Biotechnology in Functional Foods and Nutraceuticals

© 2010 Taylor and Francis Group, LLC

© 2010 Taylor and Francis Group, LLC

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

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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

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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

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© 2010 Taylor and Francis Group, LLC

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

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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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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

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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.

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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.

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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

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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

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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

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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

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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

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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

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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

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© 2010 Taylor and Francis Group, LLC

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.

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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

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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

Biotechnology in Functional Foods and Nutraceuticals

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

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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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Biotechnology in Functional Foods and Nutraceuticals

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.

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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

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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

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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.

© 2010 Taylor and Francis Group, LLC

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

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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.

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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.

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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

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Biotechnology in Functional Foods and Nutraceuticals

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

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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.)

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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

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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

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The chemical structures of terpenoid sweeteners.

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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.

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Functional Foods and Biotechnology in Japan (b) 200 a

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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

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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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Jeffrey, G.A., Huang, D.B., Pfeffer, P.E., Dudley, R.L., Hicks, K.B., and Nitsch, E., 1992. Crystal structure and NMR analysis of lactulose trihydrate. Carbohyd. Res. 226: 29–42. Jinsmaa, Y. and Yoshikawa, M. 1999. Enzymatic release of neocasomorphin and beta-casomorphin from betacasein. Peptides 20: 957–962. Kagawa, K., Matsutaka, H., Fukuhama, C., Fujino, H., and Okuda, H., 1998. Suppressive effect of globin digest on postprandial hyperlipidemia in male volunteers. J Nutr. 128: 56–60. Kagawa, K., Matsutaka, H., Fukuhama, C., Watanabe, Y., and Fujino, H., 1996. Globin digest, acidic protease hydrolysate, inhibits dietary hypertriglyceridemia and Val-Val-Tyr-Pro, one of its constituents, possesses most superior effect. Life Sci. 58: 1745–1755. Kamasaka, H., Inaba, D., Minami, K., Too, K., Nishimura, T., Kuriki, T., Imai, S., Hanada, N., and Yonemitsu, M., 2004. Application of phosphoryl oligosaccharides of calcium (POs-Ca) for oral health. J. Appl. Glycosci. 51: 129–134. Kamasaka, H., To-o, K., Kusaka, K., Kuriki, T., Kometani, T., Hayashi, H., and Okada, S., 1997. The structures of phosphoryl oligosaccharides prepared from potato starch. Biosci. Biotechnol. Biochem. 59: 238–244. Kamasaka, H., Uchida, M., Kusaka, K., Yoshikawa, K., Yamamoto, K., Okada, S., and Ichikawa, T., 1995. Inhibitory effect of phophorylated oligosaccarides prepared from potato starch on the formation of calcium phosphate. Biosci. Biotechnol. Biochem. 59: 1412–1416. Kaneko, T., Kohmoto, T., Kikuchi, H., Shiota, M., Iino, H., and Mitsuoka, T., 1994. Effects of isomalto-oligosaccharides with different degrees of polymerization on human fecal bifidobacteria. Biosci. Biotechnol. Biochem. 58: 2288–2290. Kasai, M., Nosaka, N., Maki, H., Negishi, S., Aoyama, T., Nakamura, M., Suzuki Y., et al., 2003. Effect of dietary medium- and long-chain triacylglycerols (MLCT) on accumulation of body fat in healthy humans. Asia Pac. J. Clin. Nutr. 12: 151–160. Kato, H., 2008. Nutrigenomics: The cutting edge and Asian perspectives. Asia Pac. J. Clin. Nutr. 17: 12–15. Kawashima, H., 2005. Arachidonic acid and dihomo-γ-linolenic acid-microbial production and physiological function. Foods Food Ingredients J. Jpn. 210: 106–114 (in Japanese). Kawashima, H., Akimoto, K., Higashiyama, K., Fujikawa, S., and Shimizu, S., 2000. Industrial production of dihomo-γ-linolenic acid by a Δ5 desaturase-defective mutant of Mortierella alpina 1S-4 fungus. J. Am. Oil Chem. Soc. 77: 1135–1138. Kitao, S. and Sekine, H., 1994. a-d-Glucosyl transfer to phenolic compounds by sucrose phosphorylase from Leuconostoc mesenteroides and production of α-arbutin. Biosci. Biotechnol. Biochem. 58: 38–42. Kitao, S., Ariga, T., Matsudo, T., and Sekine, H., 1993. The syntheses of catechin-glucosides by transglycosylation with Leuconostoc mesenteroides sucrose phosphorylase. Biosci. Biotechnol. Biochem. 57: 2010–2015. Kitao, S.T., Matsuda, T., Sitoh, M., and Sekine, H., 1995. Enzymatic syntheses of two stable (−)-epigallocatechin gallate-glucosides by sucrose phosphorylase. Biosci. Biotechnol. Biochem. 59: 2167–2169. Kohama, Y., Matsumoto, S., Oka, H., Teramoto, T., Okabe, M., and Mimura, T., 1988. Isolation of angiotensinconverting enzyme inhibitor from tuna muscle. Biochem. Biophys. Res. Commun. 155: 332–337. Kometani, T., Terada, Y., Nishimura, T., Takii, H., and Okada, S., 1994. Transglycosylation to hesperidin by cyclodextrin glucanotransferase from an alkalophilic Bacillus species in alkaline pH and properties of hesperidin glycosides. Biosci. Biotechnol. Biochem. 58: 1990–1994. Kurakake, M., Sumida, T., Masuda, D., Oonishi, S., and Komaki, T., 2006. Production of galacto-manno-oligosaccharides from guar gum by β-mannanase from Penicillium oxalicum SO. J. Agric. Food. Chem. 54: 7885–7889. Kurosu, J., Sato, T., Yoshida, K., Tsugane, T., Shimura, S., Kirimura, K., Kino, K., and Usami, S., 2002. Enzymatic synthesis of α-arbutin by α-anomer-selective glucosylation of hydroquinone using lyophilized cells of Xanthomonas campestris WU-9701. J. Biosci. Bioeng. 93: 328–330. Kusama, S., Kusakabe, I., Nakamura, Y., Eda, S., and Murakami, K., 1986. Transglucosylation into stevioside by the enzyme system from Streptomyces sp. Agric. Biol. Chem. 50: 2445–2451. Lakhanpal, P., 2007. Quercetin: A versatile flavonoid. Intnet J. Med. Update 2(2). Available at http://www. akspublication.com/Paper05_Jul-Dec2007_.pdf Mandai, T., Yoneyama, M., and Sakai, S., 1993. Japanese PatentH5-117290. Maruyama, S. and Suzuki, H., 1982. A Peptide inhibitor of angiotensin I converting enzyme in the tryptic hydrolysate of casein. Agric. Biol. Chem. 46: 1393–1394. Maruyama, S., Nakagomi, K., Tomizuka, N., and Suzuki, H., 1985. Angiotensin I-converting enzyme inhibitor derived from an enzymatic hydrolysate of casein. II. isolation and bradykinin-potentiating activity on the uterus and the Ileum of rats. Agric. Biol. Chem., 49: 1405–1409.

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Matsufuji, H., Matsui, T., Seki, E., Osajima, K., Nakashima M., and Osajima, Y., 1994. Angiotensin I-converting enzyme inhibitory peptides in an alkaline protease hydrolysate derived from sardine muscle. Biosci. Biotechnol. Biochem. 58: 2244–2245. Matsui, T., Yoshimoto, C., Osajima, K., Oki, T., and Osajima, Y., 1996. In vitro survey of α-glucosidase inhibitory food components. Biosci. Biotechnol. Biochem. 60: 2019–2022. Matsui, T., Yukiyoshi, A., Doi, S., Ishikawa, H., and Matsumoto, K., 2006. Enzymatic hydrolysis of ethanolinsoluble proteins from royal jelly and identification of ACE inhibitory peptides. Nippon. Shokuhin. Kagaku. Kogaku. Kaishi. 53: 200–206 (in Japanese). Matsumoto, K., Kobayashi, Y., Tamura, N., Watanabe, T., and Kan, T., 1989. Production of galacto-oligosaccharides with β-galactosidase. Denpun Kagaku. 36: 123–130 (in Japanese). McCleary, B.V. and Matheson, N.K., 1983. Action patterns and substrate binding requirements of β-mannanase with mannosaccharides and mannan-type polysaccharides. Carbohyd. Res. 119: 191–219. Mead, C.G. and Finamore, F.J., 1969. The occurrence of ascorbic acid sulfate in the brine shrimp, Artemia Salina. Biochemistry. 8: 2652–2655. Meisel, H. and Frister, H., 1989. Chemical characterization of bioactive peptides from in vivo digests of casein. J. Dairy Res. 56: 343–349. Meulenbeld, G.H., Zuilhof, H., van Veldhuizen, A., van den Heuvel, R.H.H., and Hartmans, S., 1999. Enhanced (+)-catechin transglucosylating activity of Streptococcus mutans GS-5 glucosyltransferase-D due to fructose removal. Appl. Environ. Microbiol. 65: 4141–4147. Middleton Jr, E. and Kandaswami, C., 1993. Impact of plant flavonoids on mammalian biology. In: J.B. Harborne (Ed.), The flavonoids: Advances in Research Since 1986, pp. 619–652. London: Chapman and Hall. Mitsuoka, T., 2002. Prebiotics and intestinal flora. J. Intest. Microbiol. 16: 1–10 (in Japanese). Miyoshi, S., Ishikawa, H., Kaneko, T., Fukui, F., Tanaka, H., and Maruyama, S., 1991. Structures and activity of angiotensin-converting enzyme inhibitors in an alpha-zein hydrolysate. Agric. Biol. Chem. 55: 1313–1318. Mizota, T., Suzawa, I., Seki, N., Tamura, Y., and Shimumura, S., 1994. Solubility of lactulose trihydrate. Carbohyd. Res. 263: 163–166. Montgomery, E.M. and Hudson, C.S., 1930. Relations between rotating power and structure in the sugar group. Synthesis of new disaccharide ketoses from lactose. J. Am. Chem. Soc. 52: 2101–2111. Moon, Y.H., Lee, J.H., Ahn, J.S., Nam, S.H., Oh, D.K., Park, D.H., Chung, H.J., Kang, S., Day, D.F., and Kim, D., 2006. Synthesis, structure analyses, and characterization of novel epigallocatechin gallate (EGCG) glycosides using the glucansucrase from Leuconostoc mesenteroides B-1299CB. J. Agric. Food Chem. 54: 1230–1237. Muto N., Suga, S., Fujii K., Goto, K., and Yamamoto, I., 1990. Formation of a stable ascorbic acid 2-glucoside by specific transglucosylation with rice seed α-glucosidase. Agric. Biol. Chem. 54: 1697–1703. Nagao, T., Kawashima, A., Sumida, M., Watanabe, Y., Akimoto, K., Fukami, H., Sugiura A., and Shimada, Y., 2003. Production of structured TAG rich in 1,3-capryloyl-2-arachidonoyl glycerol from Mortierella single-cell oil. J. Am. Oil Chem. Soc. 80: 867–872. Nagao, T., Watanabe, H., Goto, N., Onizawa, K., Taguchi, H., Matsuo, N., Yasukawa, T., Tsushima, R., Shimasaki, H., and Itakura, H., 2000. Dietary diacylglycerol suppresses accumulation of body fat compared to triacylglycerol in men in a double-blind controlled trial. J. Nutr. 130: 792–797. Nagaoka, S., Awano, T., Nagata, N., Masaoka, M., Hori, G., and Hashimoto, K., 1997. Serum cholesterol reduction and cholesterol absorption inhibition in CaCo-2 cells by a soy protein peptic hydrolysate. Biosci. Biotechnol. Biochem. 61: 354–356. Nagaoka, S., Futamura, Y., Miwa, K., Awano, T., Yamauchi, K., Kanamaru, Y., Tadashi, K., and Kuwata, T., 2001. Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin. Biochem. Biophys. Res. Commun. 281: 11–17. Nagaoka, S., Miwa, K., Eto, M., Kuzuya, Y., Hori, G., and Yamamoto, K., 1999. Soy protein peptic hydrolysate with bound phospholipids decreases micellar solubility and cholesterol absorption in rats and caco-2 cells. J. Nutr. 129: 1725–1730. Nakagawa, H., Yoshiyama, M., Shimura, S., Kirimura, K., and Usami, S., 1996. Anomer selective formation of l-menthyl α-D-glucopyranoside by α-glucosidase-catalyzed reaction. Biosci. Biotechnol. Biochem. 60: 1914–1915. Nakagawa, H., Yoshiyama, M., Shimura, S., Kirimura, K., and Usami, S., 1998. Anomer-selective glucosylation of l-menthol by yeast α-glucosidase. Biosci. Biotechnol. Biochem. 62: 1332–1336. Nakahara, K., Kontani, M., Ono, H., Kodama, T., Tanaka, T., Ooshima, T., and Hamada, S., 1995. Glucosyltransferase from Streptococcus sorbrinus catalyzes glucosylation of catechin. Appl. Environ. Microbiol. 7: 2768–2770.

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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.

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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

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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.)

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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

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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.)

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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.)

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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.

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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.

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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

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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.

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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

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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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Scalzo, J., Currie, A., Stephens, J., McGhie, T., and Alspach, P., 2008. The anthocyanin composition of different Vaccinium, Ribes and Rubus genotypes. BioFactors 34(1): 13–21. Scalzo, J., Mezzetti, B., Hall, H., and McGhie T., 2004. Comparing methods for evaluation of raspberry’s quality. Euroberry Symposium, Acta Hort. (ISHS) 649: 327–330. Scalzo, J., Miller, S., Edwards, C., Meekings, J., and Alspach, P., 2009. Variation in phytochemical composition and fruit traits of blueberry cultivars and advanced breeding selections in New Zealand. Acta Hort. (ISHS) 810: 823–830. Scalzo, J., Politi, A., Mezzetti, B., and Battino, M., 2005b. Plant genotype affects total antioxidant capacity and phenolic contents in fruit. Nutrition 21(2): 207–213. Schwarz-Sommer, Z., Saedler, H., Sommer, H., Russo, V.E.A. (Ed.), Brody, S., Cove, D. (Ed.), and Ottolenghi, S., 1992. Homeotic genes in the genetic control of flower morphogenesis in Antirrhinum majus. In: Development: The Molecular Genetic Approach, Springer, Heidelberg, pp. 242–256. Sellappan, S., Akoh, C.C., and Krewer, G., 2002. Phenolic compounds and antioxidant capacity of Georgiagrown blueberries and blackberries. J. Agric. Food Chem. 50: 2432–2438. Sesmero, R., Quesada, M.A., and Mercado, J.A., 2007. Antisense inhibition of pectate lyase gene expression in strawberry fruit: Characteristics of fruits processed into jam. J. Food Eng. 79: 194–199. Shin, W.-H., Park, S.-J., and Kim, E.-J., 2006. Protective effect of anthocyanins in middle cerebral artery occlusion and reperfusion model of cerebral ischemia in rats. Life Sci. 79: 130–137. Trainotti, L., Spolaore, S., Ravanello, A., Baldan, B., and Casadoro, G., 1999. A novel E-type endo-beta1,4-glucanase with a putative cellulose-binding domain is highly expressed in ripening strawberry fruits. Plant Mol. Biol. 40(2): 323–332. Tulipani, S., Mezzetti, B., Capocasa, F., Bompadre, S., Beekwilder, J., Ric de Vos, C.H., Capanoglu, E., Bovy, A., and Battino, M., 2008. Antioxidants, phenolic compounds and nutritional quality in different strawberry genotypes. J. Agric. Food Chem. 56(3): 696–704. USDA, 2005. USDA National Nutrient Database for Standard Reference, Release 18, Washington DC, US Department of Agriculture, Agriculture Research Service. USDA, 2007. USDA Database for the Flavonoid Content of Selected Foods, Release 21.1. Available at http:// www.ars.usda.gov/SP2UserFiles/Place/12354500/Data/Flav/Flav02-1.pdf Wada, L. and Ou, B., 2002. Antioxidant activity and phenolic content of Oregon caneberries. J. Agric. Food Chem. 50: 3495–3500. Wang, H., Cao, G., and Prior, R.L., 1996. Total antioxidant capacity of fruits. J. Agric. Food Chem. 44: 701–705. Wang, H., Cao, G., and Prior, R.L., 1997. Oxygen radical absorbing capacity of anthocyanins. J. Agric. Food Chem. 45: 304–309. Wang, S.Y., Chen, C.-T., Sciarappa, W., Wang, C.Y., and Camp, M.J., 2008. Fruit quality, antioxidant capacity, and flavonoid content of organically and conventionally grown blueberries. J. Agric. Food Chem. 56: 5788–5794. Wang, K., Nair, M.G., Strasburg, G.M., Chang, Y.-C., Booren, A.M., Gray, I., and Dewitt, D.L., 1999. Antioxidant and anti-inflammatory activities of anthocyanins and their aglycone, cyaniding from tart cherries. J. Nat. Prod. 62: 294–296. Wang, S.Y., Zheng, W., and Galletta, G.J., 2002. Cultural system affects fruit quality and antioxidant capacity in strawberries. J. Agric. Food Chem. 50: 6534–6542. WHO, 2003. Diet, nutrition and the prevention of chronic diseases. Report of a joint WHO/FAO expert consultation, WHO technical report series 916, Geneva, World health Organization. Wilkinson, J.Q., Lanahan, M.B., Conner, T.W., and Klee, H.J., 1995. Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display. Plant Mol. Biol. 27(6): 1097–1108. Woolley, L.C., James, D.J., and Manning, K., 2001. Purification and properties of an endo-β-1,4-glucanase from strawberry and down-regulation of the corresponding gene, cel1. Planta 214: 11–21. Wu, X. and Prior, R.L., 2005. Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/ MS in common foods in the United States: Fruits and berries. J. Agric. Food Chem. 53: 2589–2599. Wu, X., Beecher, G.R., Holden, J.M., Haytowitz, D., Gebhardt, S., and Prior, R.L., 2006. Concentrations of anthocyanins in common food in the United States and estimation of normal consumption. J. Agric. Food Chem. 54: 4069–4075.

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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

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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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McDougall, G.J. and Stewart, D., 2005. The inhibitory effects of berry polyphenols on digestive enzymes. Biofactors 23(4): 189–195. Mennen, L.I., Sapinho, D., Ho, H., Galan, P., Hercberg, S., and Scalbert, A., 2008. Urinary excretion of 13 dietary flavonoids and phenolic acids in free-living healthy subjects—variability and possible use as biomarkers of polyphenol intake. Eur. J. Clin. Nutr. 62: 519–525. Ness, A.R. and Powles, J.W. 1997. Fruit and vegetables and cardiovascular diseases: A review. Int. J. Epidemiol. 26: 1–13. O’Leary, K.A., Day, A.J., Needs, P.W., Mellon, F.A., O’Brien, N.M., and Williamson, G., 2003. Metabolism of quercetin-7- and quercetin-3-glucronides by an in vitro hepatic model: The role of human β-glucuronidase, sulfotaransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism. Biochem. Pharmacol. 65(3): 479–491. Parada, J. and Aguilera, J.M., 2007. Food microstructure affects the bioavailability of several nutrients. J. Food Sci. 72(2): R21–R32. Prasain, J.K. and Barnes, S., 2007. Metabolism and bioavailability of flavonoids in chemoprevention: Current analytical strategies and future prospectus. Mol. Pharm. 4(6): 846–864. Rahman, I., Biswas, S.K., and Kirkham, P.A., 2006. Regulation of inflammation and redox signaling by dietary polyphenols. Biochem. Pharmacol. 72: 1439–1452. Rahman, M.M., 2008. The pivotal role of anthocyanin as food factor. PhD dissertation, Niigata University of Pharmacy and Applied Life Sciences, NUPALS. Reddy, V.C., Sagar, G.V.V., Sreeramulu, D., Venu, L., and Raghunath, M., 2005. Addition of milk does not alter the antioxidant activity of black tea. Ann. Nutr. Metabol. 49: 189–195. Richelle, M., Tavazzi, I., and Offord, E., 2001. Comparison of the antioxidant activity of commonly consumed polyphenolic beverages (coffee, cocoa, and tea) prepared per cup serving. J. Agric. Food Chem. 49(7): 3438–3442. Roura, E., Andres-Lacueva, C., Estruch, R., Mata-Bilbao, M.L., Izquierdo-Pulido, M., Waterhouse, A.L., and Lamuela-Raventos, R.M., 2007. Milk does not affect the bioavailability of cocoa powder flavonoid in healthy human. Ann. Nutr. Metabol. 51: 493–498. Scalbert, A. and Willamson, G., 2000. Dietary intake and bioavailability of polyphenols. J. Nutr. 130: 2073S–2085S. Scholz, S. and Willamson, G., 2007. Interactions affecting the bioavailability of dietary polyphenols in vivo. Int. J. Vitam. Nutr. Res. 77: 224–235. Serafini, M., Ghiselli, A., and Ferro-Luzzi, A., 1996. In vivo antioxidant effect of green and black tea in man. Eur. J. Clin. Nutr. 50(1): 28–32. Sergent, T., Ribonnet, L., Kolosova, A., Garsou, S., Schaut, A., De Saeger, S., Van Peteghem, C., Larondelle, Y., Pussemier, L., and Schneider, Y.J., 2008. Molecular and cellular effects of food contaminants and secondary plant components and their plausible interactions at the intestinal level. Food Chem. Toxicol. 46(3): 813–841. Setchell, K.D., Brown, N.M., and Lydeking-Olsen, E., 2002. The clinical importance of the metabolite equol—a clue to the effectiveness of soy and its isoflavones. J. Nutr. 132: 3577–3584. Setchell, K.D., Clerici, C., Lephart, E.D., Cole, S.J., Heenan, C., Castellani, D., Wolfe, B.E., et al., 2005. S-equol, a potent ligand for estrogen receptor beta, is the exclusive enatiomeric form of the soy isoflavone metabolite produced by human intestinal bacterial flora. Am. J. Clin. Nutr. 81: 1072–1079. Shahidi, F. and Ho, C.-T., 2005. Phenolic Compounds in Foods and Natural Health Products. ACS symposium series 909, ACS. Shahidi, F. and Naczk, M., 2004. Phenolics in Food and Nutraceuticals. Boca Raton, FL: CRC Press. Singh, M., Arseneault, M., Sanderson, T., Murthy, V., and Ramassamy, C., 2008. Challenges for research on polyphenols from foods in Alzheimer’s disease; Bioavailability, metabolism, and cellular and molecular mechanisms. J. Agric. Food Chem. 56: 4855–4873. Srinivasan, K., 2007. Black pepper and its pungent principle-piperine: A review of diverse physiological effects. Crit. Rev. Food Sci. Nutr. 47: 735–748. Stahl, W., Van den Berg, H., Arthur, J., Bast, A., Dainty, J., Haenen, G., Hollman, P., et al., 2002. Bioavailability and metabolism. Mol. Aspects Med. 23: 39–100. Steinmetz, K.A. and Potter, J.D., 1996. Vegetables, fruit, and cancer prevention: a review. J. Am. Diet Assoc. 96: 1027–1039. Suda, I., Oki, T., Masuda, M., Nishiba, Y., Furuta, S., Matsugano, K., Sugita, K., and Terahara, N., 2002. Direct absorption of acylated anthocyanin in purple-fleshed sweet potato into rats. J. Agric. Food Chem. 50: 1672–1676.

