications could affect probiotic functionalities which must then be re-evaluated post facto. 21.4.1.2 Application of Sublethal Stresses Adaptation mechanisms of probiotic cells to adverse environments are based on overexpression of specific or general stress-response proteins and physiological modifications as reviewed in the previous sections. These proteins are generally not constitutively expressed or only expressed at very low levels under optimal growing conditions. In addition, physiological modifications such as changes in lipidic composition of cell membrane do not occur instantaneously and extended response times to stresses may prove too lengthy to successfully protect cells against a quick shift toward adverse conditions. Several studies reported positive effects on probiotic cells viability of sublethal stress applications prior to lethal challenging conditions (Table 21.3). LAB and probiotic cultures are typically harvested in late-log or stationary growth phase in order to achieve maximum cell yield and viability during downstream processing (Saarela et al., 2004). Nutrient starvation and/or accumulation of inhibitory fermentation end products, in particular organic acids, are the main factors leading to the entry of cells into stationary phase of growth. In response to these adverse conditions, bacterial cells develop general stress resistance mechanism that can further improve their tolerance to lethal conditions (De Angelis and Gobbetti, 2004; Gouesbet et al., 2002; Maus and Ingham, 2003; Prasad et al., 2003; Saarela et al., 2004; Teixeira et al., 1994). Specific sublethal stresses can also induce an adaptative response to protect cells against further homologous stress. For example, heat-adapted (52°C for 15 min) cells of Lb. paracasei NFBC 338 survived up to 300-fold better than unadapted control cultures when exposed to a heat stress at 60°C for 10 min (Desmond et al., 2002). Survival of Bifidobacterium adolescentis at 55°C for 10 min and 20 min increased 24-fold and 128-fold, respectively, after subjecting cells to 47°C for 15 min compared to non-pretreated cells (Schmidt and Zink, 2000). Adaptative response to acid (also called acid tolerance response) is another well-known mechanism in LAB and bifidobacteria. Park et al. (1995) observed that acid adaptation (pH 5.2 for 2 h) protected B. breve cells against subsequent exposure to lethal pH levels. Acid-adapted (30 min at pH 4.75) cells of Lb. bulgaricus were approximately 250-fold more tolerant to lethal acid challenge (pH 3.6 for 30 min) than unadapted ones (De Angelis and Gobbetti, 2004). Maus and Ingham (2003) reported that acid adaptation of B. lactis isolated from a commercial yoghurt sample protected cells against lethal pH of simulated gastric fluid. However, acid pretreatment had no significant effect for B. longum ATCC15707 (Maus and Ingham, 2003). In contrast, Saarela et al. (2004) have observed an acid adaptative response


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TABLE 21.3 Studies Reported Improvement in Cell Viability to Lethal Stressing Conditions Following Sublethal Stress Pretreatments Sublethal Stress Pretreatments

Bacterial Species

Lethal Stresses Applied to Pretreated Cells

Reference

Stationary phase, acid, and Lb. rhamnosus, Lb. brevis, Lb. reuteri, heat B. animalis, B. longum Lb. bulgaricus Acidification at the end of batch culture Lc. lactis, Lc. cremoris Heat and cold

Heat, acid, and bile salt

Saarela et al. (2004)

Freezing and frozen storage

Streit et al. (2007, 2008)

Heat, freezing, and freeze-drying

Lb. lactis

Freeze-drying

Broadbent and Lin (1999), Panoff et al. (1995) Koch et al. (2007)

Heat and spray-drying

Desmond et al. (2002)

Osmotic and heat, with or without pH control Heat, osmotic, hydrogen peroxide, and bile salts Stationary phase, cold, and acid Stationary phase, heat

Cold and acid

Maus and Ingham (2003)

Heat

B. longum, B. adolescentis B. breve

Heat, osmotic, bile salts

Heat, freeze-thawing, bile salts

Gouesbet et al. (2002), Teixeira et al. (1994) Schmidt and Zink (2000)

Acid

Park et al. (1995)

Lb. rhamnosus Lb. plantarum

Stationary phase, heat, osmotic Stationary phase, heat

Acid, bile salts, H2O2 and storage at different temperatures Storage in dried form Heat

Lb. acidophilus Lc. cremoris

Bile salts, heat, osmotic Acid

De Angelis and Gobbetti (2004) Kim et al. (2001) O’Sullivan and Condon (1997)

Lb. paracasei B. lactis, B. longum Lb. bulgaricus

Bile salts, heat, and osmotic Acid, heat, osmotic, H2O2, and ethanol

Prasad et al. (2003)

Source: Adapted from Champagne et al., 2005. Crit. Rev. Food Sci. Nutr. 45: 61–84.

in B. longum E1884 but not in B. animalis E2010. These observations suggest that acid tolerance response is strain dependent as already reported for LAB (van de Guchte et al., 2002). Conditions of the pretreatment can also affect tolerance of the cells to subsequent lethal acid challenge. For example, tolerance of Lb. rhamnosus cells to lethal pH of 2.5 was improved by pretreatment at pH 4.0 but not at pH 3.5 (Saarela et al., 2004). Sublethal stress can also confer protective effects against unrelated stresses, also called cross protection. Pretreatment of Lb. paracasei NFBC338 with sublethal concentration of NaCl, H2O2, or bile salts improve tolerance of cells to heat although less efficiently than the homologous sublethal heat treatment (Desmond et al., 2002). Heat adapted (47°C for 1 h) cells of B. animalis were more tolerant to bile exposure than control cultures (Saarela et al., 2004). In contrast, the heat pretreatment decreased the acid and, surprisingly, heat tolerance of the tested B. animalis. Nevertheless, this result obtained at laboratory scale could not be reproduced at fermenter scale due to the difficulty to find suitable conditions for the successful treatment of this strain at fermenter scale. This observation highlights one of the major hurdles for implementation of sublethal stress pretreatment at industrial scale. For example, a quick temperature upshift can be easily controlled in lab-scale vessel but not in reactors of several thousand liters.



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A general mechanism cannot be defined for sublethal stress applications since their effects are strain dependent and applied stress-specific. Moreover, a sublethal stress can improve tolerance of probiotic cells to certain lethal conditions and be detrimental to others. Application of sublethal stresses can also result in reduced cell yields, cell activity, and/or process volumetric productivity depending on the strain/species, growth phase, and the mechanism of stress-induced protection (Doleyres and Lacroix, 2005). Therefore, a labor-intensive and costly screening procedure has to be done for each bacterium to optimize sublethal stressing conditions. The use of batch cultures for the screening procedure limits considerably the number of tested variables (i.e., nature of the stress, intensity, and duration). In addition, repetitions have to be performed to compensate for large variation in results generally observed in such tests and improve the statistical power of the tests. 21.4.1.3 High-Throughput Screening Method To date, continuous culture fermentation of probiotics has been little investigated. This can be explained by the difficulty in implementing such technology at industrial scale, notably in regard to contaminant control. However, continuous cultures have important advantages over batch cultures, such as a high volumetric productivity. Kim et al. (2003) reported about fivefold higher productivity with a continuous culture of B. longum operated at a dilution rate of 0.33 h−1 than a 22 h batch fermentation. Continuous culture has recently been proposed as an alternative to batch for screening of sublethal stresses (Lacroix and Yildirim, 2007). In contrast to batch culture, the specific cell-growth rate can be easily controlled in continuous culture by adjusting the dilution rate of the system leading to a better control of cell physiology and of sublethal stress applications and consequently less variability in the results. We recently investigated the use of a two-stage continuous culture of B. longum NCC2705 as a high-throughput screening method of sublethal stresses, as illustrated in Figure 21.2 (Mozzetti et al., 2009a). In this system, the first reactor is operated at a low dilution rate to mimic physiological state of cells in late exponential growth phase. Physiological stability of continuously produced cells in this reactor was assessed as a prerequisite with regard to metabolic activities, resistance to different stress factors (heat, antibiotics, simulated gastric juice, bile salts), and genome-wide transcriptional profiles (Mozzetti et al., 2009b). Over 211 h of culture, measured physiological parameters remained stable or showed only small gradual changes which could only be detected due to the high number of time points analyzed during the culture and a highly sensitive analysis. The second stage, connected in series with the first bioreactor, was used for application of stress pretreatments. The effect of pH, temperature, and NaCl concentration stress pretreatments and their combinations were tested on cell viability and resistance to various lethal stresses (heat, osmotic, freeze-drying, gastric, and bile salts lethal tests) in a 3 by 3 factorial design (Mozzetti et al., 2009a). We showed that this two-stage continuous culture design allowed efficient screening of several sublethal stresses during the same culture experiment, since only a short time (corresponding to approximately six residence times or in the example of Figure 21.2 to ca. 4 h) is necessary to stabilize the second reactor after changing the conditions (Lacroix and Yildirim, 2007; Mozzetti et al., 2009a). Up to four different stress pretreatments could therefore be tested per day, with the conditions used in this study (mean residence time of 42 min in R2). Of all tested combinations, only those with pH 4.0 significantly decreased B. longum NCC2705 cell viability in the second reactor compared to control conditions (37°C, pH 6.0, 0% NaCl), and thus could not be considered as sublethal stresses. Pretreatments with 5% or 10% NaCl had negative effects on cell viability after gastric lethal stress. A significant improvement in cell resistance to a heat lethal stress (56°C, 5 min) was observed for cells pretreated at 47°C. In contrast, heat pretreatments negatively affected cell viability after freeze-drying and osmotic lethal stresses. Selected stress pretreatments (pH 4.0, 47°C, 47°C + 10% NaCl and pH 4.0 + 10% NaCl) were also tested during early stationary phase of batch cultures, with similar effects compared to continuous culture (Mozzetti, 2009).


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Sublethal stresses pH: 4.0, 5.0, and 6.0, T °: 37°C, 45°C and 47°C NaCl: 0%, 5% and 10%

R1 T ° = 37°C pH = 6.0 V=2L D = 0.1 h–1 Agitation: 250 RPM RT = 10 h

Lethal stresses Osmotic: 30% NaCl Freeze-drying Heat: 56°C, 5 min Simulated gastric conditions Bile salts

R2 V = 140 mL D = 1.42 h–1 Agitation: 250 RPM RT = 42 min

FIGURE 21.2 Schematic diagram of the two-stage continuous culture used to screen sublethal stresses. A first reactor (R1) is operated in fixed conditions of temperature and pH and used to produce cells with controlled physiology. Stress pretreatments (pH, heat, and osmotic) are applied in the second reactor (R2) which is connected in series with R1. The time of sublethal stress application in R2 is controlled by the fermentation volume. Cells from R1, used as control, and R2 after stress pretreatments are collected for further analysis and lethal stresses experiments. D: dilution, RT: residence time. (From Mozzetti, V. et al., 2009. Physiological stability of Bifidobacterium longun NCC2705 under continuous culture conditions. Submitted.)

21.4.2

CONTINUOUS FERMENTATION WITH ICT TO EFFICIENTLY PRODUCE PROBIOTICS

Cell immobilization consists of retaining microorganisms in a discrete region to limit their free migration and to maintain their viability and/or desired catalytic activities. Different methods have been used for immobilizing LAB and probiotic bacteria: physical entrapment in polymeric networks, attachment or adsorption to a preformed carrier, membrane entrapment, and encapsulation (see encapsulation section for details about this method and applications) (Lacroix et al., 2005). Cell entrapment in food-grade porous matrix is the most widely used technique for biomass and metabolites production in food applications (Champagne et al., 1994; Lacroix et al., 2005). ICT offers many advantages compared to free cell systems notably when combined with continuous fermentation, such as high cell density, reuse of biocatalysts, improved resistance to contamination and bacteriophage attack, enhancement of plasmid stability, prevention from washing-out, physical and chemical protection of cells, and can positively affect physiology of probiotic cells (Doleyres and Lacroix, 2005; Lacroix et al., 2005; Lacroix and Yildirim, 2007). 21.4.2.1 Cell Entrapment within Polymeric Networks Selection of cell immobilization matrices depends on several criteria (Table 21.4). Polymeric gels are among the few matrices that fulfill most of these criteria. Bacterial cells can be entrapped in several food-grade polymers that possess suitable thermal (κ-carrageenan, gellan, agarose, and gelatin) or ionotropic (alginate and chitosan) gelation properties under mild conditions. Controlledsize polymer droplets containing probiotics can be easily produced using extrusion or emulsification in a two-phase dispersion process (Lacroix et al., 2005). When immobilized cells are incubated in growth medium, diffusion limitations occurring in gel beads for both substrates and inhibitory products, mainly lactic acid in the case of LAB, confer a more favorable environment for cell



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TABLE 21.4 Selection Criteria of Matrices for Cell Immobilization According to Margaritis and Kilonzo (2005) • • • • • • • • • • • • • • • •

Retain the desired biocatalytic activity of the cells No reaction with substrates, nutrients, or products Retain their physical integrity and insoluble under the bioprocess reaction conditions Permeable to reactants and products Have a large specific area per unit of volume Have high diffusion coefficients for substrates, nutrients, and products Provide appropriate hydrophilic–hydrophobic balance for nutrients, reactants, and products Resistant to microbial degradation, and excellent mechanical strength Retain chemical and thermal stability under bioprocess and storage conditions Elastic enough to accommodate the growth cells Have functional groups for cross-linking Generally recognized as safe for food and pharmaceutical bioprocess applications Generally nontoxic and available in adequate quantities with consistent quality and acceptable price Easy and simple to handle in the immobilization procedure Environmentally safe to dispose of and/or recyclable The manufacturing system is efficient, easy to operate, and with sufficiently high yields

Source: From Margaritis, A. and Kilonzo, P.M., 2005. In: V. Nedovic and R. Willaert (Eds), Applications of Cell Immobilisation Biotechnology, pp. 375–405. Dordrecht: Springer. With permission of Springer Science and Business Media.

growth close to the bead surface than at the bead center (Arnaud et al., 1992a; Cachon et al., 1998; Doleyres et al., 2002a; Lamboley et al., 1997; Masson et al., 1994). The spatial growth leads to the formation of a high biomass-density peripheral layer at the bead surface with a high cell-growth activity. Active cell growth near the bead surface induces the spontaneous release of cells from gel beads that is favored by shear forces resulting from mechanical agitation and multiple bead contacts in the bioreactor (Arnaud et al., 1992b; Sodini et al., 1997). This phenomenon has been exploited for biomass and metabolite production (Lacroix and Yildirim, 2007). 21.4.2.2 Advantages of ICT Over Free Cell Systems for Efficient Production of Probiotics Very high cell densities ranging from 5 × 1010 to 5 × 1011 cfu/mL or g of support can be reached using ICT for LAB and bifidobacteria biomass production (Champagne et al., 1994; Lacroix et al., 2005). In comparison, maximum concentrations in traditional free cells batch culture are typically 10- to 50-fold lower. This high biomass and the use of dilution rates exceeding the maximal growth rate of cells that would lead to reactor washout with free cell continuous cultures, explain the very high productivity of immobilized cell systems. Very few studies have been carried out on the application of ICT with probiotic bacteria. (Doleyres et al., 2002b) reported cell production ranging from 3.5 to 4.9 × 109 cfu/mL of fermented broth for a dilution rate decreasing from 2 to 0.5 h−1, respectively, during a continuous culture of B. longum ATCC 15707 immobilized in gellan gum gel beads in de Man, Rogosa, and Sharpe agar (MRS) (de Man et al., 1960) medium supplemented with whey permeate. The maximal productivity of 6.9 × 1012 cfu/L of reactor and per hour was obtained at the highest tested dilution rate of 2 h−1, and was approximately 10-fold higher than during free cell batch cultures at an optimal pH of 5.5 (Doleyres et al., 2002b). High volumetric productivity of 1.9 × 1012 cells per liter of reactor and per hour measured using a real-time quantitative reverse transcriptase PCR method has also been reported during continuous fermentation of MRS operated at a dilution rate of 2.25 h−1 and with immobilized cells of B. longum NCC 2705 (Reimann, 2009). In contrast to B. longum ATCC 15707, immobilization of B. longum NCC 2705 leads to the progressive formation of macroscopic cell aggregates in the effluent.


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ICT has also been tested with mixed culture of LAB and probiotic bacteria containing competitive and noncompetitive strains during repeated batch (Doleyres et al., 2002a) and continuous culture (Doleyres et al., 2004a). Immobilization of a mixed culture containing a dominant Lc. lactis subsp. lactis biovar. diacetylactis strain and a less competitive B. longum strain allowed a stable continuous production of concentrated mixed culture in a two-stage system composed of a first reactor containing cells of the two strains separately immobilized in gel beads and a second reactor operated with free cells released from the first reactor (Doleyres et al., 2004a). Strain ratio in the concentrate was controlled by temperature. At 37°C the culture was unbalanced in favor of B. longum while at a lower temperature (32°C) Lc. diacetylactis was dominant. 21.4.2.3 Changes in Cell Physiology Induced by ICT In nature, bacteria form biofilms (i) as a defense mechanism against stress factors such as pH changes, antibiotics, disinfectants, (ii) to remain in a favorable niche for example, rich in nutrients, (iii) to exhibit cooperative behavior through quorum sensing mechanism, and/or (iv) because biofilm is a default mode of growth (Jefferson, 2004). There is abundant literature dealing with cell physiology of pathogenic bacteria in biofilm mode of growth due to their human health impact. In contrast, little is known about cell physiology of beneficial food bacteria, including probiotics, grown in biofilm. Physiological changes of potential probiotic bacteria induced by cell immobilization have been recently reviewed by Lacroix and Yildirim (2007). Immobilization of LAB and/or bifidobacteria improves technological properties such as acidifying capacity (Grattepanche et al., 2007; Koch, 2006; Lamboley et al., 1999), tolerance to freeze-drying (Doleyres et al., 2004b; Koch, 2006), and antimicrobial compounds (Doleyres et al., 2004b; Grattepanche et al., 2007; Trauth et al., 2001). Immobilized cells also exhibit enhanced tolerance to gastrointestinal conditions (i.e., low pH, bile salts, and pepsin) (Doleyres et al., 2004b). Enhanced tolerance to stressing factors could be due to nonspecific stress adaptation responses induced by the conditions encountered in the support, the selection of stress-resistant subpopulations with time, and quorum sensing effects (Lacroix and Yildirim, 2007). The effects of immobilization and long-term continuous culture were studied on probiotic and technological traits of a coculture of bifidobacteria and lactococci separately immobilized in κ-carrageenan/locust bean gum gel beads (Doleyres et al., 2004b). These authors reported that tolerance of free cells of both strains produced in the effluent medium from the immobilized cell reactor to various stresses increased progressively with culture time and was reversible after several subcultures of cells in batch mode. Improved tolerance cannot be associated with cell or strain specific mechanisms since both strains exhibited enhanced tolerance to stresses. Physical protection by cell contact and high density in the gel matrix could also be excluded since cells grown in planktonic state in the effluent. In this study, all of the tested stresses target cell membrane (nisin Z, H2O2, freeze-drying, simulated gastric, and intestinal juice) or need to pass the membrane to reach their intracellular targets (different antibiotics). Therefore, a possible explanation of the enhanced tolerance is a modification of the cell membrane composition and of its permeability properties. We recently investigated the effect of immobilization combined with continuous culture on B. longum NCC2705 cells immobilized in gellan/xanthan gel beads (Reimann, 2009). In this study, the observed increase in tolerance of continuously produced cells to bile salts, compared to free cells cultured in batch, correlated with a decrease in the ratio between unsaturated and saturated fatty acids in cell membranes as already reported for a bile resistant mutant of B. animalis (Ruiz et al., 2007). Furthermore, continuously produced cells of B. longum NCC2705 exhibited large morphological modifications toward formation of macroscopic aggregates that could also confer a protective effect against bile salts. Large changes in cell morphology and formation of macroscopic aggregates containing high viable cell and nonsoluble exopolysaccharide concentrations were already observed in a 32-day continuous fermentation of Lb. rhamnosus RW-9595M immobilized on porous silicone rubber



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supports, used for production in supplemented whey permeate of exopolysaccharides that exhibit immunostimulatory activity (Bergmaier et al., 2005). The loss of soluble-exopolysaccharide production capacity of cells in the effluent of the immobilized system was reversible and exopolysaccharide production of 1742 mg/L fully recovered after four successive pH-controlled batch cultures. Low soluble-exopolysaccharide concentration resulted in low viscosity in fermented broths, which may facilitate cell propagation and separation for exopolysaccharide producing strain. Aggregates produced by continuous immobilized cultures of B. longum NCC2705 and Lb. rhamnosus RW-9595M can be easily separated and could eventually be used as a symbiotic product with very high active cell concentrations if the prebiotic properties are demonstrated for the produced exopolysaccharides. Furthermore, the ability of probiotic to form aggregates has been associated with beneficial characteristics such as adhesion to epithelial cells (Del Re et al., 2000; Kos et al., 2003), physical protection in gastrointestinal tract and coaggregation with pathogens (Collado et al., 2007; Schachtsiek et al., 2004).

21.5

ENCAPSULATION: AN EFFICIENT STABILIZATION TECHNOLOGY TO PROTECT PROBIOTIC CELLS DURING STORAGE AND IN THE GASTROINTESTINAL TRACT FOLLOWING INGESTION

The technologies previously described aim at improving intrinsic tolerance of probiotic cells to various stresses by adding compatible solutes, applying sublethal stress, genetic manipulation, or by modifying cell physiology through ICT. Probiotic cells can also be physically protected from their external environment using encapsulation also referred as microencapsulation with respect to the size of produced capsules. Microencapsulation is defined as “the technology for packaging solid, liquid and gaseous materials in small capsules that release their contents at controlled rates over prolonged period of time” (Champagne and Fustier, 2007). Several microencapsulation techniques have been proposed and are currently used in various industrial sectors; not all are however suitable for food applications in regard to their cost and toxicity (Champagne and Fustier, 2007; Gouin, 2004). In the following section we examine different technologies suitable for probiotic encapsulation and food applications.

21.5.1

GEL PARTICLES

This technology consists of entrapment of probiotic cells within a polymeric matrix such as pectin, gellan gum, κ-carrageenan/locust bean gum, and alginate. Gel capsules containing probiotic can be produced by emulsion or extrusion techniques and various methodologies (Champagne and Fustier, 2007; Kailasapathy, 2002). High survival rates of microorganism of 80–95% were generally achieved independently of the technique (Krasaekoopt et al., 2003). Several studies reported protective effect of microencapsulation of sensitive bifidobacteria and lactobacilli in plant polymer matrices, mainly alginate, against oxygen (Talwalkar and Kailasapathy, 2003), acidic environment (Sun and Griffiths, 2000), freezing (Shah and Ravula, 2000), refrigerated storage of ice cream and milk (Hansen et al., 2002) and yoghurt (Adhikari et al., 2000, 2003; Sultana et al., 2000), and during simulated gastrointestinal tests (Lee and Heo, 2000; Ding and Shah, 2007, 2009). Advantages of using alginate as matrix includes: nontoxicity, forms gentle matrices with calcium chloride, leading to mild conditions for encapsulation of sensitive probiotic cells, and reversibility of immobilization since gels can be solubilized by sequestering calcium ions thus releasing the entrapped cells (Kailasapathy, 2002). Conversely, undesired gel dissolution can occur in food containing chelating agents. Gel particle can be coated with polycations such as chitosan and poly-l-lysine to overcome this limitation (Krasaekoopt et al., 2006). The survivability of three probiotics in uncoated alginate beads or coated with chitosan, sodium alginate, or poly-l-lysine combined with alginate was conducted in 0.6% bile salt solution and simulated gastric juice (pH 1.55) followed by incubation in simulated intestinal juice with and without 0.6% bile salt. Chitosan-coated alginate beads provided the best protection for