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Tomas-Barberan, F.A., Cienfuegos-Jovellanos, E., Marin, A., Muguerza, B., Gil-Izquierdo, A., Cerda, B., Zafrilla, P., et al., 2007. A new process to develop a cocoa powder with higher flavonoid monomer content and enhanced bioavailability in healthy humans. J. Agric. Food Chem. 55(10): 3926–3935. Tuoie, G.L., Leung, T.M., Hung, M.W., and Fung, M.L., 2007. Green tea polyphenols as an anti-oxidant and anti-inflammatory agent for cardiovascular protection. Cardiovasc. Hematol. Disord. Drug Targets 7: 135–144. Vaidyanathan, J.B. and Wall, T., 2001. Transport and metabolism of the tea flavonoid (−)-epicatechin by the human intestinal cell line Caco-2. Pharm. Res. 18: 1420–1425. Van het Hof, K.H., Kivits, G.A., Weststrate, J.A., and Tijburg, L.B., 1998. Bioavailability of catechins from tea: The effect of milk. Eur. J. Clin. Nutr. 52(5): 356–359. Venables, M.C., Hulston, C.J., Cox, H.R., and Jeukendrup, A.E., 2008. Green tea extract ingestion, fat oxidation, and glucose tolerance in healthy human. Am. J. Clin. Nutr. 87(3): 778–784. Visioll, F. and Hagen, T.M., 2007. Nutritional strategies for healthy cardiovascular aging; focus on micronutrients. Pharmacol. Res. 55: 199–206. Walle, T., 2007a. Methylation of dietary flavones greatly improves their hepatic metabolic stability and intestinal absorption. Mol. Pharm. 4: 826–832. Walle, T., 2007b. Methoxylated flavones, a superior cancer chemopreventive flavonoid subclass? Sem. Cancer Biol. 17(5): 354–362. Willamson, G. and Manach, C., 2005. Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. Am. J. Clin. Nutr. 81: 243S–255S. Wollgast, J. and Anklam, E., 2000. Review on polyphenols in Theobroma cacao: Changes in composition during the manufacture of chocolate and methodology for identification and quantification. Food Res. Int. 33: 423–447. Wu, X., Pittman, H.E. 3rd., Mckay, S., and Prior, R.L., 2005. Aglycones and sugar moieties alter anthocyanin absorption and metabolism after berry consumption in weanling pigs. J. Nutr. 135(10): 2417–2424. Yang, C.S., Sang, S., Lambert, J.D., and Lee, M.J., 2008. Bioavailability issues in studying the health effects of plant polyphenolic compounds. Mol. Nutr. Food Res. 52: S139–S151. Yung, L.M., Leung, F.P., Wong, W.T., Tian, X.Y., Yung, L.H., Chen, Z.Y., Yao, X.Q., and Huang, Y., 2008. Tea polyphenols benefit vascular function. Inflammopharmacology 16(5): 230–234. Zafra-Stone, S., Yasmin, T., Bagchi, M., Chatterjee, A., Vinson, J.A., and Bagchi, D., 2007. Berry anthocyanins as novel antioxidants in human health and disease prevention. Mol. Nutr. Food Res. 51: 675–683. Zjhang, L., Zheng, Y., Chow, M.S., and Zuo, Z., 2004. Investigation of intestinal absorption and disposition of green tea catechins by Caco-2 monolayer model. Int. J. Pharm. 287: 1–12.

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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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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

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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.

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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.

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OH O

HO

OH

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

FIGURE 6.4

OH OH

O

OH OH HO

OH O

The Function of the Next Generation Polyphenol, “Oligonol”

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.)

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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.)

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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.)

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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

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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

Oligonol LEP 20 mg/mL 20 mg/mL

3

a

a

Oligonol LEP 20 mg/mL 20 mg/mL

Relative density (control = 1)

Relative density (control = 1)

3

β-actin

a

2

a

Oligonol LEP 20 mg/mL 20 mg/mL

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

Relative density (control = 1)

a

Relative density (control = 1)

Relative density (control = 1)

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

Oligonol LEP 20 mg/mL 20 mg/mL

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.

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–

–

+

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.

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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.

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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

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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

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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

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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

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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

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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 <10% of intake to adipose and other tissue 8.4 kcal/g

Slow Slow Essential Mainly by lymph duct Once stored and oxidized if needed Equally to adipose tissue, liver, muscle 9.0 kcal/g

CoA is acyl-CoA synthetase. However, no medium-chain fatty acid-specific acyl-CoA synthetase is present in the cytoplasm. Accordingly, medium-chain fatty acids are not utilized as materials of triacylglycerol or phospholipid synthesis. Medium-chain fatty acids transferred to the portal vein are incorporated by the liver, and rapidly β-oxidized and utilized as energy. For the degradation of fatty acids as an energy source, their transport to the site of β-oxidation, the mitochondria, is necessary, for which binding with carnitine is required because long-chain fatty acids cannot pass through the mitochondrial membrane. Thus, carnitine palmitoyltransferase, which binds acyl-CoA to carnitine, is a key lipolytic rate-limiting enzyme. In contrast, medium-chain fatty acids can pass through the mitochondrial membrane without binding to carnitine. Medium-chain fatty acids transported to the liver rapidly enter the mitochondria without the influence of rate-limiting carnitine palmitoyltransferase, and undergo β-oxidation.

7.3.5

LESS BODY FAT ACCUMULATION OF MCT

Many studies reported that MCT led to the accumulation of less body fat than LCT. For example, when rats were fed an MCT-containing diet for four weeks, the visceral fat level was less than half that of rats fed a corn oil diet. Ingle et al. (1999) calculated the actual energy value of MCT from the results of 10 reported animal experiments, being 6.8 kcal/g, about 25% lower than that of general oil. It is well known that MCT does not readily accumulate as body fat in experimental animals, but the body fat accumulation-suppressing effect in humans has not been fully clarified. Thus, we performed a strict, large-scale study on the body fat accumulation-suppressing effect of MCT in humans. A double-blind study was performed in healthy subjects under strict dietary management. Seventy-eight subjects slightly fatter than average [mean body mass index (BMI) = 24.7] ate bread containing 14 g of the test oil as their breakfast every morning. A total of more than 10,000 lunches and suppers were prepared so that all subjects ate identical meals for 12 weeks. Eating between meals and beverages were included in the dietary management, and the subjects were strictly controlled to ingest 2200 kcal/day and 60 g of lipids. The body fat level was measured using the air displacement method for accuracy. Body weight and body fat were more markedly reduced in the MCT group than in the LCT (normal cooking oil) group in the subjects with a BMI of 23 or higher (Tsuji et al., 2001). Medium-chain fatty acids are not only prioritized in terms of their degradation to other fatty acids to generate energy but also increase whole body energy expenditure. When oxygen expenditure was measured in humans who ingested a beverage containing 400 kcal MCT or corn oil, the increase in oxygen expenditure was only 4% in the corn oil group, but 12% in the MCT group. One gram of MCT contains 8.4 kcal, slightly less than in general lipids, and this may be one cause of the suppression of body fat accumulation. However, body fat accumulation differed between rats

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fed the same number of calories in MCT and corn oil diets, indicating that the lower calorific value is not the main cause of the suppression of body fat accumulation.

7.3.6

APPLICATION OF MEDIUM-CHAIN FATTY ACIDS IN FOODS

The nutritional characteristics of medium-chain fatty acids described above were elucidated in the 1950–1960s. The long-term safety of medium-chain fatty acids in laboratory animals and humans was also confirmed in the same period, and a clinical application for maldigestion/malabsorption syndrome was initiated. Currently, MCT is used as powdered oil, cooking oil, therapeutic diets, enteral nutrients, and transfusion solutions. Powdered fat is prepared by coating MCT with dextrin or casein, and used by mixing with foods and beverages to improve fat absorption and to provide an energy supplement. As therapeutic foods, biscuits, cookies, and jelly are sold for calorie supplementation for patients with kidney diseases. MCT-containing enteral nutrients are also used to supplement the energy intake after surgery for digestive organ diseases. MCT has interesting nutrient and physical characteristics (colorless, tasteless, odorless, high solubility, high stability, and low viscosity), and it is also utilized as a base for spices and pigments, a lubricating oil for food-producing machines, and a mold-releasing agent.

7.3.7

APPLICATION AS AN EDIBLE OIL

MCT has disadvantages: it has a low smoking point, and easily foams. We investigated the applicability for cooking and the nutritional characteristics of oils containing MLCTs, aiming for the development of an oil and fat with the superior nutritional characteristics of medium-chain fatty acids yet with the applicability for heating as an edible oil for domestic use. MLCT represents a triacylglycerol composed of medium- and long-chain fatty acids. MLCTcontaining oil (MLCT oil) was prepared by the mixing and transesterification of MCT and LCT (Figure 7.1). Transesterification between MCT and LCT elevated the smoking point (Figure 7.2), and markedly reduced foaming.

7.3.8

LESS BODY FAT ACCUMULATION OF MLCT

The effect of MLCT oil, which is applicable for cooking, on body fat accumulation was investigated in animals and humans. When rats were fed MLCT oil and body fat was measured, the visceral fat tissue weight and carcass fat mass were lower in the MLCT group than in the control group (Takeuchi et al., 2001). Medium-chain triacylglycerol (MCT)

Medium- and long-chain triacylglycerol (MLCT)

MCFA

LCFA

MCFA

LCFA

MCFA

MCFA Transesterification

LCFA

LCFA

LCFA

MCFA

LCFA

MCFA

Long-chain triacylglycerol (LCT)

FIGURE 7.1 The manufacture of MLCT by transesterification. LCFA, long-chain fatty acid; MCFA, medium-chain fatty acid.

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Smoking point (°C)

200 150 Deep-frying temperature

100 50 0

Mixture of MCT and LCT 100

MLCT

Foam height (mm)

75 50 25 0 Mixture of MCT and LCT

MLCT

FIGURE 7.2 The improvement of the smoking point and foaming properties by transesterification. MLCT is a transesterified oil of MCT and LCT. The smoking point was measured using a Cleveland Open Cup flash point tester. In the foam height test, the oil sample was added to a test tube with an inner diameter of 28 mm, and heated to 160°C. A 1-cm potato cube was added, and the height of foam generated (maximum value) was measured.

To confirm the body fat accumulation-suppressing effect of MLCT in humans, a large-scale, long-term nutrition study was performed, similar to the MCT evaluation study. In 82 healthy subjects, diets were strictly controlled for 12 weeks under the double-blind condition, and the subjects ate bread containing MLCT or LCT (normal cooking oil) as breakfast every morning. Lunches and suppers were standardized, and eating between meals and beverages were also included in the dietary control. Losses in body weight and body fat were greater in the MLCT group than in the LCT group (Figure 7.3) (Kasai et al., 2003). The abdominal subcutaneous and visceral fat levels were measured by computed tomography, and the reductions were greater in the MLCT group than in the LCT group. Healthy male subjects ingested an emulsified beverage containing 20 g of soybean or MLCT oil daily, in addition to their usual meals, and body fat was measured before and after the experiment. The body fat level was increased by a three-week ingestion of the soybean oil beverage, but not by the MLCT oil beverage (Matsuo et al., 2001a). Twenty-three healthy subjects changed the cooking oil used at home to MLCT oil, and their body fat ratio was measured after four weeks. It was significantly reduced after the use of MLCT oil, compared to that before switching over (Figure 7.4). The increase in energy expenditure after MLCT oil ingestion (diet-induced thermogenesis) was investigated in healthy women, and the energy expenditure after MLCT oil ingestion was larger than that after LCT (soybean oil) ingestion (Matsuo et al., 2001b). The increase in energy expenditure after ingestion may be involved in the mechanism whereby MLCT oil suppresses body fat accumulation (Figure 7.5). To investigate the safety of MLCT, a four-week ingestion study (42 g/ day) was performed in 10 healthy males and females. No negative influence on liver or renal function was noted, confirming the high safety level of MLCT (Nosaka et al., 2002).

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Body fat weight (kg)

–1 Body weight (kg)

0

LCT MLCT

–2 –3

*

–4

*

–5

0

–1 –2 –3 ** –4 *

–5

*

–6

Visceral fat area (cm2)

0

4

8

–5 –10 –15

*

* –6

0

111

–20 0

12

Weeks

4

8

12

0

Weeks

4

8

* 12

Weeks

FIGURE 7.3 MLCT-induced changes in body weight, body fat weight, and visceral fat area in humans. Eighty-two healthy subjects ate bread containing MLCT or LCT for 12 weeks as breakfast under strict dietary control. The values are presented as the means ± SE. Significantly different from zero week: *p < 0.05 and **p < 0.01.

Change in body fat ratio (%)

105

100 * 95

90 0

2 Weeks

4

FIGURE 7.4 Reduction of the body fat ratio by MLCT oil ingestion. Twenty-three healthy adults changed the cooking oil used at home to MLCT oil for four weeks. The values are presented as the means ± SE. Significantly different from zero week: *p < 0.05.

4 **

kJ/kg/6 h

3

2

1

0 LCT

MLCT

FIGURE 7.5 The increase in energy expenditure after LCT or MLCT ingestion. Fifteen healthy females ingested LCT or MLCT, and energy expenditure was measured for 6 h. The values are presented as the means ± SE. **p < 0.01.

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SUMMARY

MCT is readily digested and absorbed, and increases energy expenditure, suppressing body fat accumulation. However, it has disadvantages in its use as a cooking oil, as it has a low smoking point and easily foams during deep-frying. Its applicability for heating and deep-frying could be markedly improved by transesterification. MLCT was also shown to suppress body fat accumulation by increasing energy expenditure. Since the properties of accumulating less body fat and safety were confirmed, MLCT oil was approved as a food for specified health use (product name: Healthy Resetta) by Nisshin Oillio, Japan in 2002 as a cooking oil “less likely to lead to body fat accumulation,” and sold in most supermarkets in Japan. Medium-chain fatty acids are contained in breast milk, and are a nutrient that humans ingest from birth. Conversion to MLCT broadened the range of uses, which may attract more attention to the function and application methods of medium-chain fatty acids.

7.4

CONCLUSION

The major biotechnologies utilized in the oil and fat industry were described. The progress of biotechnology and its utilization in the oil and fat industry are marked. The oil and fat industry may not be able to continue without biotechnology. Biotechnologies not mentioned in this report may spread within this industry over the next 10 years. On the other hand, some consumers are anxious and doubtful about the rapid progress and spread of biotechnology. It is necessary that the progress of biotechnology is balanced not only with productivity but also with safety, ethics, and the environment.

REFERENCES Ingle, D.L., Driedger, A., Traul, K.A., and Nakhasi, D.K., 1999. Dietary energy value of medium-chain triglycerides. J. Food Sci. 64: 960–963. Kasai, M., Nosaka, N., Maki, H., Negishi, S., Aoyama, T., Naka-mura, M., Suzuki, Y., et al., 2003. Effect of dietary medium- and long-chain triacylglycerols (MLCT) on accumulation of body fat in healthy humans. Asia Pac. J. Clin. Nutr. 12: 151–160. Matsuo, T., Matsuo, M., Kasai, M., and Takeuchi, H., 2001a. Effect of a liquid diet supplement containing structured medium- and long-chain triacylglycerols on body fat accumulation in healthy young subjects. Asia Pac. J. Clin. Nutr. 10: 46–50. Matsuo, T., Matsuo, M., Taguchi, N., and Takeuchi, H., 2001b. The thermic effect is greater for structured medium- and long-chain triacylglycerols versus long-chain triacylglycerols in healthy young women. Metabolism 50: 125–130. Nosaka, N., Abe, T., Itakura, M., Maki, H., Suzuki, Y., Kasai, M., Tsuji, H., Aoyama, T., Okazaki, M., and Kondo, K., 2002. Effects of dietary MLCT (medium- and long-chain triacylglycerols) on body composition, serum lipids, liver function, and renal function in healthy subjects. Jomyaku Keicho Eiyo 17: 99–105. Takeuchi, H., Kubota, F., Itakura, M., and Taguchi, N., 2001. Effect of triacylglycerols containing medium- and long-chain fatty acids on body fat accumulation in rats. J. Nutr. Sci. Vitaminol. 47: 267–269. Tsuji, H., Kasai, M., Takeuchi, H., Nakamura, M., Okazaki, M., and Kondo, K., 2001. Dietary medium-chain triacylglycerols suppress accumulation of body fat in a double-blind, controlled trial in healthy men and women. J. Nutr. 131: 2853–2859.

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of Nutraceutical 8 Effects Antioxidants on AgeRelated Hearing Loss Shinichi Someya, Tomas A. Prolla, and Masaru Tanokura CONTENTS 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9

Introduction .......................................................................................................................... 113 α-Lipoic Acid ....................................................................................................................... 115 Acetyl-l-Carnitine ................................................................................................................ 115 Vitamin C ............................................................................................................................. 116 Vitamin E.............................................................................................................................. 117 Folic Acid.............................................................................................................................. 117 Melatonin .............................................................................................................................. 118 Caloric Restriction ................................................................................................................ 119 Potential Mechanisms for Cochlear Protection and the Retardation of AHL by Antioxidants and CR ......................................................... 119 8.10 Summary .............................................................................................................................. 121 References ...................................................................................................................................... 122

8.1

INTRODUCTION

Age-related hearing loss (AHL), also known as presbycusis, is one of the most common features of mammalian aging and is the most common sensory disorder in the elderly (Schuknecht and Gacek, 1993; Jennings and Jones, 2001; Gates and Mills, 2005; Liu and Yan, 2007; Van Eyken et al., 2007; Yamasoba et al., 2007; Vrijens et al., 2008). AHL is characterized by an age-dependent decline of auditory function and loss of hair cells and spiral ganglion (SG) neurons in the cochlea of the inner ear. Neither of these long-lived cell types is thought to regenerate in mammals, and extensive cell loss leads to permanent hearing impairment. Schuknecht and coworkers defined sensory, neural, and strial types of AHL based on correlations between cochlear pathology and audiogram (Schuknecht and Gacek, 1993); approximately 75% of cases of AHL represent a pure sensory, neural, or strial type, or a mixture of two or more types, and in the remaining 25% of cases the cochlear pathology does not correlate with the audiogram. Therefore, the major cause of AHL is the loss of hair cells, SG neurons, and stria vascularis cells in the cochlea of the inner ear. However, despite intensive studies in the AHL field, the molecular mechanisms of AHL remain unclear. In the United States, people over 65 years of age numbered 37.9 million in 2007, representing 12.6% of the U.S. population, and this elderly population is expected to grow to 71.5 million (20% of the U.S. population) by 2030 (Administration on Aging, 2009). A study performed in Wisconsin showed that AHL affects nearly half the population between 48 and 92 years of age (Schuknecht and Gacek, 1993), and it is estimated that more than 40% of individuals over 65 years of age (15.2 million) have AHL in the United States (Seidman, 2000; Gates and Mills, 2005; Yamasoba et al., 2007). Since the overall number of people suffering from this age-associated disorder is rapidly growing as the lifespan 113 © 2010 Taylor and Francis Group, LLC

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increases in industrialized nations, AHL is considered a major social and health problem. However, there is no treatment for the disorder at this time. The oxidative stress theory of aging postulates that aging is the result of accumulated oxidative damage caused by reactive oxygen species (ROS) (Harman, 1956; Shigenaga et al., 1994; Beckman and Ames, 1998; Finkel and Holbrook, 2000). It is now widely accepted that mitochondria are a major source of ROS, and that ROS production increases during aging (Shigenaga et al., 1994; Beckman and Ames, 1998; Balaban et al., 2005; Wallace, 2005). This theory is supported by the observations that overexpressing the mitochondrial antioxidant gene MnSOD (manganese superoxide dismutase) (Sun et al., 2002) or the mitochondrial iron regulator protein frataxin significantly increases longevity in Drosophila (Runko et al., 2008), while overexpressing a mitochondrially targeted catalase gene results in reduced age-related pathology and increased lifespan in mice (Schriner et al., 2005). These results, if applicable to humans, suggest that enhancing antioxidant defenses through administration of antioxidants may slow the aging process and delay the onset of age-associated diseases. Growing evidence indicates that ROS play a physiological role through multiple cell signaling pathways (Toledano et al., 2007). These observations suggest that the activation of ROS-specific cellular pathways, as opposed to random damage, may be the critical effector of ROS in aging and age-related diseases. Previous studies have shown that antioxidants delay the onset of AHL and reduce cochlear pathology in mice and rats (Seidman, 2000; Seidman et al., 2000; Derin et al., 2004; Darrat et al., 2007; Ahn et al., 2008). The mechanisms postulated to be responsible for the beneficial effects of antioxidants on aging and AHL include reduced oxidative damage and reduced levels of mitochondrial DNA (mtDNA) mutations. Caloric restriction (CR) extends the lifespan of most mammalian species and delays the onset of age-associated degenerative diseases (Weindruch et al., 1986; Walford et al., 1987; Sohal and Weindruch, 1996; Weindruch and Sohal, 1997; Mair and Dillin, 2008). Maximum lifespan is thought to be increased by reducing the rate of aging, while the average lifespan can be increased by improving environmental conditions. Indeed, the average lifespan of humans has dramatically increased as a result of improved diet and environmental health conditions over recent decades, whereas the maximum lifespan has remained largely unchanged. CR extends the lifespan in species as diverse as birds, protozoans, water fleas, spiders, guppies, and rodents (Weindruch et al., 1986; Sohal and Weindruch, 1996; Weindruch and Sohal, 1997). In laboratory rodents, CR delays the onset of age-related diseases such as lymphomas, breast cancer, prostate cancer, nephropathy, cataracts, diabetes, hypertension, hyperlipidemia, and autoimmune diseases (Weindruch et al., 1986; Walford et al., 1987; Sohal and Weindruch, 1996; Weindruch and Sohal, 1997). CR also reduces the degeneration of neurons in animal models of Parkinson’s disease (Mattson, 2000). In monkeys, CR results in signs of improved health, including reduced body fat, higher insulin sensitivity, increased HDL, and reduced VLDL levels (Rezzi et al., 2009). CR in monkeys also reduces the incidence of diabetes, cancer, cardiovascular disease, brain atrophy, and delays mortality (Colman et al., 2009). At the molecular level, CR results in a significant reduction in the levels of oxidative protein damage in the brains, hearts, and livers of aging mice (Sohal et al., 1994). Previous studies have shown that CR delays the onset of AHL and reduces cochlear pathology in mice and rats (Seidman, 2000; Someya et al., 2007). The mechanisms postulated to be responsible for the beneficial effects of CR on aging and AHL include reduced oxidative damage and reduced levels of mtDNA mutations (Weindruch et al., 1986; Walford et al., 1987; Sohal and Weindruch, 1996; Weindruch and Sohal, 1997; Seidman, 2000; Cheng et al., 2005; Kujoth et al., 2005; Liu and Yan, 2007; Yamasoba et al., 2007). In this chapter, we review the effects of antioxidants and CR on AHL in laboratory animals and humans, and discuss potential molecular mechanisms by which antioxidants and CR slow the cochlear aging process and the progression of AHL in mammals. In this respect, the development of functional foods and nutraceuticals using biotechnological methods to reduce ROS and promote CR-like effects may present new strategies for the prevention and treatment of AHL.