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Lb. acidophilus and Lb. casei in all treatments (Krasaekoopt et al., 2004). However, Bifidobacterium bifidum did not survive the acidic conditions of gastric juice even when encapsulated in coated beads (Krasaekoopt et al., 2004). Gel beads can also be coated with proteins through a transacylation reaction. The reaction involves the formation of amide bonds between protein and alginate, producing a membrane in the bead surface which resists gastric pH and pepsin activity (Chen et al., 2006). B. bifidum encapsulated in coated or uncoated gel beads composed of alginate, pectin, and whey proteins were more resistant to simulated human gastrointestinal tract conditions (Guerin et al., 2003). Mortality in acidic solution (pH 2.5) containing pepsin was less than 2.5 log after 2 h of incubation, while free cells did not survive. In treatments with bile salts, gel beads coated with protein membrane performed better than membrane-free beads, in which cells were less resistant than free cells (Guerin et al., 2003). Gbassi et al. (2009) have investigated the survival to simulated gastric and intestinal juices of three Lb. plantarum encapsulated in whey proteins coated and uncoated alginate gel beads. A better survival rate of the three strains to simulated gastric juice was observed for coated compared to uncoated beads. In addition, only cells in the coated beads survived in the simulated intestinal juice medium following simulated gastric juice treatment. Although promising on a laboratory scale, the technologies developed to produce gel beads present serious difficulties for large-scale production such as low production capacity and large bead diameters (2–5 mm), that can affect food texture, for the droplet extrusion methods and transfer from organic solvents and large-size dispersion for the emulsion techniques (Picot and Lacroix, 2004). Moreover, the addition of these polysaccharides is not permitted in yoghurts or fermented milk in some European countries. Protein matrices can be used as an alternative to plant polymers for encapsulation of probiotics. Recently, Heidebach et al. (2009a) reported an encapsulation method of probiotic cells based on a transglutaminase-catalyzed gelation of casein suspensions containing cells of Lb. paracasei or B. lactis. This method permits production of water-insoluble spherical capsules of 165 μm diameter with encapsulation yields of 70 ± 15% and 93 ± 22% for the lactobacilli and bifidobacteria, respectively. Particle size reduction to 68 μm and an encapsulation yield of 100% for both Lb. paracasei and B. lactis can be achieved using an enzymatic-induced gelation of milk proteins with rennet (Heidebach et al., 2009b). In both studies, a protective effect on probiotic cells of the dense protein matrix against low pH values comparable with those in the human stomach was reported. Encapsulation of Lb. rhamnosus cells using extrusion combined with calcium-induced cold gelation of preheated whey protein has also been proposed (Reid et al., 2005), but large gel beads of 3 mm diameter were formed and the encapsulation yield reached only 23% due to the exposure of cells to the concentrated CaCl2 solution during the gelation process.

21.5.2

SPRAY-COATING

Most of the literature on microencapsulation of probiotics deals with gel particles while most of commercial products are prepared by spray-coating, a more suitable technique for large-scale production (Anal and Singh, 2007; Champagne and Fustier, 2007; Gouin, 2004). However, as recently highlighted by Champagne and Fustier (2007), most information on the technology, that is difficult to master, is proprietary and therefore of limited access for academic research. Spray-coating consists of suspending, or fluidizing, particles of a core material in an upward stream of air and applying an atomized coating material to the fluidized particles (Augustin and Sanguansri, 2008). Several methods differing by the injection angle of the coating material in the vessel (i.e., fluidbed, Wurster, and tangential methods) and the nature of the coating material (lipid-based, proteins, or carbohydrates) can be used for spray-coating of probiotics (Champagne and Fustier, 2007). A patent claimed that high survival rate of bacteria and yeast can be achieved during spraycoating through an appropriate selection of the operating parameters (i.e., nature of the coating material, rotor, and air speed in the vessel, pulverization pressure of the coating material, coating rate, temperature of the pulverization and incoming air, coating material, and of the product)



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TABLE 21.5 Claimed Protective Effects of Spray-Coating on Bacteria and Yeast According to Durand and Panes (2002) Microorganisms

Coating Material

Lb. acidophilus

Stearic acid

Maximum Cell Concentration in Coated Particles (cfu/g)

Stability Testsa

3.2 × 1011

Gastricb Control Candida utilis (Isenschmid et al., 1995). The leakage of cell components (amino acids, peptides, membrane lipids, and ions) into the environment was taken as an indication of membrane rupture. A loss of membrane integrity can also occur when cells are subjected to sublethal stress. During treatments with high-pressure CO2, the absorbance and loss of Mg and K ions from cells increased steeply, and then leveled off, indicating that the integrity of the cell membrane could be affected even at early processing stages. Proton permeability can be used to measure the physical damage of cell membranes, as the membrane functions as a barrier to maintain the cytoplasmic pH (Hutkins and Nannen, 1993). The release of added protons from the cytoplasm to the environment can be measured in terms of the half time (t1/2) for complete pH equilibration between the cells and the suspending medium. Whereas intact Lactobacillus plantarum cells needed a t1/2 of 50 min for a modification in pH of 0.55 units, cells exposed to high-pressure CO2 (7 MPa, 30°C, 10 min) showed almost no pH difference, complicating the determination of t1/2. Yeast disruption has been object of patenting: a yeast suspension is treated with SC-CO2 and subjected to a fast depressurization to atmospheric pressure in order to separate the CO2 from the yeast suspension, which can be further heated at 80–90°C for 5–30 min (Miyake et al., 2006). The fractionation of biomass materials with supercritical and near critical fluids is used to obtain one or more compounds, since the solvation properties can be tuned by controlling the extraction conditions (temperature, pressure, and/or modifier concentration). This technique has been applied for the recovery of intracellular components, that is, nucleic acids (Lin, 1991; Nivens and Applegate, 1996; Khosravi-Darani and Vasheghani-Farahani, 2005), off-flavors (Lin, 1991), and intracellular proteins and enzymes (Lin, 1991). Fractionation has been carried out in two steps, the first at elevated pressure (to cause disruption of the biomass and to release structural biomass constituents), and the second under supercritical or near critical fluid extraction conditions (Castor and Hong, 2003). Microbial cells were treated in countercurrent with a supercritical or pseudocritical solvent, and then the solvent solution (containing the dissolved microbial cell extract) was sent to a separation tank, where the cell extract was recovered from the bottom by gravitational precipitation (Horizoe and Makihara, 1987). In an alternative method, a liquid suspension of cultured microbial cells was mixed with a supercritical fluid, and the pressure of the mixture was suddenly released, while the temperature was kept above the critical temperature of the fluid. The mixture was introduced into a separation tank and disintegrated microbial cells were collected from the bottom, while the fluid was recovered (Horizoe and Makihara, 1987). 23.4.6.1 Recovery of Enzymes Particular enzymes do not lose their activity in SC-CO2 media, and can take advantage of the relatively low temperatures. In addition, proteases are inactivated inside the cell by the hydrophobicity of the environment and low pH as a result of the dissolved CO2, thus preserving the rest of the proteins. Highly pressurized CO2 can activate or inactivate enzymes, depending on the type of enzyme, their biochemical characteristics and structure, and the environmental conditions. Possible activity losses could be minimized by a careful selection of operational conditions, in particular: (i) pressure; (ii) depressurization rate (since rapid expansion of the CO2 dissolved in the microaqueous layer around © 2010 Taylor and Francis Group, LLC

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the enzyme can modify its structure) (Egyházi et al., 2004); and (iii) water content of the system (since the enzyme activity decreases during SC-CO2 treatment with increasing water content). Conventional methods can disrupt Saccharomyces cerevisiae cells more efficiently than highpressure CO2; however, the activity of alcohol dehydrogenase is affected by oxidation processes, whereas protease degradation and intensive localized heating effects appear in autolysis with toluene and in enzymatic lysis with β-glucuronidase. Under subcritical conditions (1000 psi, 25°C), in experiments lasting 15–24 h, proteins were not released and the alcohol dehydrogenase activity was not modified. At the same pressure and higher temperature (supercritical conditions), the rates of protein release were improved by higher temperatures, whereas the enzyme activities began to decay at temperatures above 35°C and were almost completely lost at temperatures above 55°C. Increased pressures allowed the disruption time to be reduced without affecting the enzyme activity. Operating at 5000 psi in the presence of β-glucuronidase, maximal enzyme release was attained in 90 min, and the enzyme activity was higher than with conventional methods such as β-glucuronidase, autolysis in toluene, or mechanical grinding. The cellulase enzyme complex consists of endoglucanases, exoglucanases, and β-glucosidase, which act synergically to hydrolyze cellulose. In Trichoderma cells, a considerable part of β-glucosidase remains in the intracellular space, and its recovery by disruption by SC-CO2 has been proposed. Since the resistance of this microorganism is not high, 15 min is sufficient for cell disruption at 10 MPa and 40°C (Egyházi et al., 2004).

23.5 PERSPECTIVES OF SC-CO2 IN THE BIOTECHNOLOGICAL EXTRACTION OF NUTRACEUTICALS The application of SC-CO2 for the processing of nutraceuticals is a promising alternative for extraction, sterilization, and disruption. Since the range of nutraceuticals is increasing and the production of bioactive metabolites by wild and genetically modified microorganisms will become more diverse, the future opportunities for an efficient, clean technology are expected to improve. The physicochemical properties of SC-CO2 offer advantages in relation to environmental and toxicological aspects in comparison to conventional solvent extraction or disruption techniques. SC-CO2 also offers operational advantages over high hydrostatic pressures when used for microbial and enzyme inactivation. Both environmental and toxicity aspects are key factors to address actual and future consumer demand for natural, bioactive products obtained by more efficient and less polluting processes. Due to the emerging character of this technology, it may foster developments in biotechnology to yield products of a more natural character. Increased costs inherent to sophisticated technologies would be assumed when the final product will offer additional chemical, biological, or organoleptic properties, and the process is based on an environmentally friendly technology. Even though the SCFE extraction of natural products is carried out on an industrial scale, new biotechnological applications could include aspects such as: the extraction of metabolites synthesized by genetically modified microorganisms; the fractionation of bioactive compounds from mixtures of microbial biomass and enzymes; the selective and separate recovery of compounds with different properties; and the sequential and/or simultaneous achievement of different effects (including cell disruption, extraction, fractionation, sterilization, or reaction) (Khosravi-Darani and Vasheghani-Farahani, 2005). Whereas SCFE is commercially available for obtaining bioactive extracts from a variety of substrates, the utilization of CO2 as a nonthermal pasteurization treatment is promising, but additional developments are required to achieve its industrial implementation. Since the technology is already known and dense CO2 prototypes are already available, industrial developments will probably be available in a matter of few years (Spilimbergo et al., 2003). In relation to bacterial inactivation, priority aspects to be studied include: efficient inactivation of spores (Zhang et al., 2006), retention of vitamins (Spilimbergo et al., 2003), deeper knowledge of the inactivation mechanism and process modeling (Zhang et al., 2006), expansion and scale-up of food applications, and advances in regulations (García-González et al., 2007).


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Application of 24 The Nanotechnology to Functional Foods and Nutraceuticals to Enhance Their Bioactivities Ping-Chung Kuo CONTENTS 24.1 Introduction .......................................................................................................................... 447 24.2 Nanotechnology Foods .........................................................................................................448 24.2.1 Importance of Nanotechnology ................................................................................448 24.2.2 Public Perception of Nanotechnology ...................................................................... 450 24.2.3 Acceptance of Nanotechnology Foods ..................................................................... 450 24.3 Nanonization of Functional Foods and Nutraceuticals ........................................................ 451 24.3.1 Functional Foods ...................................................................................................... 451 24.3.2 Nutraceuticals ........................................................................................................... 453 24.3.3 Traditional Chinese Medicines ................................................................................. 454 24.4 Improvements in the Bioactivity of Functional Foods and Nutraceuticals .......................... 455 24.4.1 Hepatoprotective ....................................................................................................... 455 24.4.2 Antioxidant ............................................................................................................... 455 24.5 Nanotechnology Functional Foods and Drug Delivery Systems ......................................... 456 24.6 Nanotechnology for Food and Global Climate Issues .......................................................... 457 24.7 Summary .............................................................................................................................. 458 Acknowledgments.......................................................................................................................... 458 References ...................................................................................................................................... 458

24.1

INTRODUCTION

Nanotechnology is the knowledge and control of matter at dimensions of roughly 1–100 nm, where unique phenomena enable novel applications. Encompassing nanoscale science, engineering, and technology, nanotechnology involves imaging, measuring, modeling, and manipulating matter at this scale (National Nanotechnology Initiative, 2006). Nanotechnology is a fundamental high-tech technique and is regarded as one of the key technologies of the twenty-first century. This technology holds major potential to generate new products with numerous benefits. The principle of nanotechnology is that materials with known properties and functions at their normal size will display different and often useful properties and functions at their nanosize. Thus, scientists manipulate objects of 1–100 nm in order to fabricate and identify materials and structures with novel properties and functions (Lagaron et al., 2005; Weiss et al., 2006). Nanotechnology has opened up new possibilities in various fields, including medicine, cosmetics, agriculture, and food, and has already been 447 © 2010 Taylor and Francis Group, LLC

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used as significant method in the understanding of how physicochemical characteristics of nanosized substances can alter the structure, texture, and quality of foodstuffs. Nanotechnology is increasingly being employed in a number of areas of food production, such as the processing, storage, transportation, traceability, safety, and security of food (Kuzma and VerHage, 2006; Sanguansri and Augustin, 2006). Some functional food and nutraceutical products containing nanoscale additives are already commercially available. According to an analysis of market products, the nanofood market is expected to increase from US$7.0 billion today to US$20.4 billion in 2010 (Allianz and OECD, 2005). However, there are no complete and extensive reviews relating to the application of nanotechnology in food products and the advantages and risks inherent in this technique. This chapter will discuss the present public perception and acceptance of nanotechnology foods. It also deals with the benefits of functional foods, nutraceuticals, and traditional Chinese medicines processed with nanotechnology. Moreover, the potential of nanotechnology foods in helping to resolve global warming issues such as desertification will also be highlighted.

24.2 24.2.1

NANOTECHNOLOGY FOODS IMPORTANCE OF NANOTECHNOLOGY

Nanoscale materials possess physical, chemical, optical, electrical, catalytic, magnetic, adhesive, mechanical, and most importantly biological properties that differ fundamentally from those of their macroscopic or bulk counterparts (Dresselhaus et al., 2004). Researchers usually develop new materials in two major ways. They can reduce the particles in standard materials to sizes as small as a nanometer, or about one-hundred-thousandth the width of a human hair. At nanosize, the principles of quantum physics apply and the characteristics of materials change significantly. Carbon becomes 100 times stronger than steel, aluminum turns highly explosive, and gold melts at room temperature. In addition, researchers can manipulate individual atoms and molecules to form microscopic tubes, spheres, wires, and films for specific tasks, such as generating electricity or transporting drugs in the body. Nanotechnology has the potential to revolutionize fields as seemingly disparate as medicine, food science and technology, recreation and sport sciences, and civil engineering, among others. In the field of life sciences, a comprehensive series of nanosized clusters, rods, and tubes are being examined in laboratory animals to explore their ability to target and kill cancer cell lines. For example, metal, ceramic, or polymer nanoshells and carbon nanotubes containing inert, chemopotent, or radioactive materials are injected either directly into the tumor or at the tumor site so that nanoshells then enter through microcracks in the tumor’s capillary network (Cuenca et al., 2006). Once in the tumor, nanoshells are activated and destroy cancerous cells while leaving noncancerous cells intact. In other areas of medicine, nanoshells can change the process by which skin and vein grafts, organ transplantations, and surgical suturing are completed. Antibacterial nanocoatings may prevent or treat pernicious infections when used on suture and bandage materials. Raw nanomaterials derived from vitamins and minerals and combined with carbon-based materials, such as proteins, carbohydrates, and fatty acids, can be developed to produce products that can serve as building blocks for skin, muscle, and bone tissue, among other structures—potentially revolutionizing tissue engineering. Since there are so many cases of nanotechnology improving current product items, nanosized electrodes included in nanofilms and nanocoatings have been layered onto neural, cochlear, and retinal implants to improve touch, hearing, and sight, respectively. Nanosensors that monitor a plethora of functional indicators, such as blood flow, pH, and DNA replication in specific cells can push medicine to a new frontier of prevention, diagnosis, and intervention (NickolsRichardson, 2007). Scientists, futurists, and ethicists all agree that nanotechnology will profoundly shape the human body and the physical and social environments in which humans live because of the extraordinary number of devices, processes, and applications that can be produced with this transformative technology.


Application of Nanotechnology to Functional Foods and Nutraceuticals

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An example of the application of nanotechnology to the production of food is the creation of functional foods (Sanguansri and Augustin, 2006). These are products that promise consumer improvements in targeted physiological functions (Diplock et al., 1999). Nanotechnology foods with tangible benefits for the consumer will be easier to market than nanotechnology foods without obvious consumer benefits. Consequently, it might be tempting for the industry to assume that attitudes towards nanotechnology foods will be more positive for a nanotechnology product with desirable benefits. However, novel foods even with clear health benefits may not be attractive to all consumers. Thus, introducing such new foods is generally unlikely to result in more positive attitudes toward nanotechnology food (Frewer et al., 2004). It can be concluded that nanotechnology food is accepted for some, but not all, products. Nanotechnology has the potential to alter nutrient intake by broadening the number of enriched and fortified food products. The functional food market is expected to increase as a result, in part, of the expansion of nutrient delivery systems through nanotechnology mechanisms. For example, antioxidant nutrients may be included in nanocomposites, nanoemulsions, nanofibers, nanolaminates, nanofilms, and nanotubes. Although an increase in nanonutrient intake from an enhanced food supply may be beneficial to the general population, it is also recommended that food and nutrition researchers watch closely for the overconsumption of nutrients and signs of toxicity in individuals. In addition, nanonutrient imbalances may become more prevalent. Interactions between drugs and nutrients will also require careful and detailed evaluations. Nanotechnology offers several novel vehicles for nutrient delivery. Nutrient digestion and absorption may depend on the structural, chemical, and physical states of these vehicles and the nutrients bound within them. However, food and nutrition researchers and scientists will require an understanding of the metabolic consequences of nutrients in novel food systems as nanotechnology applications expand in the food sciences. In addition, the available information on the chemical, physical, and analytical properties of nanomaterials should be evaluated to determine if there are sufficient reliable and complete data sets to establish chemical identity, characterize substance properties, determine the extent to which they clump together or remain separate, and identify potential by-products and impurities. Nanotoxicology is expected to emerge as a critical discipline in addressing the environmental, health, and occupational hazards of nanoscale substances over the course of their life cycle (Donaldson et al., 2004). Previous reports on particle toxicology have focused on such materials as coal, asbestos, mineral fibers, and ambient particulate matter (Borm, 2002). Nanomaterials are expected to exhibit different environmental transport behaviors, with colloidal aggregates expected to have the least mobility (Lecoanet et al., 2004; Lecoanet and Wiesner, 2004). In general, nanoparticles are expected to be poorly water soluble, and this low aqueous solubility may generally favor the persistence of chemicals and their absorption by biological systems. Nanomaterials are also expected to be more absorbent than other molecules. Therefore, if they were to be dispersed in the environment there are both potential positive and negative results, whether they are able to remediate or whether they absorb contaminants and disperse them more widely. Given the lack of robust environmental fate and toxicity data for nanoparticles, it would be a risky task to predict toxic potency and exposure levels with any credibility. It will also be challenging to develop an environmental fate and transport model, not only for the bulk material, but also for potential aggregates and degradation products in the environmental surroundings. Qualitative risk comparisons of expected environmental concentrations with ecologically acceptable concentrations are also not well characterized in nanomaterials. It is important to have detailed and reliable data on which to base life cycle and risk assessments. Data and facts must be provided by producers, researchers, governments, expert judgments made by competent individuals or groups, and representative stakeholders. Although adequate and representative data sets are critical, it is important to emphasize that these tools could be based on relatively incomplete data, for instance, by comparison of analogs that are similar based on size, chemical structure, and energy cost. Furthermore, the final decisions made about nanoproducts should not only be risk or environment oriented, as other aspects like cost, energy performance, life span, and customer preference are equally significant.


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24.2.2 PUBLIC PERCEPTION OF NANOTECHNOLOGY A series of research articles have examined the public perception of nanotechnology worldwide and the results of these studies show that public knowledge about nanotechnology is very limited (European Commission, 2001; Cobb and Macoubrie, 2004; Lee et al., 2005). Even though people in the United States possess little knowledge about nanotechnology, most of them are convinced that its benefits outweigh the risks (Cobb and Macoubrie, 2004). In contrast, the European public seems to be less optimistic about nanotechnology (Gaskell et al., 2005). However, most studies have examined public attitudes towards nanotechnology (Cobb and Macoubrie, 2004; Gaskell et al., 2004; Lee et al., 2005; Scheufele and Lewenstein, 2005) rather than attitudes towards realistic products (Siegrist et al., 2007). The research results into public perception of genetically modified (GM) foods finished by Tenbült et al. (2005) suggest that the more a product is seen as natural, the less acceptable will be a genetically engineered version of that product. Furthermore, the results of another recent study indicate that even when the healthfulness of natural and artificial foods are specified to be equal, most of the public with a preference for natural food continue to prefer it (Rozin et al., 2004). It could be concluded from these related studies that perceived naturalness or lack of naturalness could be a factor which also influences attitudes toward nanotechnology foods.

24.2.3

ACCEPTANCE OF NANOTECHNOLOGY FOODS

Most people are not familiar with the term nanotechnology (Cobb and Macoubrie, 2004; Gaskell et al., 2005). One way to cope with this lack of knowledge is to employ social trust when assessing the risks of a new technology (Siegrist and Cvetkovich, 2000). For example, in the domain of gene technology, results infer that people who display more trust in institutions involved in using or regulating gene technology, attribute more benefits and fewer risks to this technology (Siegrist, 1999, 2000; Tanaka, 2004). The acceptance of or willingness to buy GM foods is directly determined by the perceived risk and the perceived benefit. In other words, trust has an indirect impact on the acceptance of GM foods. Results of a Swiss study suggested that acceptance of genetically modified products was largely determined by perceived benefits (Siegrist, 2000). A Swedish study reported similar findings (Magnusson and Hursti, 2002). Tangible benefits increased people’s stated willingness to purchase GM foods. That is, products that are better for the environment, for example, or products that are healthier would increase the acceptance of consumers. Nanotechnology foods may be more acceptable to consumers who perceive tangible benefits. In a democratic society where choice exists, people will not consume foods that they associate with negative attributions. A number of factors may influence people’s concerns. These include the belief that there is a potentially negative environmental impact associated with production processes or agricultural practices, or the perception that there is uncertainty about unintended human or animal health effects. The public response to the application of technological innovations may be driven by concerns about the impact that the technology will have on social structures and relationships. For this reason, considerable effort has been directed to understanding people’s attitudes towards the emerging biosciences. There are currently no special regulations for the application or utilization of nanotechnology in foods in the United States. Although recommendations for special regulations in the European Union (EU) have been made, laws have not yet been changed. The U.S. Food and Drug Administration (FDA) states that it regulates “products, not technologies,” and anticipates that many products of nanotechnology will lie along the jurisdictions of multiple centers within FDA and will therefore be regulated by the Office of Combination Products. FDA regulates on a product-by-product basis and stresses that many products that are currently regulated produce particles in the nanosize range. FDA says that “particle size is not the issue” and stresses that new materials, regardless of the technology used to create them, will be subjected to the standard battery of safety tests (Food and Drug Administration, n.d.). In contrast to the FDA view on particle size, a recent report by the Institute of



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Food Science and Technology (IFST), a UK-based independent professional qualifying body for food scientists and technologists, states that size matters and recommends that nanoparticles be treated as new and potentially harmful materials until testing proves their safety. Nonetheless, the European Commission intends to use existing food laws with regard to food products derived through nanotechnology but acknowledges that the technology will possibly require modifications to the law. The European Commission plans to use a case-by-case approach for the risk assessments. The Royal Society and the Royal Academy of Engineering assessments of the potential impact of nanotechnology, commissioned by the UK government, recommended the identification of nanoparticle use in ingredients lists. The UK government declared that it was necessary for consumers to make informed decisions and that modifications to current labeling requirements would be necessary. The IFST suggested that when nanoparticles are used as food additives, the conventional E-numbering system for labeling be used along with the subscript “n” (IFST, 2006). The UK government also agreed to consult with EU partners relating to the IFST report’s recommendation that nanosized ingredients have to be subjected to full safety assessments before their addition to consumer products.