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8.2

115

α-LIPOIC ACID

α-lipoic acid (LA), also known as thioctic acid, is a naturally occurring dithiol compound that is synthesized by plants and animals, including humans (Linus Pauling Institute Micronutrient Information Center W 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). LA is found in a variety of foods including spinach, broccoli, kidney, and liver. There are two isomers of LA, the R-enantiomer and the S-enantiomer: only the R-isomer is endogenously synthesized and bound to proteins. Like the antioxidant glutathione (GSH), LA is an oxidized partner of the reduced form dihydrolipoic acid (DHLA), and exists as DHLA intracellularly. In animals, LA is synthesized from octanoic acid and cysteine in the mitochondria of the liver and plays an essential role in mitochondrial metabolism. At physiological conditions, LA exists as lipoamide (the functional form of LA) and is covalently attached to lysine residues of enzyme complexes as a cofactor, including dihydrolipoyl dehydrogenase and pyruvate dehydrogenase complexes in mitochondria. Liporate, a conjugate base of LA, is also a substrate for the enzyme GSH reductase. DHLA has been shown to reduce the oxidized form of endogenous antioxidants such as GSH, vitamin C (VC), α-tocopherol, and coenzyme Q10, thereby regenerating important endogenous antioxidants. In addition, DHLA reacts with ROS such as superoxide radicals, hydroxyl radicals, peroxyl radicals, and singlet oxygen. LA supplementation is thought to be beneficial to a number of oxidative stress models such as diabetes, cataracts, HIV, neurodegeneration, and radiation injury in animals (Hagen et al., 2002; Liu et al., 2002; Liu, 2008; Singh and Jialal, 2008; Linus Pauling Institute Micronutrient Information Center Web site, 2009). Nutritional LA deficiency has not been identified in healthy individuals, indicating that under normal dietary conditions, most individuals can synthesize a sufficient amount of LA in the body (Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). An increasing body of evidence indicates that supplementation with LA improves mitochondrial energy metabolism, decreases oxidative stress, and improves memory in aged rats (Hagen et al., 2002; Liu et al., 2002; Liu, 2008). Rat study: Seidman and coworkers investigated the effects of LA on AHL in aged Fischer 344 rats (Seidman et al., 2000). This rat strain displays AHL by 20 months of age (Seidman, 2000; Seidman et al., 2000). Twenty-four-month-old animals were housed individually and divided into two groups: control (N = 7) and LA (N = 7). Rats were orally supplemented with a placebo or LA (300 mg/kg/day) for six weeks. The auditory brainstem response (ABR) hearing test was used to measure hearing thresholds at 3, 6, 9, 12, and 18 kHz in the animals at the beginning and the end of the study. The cochlea tissues were collected to measure the rate of mtDNA mutations. LA supplementation significantly delayed the onset of AHL at 3 kHz, but not at the higher frequencies. Mice fed with LA also displayed reduced mtDNA deletion levels in the cochlea. Mouse study: Ahn and coworkers investigated the effects of LA on AHL in DBA/2J mice (Ahn et al., 2008). This mouse strain displays early onset of AHL by 3–5 months of age (Zheng et al., 1999; Ahn et al., 2008). The animals were divided into four groups (N = 7) and provided drinking water without LA (control) or supplemented with LA (100 mg/kg/day) beginning at 2, 4, or 8 weeks of age for 10, 9, and 4 weeks, respectively. At 12 weeks of age, ABR hearing testing was used to measure hearing thresholds at 4, 8, 16, and 32 kHz. The cochlea tissues were collected to detect 8-oxoguanine, a robust marker of oxidative DNA damage, by immunohistochemistry. LA supplementation starting from two weeks of age significantly delayed the onset of AHL at all the frequencies. Moreover, 8-oxoguanine was not detected in the cochlea of mice fed LA from two weeks of age, while 8-oxoguanine was detected in the cochlea of control mice. Collectively, these results show that dietary supplementation with LA slows the progression of AHL in rats and mice and may reduce age-associated oxidative DNA damage in the cochlea.

8.3

ACETYL-L-CARNITINE

Acetyl-l-carnitine (LC), also known as 3-acetyloxy-4-trimethylammonio-butanoate, is an acetylated derivative of l-carnitine, which is derived from the amino acids lysine and methionine (NIH;

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Linus Pauling Institute Micronutrient Information Center Web site, 2009; Dietary Supplement Fact Sheets Web site, 2009). LC is abundant in meat, poultry, fish, and dairy products, while fruits, vegetables, and grains contain relatively little LC. Only the l-isomer of carnitine is thought to be biologically active. LC is better absorbed and more efficiently crosses the blood–brain barrier compared to l-carnitine. LC is a naturally occurring compound synthesized primarily in the liver in humans and other higher organisms. LC is essential for mitochondrial β-oxidation of long-chain fatty acids for energy production. Importantly, LC is present in high concentrations in the brain and provides acetyl-equivalents for the production of the neurotransmitter acetylcholine. Indeed, LC has been shown to improve the age-associated decline of learning and memory in aged animals and to decrease mortality in rats (Hagen et al., 2002; Liu et al., 2002; Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). Moreover, LC improves nerve regeneration in rats and protects neurons from the toxicity of mitochondrial uncouplers. LC also increases levels of GSH and GABA in the brains of mice and increases the activities of NADH-cytochrome-c-oxidoreductase, succinate cytochrome-c-oxidoreducaase, and cytochromec-oxidase in synaptosomes isolated from mice. Nutritional LC deficiency has not been identified in healthy individuals, indicating that under normal dietary conditions most individuals can synthesize a sufficient amount of LC (Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). Rat studies: As in the study of the effect of LA on AHL, Seidman and coworkers also investigated the effects of LC on AHL in twenty-four-month old Fischer 344 rats (Seidman et al., 2000). The animals were supplemented with a placebo (N = 7) or LC (N = 7) (300 mg/kg/day) for six weeks. ABR hearing testing was used to measure hearing thresholds at 3, 6, 9, 12, and 18 kHz in the animals at the beginning and end of the study. In contrast to the effect of LA, LC supplementation significantly delayed the onset of AHL at 6, 9, 12, and 18 kHz, but not at the low frequency. In addition, LC supplementation significantly reduced mtDNA mutations in the cochlea. Derin and coworkers also investigated the effects of LC on AHL in aged Wistar rats (Derin et al., 2004). This rat strain displays AHL by 24 months of age (Derin et al., 2004). Twenty-four-month old Wistar rats were randomly assigned to two groups: control (N = 10) and LC (N = 10). LC (50 mg/ kg) was administered by intragastric gavage to rats once a day for 30 days, while distilled water was given orally to control rats by intragastric gavage. At the end of 30 days, ABR hearing testing was used to measure I, III, and V wave latencies and I-III, III-V, and I-V IPL (interpeak latencies). LC mice displayed reduced levels of III and V latencies and I-III, III-V, and I-V IPL latencies compared with controls. Collectively, these results show that dietary supplementation with LC slows the progression of AHL in rats.

8.4 VITAMIN C VC, also known as ascorbic acid, is a water-soluble and essential nutrient for humans (Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). Most animals and plants can synthesize VC, while humans do not have the ability to synthesize their own VC. VC is abundant in fruits and vegetables and is biosynthesized from glucose in the liver of most animals and birds. There are two isomers of VC, the l-enantiomer and the d-enantiomer; only the l-isomer is biologically active. Biologically, VC exists as l-ascorbate, that is, a reduced partner of the oxidized form dehydroascorbate, which can be reduced back to the active l-ascorbate by GSH and enzymes. Thus, VC is a powerful reducing agent well known for its antioxidant activity. VC acts as a reducing agent to maintain metals in their reduced state in most metabolic reactions. VC has also been shown to reduce gastric mucosal oxidative stress, oxidative DNA damage, and inflammation by scavenging ROS in animals (Martin, 2003; Liu and Russell, 2008). In humans, VC is required for a number of essential metabolic reactions including the synthesis of collagen and LC (Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). Indeed, VC deficiency leads to scurvy, whose

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symptoms include the weakening of blood vessels, connective tissues, and bones all of which contain collagen. Furthermore, VC plays an essential role in the synthesis of neurotransmitters, including norepinephrine and catecholamine, which are critical to brain function (Martin, 2003; Wilson, 2005; Liu and Russell, 2008). VC deficiency has also been linked to multiple age-associated diseases, including Alzheimer’s disease, cardiovascular disease, stroke, cancer, and cataracts (NIH Dietary Supplement Fact Sheets Web site, 2009; Linus Pauling Institute Micronutrient Information Martin, 2003; Liu and Russell, 2008; Center Web site, 2009). Rat studies: Seidman investigated the effects of VC on AHL in rats (Seidman, 2000). Forty Fischer 344 rats were housed individually and divided into two groups: control (N = 20) and VC (N = 20). The animals were orally supplemented with a placebo or VC (4.44 mg/day) for 22–24 months. ABR hearing testing was used to measure hearing thresholds at 3, 6, 9, 12, and 18 kHz in the animals every three months from two months of age until the death of the animals. Supplementation with VC significantly delayed the onset of AHL at 6, 9, 12, and 18 kHz, but not at the low frequency. These results indicate that dietary VC supplementation slows the progression of AHL in rats.

8.5 VITAMIN E There are eight members of the vitamin E (VE) family: α-, β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols (Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). Of these, α-tocopherol has been most widely studied and is the only form of VE that is actively maintained and has the highest bioavailability in the human body. Indeed, α-tocopherol is the only form that is maintained at levels which meets the Recommended Dietary Allowance for VE. α-Tocopherol is found in varieties of foods including vegetable oils, nuts, whole grains, and green leafy vegetables. Importantly, α-tocopherol is a potent lipid-soluble antioxidant and has been shown to protect cell membranes from oxidation by ROS, thus preventing a chain reaction of lipid destruction. α-Tocopherol is a reduced partner of the oxidized form α-tocohperoxyl radicals, which can be reduced back to the active α-tocopherol by VC. α-Tocopherol protects the lipids in low density lipoproteins (LDLs) from oxidation by ROS, and is thought to play a role in preventing cardiovascular diseases, since oxidized LDLs have been implicated in the development of the disorder. VE deficiency is known to lead to neurological symptoms, including impaired balance, damage to the retina of the eye and the sensory nerves, and muscle weakness in humans. In animal studies, VE supplementation has been shown to reduce gastric oxidative stress and gastric tumors (Liu and Russell, 2008). Previous epidemiologic studies have also shown that dietary intake of antioxidant VE is associated with a lower risk of age-associated neurodegenerative diseases, including Alzheimer’s disease (Martin, 2003). Rat studies: Seidman investigated the effects of VE on AHL in rats (Seidman, 2000). Forty Fischer 344 rats were housed individually and divided into two groups: control (N = 20) and VE (N = 20). The animals were orally supplemented with a placebo or VE (2.475 mg/day) for 24 months. ABR hearing testing was used to measure hearing thresholds at 3, 6, 9, 12, and 18 kHz every three months from two months of age until the death of the animals. Supplementation with VE significantly delayed the onset of AHL at 6, 9, and 12 kHz, but not at 3 and 18 kHz. These results show that dietary supplementation with VE slows the progression of AHL in rats.

8.6

FOLIC ACID

Folic acid (FA), also known as vitamin B9, is an essential nutrient for humans (Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). It is required for cell division and growth and to produce red blood cells to prevent anemia. The naturally occurring form of FA is folate. Folate is found in varieties of foods including citrus fruits and juice, wheat, whole grains, spinach, asparagus, lettuces, beans, peas, liver, and liver products. Humans cannot synthesize FA and thus relay on dietary intake.

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Folate acts as a coenzyme for the synthesis of purine and pyrimidine DNA bases. Thus, FA deficiency leads to inadequate DNA synthesis and cell division. Folate coenzyme is also required for the remethylation of homocysteine to methionine and is thereby essential for the synthesis of methionine. FA deficiency may tend to increase levels of homocyteine, a condition that induces oxidative stress. FA deficiency increases lipid peroxidation and decreases cellular antioxidant defenses in rats (Moens et al., 2008). Therefore, FA has been implicated as playing an antioxidant role. Folate is a substrate in a number of single-carbon-transfer reactions. Neural tube defects, which are common birth defects, result from FA deficiency (Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). Thus, women who could become pregnant are advised to eat a diet fortified with FA in order to reduce the risk of these serious birth defects. Neural tube defects result in spina bifida, orofacial cleft, congenital heart defects, and in malformation of the spine, skull, and brain. FA deficiency has also been linked to multiple age-associated diseases, including heart disease, stroke, cancer, and dementia (Durga et al., 2007). Human studies: As stated earlier, 40% of humans in the United States over 65 years of age display AHL. Durga and coworkers investigated the effects of FA on AHL in aged individuals (Durga et al., 2007). Seven hundred and twenty-eight older men and postmenopausal women (50–70 years of age) from the Gelderland province in the Netherlands were recruited and divided into two groups: placebo (N = 413) and FA (N = 406). The participants were orally supplemented with a placebo or FA (800 μg/day) for three years. An audiometer and circumaural earphones were used to measure pure-tone air conduction hearing thresholds at 0.5, 1, 2, 4, 6, and 8 kHz in the individuals. After three years, the mean thresholds of the low frequencies increased by 1.0 dB in the FA group and by 1.7 dB in the placebo group, showing that FA supplementation significantly delayed the decline in hearing associated with aging. FA supplementation did not affect the decline in high frequency hearing. These results show that FA supplementation slows the progression of AHL at low frequencies in humans.

8.7

MELATONIN

Melatonin (MT), also known as N-acetyl-5-methoxytryptamine, is a naturally occurring hormone found in the human body (Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). MT is a potent antioxidant and is known to scavenge highly toxic hydroxyl radicals. MT is lipophylic and hydrophilic, thereby diffusing into cellular compartments and preventing oxidation by ROS. MT has been shown to reduce the neurotoxicity of amyloid-β and to increase the survival of cultured neuroblastoma cells (Karasek, 2004; Linus Pauling Institute Micronutrient Information Center Web site, 2009; NIH Dietary Supplement Fact Sheets Web site, 2009). MT also improves sleep and slows the progression of cognitive impairment in Alzheimer’s patients. Parkinson’s disease is characterized by the progressive deterioration of dopamine-containing neurons in the substantia nigra in the midbrain, and the loss of these neurons is thought to be caused by auto-oxidation of dopamine by ROS (Karasek, 2004). Importantly, MT has been shown to reduce dopamine auto-oxidation and lipid peroxidation in the aged hippocampus and midbrain, and to prevent the death of cultured neuronal cells. Here we review the effects of MT on AHL in rats. Rat studies: Seidman investigated the effects of MT on AHL in aged rats (Seidman, 2000). Forty Fischer 344 rats were housed individually and divided into two groups: control (N = 20) and MT (N = 20). The animals were orally supplemented with a placebo or MT (2.475 mg/day) for 24 months. ABR hearing testing was used to measure hearing thresholds at 3, 6, 9, 12, and 18 kHz in the animals every three months from two months of age until the death of the animals. MT supplementation significantly delayed the onset of AHL at 3, 6, 9, and 18 kHz, but not at 12 kHz. These results show that dietary supplementation with MT slows the progression of AHL in rats.

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CALORIC RESTRICTION

Mouse study: The C57BL/6 (B6) mouse strain is long lived and has been widely used as a model of aging and AHL (Weindruch et al., 1986; Zheng et al., 1999; Keithley et al., 2004; Someya et al., 2007). It is also well known that B6 mice respond to CR with a robust extension of lifespan (Weindruch et al., 1986). This strain displays the classic pattern of AHL, including loss of sensory hair cells and SG neurons by 12–15 months of age (Zheng et al., 1999; Keithley et al., 2004). In a study of CR in B6 mice, animals were calorie restricted to 75% of the control calorie intake starting at two months of age and housed individually for a 13 month period (Someya et al., 2007). CR mice were fed agar-based semi-purified diets on Monday, Wednesday, and Friday, receiving 63 kcal/ week of diet, while control mice were fed on the same days and received 84 kcal/week of the diet. ABR hearing testing was used to monitor the progression of AHL at 4, 8, and 16 kHz frequencies. At 15 months of age, the mean ABR thresholds of CR mice were significantly lower than those of age-matched control mice at all the frequencies, and not significantly different from those of fourmonth-old mice at all the frequencies measured. CR mice weighed 25% less than the controls and displayed only minor cochlear degeneration; in contrast, age-matched control mice displayed severe cochlear cell loss. Rat study: In a study of CR in male Fischer rats, the animals were calorie restricted to 70% of the control intake beginning at one month of age, then housed individually for 24–25 months, after which the progression of AHL was measured by ABR hearing testing (Seidman, 2000). CR rats were fed 11.2 g of a standard rodent diet daily, while control rats were fed ad libitum. Both control and CR rats displayed large ABR threshold elevations at 3, 6, 9, 12, and 18 kHz frequencies, but the mean ABR thresholds of CR rats were significantly lower than those of controls at all the frequencies measured. CR animals also had significantly reduced hair cell loss compared to controls. Monkey study: Rhesus monkeys, a primate model of aging and AHL, display decreased auditory function by 25–31 years of age (Fowler et al., 2005). These animals are long-lived with a maximum lifespan of 40 years (Roth et al., 2004). There are two large studies ongoing at the University of Wisconsin-Madison (UW) (Ramsey et al., 2000) and the National Institute on Aging (Roth et al., 2004) designed to test the hypothesis that CR retards the rates of aging and delays the onset of AHL in a primate species. In the UW study, 33 rhesus monkeys were on CR diets (20 males and 13 females) and 35 controls were fed ad libitum (21 males and 14 females) daily; CR was maintained at 30% less than the calories consumed by the ad libitum group (Fowler et al., 2005). At the time of the hearing tests, the ages of the monkeys were between 11 and 23 years and the monkeys had been in the dietary study for 3–9 years. The animals were housed individually to control food access. All animals were daily fed pelleted, semi-purified diets. ABR and middle latency response (MLR) testing were used to monitor the progression of AHL. A previous study has shown that aged rhesus monkeys display smaller peak amplitudes of ABR waves compared to young controls (Torre and Fowler, 2000). In the UW study, the peak IV amplitude of ABR waves of CR female monkeys was significantly larger compared to control females. In addition, the binaural peak IV amplitude of ABR waves decreased significantly faster with age for control monkeys compared to CR monkeys. The mean ABR threshold (click stimuli) of CR male monkeys was lower than that of control males, but the difference was not statistically significant. Collectively, these results indicate that CR slows the progression of AHL in mice, rats, and monkeys.