24.3 NANONIZATION OF FUNCTIONAL FOODS AND NUTRACEUTICALS 24.3.1

FUNCTIONAL FOODS

Moraru et al. declared that the four major areas in the food industry to benefit from nanotechnology are the development of new functional materials, micro- and nanoscale processing, new product development, and the design of nanotracers and nanosensors for food safety and biosecurity (Moraru et al., 2003). Nanoscience approaches may be applied to the manipulation and control of the interactions between food components such as proteins, lipids, and polysaccharides and their self-assembly behavior on a molecular scale to impart desired structural and rheological properties to food (Dickinson, 2003, 2004). Nanotechnology can potentially be employed to alter food products to deliver nutrients, proteins, and antioxidants to the body more effectively and efficiently. The human body is full of nanomolecules, such as amino acids and sugars that self-assemble into proteins, enzymes, hormones, and polysaccharides. A major strategy for the delivery of these components into food is the use of microencapsulation. Microencapsulation, involving the coating or entrapment of a desired component (core) within a secondary material (encapsulant), is used to mask the color and taste of nutrients, and to protect sensitive nutrients during processing, storage, and transportation (Augustin et al., 2001). It plays a significant role in the development of the functional food industry which targets the delivery of food nutrients with positive impacts on the health condition of consumers. A food can be regarded as functional if, beyond its inherent nutritional effects, it demonstrates beneficial effects on one or more target functions in the body in ways that are relevant to either the state of well-being and health or the reduction of the risk of disease (Rowan, 2001). Other definitions concisely state that a functional food is any food that may provide a health benefit beyond the traditional nutrients it contains. In most commercial functional foods, a number of bioactive components are added that are considered beneficial to the health of the consumer. An important aspect of these functional foods is to provide the appropriate dose of these bioactive components in order to have a beneficial rather than a toxic effect on human health (Falk, 2004). Since the market and consumers have a growing interest in the health-enhancing role of specific foods and physiologically active food components, functional foods are receiving renewed attention (Hasler, 1998). Functional foods and beverages are typically developed for specific health goals such as antiaging, inflammation reducing, or cardiovascular protection. The applications of nanotechnology for targeted delivery of bioactive components such as omega-3 fatty acids, carotenes, vitamins, coenzyme Q10, or plant polyphenols, are still in their infancy, and the use of pharmaceutical products made with non-foodgrade ingredients is not acceptable for incorporation into foods. Research into food-grade encapsulants is needed to enable the delivery of desirable bioactive constituents through the food supply.


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Nevertheless, there are parallels that can be drawn between the delivery of drugs for the treatment of disease and the delivery of bioactive food components in functional food applications for the improvement of health and well-being and a reduced risk of disease. The major difference in the delivery of bioactive components through food is the use of ingredients that are generally regarded as safe for encapsulating matrices. Hence many of the encapsulation systems being developed for the delivery of bioactive principles through food have capitalized on the self-assembly behavior of natural ingredients (e.g., proteins, carbohydrates, lipids, or mixtures of these) or ingredients functionalized by physical or enzymatic means for encapsulation. Another important area where food nanotechnology is increasingly applied is in the design of functional food ingredients such as food flavors and antioxidants (Imafidon and Spanier, 1994). The goal for the application of nanotechnology is to improve the functionality of these additives in food and body systems, which may minimize the concentration and raw materials needed. Delivery and controlled release systems for the solubilization of nutraceuticals in foods have previously been mentioned (Lawrence and Rees, 2000). These new functional ingredients are increasingly being integrated into the food matrix development process (Haruyama, 2003). Food ingredients such as nanoparticulate lycopene and carotenoids are now commercially available. Their bioavailability and the ability to disperse these compounds are typically higher than that of their traditionally manufactured counterparts. Carotenoids, particularly β-carotene, the most nutritionally active carotenoid, are thereby commonly used substances in investigations into food science. In addition to their health-related properties such as the enhancement of the immune response, inhibition of mutagenesis, and blocking of free radical-mediated reactions, and antioxidant character, their coloring features are the focus of interest (Bendich and Olson, 1989; Britton, 1995a, 1995b). Due to its extremely hydrophobic character, β-carotene is insoluble in water and shows a poor bioavailability in crystalline form (Ribeiro and Cruz, 2004). Hentschel et al. (2008) formulated β-carotene in colloidal lipid particles of fat-inwater dispersions in a promising method for incorporating β-carotene into water soluble systems. A micro- to nanosized particle size of around 400 nm offers the possibility of using a nanostructural lipid carrier as a food colloid in several applications without creaming or sedimentation. Possible applications as dyes and provitamin A sources in beverages are also a focus of interest. Populations with a higher intake of seafood are known to have a lower incidence of cardiovascular diseases and certain types of cancer (Hu, 2003). The highly unsaturated fatty acids present in marine oils are responsible for their many beneficial effects. The consumption of marine oils leads to thinning of the blood, lowering of triacylglycerol and possibly of cholesterol levels and, hence, decreases clot formation as well as fat deposition. The problem with the direct incorporation of those marine oils into food products is their potency of oxidation which results in a loss of antioxidant power. They must therefore be microencapsulated for most applications. However, the solid microcapsule particles should be washed properly with a nonpolar solvent to remove any unencapsulated oil that might otherwise be oxidized in the product (Shahidi, 2004). Notwithstanding, it is envisaged that even though micro- and nanoencapsulation technologies—the latter making use of, for instance, self-assembling biopolymers (Reguera et al., 2003)—are considered as bioactive packaging systems in themselves, they may also be used in combination with the integration route mentioned above. Consequently, on their fast release immediately before packaged food consumption, the encapsulated functional substances should transfer to the food within their capsules, and thus reach the desired parts within the human body. They should also remain intact in the gastrointestinal tract ready for release and optimum bioactive functioning. The benefits of vitamin E to human health are well known to consumers. It has been shown that vitamin E has positive effects on cardiovascular disease (Stampfer et al., 1993), cancer prevention (Klein et al., 2003), the immune system (Meydani and Tengerdy, 1993), and antiageing processes (Packer and Weber, 2001). Adequate vitamin E can be ingested in a balanced diet, or the difference can be made up by vitamin E tablets or vitamin E-enriched foods or beverages. Utilization of functional beverages has grown rapidly in recent decades and there are increasing demands for vitamin



453

E products suitable for beverage applications. Because vitamin E is not soluble in water, it is necessary to convert it into a vitamin E emulsion before it can be used in beverage fortification. Many vitamin E products are already on the market, the majority in powder form and a few in liquid form. The powder forms are not designed specifically for beverage applications and therefore present problems when applied to beverages. One major problem is their poor physical stability. In beverages, vitamin E emulsion droplets rise to the top within days and form a ring around the bottleneck, so-called “ringing.” Another problem is the increase in beverage turbidity and, as a result, the change in beverage appearance. This is particularly critical for clear beverages. Liquid forms of vitamin E are mostly used for beverage applications. Liquid forms are generally stable in water, but can have stability problems in beverages containing fruit juice. They often contain more surfactant than vitamin E, which may result in sensory issues and regulatory concerns. To meet customers’ needs, an ideal vitamin E product for beverages would have physical stability in liquids and would not change the beverage appearance. In addition, it should also have no sensory or regulatory problems. No current product meets these requirements. Nanonization of vitamin E particles offers a new route which is applicable to beverage production. It has been shown that a vitamin E nanoparticle can be produced by ultra-high-pressure homogenization and stabilized by microencapsulation with starch (Chen and Wagner, 2004). The product has been shown to reduce the turbidity and physical stability problems encountered by traditional commercial products. These vitamin E nanoparticles offer the beverage industry the possibility of creating new and innovative products, which otherwise would be impossible and/or difficult.

24.3.2

NUTRACEUTICALS

Nutraceuticals are naturally occurring/derived bioactive compounds which are reported to display health benefits. The delivery systems for nutraceuticals are usually foods (functional foods), supplements, or both. The applications of nutraceuticals, such as herbal medicines, minerals, and vitamins have increased dramatically, and it is estimated that approximately one-third of Americans consume some form of dietary supplementation (Fugh-Berman, 2001). Adverse clinical side effects of nutraceutical ingestion have been well documented (Fung and Bowen, 1996), as have interferences from these agents in laboratory tests (Mantani et al., 2002). Colloidal suspensions of metal particles such as copper, gold, platinum, silver, molybdenum, palladium, titanium, and zinc have already been marketed as oral health supplements. Metal colloids are reactive and can act as reducing agents, bind to proteins, and denature enzymes, and they are efficacious as bactericides in topical formulations; however, the oral administration of metallic colloids, in particular colloidal silver protein, has been reported to have toxic effects (Fung and Bowen, 1996). In addition, a lot of commercial metal colloids are sold as nutritional supplements, and most have been rebranded as “nano” to increase consumer interest. Although clinical concern about the use of colloidal metallic compounds is longstanding, their effects on laboratory tests have not been investigated. These particles are of particular concern because their small size allows high oral bioavailability, accumulation within the blood, and excretion through the kidneys (Hillyer and Albrecht, 2001). Park et al. reported that nanosized nutraceuticals exhibited no major interferences with the tests examined. Minor interferences were noted in an LD assay on the Vitros as well as a reagent strip assay for hemoglobin. Clinical laboratories should remain vigilant for possible nanoparticle interference, as these structures, with their diverse physical and chemical properties, are being used or advocated for use in a broad range of drug delivery applications and as imaging agents (Park et al., 2006). Microfluidization produces a tighter particle size distribution than that of traditional valve homogenization. It also enhances product texture and mouthfeel. Microfluidization has already been successfully used in the production of salad dressing, syrups, chocolate and malted drinks, flavor oil emulsions, creams, yoghurts, fillings, and icings (Swientek, 1990). Microfluidics has recently introduced a multiple stream mixer reactor technology that can process at pressures of up to 2700 bars. It has been used to produce oxide particles of a few nanometers in size. This new


454


technique for nanoencapsulation could be applied to the production procedures of the food and pharmaceutical industries and thus is attracting much attention. Kwon et al. (2002) used microfluidization at 1000 bars in combination with solvent evaporation for producing poly(methyl methacrylate) nanoparticles between 40 and 260 nm containing encapsulated coenzyme Q10. Results showed an encapsulation efficiency of above 95% and a reduction in both UV and high-temperature-induced drug inactivation with this polymer nanoparticle system. Nanotechnology provides the possibility to alter nutrient intake by broadening the number of enriched and fortified food products. The functional food market is expected to increase as a result of the expansion of nutrient delivery systems through nanotechnology mechanisms. For instance, antioxidant nutrients may be included in nanocomposites, nanoemulsions, nanofibers, nanolaminates, nanofilms, and nanotubes. Although an increase in micronutrient intake from an enhanced food supply in the general population may be beneficial, researchers and scientists in food and nutrition will need to watch closely for any overconsumption of nutrients or signs of toxicity. Moreover, micronutrient imbalances may become significant as more nanotechnology nutraceuticals become available. Interactions between drugs and nutrients will also require careful evaluation. The application of nanotechnology offers many novel vehicles for nutrient delivery systems. Nutrient digestion and absorption may increase or decrease depending on the structural, chemical, and physical states of these vehicles and the nutrients bound within them. A more comprehensive interpretation of the metabolic consequences of nutrients in novel food systems will become available for food and nutrition professionals as nanotechnology applications expand in the food sciences.