8.9

POTENTIAL MECHANISMS FOR COCHLEAR PROTECTION AND THE RETARDATION OF AHL BY ANTIOXIDANTS AND CR

The molecular mechanisms by which antioxidants slow the rate of cochlear aging and the progression of AHL remain unclear and the specific molecular pathways are unknown. However, there is a growing body of evidence suggesting antioxidants protect cochlear cells and retard AHL, thereby

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implicating ROS in the mechanism of hearing loss. Several studies have shown that ROS are generated in the cochlea of mammals exposed to noise of an intensity that leads to hearing loss (Jacono et al., 1998; Ohlemiller et al., 1999). Mice lacking the antioxidant enzyme Gpx1 or SOD1 show enhanced susceptibility to noise-induced hearing loss (Ohlemiller et al., 2000; Fortunato et al., 2004). The free radical theory of aging postulates that aging is the result of accumulated oxidative damage caused by ROS (Harman, 1956; Beckman and Ames, 1998). Oxidative damage caused by ROS is also considered to play a role in AHL (Seidman, 2000; Seidman et al., 2000; Darrat et al., 2007; Liu and Yan, 2007; Van Eyken et al., 2007; Yamasoba et al., 2007). In agreement with these results, age-related cochlear hair cell loss is enhanced in mice lacking the antioxidant enzyme SOD1 (McFadden et al., 1999). Furthermore, levels of oxidative protein damage increase with age in the cochlea of mice (Staecker et al., 2001; Jiang et al., 2007). Antioxidant supplementation is thought to result in reduced levels of ROS and has been found to protect against oxidative damage and to slow AHL. Several studies have shown that supplementation with LA and LC reduces oxidative damage to DNA, proteins, and lipids in mammals (Hagen et al., 2002; Liu et al., 2002). Previous studies have also found that tissue LC levels decline with age in humans and rodents, and that supplementation with LC reverses the age-associated declines in tissue LC levels, and improves mitochondrial energy metabolism, and decreases oxidative stress in aged rats (Hagen et al., 2002; Liu et al., 2002; Liu, 2008). In agreement with this hypothesis, LA and LC reduced levels of mtDNA deletions and slowed the progression of AHL in rodents (Seidman et al., 2000; Derin et al., 2004; Ahn et al., 2008). Moreover, enhancing antioxidant defenses by supplementation with vitamin E, vitamin C, and MT significantly slowed AHL in rats (Seidman, 2000). Collectively, the mechanisms of cochlear cell protection by antioxidants may involve the decreased production of ROS with a resulting reduction in the oxidative damage to DNA (nuclear and/or mitochondrial), proteins, and lipids in the aged cochlea. Mitochondria have been postulated to play a key role in mammalian aging and AHL (Wallace et al., 1995; Kujoth et al., 2005; Wallace, 2005; Kujoth et al., 2007; Someya et al., 2008). The mitochondrial theory of aging postulates that ROS generated inside mitochondria damage key mitochondrial components such as mtDNA, membranes, and respiratory chain proteins (Wallace et al., 1995; Balaban et al., 2005; Wallace, 2005). Such damage accumulates with age, leads to mitochondrial dysfunction, and eventually to tissue dysfunction. Mitochondria are the main source of cellular energy, generating most cellular ATP. The mammalian mitochondrial genome consists of 37 genes, encoding 13 proteins of the electron transport oxidative phosphorylation system (Wallace, 2005; Kujoth et al., 2007). Because mtDNA plays essential roles in energy metabolism and cellular apoptosis, mtDNA mutations have been hypothesized to contribute to mammalian aging. Indeed, previous studies have shown that mtDNA point mutations accumulate with aging in humans (Michikawa et al., 1995), and that the accumulation of mtDNA mutations leads to premature aging in mice (Kujoth et al., 2005), indicating a causal role of mtDNA mutations in mammalian aging. It has been postulated that mtDNA mutations may also contribute to AHL. In support of this hypothesis, hearing loss is a common symptom in patients harboring inherited mtDNA mutations (Chinnery et al., 2000). Several mutations in the POLG gene (mitochondrial DNA polymerase γ) have been identified as a cause of human disorders such as Alpers syndrome and deafness (Mancuso et al., 2004; Nguyen et al., 2005). Specific mtDNA point mutations also contribute to mitochondrial disorders in humans such as MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged red fibers), the symptoms of which include hearing loss (Fischel-Ghodsian, 2003; Pickles, 2004; Kokotas et al., 2007). Furthermore, more than 100 different deletions of mtDNA have been associated with mitochondrial disorders, and of these, some mtDNA deletions cause Kearns–Sayre syndrome which involves hearing loss (Pickles, 2004). In mice, age-associated accumulation of mtDNA mutations causes AHL (Kujoth et al., 2005; Someya et al., 2008). Because mitochondria are a major source of ROS and ROS production increases with age (Wallace, 2005), antioxidant supplementation is postulated to protect mtDNA by reducing the production of ROS. In support of this hypothesis,

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supplementation with LA and LC results in reduced levels of mtDNA deletions in the auditory nerve and stria vascularis of the cochlea (Seidman et al., 2000). LA is a thiol compound and has been shown to reduce mitochondrial ROS production (Palaniappan and Dai, 2007), while LC is essential for mitochondrial β-oxidation of long-chain fatty acids for energy production (Liu et al., 2002). Taken together, the mechanisms of cochlear cell protection by antioxidants may involve reduced levels of mtDNA damage in the aged cochlea. CR extends the lifespan of numerous mammalian species examined (Weindruch et al., 1986; Walford et al., 1987; Sohal and Weindruch, 1996; Weindruch and Sohal, 1997). Previous studies have shown that rodents demonstrate a delayed onset of age-associated pathology and extension of maximum lifespan when subjected to a long-term, 20–50% reduction in caloric intake without essential nutrient deficiency (Weindruch et al., 1986; Sohal and Weindruch, 1996; Weindruch and Sohal, 1997). At the molecular level, CR results in a significant reduction in the levels of oxidative damage to DNA, proteins, and lipids in mammals (Sohal et al., 1994; Beckman and Ames, 1998; Mair and Dillin, 2008). Therefore, the mechanisms postulated to be responsible for the effects of CR include reduced oxidative stress. In mice, most of the age-related increase in oxidative DNA damage occurs in postmitotic tissues such as the brain and heart, and most of the attenuation by CR of the damage also occurs in the postmitotic tissues (Weindruch et al., 1986; Walford et al., 1987; Sohal et al., 1994; Weindruch and Sohal, 1997; Wallace, 2005; Ferguson et al., 2008). Consistently with reduced oxidative damage, CR reduces levels of mtDNA deletions in rats (Seidman, 2000) and delays the onset of AHL in mice, rats, and monkeys (Seidman, 2000; Fowler et al., 2005; Someya et al., 2007). CR is also postulated to reduce levels of apoptotic cell death in the cochlea tissue. Previous studies demonstrated that TUNEL-positive hair cells and SG neurons were distinctly evident in the cochlea of aged gerbils (Zheng et al., 1998) and mice (Usami et al., 1997), suggesting that apoptosis may be associated with the death of these cells in aged animals. In the animal model of Parkinson’s disease, CR lowers symptom severity and increases the resistance of neurons to apoptosis (Mattson, 2000). CR also suppresses the age-related increase in caspase-3 and caspase-9 in the brains of aged rats (Shelke and Leeuwenburgh, 2003), suggesting that CR is neuroprotective. We have shown previously that CR decreases the expression of proapoptotic genes, including the Bcl-2 family members Bak and Bim and that CR reduces the levels of TUNEL-positive cells in the cochlea (Someya et al., 2007). Collectively, the mechanisms of cochlear cell protection by CR may involve decreased production of ROS and decreased levels of apoptotic cell death in the cochlea. We note that avoiding weight gain may also be an important factor in the prevention of AHL since overweight conditions in humans are significantly associated with diabetes (Mokdad et al., 2003), which is a risk factor for human AHL (Vaughan et al., 2006). Therefore, staying lean by CR may have the benefit to humans of reducing the risk of AHL.

8.10 SUMMARY An increasing body of evidence suggests that oxidative damage contributes to the development of age-associated neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and AHL (Weindruch et al., 1986; Beckman and Ames, 1998; Mattson, 2000; Liu et al., 2002; Darrat et al., 2007; Liu and Yan, 2007; Yamasoba et al., 2007). As discussed earlier, organs such as brain and cochlea which consist of nonregenerating postmitotic cells are particularly susceptible to oxidative damage, since most of the aged neurons and sensory cells are thought not to regenerate in mammals, and extensive cell loss leads to permanent postmitotic tissue dysfunction. Therefore, we speculate that oxidative damage caused by ROS may play a causal role in AHL, leading to loss of sensory hair cells and neurons in the cochlea. Here we have presented some candidate agents that show potential in the attenuation of AHL. Future directions include the development of bioactive nutraceuticals which may reduce the oxidative damage to nuclear DNA, mtDNA, proteins, and lipids in the cochlea, thereby protecting the cochlear cells, slowing cochlear aging and the progression of AHL.

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Someya, S., Yamasoba, T., Kujoth, G.C., Pugh, T.D., Weindruch, R., Tanokura, M., and Prolla, T.A., 2008. The role of mtDNA mutations in the pathogenesis of age-related hearing loss in mice carrying a mutator DNA polymerase gamma. Neurobiol. Aging 29: 1080–1092. Someya, S., Yamasoba, T., Weindruch, R., Prolla, T.A., and Tanokura, M., 2007. Caloric restriction suppresses apoptotic cell death in the mammalian cochlea and leads to prevention of presbycusis. Neurobiol. Aging 28: 1613–1622. Staecker, H., Zheng, Q.Y., and Van De Water, T.R., 2001. Oxidative stress in aging in the C57B16/J mouse cochlea. Acta. Otolaryngol. 121, 666–672. Sun, J., Folk, D., Bradley, T.J., and Tower, J., 2002. Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster. Genetics 161: 661–672. Toledano, M.B., Kumar, C., Le Moan, N., Spector, D., and Tacnet, F., 2007. The system biology of thiol redox system in Escherichia coli and yeast: differential functions in oxidative stress, iron metabolism and DNA synthesis. FEBS Lett. 581: 3598–3607. Torre, P., 3rd and Fowler, C.G., 2000. Age-related changes in auditory function of rhesus monkeys (Macaca mulatta). Hear Res. 142: 131–140. Usami, S., Takumi, Y., Fujita, S., Shinkawa, H., and Hosokawa, M., 1997. Cell death in the inner ear associated with aging is apoptosis? Brain Res. 747: 147–150. Van Eyken, E., Van Camp, G., and Van Laer, L., 2007. The complexity of age-related hearing impairment: Contributing environmental and genetic factors. Audiol. Neurootol. 12: 345–358. Vaughan, N., James, K., McDermott, D., Griest, S., and Fausti, S., 2006. A 5-year prospective study of diabetes and hearing loss in a veteran population. Otol. Neurotol. 27: 37–43. Vrijens, K., Van Laer, L., and Van Camp, G., 2008. Human hereditary hearing impairment: Mouse models can help to solve the puzzle. Hum. Genet. 124: 325–348. Walford, R.L., Harris, S.B., and Weindruch, R., 1987. Dietary restriction and aging: Historical phases, mechanisms and current directions. J. Nutr., 117: 1650–1654. Wallace, D.C., 2005. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: A dawn for evolutionary medicine. Annu. Rev. Genet. 39: 359–407. Wallace, D.C., Shoffner, J.M., Trounce, I., Brown, M.D., Ballinger, S.W., Corral-Debrinski, M., Horton, T., Jun, A.S., and Lott, M.T., 1995. Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim. Biophys. Acta. 1271: 141–151. Weindruch, R. and Sohal, R.S., 1997. Seminars in medicine of the Beth Israel Deaconess Medical Center. Caloric intake and aging. N. Engl. J. Med. 337: 986–994. Weindruch, R., Walford, R.L., Fligiel, S., and Guthrie, D., 1986. The retardation of aging in mice by dietary restriction: Longevity, cancer, immunity and lifetime energy intake. J. Nutr. 116: 641–654. Wilson, J.X., 2005. Regulation of vitamin C transport. Annu. Rev. Nutr. 25: 105–125. Yamasoba, T., Someya, S., Yamada, C., Weindruch, R., Prolla, T.A., and Tanokura, M., 2007. Role of mitochondrial dysfunction and mitochondrial DNA mutations in age-related hearing loss. Hear Res. 226: 185–193. Zheng, Q.Y., Johnson, K.R., and Erway, L.C., 1999. Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear Res. 130: 94–107. Zheng, Y., Ikeda, K., Nakamura, M., and Takasaka, T., 1998. Endonuclease cleavage of DNA in the aged cochlea of Mongolian gerbil. Hear Res. 126: 11–18.

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Part II The Impact of Genetic Modification on Functional Foods

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Production of 9 Increased Nutriments by Genetically Engineered Bacteria Kazuhiko Tabata and Satoshi Koizumi CONTENTS 9.1 9.2

Introduction .......................................................................................................................... 127 Production of Nutriments by Genetically Engineered Bacteria ........................................... 128 9.2.1 Glutathione ............................................................................................................... 128 9.2.2 Ala–Gln .................................................................................................................... 129 9.2.3 Hydroxyproline ......................................................................................................... 130 9.2.4 Hyaluronic Acid ........................................................................................................ 131 9.2.5 N-Acetylglucosamine................................................................................................ 132 9.2.6 Cytidine 5′-Diphosphate Choline ............................................................................. 134 9.3 Future Perspectives ............................................................................................................... 135 References ...................................................................................................................................... 136

9.1 INTRODUCTION Over recent decades our lifestyle has changed and the elderly population has grown. An increased interest in obesity and other diet-related health problems has prompted consumers to take nutritional supplements in order to avert health problems before they occur. The nutritional supplement market has been on an upswing, growing by 5% in 2006 and 8% in 2007. As well as vitamins, minerals, homeopathics, and herbals, nutritional products targeting specific health conditions such as age-related treatments, gender- and child-targeted products have all been launched into the market. There are many metabolites in animals, plants, and microorganisms that are expected to have some nutritional functions in humans. However, the quantities are often very low and it is difficult to produce enough of these compounds at a reasonable cost. These potential nutriments can be obtained by means of extraction, chemical synthesis, and biocatalysis. Among these methods, production by biocatalysis using enzymes has been dramatically improved along with the development of genetic engineering. In addition, microbial fermentation and metabolic engineering offer advantageous alternative ways to produce the numerous chemicals which have been traditionally manufactured by chemical synthesis or by extraction from raw materials. It has become possible to produce potential nutriments using genetically engineered bacteria at a cheaper cost and some of these nutriments have been introduced into the market as novel nutritional supplements. Here we describe some examples of recent successes in establishing production methods using genetic engineering in order to enter new areas of the market for nutritional supplements. 127 © 2010 Taylor and Francis Group, LLC

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9.2 9.2.1

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PRODUCTION OF NUTRIMENTS BY GENETICALLY ENGINEERED BACTERIA GLUTATHIONE

Glutathione (γ-glutamyl-l-cysteinylglycine, GSH), a tripeptide composed of l-glutamic acid, l-cysteine, and glycine, is the most abundant nonprotein thiol compound widely found in living organisms, predominantly in eukaryotic cells (Meister and Anderson, 1983). It plays a role in the detoxification and excretion of drugs and xenobiotics (Chasseaud, 1979). The reduced form of GSH acts as a hydrogen donor in the reductive detoxification of hydrogen peroxide and lipid peroxide (Chance et al., 1979; Burk, 1983). In Japan, GSH has been used as a pharmaceutical in the form of oral drugs and injections, for therapies related to drug intoxication, metal intoxication, toxemia due to late pregnancy, liver function disorders in chronic liver diseases, dermatitis, corneal injuries, and leucopenia from radiotherapy. GSH has also been used in eye drops for the treatment of early senile cataracts, corneal ulcers, corneal erosion, and keratitis. In the United States, supplements containing GSH have been marketed. Since the mechanisms of the action of GSH are different from other antioxidants such as vitamin C, vitamin E, and so on, it might occupy an important position in the prevention of lifestyle-related diseases and health promotion. GSH has been produced by direct fermentative methods from sugar, and Saccharomyces cerevisiae and Candida utilis are most commonly used for its industrial production. Several production methods using genetically engineered microorganisms have been developed (Figure 9.1). GSH is synthesized in two consecutive adenosine 5′-triphosphate (ATP)-dependent reactions (Meister and Anderson, 1983). The dipeptide γ-glutamylcysteine (γ-GC) is first synthesized from l-glutamic acid and l-cysteine by γ-glutamylcysteine synthetase (GSHI). In the second step, glycine is added to the C-terminal site of γ-GC to form GSH catalyzed by glutathione synthetase (GSHII). The activity of GSHI is feedback-inhibited by GSH to avoid its overaccumulation, which therefore indicates that GSHI is the rate-limiting step in GSH biosynthesis (Richman and Meister, 1975; Murata and Kimura, 1990). The genes gshA and gshB encoding GSHI (Murata et al., 1981) and GSHII (Murata et al., 1983), respectively, have been cloned from Escherichia coli and overexpressed in E. coli, Saccharomyces cerevisiae, and Lactococcus lactis. In addition, the genes of GSHI (gshA*), which were insensitive to feedback inhibition by GSH, were cloned through screening in E. coli (Murata and Kimura, 1982). The introduction of the recombinant plasmid harboring the gshA* and gshB genes into E. coli resulted in a simultaneous increase in the activities of GSHI (10.0-fold) and GSHII E. coli, S. cerevisiae, L. lactis

l-Cys

Gly γ-Glu-Cys

l-Glu

GSH

gshA ATP

gshB ADP + Pi

ATP

ADP + Pi

(GSH)

FIGURE 9.1

An outline of the glutathione producer strain.

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(14.5-fold) (Gushima et al., 1983). However, the intracellular GSH concentration in the recombinant E. coli cells only increased 1.3-fold compared with the wild-type strain. When intact recombinant E. coli cells were used as enzyme sources for GSH biosynthesis, 5 g/L of GSH was produced in the presence of three precursor amino acids and ATP. Similar work was done in Saccharomyces cerevisiae, where the expression of GSHI and GSHII increased 1039- and 33-fold, respectively, and intracellular GSH content increased 2.8-fold (Ohtake et al., 1988, 1989). Overexpression of gshA and gshB did not lead to a significant increase in GSH content in either E. coli or Saccharomyces cerevisiae. This might be due to the feedback inhibition on GSHI by GSH or the degradation of GSH by γ-glutamyl transpeptidase. Recently, an extremely high intracellular concentration of GSH, up to 140 mM upon addition of 5 mM l-cysteine, was achieved in L. lactis by expressing the gshA and gshB from E. coli under the control of a nisin-induced expression system. This is the highest intracellular GSH content achieved in bacteria to date (Li et al., 2005).

9.2.2

ALA–GLN

l-Glutamine is the most common amino acid found in the blood or intracellular pool, accounting for 50–60% of the amino acids in skeletal muscle and 20% or more in plasma. l-Glutamine demonstrates important physiological effects on the human body such as energy supply, stimulation of protein synthesis, wound healing, and immunostimulation, and therefore a lot of nutritional supplements containing l-glutamine are commercially available in the market. However its poor stability and low solubility in water limit its application. l-Alanyl–l-glutamine (Ala–Gln), a dipeptide composed of l-alanine and l-glutamine, circumvents the above drawbacks of l-glutamine. Ala–Gln is used in clinical nutrition as a source of l-glutamine due to its high solubility and stability in aqueous solution. Recently a drink containing Ala–Gln was launched in the U.S. market. Despite its usefulness, the commercial usage of Ala–Gln has been limited due to its unavailability at a reasonable price. Chemical and enzymatic synthesis are already known as production methods for Ala–Gln. Chemical synthesis requires protection and deprotection steps and its complicated operation pushes production costs very high for wider commercial use (Yagasaki and Hashimoto, 2008). Enzymatic methods usually employ the reverse reaction of proteases or peptidases to connect amino acids (Kumar and Bhalla, 2005; Yagasaki and Hashimoto, 2008), and therefore the protection of the amino- or carboxyl-group of the amino acids is required as well as chemical synthesis to suppress the hydrolysis of the peptide bond. Thus, the application of enzymatic methods has also been limited. The ideal way to synthesize Ala–Gln is by connecting unmodified amino acids in an irreversible reaction. Recently a novel enzyme that could synthesize dipeptides directly from amino acids was found through a smart approach for enzyme screening based on the protein structure (Tabata et al., 2005). Since all activities in nature that form peptide bonds use ATP, a dipeptide-synthesizing enzyme would be registered in protein databases as a function-unknown protein with ATP-grasp-domain and it would share weak homology with d-alanine–d-alanine (d-Ala–d-Ala) ligase which catalyzes the formation of d-Ala–d-Ala from two molecules of d-alanine. According to these hypotheses, a candidate, YwfE of Bacillus subtilis, was selected. The gene of YwfE was expressed in E. coli and the purified enzyme was confirmed to synthesize Ala–Gln from unprotected l-alanine and l-glutamine in the presence of ATP. YwfE was named l-amino acid α-ligase (Lal) since it was shown to belong to ATP-dependent carboxylate-amine–thiol ligase superfamily by the similarity of its amino acid sequence (Galperin and Koonin, 1997). Although Lal has the activity to synthesize Ala–Gln from unmodified amino acids, the expression of Lal in E. coli cells brought no accumulation of Ala–Gln in the cultivation fluid (Tabata and Hashimoto, 2007). It was assumed that it would be necessary to solve two problems: firstly the low concentration of the substrates l-alanine and l-glutamine in the cells, and secondly the dipeptide degrading activity of the cells. In order to elevate the intracellular concentration of the substrate amino acids, knowledge of the biosynthetic pathways and regulations of amino acids in bacteria was applied. To increase the

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E. coli Δpeps Ala–Gln

Ala–Gln

Ala Δdpp

Gln ΔglnEB

YwfE ATP

ADP

Metabolism

FIGURE 9.2

An outline of the Ala–Gln producer strain.

intracellular pool of l-glutamine in E. coli, genes for the negative regulator of l-glutamine biosynthesis (glnE and glnB) were disrupted. In addition to the deregulation of l-glutamine biosynthesis, enhancement of the metabolic flux toward l-alanine by expressing l-alanine dehydrogenase (Ald) from B. subtilis which catalyzed the formation of l-alanine from pyruvate resulted in the accumulation of Ala–Gln. Thus, simultaneous elevation of the supply of its substrate amino acids was proven to be effective for Ala–Gln production. Since E. coli possesses more than 50 kinds of peptidases, it was impractical to destroy all the genes of peptidases and therefore E. coli cells deficient in their three or four major peptidase genes (pepA, pepB, pepD, or pepN) were constructed. In addition to the disruption of major peptidase genes, disruption of the dipeptide import system gene (dpp) showed a synergistic effect on Ala–Gln accumulation. Finally, mutations that contributed toward increasing the intracellular concentration of the substrates and decreasing the dipeptide degrading activities were integrated. E. coli cells deficient in pepA, pepD, pepN, dpp, glnE, and glnB were transformed by the plasmid constitutively expressing Ald and Lal (Figure 9.2). When recombinant E. coli was cultivated on a glucose–ammonium salt medium without the addition of l-alanine and l-glutamine, Ala–Gln accumulated at the level of 100 mM after 47 h.