24.3.3 TRADITIONAL CHINESE MEDICINES The nanonization of traditional Chinese herbal medicines has attracted much attention recently (Yang et al., 2003). Various approaches have been proposed, such as the use of polymeric microand nanoparticles, micro- and nanoemulsions, or liposomes (Singla et al., 2002). Nanoparticles based on solid lipids have also been demonstrated as a promising alternative drug delivery system (Miglietta et al. 2000; Cavalli et al., 2001; Chen et al., 2001; Serpe et al., 2004). Nanospheres are particles with a matrix-type structure in which the active ingredient is dispersed throughout, whereas nanocapsules have a polymeric membrane and an active ingredient core. Nanoparticle systems with average particle size slightly above the 100 nm border have also been reported in literature, including nanonized curcuminoids (Tiyaboonchai et al., 2007), paclitaxel (Arica Yegin et al., 2006), and praziquantel (Mainardes and Evangelista, 2005) which have mean particle sizes of 450, 147.7, and even higher than 200 nm, respectively. Hence, in the food and drug systems, nanoparticles could also be defined as being submicronic (rotective, 455 lipid. 457 Salvia miltiorrhiza, 454 stabilizers, 457 Nanospheres. 454 Nanotechnology, 447 applications, 448 bioactive components delivery, 451-452 drug delivery systems, 456 global climate issues, 457-458 nanoparticle formulation, 454 principle, 447 public perception. 450 regulations, 450-451 traditional Chinese medicines, 454 Nanotechnology foods, 448. See also Food biotechnology; Functional foods acceptance. 450 functional foods. 451,455, 456 functional food ingredients design, 452 importance, 449 microencapsulation, 451 nutraccuticals. 453, 455 1-Naphthaleneacetic acid (NAA). 72 National Institutes of Health (NIH). 525 Natto. 332. See also Functional food: Nattokinase (NK) functions, 333 nattokinase (NK), 333 preparation, 335 Natto bacteria. See Bacillus.subtitis-var.natto Nattokinase (NK). 333. 334,338 activities. 336 activity unit. 337 animal studies. 338-339, 341-342 antiplatelet activity, 344 assay, 337, 337 bioavailability, 338 blood viscosity. 344 B.subtilis var.natto. 333 efficacy, 340 fibrinolytic activity, 334 fibrinolytic effect, 342-343 fibrinolytic enzyme, 333 human studies, 339-340,342 hypertension reduction, 343-344 mutagenicity, 339 NSK-SD production. 335, 336 pathogenicity study, 339 primary structure. 333 production, 335 safety, 338 scope. 344-345

Index vitamin K2 concentrations, 337 in vitro assessment, 341 Natural dietary components, 256 Natural killer (NK), 54 cell activity, 56 Natural toxicants. See Antinutrients NDI. See New dietary ingredient (NDI) Neomycin phosphotransferase (nptll) gene, 147 Neural tube defects. 118 New dietary ingredient (NDI). 96. 284 NHB. See Northern highbush (NHB) Nif genes, 199 NIH. See National Institutes of Health (NIH) Nitric oxide (NO), 57,96 Nitrogen fixation, 198-199 ability, 148, 200 asymbiotic bacteria, 200 genetic manipulations, 199 Gluconacetobacier diazotrophicus, 199 microorgansms, 200, 201 nif genes isolation. 199 organisms, 198-199 requirement, 199 Nitrosoguanidine (NTG), 269,293 NK. See Nattokinase (NK): Natural killer (NK) NLEA. See Nutrition Labeling and Education Act (NLEA) NMR. See Nuclear magnetic resonance (NMR) NO. Sec Nitric oxide (NO) Nod genes, 199 Nonphotochemical quenching. See Xanthophyll-cycle Nonthermal treatments, 425,497 C0 2 . 438 PEF process. 471 Northern highbush (NHB), 64 No-till farming, 202 Novel foods, 284, 449, 514,536. See also Food biotechnology Novel proteins safety. 514 bioinformatics. 515 dietary intake. 516 hazard characterization. 516 HOSU, 515 mode of action, 514 stability, 515 nptll gene. See neomycin phosphotransferase (nplll) gene NTG. See Nitrosoguanidine (NTG) Nuclear magnetic resonance (NMR), 272 Nutraceuticals, 252,280,453. See also Food biotechnology; Functional foods; Nanotechnology foods apoptosis. 256 biological systems, 253 carotcnoids, 289 diseases. 256 extraction, 416 foods, 4 genes regulation. 252, 253 metal colloids, 453 methods. 252 microalgal,281,281,307 microfluidization, 453 miRNA, 252-253 nutritional requirements, 252

Index post-translational modification, 253 relationship. 259 Nutrigenomics, 185,252-253,258 Nutriments, 127 N-acetylglucosamine, 132, 133-134 Ala-Gin, 129-130 CDP-cholinc. 134-135 GSH. 128. 129 hyaluronic acid (HA), 131, 132, 133 hydroxyproline, 130-131, 132 production, 128 Nutritional genomics. See Nutrigcnomics "nutritionally enhanced" foods, 509 Nutrition Labeling and Education Act (NLEA). 523

Obligate autotroph, 295. See also Autotrophic nutrition Oenology, 400. See also Viticulture Office of the Gene Technology Regulator (OGTR), 534 OGTR. See Office of the Gene Technology Regulator (OGTR) OHV-2. See Ovine herpesvirus-2 (OHV-2) Oil(s), 11 arachidonic acid, 106 aslaxanthin, 320-322 biomass fuels. 106 and biotechnology, 104. 162-165 canola oil extraction, 14 inDHAandARA,43,422 dietary, 106 crucic acid. 13 by fermentation, 44, 106 fish, 43 y-linolenic acid, 106 healthier cooking, 13-14 healthier FA profile, 12 microalgal. 281 MLCT. 109 modified. 162 oleic acid-rich. 105

565 Oligonol, 91-95 absorption, 95 antifatiguc effect. 96. 97.98 beauty effects, 99 blood flow improvement. 96 blood polyphenol level. 95 flavanols and proanthocyanidins, 94 functions, 96 safety assessments, 95, 96 visceral fat reducing action, 98 Oligosaccharides, 33 active ingredients development, 33 FOSHU approval, 36 fructo-oligosaccharides production, 34 gentiobiosc, 35 GOS preparation, 34 IMO production. 34 lactulose synthesize, 33-34 raffinose production, 34 xylo-oligosaccharid&s preparation, 34 One unit (1FU), 337 Open pond microalgal cultivation, 300. See also Microalgal production methods drawbacks. 301 Dunaliclla salina, 301 Haematococcus, 301 raceway ponds, 300 Spindina, 300 Opioid peptides. 32 Organic fertilizers. 201 Organization for Vine and Wine (OIV), 393 Oryza sativa. See Rice Output traits, 518 claims, 527 crops GM. 519-521 Ovine hcrpcsvirus-2 (OHV-2), 243 Oxidative stress FA deficiency. 118 gastric, 116-117 models, 115 theory of aging, 114 by ultraviolet ray. 99

palm, 42

PUFA, 422 rapeseed, 13 SFAs. 13 soybean. 105 stearic acid rich. 12-13 Thalassiosira pscudonana. 12 thermal stability, 13 transcsterification, 105-106 Oilseed crops gene transfer. 165 improving FA, 162 LC-PUFA content. 164 MCFA content, 162 modified oils, 162 oleic acid content, 164 saturated fatty acid content, 163 OIV. See Organization for Vine and Wine (OIV) 2-0-01eoyl-l-0-palmitoyl-3-0-stearoyIglycerol (POS). 42 2-0-Oleoyl-3-0-palmitoyl-l-0-stearoylglycerol (POS), 42

p PAI-1. See Plasminogen activator inhibitor type 1 (PAI-1) PAL. See Phenylalanine ammonia-lyase (PAL) Palmitic acid, 105-106 1.3-di-0 Palmitoyl-2-O-oleoylglycerol (POP), 42 Parasitic witch weed, 202

Parasporal crystals, 206. See also Bacillus thuringiensis Parietal adsorption capacity, 397, 400 Parkinson's disease. 118 PAs. See Proanthocyanidins (PAs) Pasteurization, 498 Pathogenesis-related (PR) proteins, 391,392 Botrytis cinema. 392 PBN. See Prcmarkct biotechnology notification (PBN) PCR. See Polymerase chain reaction (PCR) PCV-2. See Porcine circovirus-2 (PCV-2) Pea, 139, 142 a-amylase gene, 142 coat protein gene, 143 immature cotyledons, 142

566 Pea (Continued) PEMV, 143 PSbMV, 143 transgenic plants. 142, 143 Pea Enation Mosaic Virus (PEMV), 143 Pea seed borne mosaic virus (PSbMV), 143 Pcctinascs. 394. 466 anthocyanin recovery. 468 Aspergillus niger, 466 berry juices. 467 blackcurrant, 467,468 commercial, 466 haze-active proteins, 468-469 pectinex maceration, 467-468 preparation. 466. 467.468 relative glycosidase activities. 467 side activities, 468 PER See Pulsed electric field (PEF) PEG. See Polyethylene glycol (PEG) PEMV. See Pea Enation Mosaic Virus (PEMV) Peptides, ACE activity, 31. 32 Pesticide, 205, 211,534, 535 Pesticide resistance. 205 agricultural productivity. 205 Bacillus thuringiensis (BT), 206-207 bioinsecticides, 206 biolcch crops, 208, 210 commercial use. 208. 209 genetic engineering strategies. 205-206 insect-protected crops, 209 insect resistance, 206 pfe genes. See Ferritin (pfe) genes PGA. See Poly-y-glutamic acid (PGA) pH, 428 Bacillus subtilis, 428 cellular activity. 428 Phaffia rhodozyma* 322,418. 419 astaxanthin, 418 two-stage extraction, 421 Phaseolus vulgaris. 143, 147 a-amylase gene, 142 Phenylalanine ammonia-lyase (PAL). 190 Phenylpropanoid. 181. 182. 183. 186. 187-189 anthocyanins, 184,188 biosynthetic pathway, 181, 182 CGA, 184 coumarins, 183 flavoncs. 184, 187 flavonoid. 181, 183. 184-185, 189 flavonots. 184, 187 lignans, 182, 183, 187 lignins, 182,183 metabolic engineering, 186-189 pas. 184 resveratrol. 183-184, 187 stilbenes. 183 Phenylpropanoid biosynthesis. 189 allele-specific inhibition, 189 downrcgulalion, 190 tissue-specific inhibition. 189 pHi. See Intracellular pH (pHi) Phosphate-solubilmng bacteria. 201 Phosphinothricin, 203-204 Phosphorylated oligosaccharides (POs), 35

Index Photobioreactor, 283, 293, 301-306, 315. See also Fcrmcntcrs air-lift reactors. 305 areal productivity, 302 bubble column. 304. 305 Chaetoceros caleitrans, 317 computational fluid dynamics analysis. 317 dome-type, 316 flat-panel reactors, 303,305 pipe-type, 316 productivity in outdoor, 302 steam sterilizable reactor. 305 tubular reactors, 302, 303. 304 verticalflat-plate-type,318 volumetric productivity, 301-302 Yamaha high-efficiency. 318 Photorcspiration, 2%. See also Photosynthesis Photosynthesis, 295 antenna complexes, 295 dark reactions, 295, 296 efficiency, 297 electron transport chain, 296 light intensity, 297 light reactions. 295 ribu lose-diphosphate. 2% thylakoids. 296 Photosystem II (PSII), 290. See also Photosynthesis phyA. See phytase (phyA) genes Phycobiliprotcins. 418 phytase (phyA) genes, 9 phytochemicals. 10-11 alliums consumption. 11 composition, 62, 65,68, 69 compounds efTect, 61 epidemiologic studies, 11 traits, 68, 70 Phytoene desaturase. 166 Phytoene synthase (psy), 8, 165. 166. 288 Phytosiderophores. 10 Pigeon pea, 139. 143 Cattosobruchus maculates, 143 regeneration. 143 transgenic plants, 143 Piperine, 86 Plant breeding, 4. 140 Plant expression platforms, 169 Plant insecticidal genes. 209 Plant regeneration, 140, 141-143 genes introduction. 144 pathways. 143-144 somatic embryogenesis, 144 Plants with novel traits (PNT), 531 Plasminogen activator inhibitor type 1 (PAI-1), 334 pmf. See Proton motive force (pmf) PNT. See Plants with novel traits (PNT) Polyethylene glycol (PEG). 140 Poly-Y-glutamic acid (PGA). 335. 345 Polymerase chain reaction (PCR), 72, 148 Polyphenol oxidase (PPO). 469 Polyphenols. 81, 91,95 Polysaccharide hydrolases, 393 Polyunsaturated fatty acid (PUFA), 12, 164,286.422 processing. 422—423

Index producing organisms, 422 recovery. 423 Polyunsaturated oils hydrogenation, 163 Poor man's meat, the, 139 POP. See l,3-di-0-palmitoyl-2-Oo!eoylglycerol (POP) Porcine circovirus-2 (PCV-2), 242 POS. See 2-O-oIcoyl-l-0-palmitoyl-3-0-stcaroylglyccrol; 2-0-oleoyl-3-0-palmitoyl-l-0stearoylglycerol (POS) POs. See Phosphorylated oligosaccharides (POs) Post-translational modification, 253 Potato. 160, 213 PP. See Precautionary principle (PP) PPO. See Polyphenol oxidase (PPO) Prawn muscle hydrolysate, 33 Prebiotics, 33, 424 Precautionary principle (PP), 538 Premarket biotechnology notification (PBN), 508 Presbycusis. See Age-related hearing loss (AHL) Pressurized C02 treatment. See also Dense C02 Proanthocyanidins (PAs). 63. 91. 181. 188-189 low-molecular weight conversion, 91-92 thiolysis method, 91-92 Probiotics.351,362,485 antibiotic tolerance, 363-364 applications, 363 bacteria, 486. 487, 488 commercial. 485 continuous culture. 373. 374 doubtful strains, 492 functional properties. 363 genetic manipulation, 370 health effects, 491 high-throughput screening method, 373 ICT, 374-380 intrinsic technological properties. 370 iMctobacillus, 486, 487-488 osmotic stress, 369 oxidative stress, 365-366 risky strains, 492 safe strains. 492 safety. 363. 492 selection, 363,492 stresses, 364-370 sublethal stresses application, 371-373 technological criteria. 364 Probiotics. benefits. 488 anticarcinogens. 490 antimutagens. 490 diarrhea, 489 immune system stimulation, 491 inflammatory bowel disease, 491 lactose metabolism, 489-490 serum cholesterol. 491 Procyanidin. 93.94 Programmed cell death. 15,256 Proline 4-hydroxylase. 131 L-Proline hydroxy lation, 131 Propionic acid, 268 metabolic pathways. 267 Propionibacteriunu 268 Proteinase inhibitors. 207. 208 Proteins, 14, 206, 514 corn, 14