9.2.3

HYDROXYPROLINE

Hydroxyproline (trans-4-hydroxy-l-proline) is an amino acid that exists specifically in collagen, accounting for about 10–15% of the amino acids composing collagen. It is biosynthesized by the hydroxylation of l-proline residue in procollagen by the action of procollagen–proline dioxygenase. The physiological function of hydroxyproline is to stabilize the triple-helix structure of collagen in order to protect against digestion by proteolytic enzymes (Burjanadze and Veis, 1997), and it is known that hydroxyproline enhances the proliferation of epidermal cells and retains skin water content (Kawabe et al., 2009). In the European Union, hydroxyproline has been used as a raw material for anti-inflammatory agents in the form of an N-acetyl derivative. It is also used in cosmetics because of its moisturizing activity. In Japan, hydroxyproline has been used as a food additive in fruit drinks and refreshing beverages as an additive to adjust the taste and flavor in foods in general. Hydroxyproline has been manufactured by the acid hydrolysis of collagen; however, the collagen hydrolysis method requires a complex purification process and generates a large amount of waste

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material. Although enzymatic production of hydroxyproline from l-proline could overcome these drawbacks, no biocatalytic method has been discovered. Hydroxyproline has also been found in some peptide antibiotics such as etamycin (Katz et al., 1962; Sheehan et al., 1958). Procollagen–proline dioxygenase that hydroxylates l-proline to hydroxyproline in mammalian cells accepts only peptidyl proline as a substrate, and free l-proline is not accepted. In contrast, proline 4-hydroxylase activity, which hydroxylates free l-proline to hydroxyproline, has been reported in etamycin biosynthesis of Streptomyces griseoviridis P8648 (Onishi et al., 1984; Baldwin et al., 1993; Lawrence et al., 1996). However, the activity was very weak and the gene has never been cloned. Extensive screening of microbial proline 4-hydroxylases was carried out by the whole-cell reaction method focusing on Actinomyces (Shibasaki et al., 1999). More than 3000 microbial strains isolated from soil were examined and five strains, RH1, RH2, RH3, RH4, and RH5, were selected as producers of proline 4-hydroxylase which hydroxylates l-proline to hydroxyproline as well as an etamycin-producing Streptomyces strain. Strains RH1, RH3, and RH4 were identified as Dactylosporangium strains and strains RH2 and RH5 were identified as Amycolatopsis strains. The proline 4-hydroxylase of Dactylosporangium sp. RH1, which hydroxylates free l-proline to hydroxyproline, was purified and characterized. The enzyme was classified as a 2-oxoglutaratedependent dioxygenase because it required 2-oxoglutarate and dioxygen as substrates and ferrous ion as a cofactor. A proline 4-hydroxylase gene was cloned by the hybridization method using the probe corresponding to the N-terminal amino acid sequence (Shibasaki et al., 1999). The cloned gene encodes a 272 amino acid polypeptide containing amino acid sequences that had the His motif that was common to 2-oxoglutarate-dependent dioxygenase (Shibasaki et al., 1999). There were two hurdles in the construction of a manufacturing process for hydroxyproline: firstly the enzyme activity and secondly the supply of 2-oxoglutarate as a substrate. The cellular activity of proline 4-hydroxylase of Dactylosporangium sp. RH1 was so weak that the activity had to be overexpressed by gene amplification. However, it was not possible to overexpress the proline 4-hydroxylase gene in E. coli by simple application, since the gene had high G + C content (74%) and contained codons rare for E. coli, which are considered to be unfavorable to gene expression in E. coli. Therefore, the codon usage of the 5′ end of the gene was optimized for the expression in E. coli. The E. coli strain, having optimized the plasmid, pWFH1, showed a 1400-fold higher activity than that of the original Dactylosporangium sp. RH1 (Shibasaki et al., 1999). Since 2-oxoglutarate is a metabolic intermediate of tricarboxylic acid (TCA) cycle and its production from glucose has been reported (Yokota et al., 1994), the 2-oxoglutarate required for the reaction was expected to be supplied from glucose through the metabolic pathway. When E. coli harboring the plasmid pWFH1 was cultivated on a medium containing l-proline, glucose, and ferrous ion, hydroxyproline was produced in a medium at an amount of 41 g/L and the yield from l-proline was 87% (Shibasaki et al., 1999). The hydroxylation of l-proline to hydroxyproline did not proceed quantitatively because of the degradation of l-proline to l-glutamate through the action of a bifunctional enzyme encoded by putA. The introduction of a putA mutation into the host cells of E. coli improved the yield of hydroxylation of l-proline and it was hydroxylated to hydroxyproline quantitatively (Shibasaki et al., 2000) (Figure 9.3).

9.2.4

HYALURONIC ACID

Hyaluronic acid (hyaluronan, HA) is a polymer of disaccharide, composed of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) linked together via alternating β-1,4 and β-1,3 glycosidic bonds and it can reach chain lengths of up to 20,000 disaccharide units or higher (8 MDa). HA is naturally found in various tissues of the body, such as skin, cartilage, and vitreous humor and is one of the chief components of the extracellular matrix. It is known that HA contributes to cell proliferation and migration and may also be involved in the progression of some malignant

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Biotechnology in Functional Foods and Nutraceuticals E. coli l-Glu putA

l-Pro

2OG Glucose

TCA cycle Succinate

FIGURE 9.3

l-Pro

Hydroxylase Hydroxy proline

Hydroxyproline

An outline of the hydroxyproline producer strain.

tumors. It is therefore well suited to biomedical applications targeting these tissues. The first biomedical product containing HA was approved for use in eye surgery and it is also used to treat osteoarthritis of the knee (Puhl and Scarf, 1997). Oral use of HA has been suggested, with its use as a dietary supplement (Kajimoto et al., 2001; Satoh et al., 2002). HA is a common ingredient in skin care products. HA can be obtained by extraction from rooster combs or through fermentation with strains of group C Streptococcus. In rooster combs, HA forms a complex with proteoglycans, which makes the isolation of HA with high purity and high molecular weight costly (O’Regan et al., 1994). In addition, animal-derived products carry the risk of transmitting viruses and other adventitious agents as well as inducing immunogenic and inflammatory responses. The fermentation method using Streptococcus strain is far from ideal despite its long history because of the difficulties in genetic engineering. HA is produced from uridine 5′-diphosphate–N-acetylglucosamine (UDP–GlcNAc) and UDP– GlcUA by hyaluronan synthase. UDP–GlcNAc is a precursor of cell wall biosynthesis and UDP– GlcUA is synthesized from UDP-glucose by NADH-dependent UDP-glucose dehydrogenase. Recently, metabolically engineered B. subtilis strains have been constructed for HA fermentation (Widner et al., 2005). In these strains, the hasA gene from Streptococcus equisimilis, which encodes hyaluronan synthase, has been expressed under the mutated version of the amyQ promoter from Bacillus amynoliquefaciens. Artificial operons, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the biosynthesis of UDP–GlcNAc and UDP–GlcUA, were assembled and introduced on the chromosome of B. subtilis (Figure 9.4). It was determined that the production of UDP–GlcUA is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene encoding UDP-glucose dehydrogenase is sufficient for highlevel production of HA. When the Bacillus strains were cultivated on a minimal medium with sucrose as the carbon source, multigrams of HA were produced in the medium and the molecular weight was in the 1.1–1.2 MDa range. Although there are no major differences in productivity and molecular weight as compared to the Streptococcus fermentation, Bacillus would be a better production strain because: (1) B. subtilis is generally recognized as safe (GRAS), (2) tools for genetic engineering are well developed in Bacillus, and (3) HA produced is not cell associated, facilitating the downstream processing.

9.2.5 N-ACETYLGLUCOSAMINE Glucosamine and GlcNAc are monosaccharide and are known to be precursors in humans of the disaccharide unit in glycosaminoglycans such as HA, chondroitin sulfate, and keratan sulfate, which are necessary to repair and maintain healthy cartilage and joint function. It is estimated that 33 million people suffer from osteoarthritis in the United States, and glucosamine has been

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B. subtilis

Glc-6-P

Fru-6-P

Glc-1-P

GlcN-6-P

GlcN-1-P

UDP-Glc tuaD

GlcNAc-1-P UDP-GlcUA UDP-GlcNAc hasA

Hyaluronic acid

Hyaluronic acid

FIGURE 9.4

An outline of the hyaluronic acid producer strain.

widely used as a dietary supplement for joint health (Hungerford and Jones, 2003, Towheed, 2003). Currently, glucosamine is produced by the acid hydrolysis of chitin, a linear polymer of GlcNAc, which is extracted from crab and shrimp shells. Concentrated hydrochloric acid breaks down the polymer and deacetylates GlcNAc to form glucosamine. Glucosamine production could become limited by variable raw material supply as its demand continues to increase. Moreover, it is pointed out that glucosamine from shellfish may not be suitable for people with shellfish allergies. Some filamentous fungi contain chitosan (a linear copolymer of glucosamine and GlcNAc) in their cell walls, and a method for glucosamine production from the fungal biomass has also been developed. GlcNAc supplements for joint health are also available, although the market is much smaller than that for glucosamine. GlcNAc is now produced by chemical acetylation of glucosamine using acetic anhydride; however, GlcNAc is synthesized in bacteria, and therefore a novel fermentation method for GlcNAc has been expected. A metabolically engineered E. coli for the production of GlcNAc was constructed through (1) the overexpression of the biosynthetic genes and (2) the inactivation of the degradation of intermediates for GlcNAc synthesis and GlcNAc transporters (Deng et al., 2005, 2006a,b) (Figure 9.5). Glucosamine synthase (GlmS), which catalyzes the synthesis of glucosamine-6-phosphate (GlcN-6-P) from fructose-6-P and glutamine, is known to be inhibited by GlcN-6-P. Therefore a mutant enzyme whose sensitivity to GlcN-6-P was significantly reduced was generated. The gene for GlcN-6-P acetyltransferase (GNA1), which converts GlcN-6-P into GlcNAc-6-P, was cloned from Saccharomyces cerevisiae and overexpressed in E. coli. GlcNAc-6-P is then dephosphorylated and secreted into the medium. In the producer strain of GlcNAc, transporter proteins of GlcNAc as well as GlcNAc-6-P deacetylase and GlcN-6-P deaminase were deleted. When the metabolically engineered E. coli

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Biotechnology in Functional Foods and Nutraceuticals Glucose

Glc-6-P

E. coli

Fru-6-P

ΔnagB

glmS GlcN-6-P GNA1

ΔnagA

GlcNAc-6-P

GlcNAc ΔmanXYZ, ΔnagE

GlcNAc

FIGURE 9.5

An outline of the GlcNAc producer strain.

was cultivated on a minimal medium with glucose as the carbon source, GlcNAc accumulated at concentrations greater than 110 g/L for 72 h.

9.2.6

CYTIDINE 5′-DIPHOSPHATE CHOLINE

Cytidine 5′-diphosphate choline (CDP-choline) is an intermediate in the biosynthesis of phosphatidylcholine, one of the phospholipids that form the main constituents of cell membranes, and acetylcholine which is known as a neurotransmitter. Both compounds are important for proper brain function. Ischemia causes an increased consumption of ATP, raising the level of adenosine 5′-monophosphate and releasing cytotoxic free fatty acid such as arachidonic acid and diacylglycerols from phosphatidylcholine, leading to further lesions. CDP-choline traps arachidonic acid by promoting the synthesis of phosphatidylcholine, and suppresses the occurrence of lesions by maintaining the structure of nerve cells (Trovarelli et al., 1981). It has also been reported that CDP-choline elevated the cholinergic activity by activating choline acetyltransferase (Dixon et al., 1997; Amenta et al., 2001). Furthermore, CDP-choline elevated the levels of noradrenaline and dopamine (Secades and Frontera, 1995). It is considered that CDP-choline is efficacious in improving brain function through the mechanisms described above. It is expected that the administration of CDP-choline as a supplement would be useful for patients who are not subjected to therapy for traumatic brain disorders or Alzheimer’s disease. In fact, it was reported that oral administration of CDP-choline increased the amounts of phosphatidylcholine in the brains of healthy rats (Lopez-Coviella et al., 1995). It was also reported that when CDP-choline was administered to elderly subjects suffering from senile disturbance of the memory, but not from dementia, the subjects’ memories were improved (Alvarez et al., 1997). CDP-choline has been used as a pharmaceutical for activating brain function in Japan and the European Union, and is marketed as a dietary supplement ingredient. Because strokes and Alzheimer’s disease are serious problems throughout the world, a CDP-choline product as a supplement that can be taken easily might have great social and economical value. CDP-choline has been produced by chemical synthesis or enzymatic methods from cytidine 5′-monophosphate (CMP) or cytidine using yeast cells as the enzyme sources. Enzymatic production

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C. ammoniagenes

UMP

Orotic acid PRPP PPi

UDP ATP ADP

UTP

UTP

ATP ADP

Glucose

E. coli UTP

pyrG

cki Choline

CTP cct

P-choline

CDP-choline

Cells are permeabilized by an organic solvent

FIGURE 9.6

An outline of CDP-choline production.

of cytidine diphosphate choline (CDP-choline) from orotic acid and choline chloride was established by coupling genetically engineered bacteria. Corynebacterium ammoniagenes cells were able to produce uridine nucleotides from orotic acid, a cheap starting material for CDP-choline; however, the accumulation of cytidine 5′-triphosphate (CTP), a direct precursor of CDP-choline, was very small. Therefore it was considered that three enzyme activities were needed for CDP-choline production. E. coli strains overproducing CTP synthetase gene (pyrG) from E. coli, choline kinase gene (cki) from Saccharomyces cerevisiae, and cholinephosphate cytidylyltransferase (cct) from Saccharomyces cerevisiae, respectively, were constructed. These three E. coli cells were coupled with Corynebacterium ammoniagenes cells, and 7.7 g/L of CDP-choline accumulated from orotic acid and choline chloride as substrates using glucose as an energy source after 23 h (Fujio and Maruyama, 1997). An improved method for the production of CDP-choline was developed (Figure 9.6). A plasmid that simultaneously overexpressed pyrG, cki, and cct was constructed. In this construct, cct and cki were designed to be expressed as a single cct/cki-fused protein. When E. coli cells expressing the three genes were coupled with Corynebacterium ammoniagenes, 11 g/L of CDP-choline accumulated from orotic acid and choline chloride after 23 h (Fujio et al., 1997).

9.3 FUTURE PERSPECTIVES The development of technology for the production of potential nutriments through the isolation of enzymes and genetic engineering enables large-scale production of compounds that were previously rare and difficult to obtain in sufficient quantity at a reasonable cost. More and more healthpromoting compounds could be produced by genetic engineering technology. However, at present, consumers tend to dislike products that have been produced by genetically modified organisms (GMO), particularly in the European Union and Japan. Continuous efforts to establish the safety of products produced by GMO aim to gain consumers’ understanding and acceptance of nutriments produced by genetic engineering. We hope that nutriments produced by genetically engineered bacteria will be launched onto the market and will contribute to the health and wealth of the people of the world.

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Murata, K. and Kimura, A., 1990. Overproduction of glutathione and its derivatives by genetically engineered microbial cells. Biotechnol. Adv. 8: 59–96. Murata, K., Miya, T., Gushima, H., and Kimura, A., 1983. Cloning and amplification of a gene for glutathione synthetase in Escherichia coli B. Agric. Biol. Chem. 47: 1381–1383. Murata, K., Tani, K., Kato, J., and Chibata, I.., 1981. Glutathione production by immobilized Saccharomyces cerevisiae cells containing an ATP generation system. Eur. J. Appl. Microbiol. Biotechnol. 11: 72–77. Ohtake, Y., Watanabe, K., Tezuka, H., et al., 1988. The expression of gamma-glutamylcysteine synthetase gene of Escherichia coli in Saccharomyces cerevisiae. Agric. Biol. Chem. 52: 2753–2762. Ohtake, Y., Watanabe, K., Tezuka, H. et al., 1989. Expression of glutathione synthetase gene of Escherichia coli B in Saccharomyces cerevisiae. J. Ferment. Bioeng. 68: 390–399. Onishi, M., Okumura, Y., Okamoto, R., and Ishikura, T., 1984. Proline hydroxylation by cell free extract of a streptomycete. Biochem. Biophys. Res. Commun. 120: 45–51. O’Regan, M., Martini, I., Crescenzi, F., DeLuca, C., and Lansing, M., 1994. Molecular mechanisms and genetics of hyaluronan biosynthesis. Int. J. Biol. Macromol. 16: 283–286. Puhl, W. and Scharf, P., 1997. Intra-articular hyaluronan treatment for osteoarthritis. Ann. Rheum. Dis. 56: 637–640. Richman, P.G. and Meister, A., 1975. Regulation of gamma-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J. Biol. Chem. 250: 1422–1426. Satoh, H., Sakamoto, W., Odanaka, W., Yoshida, K., and Urushihata, O., 2002. Clinical effects of foods containing hyaluronic acid on dry and rough skin. Aesthetic Dermatol. 12: 109–120. Secades, J.J. and Frontera, G., 1995. CDP-choline: Pharmacological and clinical review. Methods Find. Exp. Clin. Pharmacol. 17: 1–54. Sheehan, J.C., Zachau, H.G., and Lawson, W.B., 1958. The structure of etamycin. J. Am. Chem. Soc. 80: 3349–3355. Shibasaki, T., Mori, H., Chiba, S., and Ozaki, A., 1999. Microbial proline 4-hydroxylase screening and gene cloning. Appl. Environ. Microbiol. 65: 4028–4031. Shibasaki, T., Mori, H., and Ozaki, A., 2000. Enzymatic production of trans-4-hydroxy-l-proline by regio- and stereospecific hydroxylation of l-proline. Biosci. Biotechnol. Biochem. 64: 746–750. Tabata, K. and Hashimoto, S., 2007. Fermentative production of l-alanyl–l-glutamine by a metabolically engineered Escherichia coli strain expressing l-amino acid α-ligase. Appl. Environ. Microbiol. 73: 6378–6385. Tabata, K., Ikeda, H., and Hashimoto, S., 2005. ywfE in Bacillus subtilis codes for a new enzyme, l-amino acid ligase. J. Bacteriol. 187: 5195–5202. Towheed, T.E., 2003. Current status of glucosamine therapy in osteoarthritis. Arthritis Rheum. 49: 601–604. Trovarelli, G., de Medio, G.E., Dorman, R.V., Piccinin, G.L., Horrocks, L.A., and Porcellati, G., 1981. Effect pf cytidine diphosphate choline (CDP-choline) on ischemia-induced alterations of brain lipid in the gerbil. Neurochem. Res. 6: 821–833. Widner, B., Behr, R., Dollen, S.V., et al., 2005. Hyaluronic acid production in Bacillus subtilis. Appl. Environ. Microbiol. 71: 3747–3752. Yagasaki, M. and Hashimoto, S., 2008. Synthesis and application of dipeptides; current status and perspectives. Appl. Microbiol. Biotechnol. 81: 13–22. Yokota, A., Shimizu, H., Terasawa, Y., Takaoka, N., and Tomita, F., 1994. Pyruvic acid production by a lipoic acid auxotroph of Escherichia coli W1485. Appl. Microbiol. Biotechnol. 41: 638–643.

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Advances in 10 Recent the Development of Transgenic Pulse Crops Susan Eapen CONTENTS 10.1 Introduction .......................................................................................................................... 139 10.2 Transgenic Pulse Crops ........................................................................................................ 140 10.2.1 Chickpea (Cicer arietinum L.) .................................................................................. 140 10.2.2 Moth Bean (Vigna aconitifolia L. Jacq. Marechal) .................................................. 141 10.2.3 Blackgram (Vigna mungo L. Hepper)....................................................................... 141 10.2.4 Greengram (Vigna radiata) ...................................................................................... 142 10.2.5 Cowpea (Vigna unguiculata) .................................................................................... 142 10.2.6 Vicia Species............................................................................................................. 142 10.2.7 Pea (Pisum sativum L.) ............................................................................................. 142 10.2.8 Common Bean (Phaseolus vulgaris L. and Phaseolus acutifolius L. Gray)............ 143 10.2.9 Pigeon Pea (Cajanus cajan L. Millsp.) ..................................................................... 143 10.3 Improving Plant Regeneration .............................................................................................. 143 10.4 Improving Gene Transfer Efficiency .................................................................................... 144 10.5 Marker Genes for Selection of Transformants ..................................................................... 147 10.6 Preventing Horizontal Gene Transfer by Chloroplast Transformation................................. 148 10.7 Transgenic Plant Development: Possible Targets ................................................................. 148 10.8 Translating Model Legume Genomics to Pulse Crops ......................................................... 149 10.9 Conclusions ........................................................................................................................... 149 References ...................................................................................................................................... 150

10.1 INTRODUCTION Pulse crops, belonging to the family Leguminosae, are harvested solely for their dry grains and are known to play a pivotal role in improving the fertility of the soil by nitrogen fixation. They are of immense importance to humanity and are a key to sustainable agriculture. Pulses, due to their high protein content, are important for the vegetarian population of developing countries and are known as “the poor man’s meat.” Pulse proteins are generally deficient in the essential amino acid methionine. The digestibility of pulse protein is high and consumption of pulses is known to reduce mortality from coronary heart diseases (Menotti et al., 1999). India is the world’s largest producer and consumer of pulse crops. The major pulse crops include pigeon pea or red gram (Cajanus cajan), chickpea (Cicer arietinum), kidney bean (Phaseolus vulgaris), mung bean or green gram (Vigna radiata), black gram or urd bean (Vigna mungo), azuki bean (Vigna angularis), moth bean (Vigna aconitifolia), field bean (Vicia faba), lima bean (Vigna lunatus), dry cowpea (Vigna unguiculata), lentil (Lens culinaris), and dry peas (Pisum sativum). Minor pulses include winged bean (Psophocarpus tetragonolobus), velvet bean (Mucuna spp.), and so on. 139 © 2010 Taylor and Francis Group, LLC

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Although conventional plant breeding has contributed to the improvement of crops in the past, transgenic technology has immense potential to achieve this objective as an additional/supplementary method. Genetically modified crops represent one of the most rapidly adopted technological innovations that have been commercialized in the history of agriculture, in spite of significant political and regulatory barriers (Dunwell, 2000; Fernandez-Cornejo, 2005). Pulse crops, well known for their inherent ability to fix nitrogen and the pivotal role they play as the major source of protein for the population of developing countries, ranking third in world food production after cereals and oilseed crops, need further genetic improvement by the incorporation of heterologous genes using transgenic technology to meet the increase in demand for food along with improved quality (Christou, 1997; Popelka et al., 2004; Eapen, 2008). Crop yields can be improved by manipulating the physiological processes and the ability to withstand biotic and abiotic stresses. Despite the development of transgenic technology in the 1980s and the first report on the development of a transgenic pulse crop Vigna aconitifolia in the same decade (Eapen et al., 1987; Kohler et al., 1987a, 1987b), the progress achieved is not satisfactory. Transgenic pulse crop research, although it has moved forward, is still confined to the four walls of the laboratory. Since these tropical grain legumes are of prime importance in developing countries, less effort has been made compared to crop species with importance for developed countries. Different factors such as the recalcitrance of pulses for regeneration, low competency of regenerating cells for transformation, and lack of a reproducible in planta transformation system have been pointed out as reasons for the lack of development of transgenic pulse crops with high efficiency (Somers et al., 2003; Popelka et al., 2004; Dita et al., 2006; Eapen, 2008). However, soybean—a leguminous oil crop and a source of protein—is a successful example, where transgenics have been developed and commercialized.

10.2

TRANSGENIC PULSE CROPS

Different methods can be employed for the production of transgenic pulse crops. They have been produced using Agrobacterium-mediated transformation (Eapen et al., 1987; Krishnamurthy et al., 2000; Sharma et al., 2006), by particle gun bombardment (Kamble et al., 2003; Indurker et al., 2007), by electroporation of intact axillary buds (Chowrira et al., 1996), and by electroporation and polyethylene glycol (PEG) mediated transformation using protoplasts (Kohler et al., 1987a, 1987b). Of all the methods, Agrobacterium-mediated transformation of explants is the most popular method for the development of transgenics in pulse crops. While, in the early years, antibiotic marker genes were introduced to test the feasibility of transformation (Eapen et al., 1987; Saini and Jaiwal, 2007), other genes of economic importance such as Cry genes (Sanyal et al., 2005; Indurker et al., 2007), sunflower albumin gene (Molvig et al., 1997), α-amylase inhibitor gene (Sonia et al., 2007), and chitinase gene (Kumar et al., 2004) have also been introduced into major pulse crops. Since most legumes regenerate from young embryonic tissues, embryonic axes (Krishnamurthy et al., 2000) and cotyledonary nodes (Kumar et al., 2004) are the preferred explants for transformation. Results of transgenic plant development in some of the pulse crops are detailed below.

10.2.1 CHICKPEA (CICER ARIETINUM L.) Chickpea is the second most important grain legume in the world and is widely grown in Asia, Africa, parts of America, Europe, and Australia. Chickpea is vulnerable to many insect pests such as Heliothis armigera, a lepidopteran pest, seed beetle (Callosobruchus chinesis), and fungal diseases caused by Ascochyta rabiei and Fusarium oxysporum, which result in a drastic reduction in yield. Although wild relatives of chickpea possess genes for resistance to some biotic and abiotic stresses, classical breeding attempts to introgress resistance into cultivated species have not led to any breakthroughs and hence genetic engineering of plants may provide a good scope for the introduction of genes for the improvement of chickpea. Plant regeneration via organogenesis (Shri and Davis, 1992) and somatic embryogenesis has been reported, but stable transformation reports using these methods of regeneration are limited.

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Most reports on genetic transformation in chickpea have employed Agrobacterium as a vector for the introduction of DNA (Fontana et al., 1993; Kar et al., 1996; Krishnamurthy et al., 2000; Sanyal et al., 2003; Senthil et al., 2004; Tewari-Singh et al., 2004) and the frequency of transformation ranged from 0.2% to 4%. In our laboratory, we were able to obtain high-frequency transformation in chickpea using particle gun bombardment (Indurker et al., 2007) and a CryIAc gene was introduced. For the majority of work on chickpea transformation, embryonal axes devoid of shoot meristems have been used as the explant. The rooting and survival of transgenic chickpea has been problematic and some authors have tried in vitro grafting in order to get complete plants (Krishnamurthy et al., 2000). Chakraborti et al. (2009) developed transgenic chickpea with Allium sativum leaf agglutinin. Sarmah et al. (2004) and Ignacimuthu and Prakash (2006) introduced an α-amylase inhibitor gene from Phaseolus vulgaris and the transgenic plants showed resistance to bruchid weevil, while Indurker et al. (2007) introduced the CryIAc gene for resistance to insect pests. An osmoregulatory gene P5CSF129A, when introduced into chickpea, resulted in the enhanced accumulation of proline (Bhatnagar-Mathur et al., 2009). Scope for the improvement of chickpea through genetic transformation exists for the introduction of genes for insect resistance (Cry genes, protease inhibitor genes), fungal resistance (chitinase gene), and different biotic and abiotic stresses such as drought and temperature.

10.2.2 MOTH BEAN (VIGNA ACONITIFOLIA L. JACQ. MARECHAL) Moth bean is a drought-tolerant pulse crop in India. The plant is susceptible to leaf spot Cercospora cruenta, mildew Sparotheca humuli, a blight that affects leaves, stalks, and pods caused by Phyttotica phaseolence and Phyttotica anthracene. It is also susceptible to pod borer insects (Heliothis armigera). Plant regeneration in this species was reported in the 1980s. Regeneration can be obtained through organogenesis (Eapen et al., 1986) or through somatic embryogenesis. This is the only pulse crop in which regeneration has been obtained from protoplasts (Shekhawat and Galston, 1983; Eapen et al., 1987); hence, protoplasts were used for Agrobacterium-mediated gene transfer (Eapen et al., 1987) and direct DNA transfer using the incubation of protoplasts with PEG and electroporation (Kohler et al., 1987a, 1987b) and complete plants were obtained. The plant cultivar type and plasmid type were found to determine the transformation frequency (Kohler et al., 1987a). Direct DNA transfer using particle gun bombardment has been reported by Kamble et al. (2003). Malabadi and Nataraja (2007) introduced marker genes, developed somatic embryogenesis and plant regeneration, and showed transmission of transgenes. Vigna aconitifolia can serve as a model plant for genetic transformation in pulse crops, since regeneration can be achieved from almost all explants and isolated protoplasts.

10.2.3 BLACKGRAM (VIGNA MUNGO L. HEPPER) This is an important grain legume of the tropics. It is susceptible to a wide range of insect pests and is also susceptible to many viruses. Regeneration of Vigna mungo has been reported from cotyledonary nodes and from immature cotyledons (Tivarekar and Eapen, 2001); Geetha et al. (1997) could regenerate shoots from callus cultures of epicotyl, cotyledonary nodes, and the immature leaf of Vigna mungo, while Das et al. (1998) obtained shoots from petiole explants. Somatic embryogenesis was reported in Vigna mungo derived from immature explants (Eapen and George, 1990), but further regeneration to plants was found to be difficult; Karthikeyan et al. (1996) obtained transgenic calli of Vigna mungo by cocultivating primary leaves and stable transformation was obtained. Stable genetic transformation using Agrobacterium tumefaciens has been reported by Saini et al. (2003) and Saini and Jaiswal (2005). Phosphoinothricin was used as a selectable marker for Vigna mungo transformation by Murugananthum et al. (2007). Overexpression of the glyoxalase 1 gene into Vigna mungo resulted in salt tolerance (Bhomkar et al., 2008). The characteristics which need to be improved in Vigna mungo include disease resistance, particularly resistance to viruses.

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10.2.4 GREENGRAM (VIGNA RADIATA) Regeneration in Vigna radiata is from preexisting meristems of cotyledonary nodes which produce axillary buds by de novo regeneration. Pal et al. (1991) reported primary transformants of Vigna radiata using cotyledons and using Agrobacterium as a vector. Jaiwal et al. (2001) reported transgenic Vigna radiata using cotyledonary nodes and using Agrobacterium as a vector. Sonia et al. (2007) transferred a Phaseolus vulgaris α-amylase gene to Vigna radiata. There is a lot of scope for improvement in this crop for disease resistance using transgenic technology.

10.2.5 COWPEA (VIGNA UNGUICULATA) Cowpea is one of the most important pulse crops. Muthukumar et al. (1996) reported shoot regeneration from intact cotyledons of cowpea. The biolistic method of genetic transformation was used to transform Vigna unguiculata (Ivo et al., 2008). The system is based on using of the herbicide imazaphys to select transformed meristem cells after the physical introduction of the ahas gene. The transgenes (gus and ahas) segregated in a Mendelian fashion. Popelka et al. (2006) reported the development of transgenic plants using Agrobacterium-mediated gene transfer, while particle gun bombardment was used by others (Kononowicz et al., 1993; Matsuoka et al., 1997; Ikea et al., 2003). Solleti et al. (2008) developed transgenic cowpea seeds expressing a bean α-amylase I inhibitor gene which improved resistance to storage pests such as Callosobruchus maculates and Callosobruchus chinensis. The traits that need improvement include resistance to biotic and abiotic stresses, especially the development of cowpea resistant to cowpea pod borer aphids and weevils.

10.2.6

VICIA SPECIES

Vicia consists of 150 species and Vicia faba is the most important of all Vicia species. Other Vicia species which are used include Vicia narbonensis, Vicia sativa, Vicia villosa, and so on. In Vicia faba, somatic embryogenesis has been reported by Griga et al. (1987), but they did not develop into plants. Somatic embryogenesis has been described in the leaf-derived callus of Vicia narbonensis (Albrecht and Kohlenbach, 1989). Plant regeneration through somatic embryogenesis was described by Pickardt et al. (1989) in Vicia narbonensis. Pickardt et al. (1991) reported the development of transgenic plants in Vicia narbonensis by selecting for hygromycin resistance and using in vitro grafting to obtain complete plants. About 200 independent transgenic plants containing various genes such as nptII, hptII, PAT, and methionine-rich proteins (Saalbach et al., 1994; Pickardt et al., 1995) have been produced. Czihal et al. (1999) also obtained transgenic Vicia narbonensis plants. Transgenic Vicia faba plants were developed by Bottinger et al. (2001). Possible applications of genetic transformation for Vicia species should aim at developing fungal resistance, generating male sterile lines, and controlling Orobanche infestation.

10.2.7 PEA (PISUM SATIVUM L.) Pisum sativum L. is used as a food for human consumption and also as an animal feed. PuontiKaerlas et al. (1990) developed transgenic plants using Agrobacterium tumefaciens after the cocultivation of explants from the shoot cultures and epicotyl of 10-day-old seedlings. Schroeder et al. (1993) and Bean et al. (1997) used cotyledonary nodes as explants for obtaining transgenic plants of Pisum sativum. Immature cotyledons were used as explants for the transformation of Pisum sativum by Grant et al. (1995). Other reports on Agrobacterium tumefaciens-mediated gene transfer in Pisum sativum include those of Miller et al. (1999). Chowrira et al. (1996) reported the production of transgenic pea plants using the electroporation of apical nodes. Like other pulse crops, Pisum sativum is also susceptible to bruchids, which cause major grain losses throughout the world. Shade et al. (1994) introduced a bean α-amylase gene under the control of a bean phytohemagglutinin gene promoter. The transgenic plants and their progeny were found to

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be resistant to Azuki bean weevil. Pusztai et al. (1999), after feeding tests using transgenic pea with α-amylase, showed that it caused no adverse effects in farm animals. A coat protein gene for alfalfa mosaic viruses was transferred to Pisum sativum (Grant et al., 1995, 1998) and glass house tests were conducted (Timmerman-Vaughan et al., 2001). Timmerman-Vaughn et al. (2001) found that only plants that had accumulated detectable amounts of the transgene coat protein product were found to be resistant. A transgenic plant resistant to another virus—Pea seed borne mosaic virus (PSbMV)— was also developed by Grant and his colleagues. A replicase gene was transferred for obtaining plants resistant to PSbMV. Chowrira et al. (1996) obtained plants resistant to Pea Enation Mosaic Virus (PEMV) by using transgenic technology. Introduction of a bean α-amylase inhibitor gene in Pisum sativum inhibited the development of pea weevil larvae (De Souse-Major et al., 2007).

10.2.8 COMMON BEAN (PHASEOLUS VULGARIS L. AND PHASEOLUS ACUTIFOLIUS L. GRAY) There are five cultivated species within the genera Phaseolus, of which Phaseolus vulgaris and Phaseolus acutifolius are economically the most important. The common bean (Phaseolus vulgaris) is one of the most important grain legumes of India, Latin America, and Africa. The susceptibility of Phaseolus vulgaris to Agrobacterium has been demonstrated by using different explants such as leaves, meristems, cotyledons, and hypocotyls (Mc Clean et al., 1991; Franklin et al., 1993; Becker et al., 1994; Lewis and Bliss, 1994; Nagl et al., 1997). However, regeneration from transgenic callus was found to be difficult. Mature embryos of Phaseolus were transformed with the methionine-rich albumin gene of Brazil nuts. However, no stable transformant was recovered (Aragao et al., 1992). Later, a 2S albumin gene from Brazil nuts was introduced by Aragao et al. (1999) and transgenic plants were obtained. Russell et al. (1993) introduced a bar gene that confers resistance to herbicide phosphinothricin and also a coat protein gene for BGM virus resistance. Two transgenic lines showed delayed and attenuated viral symptoms (Aragao et al., 1998). In Phaseolus vulgaris, Dillen et al. (1995) introduced transgenes using electroporation. De Clerq et al. (2002) have demonstrated in Phaseolus acutifolius transformation using regeneration competent tissues. Once transgenic technology is developed, one can introduce several genes of interest for the improvement of Phaseolus.

10.2.9 PIGEON PEA (CAJANUS CAJAN L. MILLSP.) Pigeon pea (Cajanus cajan L. Millsp.), also known as red gram, is a major pulse crop of the tropics and subtropics. It is widely cultivated on the Indian subcontinent and accounts for 90% of the world’s pigeon pea cultivation. The crop is susceptible to insects like pod fly, pod borer (Heliothis armigera), storage pests such as Callosobruchus maculates, and fungal diseases such as wilt caused by the sterility mosaic virus. Regeneration in Cajanus has been reported from cotyledons (Mehta and Mohan Ram, 1980; George and Eapen, 1994; Geetha et al., 1998) and primary leaf explants (Eapen and George, 1993; Geetha et al., 1998). Geetha et al. (1999) and Lawrence and Koundal (2001) reported on the development of transgenic plants of pigeon pea. Satyavathi et al. (2003) produced transgenic plants of pigeon pea which expressed the hemagglutinin protein of Rinderpest virus, while rice chitinase gene was introduced into the plant by Kumar et al. (2004). Sharma et al. (2006) developed transgenic pigeon pea plants with a synthetic Bt CryIAb gene for tolerance to insects. Overexpression of dhdps gene in Cajanus cajan resulted in enhanced lysine content in seeds (Thu et al., 2007). In planta transformation of pigeon pea was developed by Rao et al. (2008), but needs to be repeated by other laboratories.

10.3

IMPROVING PLANT REGENERATION

Plant regeneration in pulse crops just as in other plants can occur through three pathways, namely, de novo organogenesis, somatic embryogenesis, or through the proliferation of shoot meristems from areas surrounding a shoot bud (see Jaiwal and Singh, 2003). Among the three modes of regeneration

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as target cells for transformation, meristematic areas of cotyledonary nodes are the preferred explant source as in Cajanus cajan (Dayal et al., 2003; Thu et al., 2003), Cicer arietinum (Sarmah et al., 2004), Vigna mungo (Saini et al., 2003), and Pisum sativum (Pniewski and Kapusta, 2005). In some cases, de novo organogenesis from leaf (Dayal et al., 2003) and epicotyl (Barik et al., 2005; Indurker et al., 2007) was utilized for developing transformants, whereas somatic embryogenesis was employed for the development of transgenic plants as in the case of Vicia narbonensis (Pickardt et al., 1991). The introduction of genes which will induce somatic embryogenesis into grain legumes may further improve the target source with competent cells capable of regeneration and transformation. The introduction of genes such as Wuschel (Zuo et al., 2002), Baby boom (Boutilier et al., 2002), and esr-1 (Bano and Chua, 2002) into pulse crops may enhance their regeneration potential and the regenerating tissues can subsequently be used as targets for the introduction of transgenes.

10.4

IMPROVING GENE TRANSFER EFFICIENCY

A quick method of delivering foreign genes with high transformation efficiency is required for the development of transgenic pulse crops. Agrobacterium-mediated gene transfer is the most common method of transformation of tropical grain legumes, although particle gun bombardment is also used to develop transgenic plants. Among the pulse crops, Vigna aconitifolia is the only pulse crop reported to produce regeneration of complete plants from isolated protoplasts (Eapen et al., 1987), which were exploited for genetic transformation using Agrobacterium (Eapen et al., 1987) and direct DNA transfer using PEG and electroporation (Kohler et al., 1987a, 1987b). However, these methods of gene transfer into protoplasts/cells cannot be applied to other pulse crops, where totipotency of protoplasts has not yet been demonstrated. In planta infection/electroporation of axillary meristems of pea, cowpea, and lentil for transformation, although reported by Chowrira et al. (1996, 1998), needs to be repeated by other laboratories, and also extended to other grains legumes. In lentil, vacuum infiltration with Agrobacterium was found to enhance transient gene expression (Mahmoudian et al., 2002). Although Agrobacterium is the most commonly used vector for transformation, it is worth trying other vectors such as Rhizobium (Broothaerts et al., 2005) for genetic transformation of pulse crops, since Rhizobium is a natural agent, which causes nodulation and nitrogen fixation in leguminous plants. The development of quick transformation protocols—such as the imbibing of germinating seeds with Agrobacterium and the floral dip method as in the case of Arabidopsis (Clough and Bent, 1998)—is ideal and should be tested for the transformation of pulses. However, it is unlikely that the floral dip method of transformation will be successful in case of pulse crops due to an inability to reach the gametic cells because of the closed floral structure of legumes. Liu et al. (2005) developed a nontissue culture method for Phaseolus vulgaris using sonication and the vacuum infiltration assisted Agrobacterium-mediated method for transformation. Development of an in planta transformation method will overcome the tedious procedure of tissue culture and unwanted variation—somaclonal variation. Only one of the pulse crops, pigeon pea, is reported to have an in planta transformation (Rao et al., 2008). The protocol involves developing whole plant transformants (to plants) directly from Agrobacterium-infected young seedlings. The apical and intercotyledonary regions are pricked with a needle, treated with Agrobacterium, and plants developed. The seeds produced were analyzed further and transgenics developed. This technique of gene transfer looks simple, but has to be repeated in other laboratories and with other pulse crops for the protocol to be of practical use. Microprojectile bombardment is also an efficient method for developing transgenic plants, as has been demonstrated in the case of Vigna aconitifolia (Kamble et al., 2003), lentil (Gulati et al., 2002), and chickpea (Indurker et al., 2007). The development of transgenic pulse crops using Agrobacterium, particle gun bombardment, and other direct DNA transfer methods is shown in Tables 10.1, 10.2, and 10.3, respectively. Despite several reports on the development of transgenic plants, transformation frequency in pulse crops has been very low. The selection of supervirulent strains of Agrobacterium with improved vir genes and the addition of phenolic compounds like acetosyringone, thiol compounds

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TABLE 10.1 Selected Examples of Pulse Crop Transformation Using Agrobacterium tumefaciens Plant Species Cicer arietinum

Cajanus cajan

Explant Used

Lupinus angustifolius L.