567 programmed cell death, 15 QMP, 14-15 synthetic methionine. 15-16 Proteomic analysis. 168 Proteomics, 171, 185-186,401,546. SeealsoDNAchip Proton motive force (pmf). 366 Provitamin A, 165-166. 171. See P-carotcnc. See also Carcinoids; Vitamin A C, 40. 41 PR proteins. See pathogenesis-related (PR) proteins PRV. See Pseudorabies virus (PRV) PSI1. See Photosystem II (PSII) PSbMV. See Pea seed borne mosaic virus (PSbMV) Pseudorabies virus (PRV), 242 psy. See phytocne synthase (psy) PUFA. See Polyunsaturated fatty acid (PUFA) Pulse crops. 139, 140, 148 genetically modified crops. 140 plant breeding, 140 transformation, 145-146 transgenic technology, 140 Pulse proteins, 139 Pulsed electric field (PEF). 427. 470. See also Nonthermal—inactivation method carotenoid retention. 472 factors affecting, 470 heterogeneity, 471 microbial inactivation. 470 microcidal activity, 471

plant material. 471 sterilizing fruit juices. 471

Q QA. See Quality assurance (QA) QacT. See Quaternary ammonium compound transport (QacT) QMP. See Quality maize protein (QMP) QOL. See Quality of life (QOL) QTLs. See Quantitative trait loci (QTLs) Quality assurance (QA). 241 GMP conditions, 320 management quality assurance standards, 285 microbial food, 283 Quality maize protein (QMP), 14 benefits, 15 hybrid. 15 Quality of life (QOL), 53-54,323 Quantitative trait loci (QTLs), 245 Quaternary ammonium compound transport (QacT). 369 R Rabbiteye(RE),64 Rabies, 240 combined rabies vaccines. 242 glycoprotein. 241 pseudorabies virus (PRV). 242 rabies virus. 240 vaccine production, 240, 241 veterinary rabies vaccine. 241 Raffinose, 33, 34

Index

568 Rapcsccd. 13. 105. 158 lauric acid-rich, 105 oil. 13 Ratings of perceived exertion (RPE), 98 RBC. See Red blood cell (RBC) RDA. See Recommended daily allowance (RDA); Recommended Dietary Allowance (RDA) RDI. See Reference daily intake (RDI) RDNA. See Recombinant deoxyribonucleic acid (rDNA); ribosomal deoxyribonucleic acid (rDNA) RE. See Rabbiteye (RE) Reactive oxygen species (ROS), 114, 291, 365,455. See also Antioxidants aging, 114 p-carotene, 291 cochlear pathology. 114 oxidative stress theory, 114 role. 114 UV-induced, 99, 100 Recombinant deoxyribonucleic acid (rDNA), 508. 509 DNA technology, 4, 205, 547. See Genetic engineering enzymes, 39 insulin. See Humulin. See also Insulin plants, 104-105 strains, 371,400 tRNA. 158-159 Recombinant protein, 169.236. See also Genetic engineering erythropoietin, 237 interferons, 236 Molecular Pharming* 169 pharmaceutical products, 236 recombinant insulin, 236 transgenic animals, 231.236 Recommended daily allowance (RDA), 166.472 Recommended Dietary Allowance (RDA), 8,117 Red blood cell (RBC), 228, 340 folic acid, 117 hemoglobin. 228 iron, 523 serine protease, 340 Red gram. See Pigeon pea Reducing agent, 116 metal colloids, 453 vitamin C. 116 Reference daily intake (RDI). 521 Regenerative medicines. 256-257 categories, 256 miRNA and, 254 Regulations Australia regulations. 534 biotechnology products. 507-527.532-539 biotechnology-derived crops.. 212 Canada regulations. 536 EU regulations. 534-535 functional food, 546-547 Japan regulations, 536 New Zealand regulations. 534 US regulations, 535 Resistance genes a-amylase inhibitor, 208 animals, from, 208

CO protein, 207 CpTI gene, 207-208 higher plants, from. 206, 207 lectins, 208 microorganism derived, 207 plant insecticidal genes, 209 proteinase inhibitors, 207 transgenic plants, 209 types, 206 Rcsvcratrol, 182. 183-184. 187. ê«/joStilbcnc(s) production. 186.190 Vaccinium spp., 63 Retroviral vector method. 228 RgMT genes. See metal lothionein-1 ike (rgMT) genes Rhizobium strains, 200 genetic transformation, 144 nitrogen fixers, 200, 201 nod genes, 199 Ribosomal deoxyribonucleic acid (rDNA). 399 Ribulose-diphosphate, 296 Rice, 5, 161, 168 allergenic protein, 168 endosperm. 166 GLPA. 17 golden rice (GR). 8, 166 iron content. 9 low phytic acid mutant, 17 transgenic, 158 zinc content. 10 Ringing. 453 RNA interference technology, 149 ROS. See Reactive oxygen species (ROS) Rotavirus diarrhea, 489 Roundup, 104, 202. See Glyphosatc RPE. See Ratings of perceived exertion (RPE) Rutin glucosides. 38

s SAAT. See Strawberry—alcohol acyitransferase (SAAT) Saccharides. See also Active hexose correlated compound (AHCC) BE action model, 37 botanical polysaccharides, 54 Ca-Spirulan (Ca-SP). 294 dietary fibers. 35 exopolysaccharides. 377 HBCD preparation. 36 oligosaccharides, 33-35, 36 phosphoryIated oligosaccharides (POs), 35, 36 polysaccharides, 391, 393,396 soybean, 33, 34 xylo-oligosaccharides. 34 Saccharomyces cerevisiae altering properties. 399 p-glycosidase, 396 cell disruption, 437-438 cell wall, 399 cell wall properties, 397 de novo compounds. 397 engineered strains, 400 ethanol, 434 fermentation phenotypes, 397 flocculation, 399-400

Index dor-forming strains, 399 GSH. 128-129 health benefits. 488,489 mannoproteins, 397, 399 role, 396,397 SAG. See Senescence associated gene (SAG) Salmonella typhi murium, 490 EPSPS mutant gene. 203 pressure effect, 432 reverse-mutation assay, 339 TA-100 mutant, 490 Sardine muscle hydrolysatc, 30, 33 Saturated FAs (SFAs), 12 SBPs. See Systolic blood pressures (SBPs) Scanning electron microscopy (SEM), 435-436 SC-C0 2 . solubility in, 409-410 dynamic methods, 412 natural cntraincrs, 412 nutraceuticals, 409,410 polar cntraincrs, 410 pressure and temperature effect, 411 static methods, 412 SC-C0 2 . See Supercritical C0 2 (SC-C02) SC-COjE. See Supercritical carbon dioxide extraction (SC-C02E) SCD. See Stcaroyl-CoA desaturase (SCD) SCF. See Supercritical fluid (SCF) SCFE. See Supercritical fluid extraction (SCFE) SCHF. See So-called healthy food (SCHF) SCNT. See Somatic cell nuclear transfer (SCNT) SCORAD, See Scoring atopic dermatitis (SCORAD) Scoring atopic dermatitis (SCORAD), 323 SDA. See Stcaridonic acid (SDA) Seed proteins. 148 SEM. See Scanning electron microscopy (SEM) Senescence associated gene (SAG), 15 Serum-free medium, 222 SFA. See Subcutaneous fat areas (SFA) SFAs. See Saturated FAs (SFAs) SG neurons. See Spiral ganglion (SG) neurons SGAs. See Steroidal glycoalkaloids (SGAs) SHB. See Southern highbush (SHB) SHR. See Spontaneously hypertensive rats (SHR) SHsp. See Small heal shock proteins (sHsp) SI. See Spleen inhibitor (SI) Significant scientific agreement (SSA). 525 Single nucleotide polymorphisms (SNP). 253 Sir2. See Sirtuin gene (Sir2) Sirtuin gene (Sir2\ 183 Small heat shock proteins (sHsp), 370. See also Chapcrone proteins Smallpox, 238, 239 vaccine production, 238.239 variola virus. 239 variolation, 239 SMCS. See S-methylcysteine sulfoxide (SMCS) S-mcthylcyslcinc sulfoxide (SMCS), 11 Smoking point, 110 SNP. See Single nucleotide polymorphisms (SNP) So-called healthy food (SCHF). 333, 337. See also Nattokinase (NK) SOD. See Superoxide dismutase (SOD) Solatium tuberosum. See Potato Solid content (SS), 70

569 Solid-liquid extraction, 413,414 Solubility. 409-410 determination. 412 mixed-solid systems, 412 modifier, 415, 420 nutraceuticals. 410, 411 temperature effect, 420 Somatic cell nuclear transfer (SCNT). See Cloning Somatic embryogencsis. 141. 144 Viciafaba. 142 Vicia narbonensis. 142, 144 Vigna mungo, 141 SOPs. See Standard operation procedures (SOPs), 3-di-Ostcaroyl-2-O-olcoylglyccrol (SOS) SOS. See I Southern highbush (SHB), 64 Soybean, 105, 140,158 ferritin gene, 9. 167 glyphosate-tolerant, 210 herbicide resistant, 104 hypoallergenic foods. 16 modified fatty acid composition, 105 natto, 332. 335 oil, 42,421, 518

oligosaccharides, 34,36 oral vaccine, 170 proteases, 33 seeds. 16. 170-171 soy protein. 32 storage protein P34, 168 SPI-H. See Digested soy peptides (SPI-H) Spiral ganglion (SG) neurons, 113 Spirulina, 418 biomass. 425 Ca-Spirulan, 294 GLA extraction, 423 health concerns, 285 nutraceutical production, 281 open pond cultivation, 282. 300 phycobiliprotcins. 418

productivity, 298. 302 quality standards for cultured. 284 Spirulina maxima, 421,423 two-stage extraction, 421 Spirulina platens is, 416 ot-tocophcrol from, 422

antioxidant activity. 424 contents. 416 extracts. 424 GLA extraction, 423 Spleen inhibitor (SI), 208 Spontaneously hypertensive rats (SHR), 31, 351 Spore, 426, 498. See also Bacterial morphology; Spore-forming bacteria Alicyclobacillus acidoterretis. 475 Bacillus subtilis, 427 C0 2 cfTcct,427 dehydrated form, 426 germination, 499 high-pressure treatments. 499-500 inactivation, 427. 499 pressure-induced germination, 499-500, 502 Pseuthmonas aeruginosa* All Saccharomyces cerevisiae. 475