Phaseolus acutifolius

Gene

Reference

Leaf and stem

Callus

nptII

Embryos Embryonic axis

Plant Plant

uidA, nptII cryIAc gene

Embryonic axis

Plant

nptII, uidA

Embryonic axis and cotyledonary node Plumule Half embryo with one cotyledon Embryo explant

Plant

uidA

Plant Plant

Senthil et al. (2004) Sarmah et al. (2004)

Plant

Embryo slice Embryonic axis Embryos

Plant Plant Plant

Single cotyledon, half embryo explant Axillary meristems

Plant

bar α-amylase inhibitor aspartate kinase gene uidA/nptII cryIAc gene α-amylase inhibitor gene, nptII Allium sativum leaf agglutinin

Plant

PSCSF129A

Plant

uidA, nptII

Bhatnagar-Mathur et al. (2009) Geetha et al. (1999)

Organogenesis, and callus Organogenesis

nptII

Shoot apices, cotyledonary node Embryonic axis

Srinivasan et al. (1988, 1991) Fontana et al. (1993) Kar et al. (1996, 1997) Krishnamurthy et al. (2000) Sanyal et al. (2003)

Tewari-Singh et al. (2004) Polowick et al. (2004) Sanyal et al. (2005) Ignacimuthu and Prakash (2006) Chakraborti et al. (2009)

Lawrence and Koundal (2001) Satyavathi et al. (2003)

Leaf Cotyledonary node Cotyledonary node

Plant Plant Plant

Axillary bud of germinating seed Plumular, intercotyledonary region Cotyledonary node, decapitated embryo Cotyledonary node Embryonal axis slices Shoot apieces Callus

Plant

nptII hemagglutinin protein gene uidA, nptII uidA hptII, rice chitinase cryIAc gene

Plant

uidA, nptII

Rao et al. (2008)

Plant

nptII, uidA

Sarkar et al. (2003)

Plant Plants

nptII, uidA Sunflower seed albumin gene bar gene uidA/nptII

Celikkol et al. (2009) Molvig et al. (1997)

Embryonic axis and cotyledonary nodes

Lens culinaris

Fate of Explant

Plant Plant

Dayal et al. (2003) Thu et al. (2003) Kumar et al. (2004) Sharma et al. (2006)

Pigeaire et al. (1997) De Clerq et al. (2002) continued

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TABLE 10.1 (continued) Selected Examples of Pulse Crop Transformation Using Agrobacterium tumefaciens Plant Species Pisum sativum

Explant Used

Fate of Explant

Gene

Shoot cultures

Plant

hptII

Immature embryo slices

Plant

bar gene

Immature embryo slices Cotyledonary node

Plant

bar gene

Plant Plant

Embryonic segments Stem

Plant Plant

Embryo axes

Plant

Vicia narbonensis

Emryogenic callus Emryogenic callus

Plant Plant

Vigna aconitifolia

Protoplast Leaf explant

Plant Plant

nptII, uidA BAR, PGIP, VST1 uidA, bar gene uidA, lysC, methioninerich sunflower 2S albumin gene SFAs gene, bar, lysC hptII nptII, 2S albumin gene, PAT nptII uidA, nptII

Vigna angularis

Epicotyl

Plants

hptII

Vigna mungo

Cotyledonary nodes Embryonic axes

Plant Plants

uidA, nptII uidA, nptII

Shoot apex

Plant

uidA, nptII

Immature cotyledonary node Cotyledons Hypocotyl, cotyledonary node, primary leaves Leaf

Plants

nptII, uidA, bar

Plants Callus, plants

nptII, uidA uidA, nptII

Plants

nptII, uidA

Cotyledonary node

Plant

Cotyledonary node Cotyledonary node

Plants Plants

bar, α-amylase inhibitor nptII, uidA uidA, nptII

Cotyledonary node

Plants Plant

Vicia faba

Vigna radiata

Vigna sequidepalis Vigna unguiculata

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bar gene α-amylase inhibitor gene

Reference Puonti-Kaerlas et al. (1990) Schroeder et al. (1993)

Grant et al. (1995, 2003) Svabova et al. (2005) Richter et al. (2007) Krejci et al. (2007) Bottinger et al. (2001)

Hanafy et al. (2005) Pickardt et al. (1991) Saalbach et al. (1994), Pickardt et al. (1995) Eapen et al. (1987) Malabadi and Nataraja (2007) EL-Shemy et al. (2002) Saini et al. (2003) Saini and Jaiwal (2005) Saini and Jaiwal (2007) Murugananthum et al. (2007) Pal et al. (1991) Jaiwal et al. (2001)

Tazeen and Mirza (2004) Sonia et al. (2007) Ignacimuthu (2000) Chaudhary et al. (2006) Popelka et al. (2006) Solleti et al. (2008)

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TABLE 10.2 Selected Examples of Transgenic Pulse Crops Developed Using Particle Gun Bombardment Plant Species

Explant Used

Fate of Explant

Gene of Interest

Cicer arietinum Cajanus cajan

Epicotyl Embryo axis

Plant Callus

Lens culinaris

Cotyledonary node Embryo axis

Organogenesis

cryIAc, nptII, uidA hptII, uidA DHDPS gene Als (Lou Gherig’s disease) uidA, BAR coat protein

Indurker et al. (2007) Thu et al. (2003) Thu et al. (2007) Gulati et al. (2002)

Embryo axis

Plant

Aragao et al. (1996)

Embryonic axes

Organogenesis Plant Plant Plant

uidA, nptII, methioninerich albumine bar cryIAc, nptII, uidA uidA, bar uidA, ahas

Kamble et al. (2003) Ikea et al. (2003) Ivo et al. (2008)

Phaseolus vulgaris

Vigna aconitifolia Vigna unguiculata

Plant

Hypocotyl Embryo axis Meristematic cells

Reference

Russell et al. (1993)

Aragao et al. (2002)

TABLE 10.3 Selected Examples of Genetic Transformation in Pulse Crops Using Other Direct DNA Transfer Methods Plant Species

Method

Explant Used

Fate of Explant

Gene of Interest

Pisum sativum

Injection

Axillary meristems

Plant

Vigna aconitifolia

PEG + DNA, electroporation Electroporation

Protoplast

Plant

PEMV virus coat protein nptII

Intact axillary bud

Plant

uidA

Vigna unguiculata, Lens culinaris, Pisum sativum

Reference Chowrira et al. (1998) Kohler et al. (1987a, 1987b) Chowrira et al. (1996)

such as l-cysteine (Olhoft and Somers, 2001; Olhoft et al., 2001) and the supplementation of the medium with osmotic agents may improve the transformation frequency in these important crop species. Some of the methods which can be used to improve the efficiency of transformation include supplements of surfactants, controlling the density of Agrobacterium, altering the period and temperature of Agrobacterium cocultivation, vacuum infiltration with Agrobacteria, sonication, subjecting the explants to heat shock, and particle gun bombardment for inducing microwounds to help in Agrobacterium-mediated gene transfer.

10.5 MARKER GENES FOR SELECTION OF TRANSFORMANTS For the selection of transgenic cells and plants, selectable marker genes have been included in the plasmid along with the gene of interest. The neomycin phosphotransferase (nptII) gene conferring resistance to kanamycin is the most widely used selectable marker gene for pulse crop transformation. Other markers like the hpt gene (Puonti-Kaerlas et al., 1990), herbicide resistance (bar) gene (Richter et al., 2007), and anthranalate synthase gene (asa2) (Chen, 2004) have also been used for the selection of genetic transformants. Genes of plant origin such as twc 19 from Arabidopsis (Mentewab and Stewart, 2005), known to confer antibiotic resistance, should also be tested for the selection of transgenic pulses. Another method of overcoming concerns over genetically modified

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(GM) crops is to use positive selectable marker genes such as phosphomannose isomerase (Boscariol et al., 2003; Aswath et al., 2006). Methods used for the production of marker-free transgenic plants, already used in crop plants (Hohn et al., 2001; Hare and Chua, 2002; Darbani et al., 2007), can be extended to pulse crops too. The development of transformation strategies without the use of selectable marker genes, such as the use of virulent Agrobacterium strains followed by screening for transformed shoots by polymerase chain reaction (PCR) (de Vetten et al., 2003), as used for potato and cassava, can also be adopted for pulse crops.

10.6

PREVENTING HORIZONTAL GENE TRANSFER BY CHLOROPLAST TRANSFORMATION

One of the primary concerns about transgenic plants is that they will hybridize with wild relatives, permitting transgenes to escape into the environment. One strategy to prevent the escape of transgenes is to target the foreign gene into the chloroplast. Transgene targeting to chloroplast has not only the advantage of high-level expression, but also transgene containment by maternal inheritance. However, transplastomic plants are yet to be developed in pulse crops.

10.7 TRANSGENIC PLANT DEVELOPMENT: POSSIBLE TARGETS In spite of the importance of pulse crops as a key to sustainable agriculture, increases in yield through breeding over the last few decades have lagged behind cereals. Biotic impediments like soil salinity, acidity, drought, nutrient limitations, diseases, and pests limit the yield of pulse crops. The main targets for improvement are the following (Eapen, 2008): 1. Pulse crops are susceptible to several insect pests which bring about great losses in yield. The genes which can be transferred to grain legumes to achieve insect pest resistance include Cry genes from Bacillus thuringiensis, protease inhibitor genes, α-amylase inhibitor genes, and lectin genes (Dita et al., 2006). 2. Pulses are also susceptible to diseases caused by bacteria, fungi, and viruses, and hence transfer of genes such as chitinase genes, stilbene synthase genes or antifungal protein genes for fungal resistance, coat protein genes of viruses for viral resistance, and the T4 lysozyme gene for bacterial resistance may benefit plants facing infection by these biotic agents. 3. Abiotic stresses such as drought, salinity, water-logging, mineral toxicities, temperature, and so on, also bring about a decline in yield in pulses. Genes involved in developing abiotic stress tolerance from heterologous sources to pulse crops will improve their ability to withstand stress (Dita et al., 2006). 4. Seed proteins of pulse crops are deficient in sulfur-containing amino acids and the transfer of such genes from other plants such as sunflower or cereals will be beneficial in this respect. However, care should be taken to see that the introduced gene does not cause any allerginicity to humans and animals. 5. Improving the N2 fixing ability of grain legumes and the translocation of fixed N2 to the developing seeds should also be the focus of transgenic studies. Increasing N2 fixation and its accumulation in legume seeds is a crucial feature for increasing yield. Once N2 is fixed, it should be routed toward the grains. Specific enzymes involved in nitrogen metabolism should be the targets of transformation. 6. One of the major problems that grain legumes face is their low yield. Flower drop is a serious problem which reduces grain yield. The introduction of phytohormone genes would probably enhance the biomass and inhibit flower drop. 7. Changing plant architecture can be achieved by the identification and introduction of genes controlling growth and development to produce dwarf or semidwarf plant types. Genes

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such as the gai gene from Arabidopsis or rol genes from Arabidopsis rhizogenes can be introduced into pulse crops such as Psophocarpus, which has a climbing habit, to develop genotypes with changed plant architecture. 8. Some grain legumes are long-duration crops like pigeon pea, which may take about 4–5 months before harvest. An introduction of some early flowering genes would help in reducing the time needed for flowering and seed set. 9. RNA interference technology can be used to engineer metabolic pathways to suppress endogenous genes in order to improve carotenoides, flavanoides, and also for removing antinutritional factors. This technology is yet to be tried out in pulse crops.

10.8 TRANSLATING MODEL LEGUME GENOMICS TO PULSE CROPS Since the first whole-genome sequencing report of Haemophilus influenzae (Fleishmann et al., 1995), we have witnessed an exponential progression in whole-genome sequencing of prokaryotic and eukaryotic organisms including plants and animals. The biological processes pertinent to legumes are unique and Arabidopsis cannot be used for the purpose. Legume research, both fundamental and applied, is undergoing a revolution in the field of genomics, ever since legumes such as Medicago truncatula (Bell et al., 2001; National Center for Genome Resources, n.d.; Tadege et al., 2005; The Samuel Roberts Noble Foundation, n.d.; Young and Udvardi, 2009) and Lotus japonicus (Udvardi et al., 2005) were adopted internationally as model legumes. The availability of genomic sequences and DNA markers has accelerated the process of identification and the isolation of genes responsible for legume-specific characters. The combination of the two genome sequencing projects in Lotus japonicus and Medicago truncatula and expressed sequence tag (EST) projects in these and other legumes such as soybean, pea, and peanut may offer a unique opportunity for comparative studies using pulse crops. Genomic resources such as whole-genome arrays make it possible to pursue detailed questions regarding gene expression in model legumes. Tagged mutant populations simplify the process of determining gene functions. The information gained from studies on the genomics and functional genomics of model legumes in different areas such as symbiotic nitrogen fixation, drought tolerance, plant pathogen interaction, phosphorus uptake, seed development, and storage protein should be translated successfully for the improvement of pulse crops.

10.9 CONCLUSIONS At present, transgenic pulse crops are mostly produced only up to T0 levels and rarely up to T1 and T2. Most of the earlier transgenes which were introduced into pulse crops included antibiotic markers and herbicide-resistant genes. Later, genes for insect and pest resistance and fungal resistance have been introduced. The second generation of transgenics to be developed should have improved nutritional factors, quality, and yield parameters. However, these are yet to be attempted in pulse crops. For successful commercialization, the stability and inheritance of the transgene first have to be ascertained under field conditions. The transgene should preferably be contained in the chloroplast by producing transplastomic plants to prevent spread by pollen grains to related species. The postgenomic era offers opportunities for a better understanding of metabolic pathways and the identification of genes involved in different pathways. This information can be utilized for the development of pulse crops, which will have higher nutritive, higher-yielding, and biotic and abiotic stress-tolerant plants. RNA interference (RNAi) technology can be used for suppression of antinutritional chemicals or compounds. Plant breeders and farmers are looking forward to growing transgenic pulse crops on large farmlands, but to date the progress achieved has been very limited (Dita et al., 2006; Eapen, 2008). Technological advances in the development of transgenic pulse crop technology are major challenges facing twenty-first century plant biotechnologists and rapid strides have to be taken in this

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direction. However, sustained, coordinated research coupled with long-term funding for the support of research into the development of transgenic pulse crops will help to hasten this objective. Considering the slow pace with which transgenic pulse crop research is moving, the commercialization of this important group of plants still lies a long way ahead. However, due to the importance of this group of crop plants as the main source of protein for the population of developing countries, more efforts need to be focused on the development of transgenic pulse crops.

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Improvement 11 The and Enhancement of Phyto-Ingredients Using the New Technology of Genetic Recombination Hisabumi Takase CONTENTS 11.1 Improving the Amino Acid Balance in Crop Plants by Genetic Engineering ..................... 157 11.1.1 Improving Lys Content by Genetic Engineering ...................................................... 158 11.1.2 Improving Met Content by Genetic Engineering ..................................................... 159 11.1.3 Improving Trp Content by Genetic Engineering ...................................................... 161 11.2 Improving the Fatty Acid Composition in Oilseed Crops by Genetic Engineering ............. 162 11.2.1 Improving the Medium-Chain Fatty Acid Content by Genetic Engineering ........... 162 11.2.2 Improving Saturated Fatty Acid Content by Genetic Engineering .......................... 163 11.2.3 Improving Oleic Acid Content by Genetic Engineering .......................................... 164 11.2.4 Improving Long-Chain Polyunsaturated Fatty Acid Content by Genetic Engineering ............................................................................................ 164 11.3 Improving the Bioavailability of Minerals and Vitamins by Genetic Engineering ............. 165 11.3.1 Improving Vitamin Contents in Crop Plants by Genetic Engineering ..................... 165 11.3.2 Improving the Mineral Bioavailability of Crop plants by Genetic Engineering ...... 166 11.4 Eliminating Allergens by Genetic Engineering ................................................................... 168 11.5 Eliminating Toxins by Genetic Engineering ........................................................................ 168 11.6 Production of an Edible Vaccine by Genetic Engineering ................................................... 169 11.7 Future Perspectives ............................................................................................................... 171 References ...................................................................................................................................... 172

11.1

IMPROVING THE AMINO ACID BALANCE IN CROP PLANTS BY GENETIC ENGINEERING

Animals, including humans, are incapable of synthesizing 10 of the 20 amino acids required for protein synthesis. Consequently, these essential amino acids must be obtained from the diet. A deficiency in essential amino acids leads to malnutrition, resulting in growth retardation and/or even death. However, most crop plants have a poor balance of the amino acids essential for the needs of humans and animals. Among the essential amino acids, lysine (Lys), tryptophan (Trp), methionine (Met), and cysteine (Cys) have received the most attention because they are the most limiting in cereal (Lys and Trp) and legume crops (Met and Cys). To enrich crop plants with the desired amino acids, three approaches have been used: (1) the selection of mutants with an increased desired 157 © 2010 Taylor and Francis Group, LLC

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amino acid content, (2) genetic modification to promote the biosynthesis of the desired amino acids, and (3) genetic modification to increase the contents of the desired amino acids in storage proteins. Unfortunately, conventional genetic approaches have met with limited success due mostly to the limited availability of genetic resources for breeding. In addition, genetic traits for a high content of Lys, Trp, or Met are generally associated with abnormal growth and development. Recent advances in genetic engineering appear to offer much promise because they allow the seed-specific expression of specific traits of interest using seed-specific promoters. Another advantage of genetically engineered traits is that they can be transformed into multiple plant species and genotypes and can function synergistically with many other agronomically important traits. The current status of and potential developments in the improvement of Lys, Met, and Trp content in crop plants using a transgenic approach are presented here.

11.1.1

IMPROVING LYS CONTENT BY GENETIC ENGINEERING

Lys is the most limiting essential amino acid in cereal grains. Therefore genetic engineering has been devoted to understanding the regulation of Lys metabolism and its exploitation in order to increase free Lys content in seeds. Early studies of model plants, including tobacco and Arabidopsis (Arabidopsis thaliana), have revealed that (1) Lys is synthesized by a branch of the asparagine (Asp) family pathway that also leads to the synthesis of two essential amino acids [Met and threonine (Thr)], and (2) the flux through the Lys biosynthetic branch is strongly regulated by a feedback inhibition loop in which Lys inhibits the activity of dihydrodipicolinate synthase (DHDPS) (Galili, 2002). Following studies of model plants, it has been demonstrated in soybean (Glycine max), rapeseed (Brassica napus), and maize (Zea mays) that the expression of a bacterial Lys-feedbackinsensitive DHDPS in a seed-specific manner has resulted in the elevation of free Lys content in mature seeds (Falco et al., 1995; Mazur et al., 1999; O’Quinn et al., 2000; Frizzi et al., 2008). Based on this simple approach, the high-Lys maize line LY038 was developed, which is the first genetically modified (GM) crop with high nutritive value to be approved for commercial use in a number of countries (Agbios, 2008; Dizigan et al., 2007; ILSI, 2008c). In this case, the cordapA coding sequence transcribed under the control of the maize Glb1 promoter was introduced into maize to direct the expression of the Corynebacterium glutamicum derived Lys-feedback-insensitive DHDPS protein predominantly to the germ portion of maize kernels. The resulting transgenic line LY038 produced grain with high free and total Lys content, improving its nutritive value for use as a feed ingredient in broiler diets (Lucas et al., 2007). Interestingly, the bacterial DHDPS caused Lys overproduction only when expressed in the embryo and not in the endosperm (Frizzi et al., 2008), suggesting that Lys was efficiently catabolized in the endosperm. In addition, Lys catabolism occurred actively in the maize endosperm (Arruda et al., 2000). To overcome Lys catabolism in the endosperm, maize plants were therefore transformed with a single endosperm-specific expression/ silencing transgene cassette that simulated the expression of deregulated CordapA and silenced the Lys degradation enzyme LKR/SDH (Frizzi et al., 2008). The combination of CordapA expression and LKR/SDH silencing led to the accumulation of more than 4000 ppm free Lys in transgenic grain, compared to less than 100 ppm in wild-type controls. Although increases in free Lys in the embryos of transgenic plants expressing the bacterial feedback-insensitive DHDPS alone were often associated with abnormal seed germination (Falco et al., 1995; Mazur et al., 1999), the endospermspecific CordapA-expressing and LKR/SDH-silencing transgenic maize plant exhibited no detectable negative effect on seed germination (Frizzi et al., 2008). Recently a novel approach has been adopted to increase Lys content in cereal seeds, utilizing a recombinant tRNA (lys) species that introduces Lys at alternative codons during protein synthesis (Wu et al., 2003). The expression of the recombinant tRNA (lys) gene in transgenic rice (Oryza sativa) induced the enrichment of Lys content in seed proteins (Wu et al., 2003). Notably, even the stable expression of a wild-type Arabidopsis gene encoding a Lys tRNA synthase in transgenic maize caused the translational recoding of Lys into Lys-deficient zein storage

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proteins, resulting in significantly enriched Lys content in the grain (Wu et al., 2007). It will be interesting to test the impact of this approach on the performance of the crop plants and on their safety regulations.