Index

570 Spore-forming bacteria, 498. See also Bacterial morphology B.anthracis. 498 B.cereus, 498 B.coagulans, 498 B.subtitis, 498 B. thu ringiensis, 498 C.acetobutyticum, 498 C.botutinum. 498 C.perfringens, 498 C.thermocellum, 498 SS. See Solid content (SS) SSA. See Significant scientific agreement (SSA) STA1, See Slate trait anxiety inventory (STA1) Standard operation procedures (SOPs), 320 State trait anxiety inventory (STAI), 323 Stearidonic acid (SDA), 164 Stearoyl-CoA desaturase (SCD). 21 l,3-di-0-Stearoyl-2-0-oleoylglycerol (SOS), 42 Steroidal glycoalkaloids (SGAs), 168 Stevioside. 41 glucosylation, 42 preparation. 41-42 stevia glycosides. 509 Stilbcne (s), 183 resveratrol, 183, 190 synthases (STSs), 148, 182. 183 Storage pests. 142. 143 Strawberry. 68 alcohol acyltransferase (SAAT), 73 antioxidant activity. 68,70 ascorbic acid degradation, 477 mRNA populations, 72 negative correlation. 70.71 nutritional quality improvement. 70, 71. 74-75 PEF treatment, 471-472 phylochemical composition, 68.69 phytochemical traits, 68,70 total ACY quantification. 69 wild, 71-72 Streptococcus mutans mutans streptococci, 38 surface protein antigen, 170 STSs. See Stilbene-synthases (STSs) Subcutaneous fat areas (SFA), 325 Subtilisin NAT. See Nattokinasc (NK) Sulfonylureas resistance. 204 ALS, 204 herbicide mechanisms. 205 herbicide-resistant plants. 204 transferring mutant genes, 204 Supercritical carbon dioxide extraction (SC-C02E), 408.409 Supercritical C0 2 (SC-C02). 409 advantages. 408.436 entrainer effect, 420 enzymes activity, 429 extraction conditions, 419 flavonoids, 424 higher severity. 420 intracellular substances extraction, 430 lipid extraction. 422-424 mechanical treatment, 419 microbial inactivation, 427, 431 nutraceuticals solubility in. 409

perspectives, 438 prebiotics. 424 pressure, 419 products extracted. 417-418 temperature, 419 tocopherol analysis. 422 two-stage extraction, 421 undesired compound removal, 425 vanillin extraction, 424 Supercritical fluid (SCF), 408. See also Supercritical C02 (SC-C02) advantages, 409 applications, 409 density. 408 disadvantages. 409 extraction set-up. 414 physical properties. 408 processing, 409 solvent properties, 408 Supercritical fluid extraction (SCFE). 412 astaxanthin, 418 Blakeslea trispora, 418 carotenoids extraction. 416, 418, 419 Chloretia pyrenoidosa, 418 CMorella vulgaris. 418 cis-trans isomers extraction, 419 C0 2 flow rates. 415. 420 control extractions. 416 equipment. 414 fermentation metabolites, 412 H.pluvialis, 418 kinetics, 413 lutein, 418 lycopene. 418 major variables affecting, 415 mechanism, 413 modifier, 415 Nannochbropsis gaditana, 418 nutraceuticals from microbial sources. 416 phycobiliproteins, 418 pressure and temperature. 415 Spirit Una, 418 Synechococcus strains, 418 Superoxide dismulasc (SOD), 365, 456 Bifidobacterium longum, 365 Lactococcus lactis* 365 manganese. 114 Symbiotic nitrogen fixers, 200.201. See also Nitrogen fixation biofertilizers, as, 201 diazotrophic microorganisms, 200 green manuring, 200 Rhizobium strains. 200 Synechococcus strains. 418 carotenoids extraction. 419 desorption of pigments, 421 disruption by sonication, 419 systems biology, 186,400, 401 Systolic blood pressures (SBPs), 356

T TA. See Titratablc acidity (TA) TCA cycle. See Tricarboxylic acid (TCA) cycle

Index TEAC. See Trolox equivalent antioxidant capacity (TEAC) TEM. See Transmission electron microscopy (TEM) Tetrahydrofolate (THF), 473 TF transgenes, 191 TFA. See Total fat areas (TFA) TGA. See Therapeutic Goods Administration (TGA) Therapeutic Goods Administration (TGA), 534 THF. See Tetrahydrofolate (THF) Thioctic acid. See a-lipoic acid (LA) Thraustochythrid. See Schizochytrium Thrombus, 333, 334 Tier I safety evaluation. See Novel Proteins Safety Tier II safety evaluation. See Novel Proteins Safety Tissue culture, 223 animal. 220 cell culture, 223 explants culture. 223 organ culture, 223 Tissue softening, 72 Titratable acidity (TA). 70 TNBS. See Trinitrobcnzenesulfonic acid (TNBS) TNF. See Tumor necrosis factor (TNF) Tobacco hornworm, 206, 207, 208 Total ACY. See total anthocyanin content (total ACY) Total anthocyanin content (total ACY), 64, 68 berry fruits, 67 quantification. 69 Total DC. See total dendritic cells (total DC) Total dendritic cells (total DC), 55 Total fat areas (TFA). 325 Total polyphenols (TPH), 68,74 Total soluble protein (TSP), 170 Toxins elimination, 168 antinutricnts. 168 antisense-mediated gene silencing. 168-169 cassava. 168 cyanogenic glucoside synthesis, 169 GM traits, 169 HNL activity, 169 transgenic cassava plants. 169 Toxoids. 238,241 TPH. See Total polyphenols (TPH) Transcriptomics. 185, 401. 546 Transesterification, 104,105,109 application. 105 cacao butter substitution. 105 foaming properties. 110 MLCT. 109 oils and fats, 105 palmitic acid, 105-106 reaction, 105 smoking point, 110 Transformants plant, 144 selection, 147 Transgenesis. See Transgenic technology Transgenic animals, 220, 227. See also Animal biotechnology; Transgenic technology benefits, 232 chicken, 231 fish, 229-231 human gene therapy, 233 mice, 227,230

571 pig, 228 production, 229 recombinant protein, 231-232 Transgenic crops, 73. 147, 202, 209.210. See also Transgenic animals; Transgenic plant development Agrobacterium-medtnled transformation, 140 benefits. 211 black gram, 139, 141 Bt cotton, 210 canola plants. 162 cassava plants. 169 chickpea, 140 fu mom sin, 211 green gram, 139, 142 herbicide-resistant. 204, 211 insect-resistant, 206 lupine, 159 moth bean. 139. 141 no-till crop acreage, 211 pea, 139, 142 pesticides, 211 pigeon pea, 139, 143 Viciafaba. 139. 142 weed management. 210-211 Transgenic plant development. 144. 147. 148 See Transgenic crops abiotic stresses, 148 changing plant architecture, 148-149 grain legumes face. 148 long-duration crops. 149 N2 fixing ability, 148 pulse crops, 148 pulses. 148 RNA interference technology, 149 seed proteins, 148 Transgenic technology, 140, 226 approaches, 71 definition. 227 embryonic stem cell method. 228 microinjection method. 228 principle. 226 procedures, 226 retroviral vector method, 228 techniques. 227 Transmissible spongiform encephalopathies (TSEs), 246 Transmission electron microscopy (TEM). 435 Traveler's diarrhea, 489 Triacylglycerols, 43 lipase, 43 medium-and long-chain, 42 transesterification. 105 Tricarboxylic acid (TCA) cycle. 131.266, 267,295 citric acid, 266

2-oxoglutarate. 131 Trinitrobcnzenesulfonic acid (TNBS), 57 Trolox equivalent antioxidant capacity (TEAC), 68,97,474 antioxidant activity assessment. 62.68 Pearson's correlation and significance, 71 phytochemical composition, 69 TSEs. See Transmissible spongiform encephalopathies (TSEs) TSP. See Total soluble protein (TSP)

572 Tumor necrosis factor (TNF), 324,491 Tumor suppressors. 254. See also miRNA Two-step pressure gradient operation, 421 astaxanthin, 421 process, 421

u UDP. See Uridine 5'-diphosphatc (UDP) UDP-GlcNAc. See 5'-diphosphate-Af-acetylglucosamine (UDP-GlcNAc) UFAs. See Unsaturated FAs (UFAs) Ulmtts americana L. See Elm Ultrasound, 476 drug release, 457 food industry applications. 476 juice processing. 477 microbial load reduction. 476 Ultraviolet (UV) light, 99,477 limitations, 478 microbial inactivation. 477 UN. See United Nations (UN) United Nations (UN). 14. 546 United States Department of Agriculture (USDA). 509, 535,539 University of Wisconsin-Madison (UW), 119 Unsaturated FAs (UFAs), 12 Uridine 5'-diphosphatc (UDP). 86 N-acetylglucosamine (UDP-GlcNAc). 132 HA production, 131-132 USDA. See United States Department of Agriculture (USDA) U V light. See Ultraviolet (U V) light UW. See University of Wisconsin-Madison (UW)

V Vaccine immunopotcntiators. See Adjuvants Vaccine, killed. 244 adjuvants. 244 subunit vaccines, 244 whole organisms, 244 Vaccine, live. 241. 243. 244 infection treatment. 243

less virulent strains, 243 life cycle, 243-244 temperature-sensitive strains, 243 transient infections, 243 Vaccine production, 238 dead or inactivated, 238 equine herpesvirus vaccine (EHV). 240 influenza virus. 239 live or attenuated. 238 rabies, 240 smallpox vaccine, 239 subunit vaccines, 238 Vaccinia-based recombinant glycoprotein (VRG), 241 VAD. See Vitamin A-deficiency (VAD) Vanillin. 411. 424 antioxidant properties, 424 SC-C0 2 extraction, 424 Variolation. 239

Index Variety, 513n Variola virus. 239 VAS. See Visual analog scale (VAS) VC. See Vitamin C (VC) VE. See Vitamin E(VE) Very long chain PUFAs (VLCPUFAs), 12 Veterinary vaccines, 240. See also Vaccine production; Viral vaccines bacterial vaccines. 241 killed vaccines. 244 live vaccines, 243 viral vaccines 241 VFA. See Visceral fat—areas (VFA) Viciafaba, 139. 142 plant regeneration, 142 Vicia species. 142 Viral vaccines 241 a-herpesviruses, 242 circoviruses, 242

combined vaccines. 242 flaviviruses, 242 influenza. 242 iridoviruses, 242 orbiviruses, 242 paramyxoviruses, 242

parasite vaccines, 243 rabies vaccines, 241 retroviruse. 242 viruses without vaccines, 242 Visceral fat adipokine regulation, 99 areas (VFA), 111,325 clinical trial effects. 98, 99 high-fat diet. 98 oligonol, 99 reducing action, 98 Visual analog scale (VAS), 323 Vitamin A, 8. See also P-carotene archetypal technology, 8 deficiency (VAD), 8. 165-166 GR technology. 8 Vitamin B9. See Folic acid (FA) Vitamin C (VC), 115, 116. See Glycosylated products— L-ascorbic acid glucosidcs deficiency. 116-117 multiple age-associated diseases, 117 reducing agent, 116 Vitamin E (VE). 117,452 a-tocopherol, 117,421 benefits, 432 bioavailability, 117 deficiency. 117 functional foods. 453 Vitamin K2, 345 concentrations in NK products, 337 Vitamin P. See Hesperidin Vitamins, 165. See also Antioxidants 0-carotcnc conversion, 166 fortification routes. 165 Golden Potato. 166 Golden Rice. 166 improving vitamin contents, 165

medium, 221 Vitamin A deficiency, 165-166

573

Index Viticulture, 400 Vitis vinifera. 389. 391. See also Must; Wine polysaccharides, 392 role, 391 VLCPUFAs. See Very long chain PUFAs (VLCPUFAs) VRG. See Vaccinia-based recombinant glycoprotein (VRG)

W Weed management. 202. 210-211 glyphosate-tolerant soybeans, 210 problems, 202 West Nile virus (WNV), 56 WHO. See World Health Organization (WHO) Whole-genome sequencing, 149 Wild-type Lactobacillus (LB-WT). 269 Wine, 389. See also Must; Winemaking chemical constituents. 390 flor formation, 399 Oenococcus oem\ 390 polysaccharides, 392. 393 Saecharomyees cerevisiae* 390 Vitis vinifera. 389 Winemaking biological spectral profiling, 401 chcmomctrics, 401 fining-rclatcd treatments, 398 future trends, 400 processing and aroma. 393 remove off-flavor compounds, 399 stages. 400 yeast cell wall, 397 yeast strains, 393 WNV. See West Nile virus (WNV) World Health Organization (WHO). 239

World Trade Organization (WTO), 533 WTO. See World Trade Organization (WTO) X Xanthophyll. 288.321. See also Astaxanthin: Carotenoids: Chlorophyll cycle. 290 humans, in, 321 Xenotransplantation, 227, 232, 233,234. See also Transgenic technology animal organ donors. 235 drawbacks. 235 historical perspectives. 234—235 hyperactive organ rejection, 235 Y Yamaha Motor Co. Ltd. 313 astaxanthin manufacturing process, 319-320 dome-type photobioreactor, 316 Fukuroi Factory II, 318-319 GMP-gradc astaxanthin oil product, 321 high-efficiency photobioreactors, 318 H.pluvialis mass cultivation. 317 microalgae commercial production. 316 microalgal biotechnology, 314 pipe-type photobioreactor, 316 quality assurance system, 320 vertical flal-plate-type photobioreactor, 318

z Zea mays. See Maize Zinc, 10 cereal enrichment, 10 deficiency. 10, 167

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