11.1.2

IMPROVING MET CONTENT BY GENETIC ENGINEERING

Early attempts to manipulate Met sources by transgenes encoding key enzymes of Met biosynthetic pathways met with limited success, either because they were associated with severe abnormal phenotypes or because Met content exhibited little increase due to the extensive catabolism of Met (Amir et al., 2002; Hesse et al., 2004). In contrast, the expression of a heterologous Metrich protein has been a successful strategy for modifying plant Met content. For example, the expression of the 2S albumin seed storage protein from sunflower (Helianthus annuus) under the control of a strong seed-specific promoter in the transgenic narrow leaf lupine (Lupinus angustifolius) resulted in increases in total seed Met content of up to 100%, and the additional Met was demonstrated to be bioavailable to rats and chickens (Molvig et al., 1997; Ravindran et al., 2002). Expressing the Brazil nut and sesame 2S albumins in seeds, including tobacco, canola, narbon bean, soybean, and rice, also increased total seed Met content by up to 30–100% (Altenbach et al., 1989, 1992; Tabe and Higgins, 1998; Hagan et al., 2003; Lee et al., 2003b). Nutritional evaluation studies of transgenic lupine expressing the sunflower 2S albumin demonstrated that the additional Met was bioavailable in rat, broiler chicken, and Merino sheep (Molvig et al., 1997; Ravindran et al., 2002; White et al., 2001). It was observed in Merino sheep that rumen stability induced by the added Met-rich sink protein increased the efficiency of both wool growth and live weight gain (White et al., 2001). Unfortunately most 2S albumin seed storage proteins of transgenic seeds exhibited allergenicity (Nordlee et al., 1996; Pastorello et al., 2001), reducing their usefulness as additive Met-sink proteins for human nutrition. In order to overcome this problem, a gene that encodes a nonallergenic seed-specific protein, amaranth seed albumin (AmA1), from Amaranthus hypochondriacus was introduced into potato (Chakraborty et al., 2000). AmA1 has great potential as a donor protein because it is well balanced in terms of amino acid composition and is nonallergenic in its purified form (Raina and Datta, 1992; Datta et al., 1997). Expressing AmA1 in a tuber-specific manner resulted in 4- to 8-fold increases in Lys, Met, Cys, and tyrosine (Tyr) contents in tubers that have limited nutritive value. Total protein content in transgenic tubers showed a 35–45% increase, and this increase was correlated with the increase of most essential amino acids. Unexpectedly there was a more than 2-fold increase in the tuber number of transgenic potatoes with high transgene expression levels, as well as a 3- to 3.5-fold increase in tuber yield in terms of fresh weight (FW). Besides these nutritional improvements, AmA1 did not evoke any IgE response, proof that the protein does not induce any allergic reactions in human. Taken together, these findings suggested the feasibility of using the AmA1 gene in genetic engineering to improve the nutritive value of nonseed crops. In addition to these natural sources, a synthetic gene encoding a completely artificial protein was introduced into crop plants to improve their nutritive value. For example, the artificial storage protein ASP-1 was introduced into sweet potato (Ipomoea batatas). Field cultivation trials of transgenic sweet potatoes revealed that asp-1 gene expression increased the contents of essential amino acids, including Met, Thr, Trp, isoleucine (Ile), and Lys, and protein contents in storage roots, resulting in a 3- to 5-fold increase in total proteins (ILSI, 2008a). Although the reason was unknown, a 2- to 3-fold increase in sucrose, glucose, and fructose contents was also observed in transgenic sweet potato. In contrast, the agronomic characteristics were equivalent to those of isogenic conventional sweet potato; for example, no significant differences were observed in growth pattern and photosynthetic efficiency. ASP-1 is a novel protein that has not been previously fed to animals, including humans. Bioinformatics analyses revealed no homology to any known allergen. Feeding trials showed that the increased protein and amino acid content in transgenic sweet potato were bioavailable in hamsters and that they had no adverse effects on the hamster’s brain, liver, kidney

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weight, and bone calcification, as well as total cholesterol, triglyceride, and low density lipoprotein (LDL)-cholesterol levels in plasma and liver (ILSI, 2008a). Taken together, the results suggested the feasibility of using the completely artificial protein ASP-1 in genetic engineering to improve the nutritive value of nonseed crops. Based on a similar strategy, the amino acid balance of cassava (Manihot esculenta Crantz) was also improved by introducing ASP1 (Zhang et al., 2003). As described above, Met-sink manipulations by expressing genes encoding Met-rich proteins seemed to be a promising approach to increase overall Met availability in foods and feeds. However, the amount produced was still less than the optimal level required for human food and animal feed. It was also pointed out that the expression of Met-rich foreign proteins often caused a down-regulation of the formation of endogenous sulfur-containing compounds (Tabe and Droux, 2002; Hagan et al., 2003). These results suggested that foreign protein synthesis competed with endogenous compound synthesis for the limited Met supply. Furthermore it was demonstrated in lupine (Lupinus angustifolius) expressing the sunflower 2S albumin that the Met and Cys contents in seeds were dependent not only on their transport into developing seeds but also on their de novo synthesis in developing seeds (Tabe and Droux, 2002). Based on these findings, the combination of Met-sink manipulation and Met-source manipulation was examined. As expected, the expression of both the Brazil nut 2S albumin and a feedback-insensitive bacterial aspartate kinase (the first enzyme of the Asp family pathway) increased in total Met content in transgenic narbon bean seeds (Demidov et al., 2003). The coexpression of maize Met-rich zein and Arabidopsis cystathionine γ-synthase (the major regulatory enzyme of Met biosynthesis) in alfalfa (Medicago sativa) leaves and potato tubers also increased the accumulation level of zein protein when compared with its expression alone (Bagga et al., 2005; Amira et al., 2005; Avraham et al., 2005; Dancs et al., 2008). Taken together, it is likely that the synergistic action of Met-sink trait and Met-source trait is required to genetically modify Met content to improve the nutritive value of crop plants. Recent molecular investigations of the sulfur-containing amino acid biosynthetic pathway and its regulatory mechanisms have pointed to the possibility that Met-source manipulation boosts Met content. A successful technical example was reported in potato (Solanum tuberosum) (Zeh et al., 2001). Partial inhibition of Thr synthase through antisense-mediated down-regulation resulted in an 8- to 17-fold increase in Met content, corresponding to 10–20 μmol/g of FW of free Met, in transgenic potato tubers. The most remarkable point is that the significant increase in Met content was not accompanied by severe phenotypic changes, including growth retardation and acute reduction in the tuber yield. The contents of most other amino acids did not change significantly in the transgenic lines; for example, a reduction in Thr content was not observed even though Thr synthase activity, which is involved in synthesizing Thr from O-phosphohomoserine, was reduced; Lys and Asp contents were not altered; the amount of Ile synthesized from Thr was increased; amino acids not related to the Asp family (Val, Ala, and Glu) were not altered (Zeh et al., 2001). These findings in transgenic potato have sparked moves toward the development of an alternative strategy to increase Met content, thus improving the nutritive value of crop plants. Recent studies of mutants with increased Met content have also provided a novel strategy to increase the Met content of crop plants. Analysis of a natural maize mutant with a high content of the Met-rich δ-zein seed storage protein Dzs10 revealed a lesion in the dzr1-locus-mediated posttranscriptional control mechanism that normally suppressed Dzs10 transcript accumulation (Lai and Messing, 2002). The introduction of mutated Dzs10 gene removed the target site for negative regulation through the dzr1 locus in maize, resulting in an elevated Dzs10 mRNA accumulation in immature seeds and a high accumulation of Dzs10 protein in seeds. Feeding trials of chicks demonstrated that the additional Met in the transgenic maize kernel was functionally equivalent to supplemental Met, indicating that the increased Met content in the transgenic maize kernels allowed the formulation of a useful animal feed ration without having to add synthetic Met (Lai and Messing, 2002).

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IMPROVING TRP CONTENT BY GENETIC ENGINEERING

Trp is the second most limiting essential amino acid in cereals such as rice and maize. In plants Trp synthesis is strongly feedback regulated by inhibiting its biosynthetic enzyme, anthranilate synthase (AS), which catalyzes the conversion of chorismate into anthranilate. The discovery that a mutation in the model plant Arabidopsis α-subunit of AS renders AS insensitive to feedback inhibition by Trp and enhances Trp accumulation has led to a number of studies using this trait to improve Trp content in crop plants (Kreps et al., 1996; Li and Last, 1996). To date the most dramatic improvement in free Trp content in crop plants has been achieved by introducing a feedback-insensitive mutant of the AS α-subunit of the rice gene (OASA1D) into rice (Oryza sativa) (Tozawa et al., 2001). The introduction of the rice OASA1D gene raised the free Trp content in potato (Solanum tuberosum) and adzuki bean (Vigna angularis) (Yamada et al., 2004; Hanafy et al., 2006). In addition to the rice OASA1D gene, it was reported that introduction of a feedback-insensitive tobacco AS gene resulted in an increase in free Trp content in forage legume (Astragalus sinicus) and soybean (Cho et al., 2000; Inaba et al., 2007). The effect of rice OASA1D expression under the control of the constitutive ubiquitin promoter in rice was studied, both on the amounts of Trp and other amino acids in seeds and on the agronomic traits of plants cultivated in isolated field trials (Wakasa et al., 2006). OASA1D transgenic rice seeds showed a marked increase in free Trp content, with the mean free Trp content ranging from 3300 to 5300 nmol/g of dry seed weight. Those values corresponded to 190- to 310-fold increases compared with the free Trp content of nontransgenic seeds (17 nmol/g of dry seed weight). The amount of free Trp, which was expressed as the percentage of total Trp in the transgenic seeds (43–48%), was also greatly increased compared with that in wild-type seeds (0.8%). Total Trp content (both free Trp and Trp in proteins) of transgenic seeds ranged from 5700 to 8500 nmol/g of dry seed weight, which corresponded to increases of 9- to 14-fold compared with that of nontransgenic seeds (600 nmol/g of dry seed weight). The free Trp accumulation in the transgenic seeds grown under field conditions was accompanied by an increase in the amounts of other amino acids to some extent, and the total amount of free amino acids was also increased by 3- to 5-fold in transgenic seeds (Wakasa et al., 2006). The significant increase in free Trp content caused by a GM trait is often accompanied by negative effects on some agronomic traits, including low germination, retarded seedling growth immediately after germination, spikelet fertility, and yield loss (Wakasa et al., 2006; Dubouzet et al., 2007). Besides being an essential building block of proteins, Trp is a precursor for a variety of secondary metabolites, such as anthranilate, tryptamine, serotonin, melatonin, and indole. This fact, and the observation that the enhanced AS activity accompanied the production of Trp-derived secondary metabolites in response to exogenous stimuli, indicate the concerted regulation of Trp production and the downstream secondary metabolism (Ishihara et al., 2008). Therefore, activation of the Trp pathway through rice OASA1D gene expression may increase the amount of Trp-pathway-derived secondary metabolites derived from the Trp pathway. Indeed, it was observed that constitutive OASA1D expression resulted in a nearly 2-fold increase in the content of the plant hormone indole acetic acid (IAA) in rice seeds (Wakasa et al., 2006) and high levels of Trp, anthranilate, tryptamine, and serotonin in leaves collected from 8-day-old seedlings (Dubouzet et al., 2007). However, the lack of new peaks or changes in peak heights in the chromatograms generated by HPLC–PDA analysis suggested that unexpected Trp metabolism does not occur in OASA1D rice lines (Dubouzet et al., 2007). This is a favorable phenotype from the viewpoint of metabolic engineering because it may support the substantial equivalence of the transgenic lines. Further analysis of the relationship between the increase in the amount of Trp-pathway-derived secondary metabolites and plant development could unravel the mystery of how the rice plant deals with the abnormal situation caused by the excessive accumulation of Trp and Trp-pathway-derived secondary metabolites. It is interesting to determine whether the spatial- and temporal-specific expression of OASA1D gene in the rice grains may reduce the negative effects on some agronomic traits. Embryo-specific and endosperm-specific promoters from rice and other cereals are currently available, rendering such an approach feasible.

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11.2

Biotechnology in Functional Foods and Nutraceuticals

IMPROVING THE FATTY ACID COMPOSITION IN OILSEED CROPS BY GENETIC ENGINEERING

Fats and oils account for a substantial portion of the calorific value of an animal’s diet, and animals must ingest certain fatty acids through their diets for sustenance. In addition to their nutritive impact, considerable attention has been given to the influence of the constituents of fats and oils on cardiovascular diseases, cancers, and inflammatory conditions. These nutritive and industrial values as well as health benefits are determined by a fatty acid profile that is made up of several fatty acids with distinct carbon chain lengths and unsaturation levels. Most fats and oils consumed by humans are derived from plant sources and major alterations in the proportions of individual fatty acids have been achieved in a range of oilseeds using conventional selection and induced mutation (Scarth and Tang, 2006). Examples of such modified oils include low-erucic rapeseed (CanolaTM), low-linolenic linseed (LinolaTM), high-oleic sunflower (SunolaTM), and high-oleic canola (NatreonTM). In spite of these successes, it is still not possible to develop species with the required fatty acid compositions by using conventional breeding approaches alone, because some species are not readily amenable to mutation breeding due to their complex genomic structures, and because the fatty acid content in nonseed organs often results in severe agronomic issues (Miquel and Browse, 1994). Recent progress in genetic engineering has provided an opportunity to overcome these limitations and inspired attempts at the radical alteration of the fatty acid composition in major oilseed crop species mutation (Scarth and Tang, 2006). The transgenes used in genetic modification include those coding for the enzymes that play important roles in the formation of fatty acids, such as 3-ketoacyl-acyl-carrier protein synthase (KAS), acyl-ACP thioesterase, desaturase, elongase, and acyltransferase. Various gene sources from plants, yeasts, bacteria, and animals have been isolated and introduced into oilseed crops in combination with a seed-specific promoter to direct their expression in the developing seeds. As a result, the genetic modification of oilseed crops has become a powerful means to modify the composition of oilseeds to improve their nutritive value, thereby providing an abundant yet relatively inexpensive source of dietary fatty acids with wide-ranging health benefits.

11.2.1

IMPROVING THE MEDIUM-CHAIN FATTY ACID CONTENT BY GENETIC ENGINEERING

Medium-chain fatty acids (MCFAs) are 6–12 carbons long and are minor components of natural foods, with the exception of coconut and palm kernel oils. Medium-chain triglycerides (MCTs) together with MCFAs are rapidly oxidized as a quick source of energy. The substitution of MCTs with long-chain triglycerides (LCTs) in the diet resulted in low weight, less adipose tissue, increased metabolic rate, and endurance enhancement (Baba et al., 1982; Geliebter et al., 1983; Fushiki et al., 1995). Thus MCFA contents in oilseed crops have been improved by genetic engineering. For example, the expression of the acyl-ACP thioesterase ChFadB2 from a Mexican shrub (Cuphea hookeriana) in seeds of transgenic canola, an oilseed crop that normally does not accumulate any caprylic acid (C8:0), capric acid (C10:0), or lauric acid (C12:0), resulted in a dramatic increase in the contents of caprylic and capric acids and a decrease in the contents of linoleic acid (C18:2) and α-linolenic acid (C18:3) (Dehesh et al., 1996). Most transgenic canola plants accumulated 4–7 mol% C8:0 and 12–16 mol% C10:0, and the highest expressing ChFatB2 transgenic line accumulated 11, 27, and 2 mol% C8:0, C10:0, and C12:0, respectively. The coexpression of Cuphea ChKAS IV and ChFadB2 resulted in a 30–40% increase in MCFA content compared with the seed oil of plants expressing the thioesterase ChFadB2 alone (Dehesh et al., 1998). In addition, it was reported that the expression of UcFatB1 12:0-ACP thioesterase form California bay (Umbellularia californica) in canola increased C12:0 (laurate) content in seed oil (Voelker et al., 1996). The transgenic canola produced seed oil with up to 56 mol% C12:0, compared to 0.02 mol% C12:0 in conventional canola. Detailed analysis of the high-laurate canola oil showed that C12:0 was found almost exclusively at the sn-1 and sn-3 positions of triacylglycerol. In addition the coexpression of a coconut (Cocos nucifera) 12:0-CoA-preferring lysophosphatidic acid

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acyltransferase CLP and UcFatB1 in canola led to a drastic increase of C12:0 at the sn-2 position of triacylglycerol, inducing an approximately 5% C12:0 increase in mature seed lipid composition (Knutzon et al., 1999).

11.2.2 IMPROVING SATURATED FATTY ACID CONTENT BY GENETIC ENGINEERING Highly saturated oils are stable for use in cooking, particularly for commercial deep-frying, where they are exposed to high temperatures and oxidative conditions for long periods. Under such conditions, unsaturated oils are oxidatively broken down at the carbon double bonds into short-chain aldehyde, hydroperoxide, and keto derivatives, imparting undesirable flavor and reducing the frying performance of the oils. Furthermore, some of the breakdown products present in thermooxidized fats and oils are readily absorbed into the bloodstream and have been reported to exert undesirable effects, including impaired arterial endothelial function and accelerated antherogenesis. Polyunsaturated oils can be converted into stable cooking oils by industrial hydrogenation in which carbon double bonds (unsaturated) are reduced to single bonds (saturated) by the action of hydrogen in the presence of a catalyst (reviewed in Liu et al., 2002a). Recently genetic engineering has been added as an alternative approach to alter the number of carbon double bonds in fatty acids in oilseed crops. For example, an increase in C16:0 (palmitic acid) content in seed oil was reported in transgenic canola seeds expressing acyl-ACP thioesterase from Cuphea hookeriana (ChFadB1) (Jones et al., 1995). The C16:0 content in transgenic canola seeds was distributed predominantly between 13 and 27 mol%, compared to 6 mol% in conventional canola seeds. The highest C16:0 accumulating transgenic seed has C16:0 content of up to 34 mol%. Alteration of C16:0 content was also demonstrated in seed oils of transgenic canola plants expressing FatB genes from elm (Ulmus americana L.) and nutmeg (Myristica fragrans Houtt.) (Voelker et al., 1997). In elm UaFatB1 transformed seeds, C16:0 content ranged from 15 to 33 mol%. Interestingly the contents of other saturated fatty acids ranging from C10:0 to C18:0 were positively correlated with C16:0 content, and total saturated fatty acid content in transgenic seeds reached 54 mol%, compared to 7 mol% in conventional canola seeds. Therefore, it is likely that the increase in saturates is balanced predominantly by the reduction in C18:1. A similar alteration in fatty acid composition was observed in nutmeg MfFatB1 transformed canola seeds (Voelker et al., 1997). Another successful example is the increased C18:0 (stearate) content in seed oil. This has been achieved by several strategies based on the knowledge that the C18:0 content in seed oil is controlled by at least four enzymes: KASII, which elongates C16:0 to stearoyl-ACP; stearoyl-ACP Δ9-desaturase, which converts C18:0-ACP into C18:1Δ9-ACP; acyl-ACP thioesterases, which release acyl-ACP formed in the plastid to the cytosol; and acyltransferases. For example, the overexpression of an acyl-ACP thioesterase GarmFatA gene of mangosteen (Garcinia mangostana) in canola seeds has led to the accumulation of C18:0 to as high as 22% in the transgenic seed oil, although nontransgenic canola seed has C18:0 content generally ranging from 1.2% to 2.5% (Hawkins and Kridl, 1998). Furthermore, the expression of a mutated GarmFatA gene generated by site-specific mutagenesis in canola led to a 55–68% increase in C18:0 content, compared with the wild-type GarmFatA version (Facciotti et al., 1999). The second strategy is the reduction of Δ9-desaturase activity, which directs the carbon flux to C18:1-ACP production. Antisense suppression using Brassica rapa Δ9-desaturase gene increased C18:0 content to greater than 32% in transgenic Brassica rapa and to 40% in Brassica napus (Knutzon et al., 1992). Recently hairpin RNA (hpRNA) mediated gene silencing was applied to generate high-steric cottonseed (Liu et al., 2002b). The resulting substantial down-regulation of the Δ9-desaturase gene ghSAD-1 in cotton (Gossypium hirsutum) increased C18:0 (stearic acid) content from the normal 2% to as high as 40%, while C18:1 (oleic acid) content was reduced from 13% to 4%, and C18:2 (linoleic acid) content was down from 59% to 39%. Interestingly C16:0 (palmitic acid) content was reduced from 26% to 15% (Liu et al., 2002b).

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The third strategy is the simultaneous manipulation of the activities of the two enzymes. The combination of FatA thioesterase overexpression and Δ9-desaturase down-regulation increased C18:0 content to as high as 45%, which was higher than the case where FatA thioesterase transgene (11% C18:0) and Δ9-desaturase transgene (13% C18:0) were separately expressed (Töpfer et al., 1995). An additional approach for developing high-C18:0 Brassica oil could be the down-regulation of the activities of both Δ9- and Δ12-desaturases, as shown in cottonseed engineered with hp RNA silencing constructs for the two desaturases that increased C18:0 content from 2% to 3% to 40% (Liu et al., 2002b).

11.2.3

IMPROVING OLEIC ACID CONTENT BY GENETIC ENGINEERING

Edible oils rich in monosaturated fatty acids exhibit improved oil stability, flavor, and nutritive value for human and animal consumption. Monounsaturated oleic acid (C18:1) provides more stability than polyunsaturated linoleic (C18:2) and α-linolenic (C18:3) acids. The presence of high monounsaturated fatty acid is also preferred from a health perspective. Considerable progress has been made in developing high C18:1 oilseed by engineering Δ12-desaturase and FatB thioesterase. Transgenic Brassica napus could accumulate as high as 89% C18:1 with the polyunsaturated fatty acid (PUFA) fraction being reduced in the seed oils by post-transcriptional gene silencing of Δ12-desaturase constructs (Kinney, 1994; Stoutjesdijk et al., 2002). Transgenic Brassica juncea engineered with a Δ12-desaturase antisense gene of Brassica rapa exhibited increased C18:1 content from 53% to 73% (Sivaraman et al., 2004). Recently, hpRNA-mediated silencing of Δ12-desaturase gene ghFAD2-1 in cottonseed resulted in an increase in C18:1 content from the normal value of 13% up to 78%, and correspondingly, C18:2 content was reduced from the normal value of 59% down to 4% (Liu et al., 2002b). Interestingly C16:0 content was reduced from 26% to 15% (Liu et al., 2002b). In order to generate further variability of fatty acid composition, the possibility of combining the silencing of Δ9-desaturase and Δ12-desaturase genes into a single line was examined by intercrossing ghSAD-1 silencing cotton lines and ghFAD2-1 silencing ones (Liu et al., 2002b). The simultaneous gene silencing of ghSAD-1 and ghFAD2-1 in the cottonseeds resulted in unique intermediate combinations of C16:0, C18:1, and C18:2 contents; for example, 40% C18:0 and 37% C18:1, and retained the reduced level of C16:0. Based on the pattern of variation in the crossed lines showing gene silencing of both Δ9- and Δ12-desaturases, it now appears possible to develop a wide range of improved fatty acid profiles in cottonseed oil having novel combinations of C16:0, C18:0, C18:1, and C18:2 contents that can be used in making margarine and in deep-frying without hydrogenation, and potentially in high-added-value confectionery applications. Furthermore, it is possible to extend this variation in fatty acid composition by choosing parental lines that have appropriate intermediate degrees of silencing of Δ9- and Δ12-desaturase genes.

11.2.4

IMPROVING LONG-CHAIN POLYUNSATURATED FATTY ACID CONTENT BY GENETIC ENGINEERING

γ-Linolenic acid (GLA; C18:3 n-6) is a PUFA and is a member of the n-6 family of essential fatty acids. GLA is a nutritionally important PUFA in human and animal diets and a precursor of longchain polyunsaturated fatty acid (LC-PUFA). Considerable progress has been made in engineering oilseed crops for GLA production, including Brassica napus, Brassica juncea, soybean, and safflower (Carthamus tinctorius). The coexpression of a Mortierella alpina Δ6-desaturase gene and Δ12-desaturase gene in low C18:3 Brassica napus resulted in the generation of more than 40% GLA (Huang et al., 2004). The expression of a Pythium irregulare Buis. Δ6-desaturase gene in Brassica juncea also generated 25–40% GLA in the seed oil (Hong et al., 2002). The Western diet is often deficient in n-3 LC-PUFAs, including eicosapentaenoic acid (EPA; C20:5 n-3) and docosahexaenoic acid (DHA; C22:6 n-3) (Hibbeln et al., 2006). This is due in part to the high intake of vegetable oils that are rich in n-6 fatty acids and the low intake of fish and their

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oils rich in n-3 fatty acids. The inadequate levels of EPA and DHA in Western-style diets have been associated with increased incidences of cardiovascular disease, cancer, stroke, diabetes, inflammatory disease, and neuropsychiatric disorders, and nutritionists and health authorities regularly recommend significant increases in the consumption of fish and other seafood rich in EPA and DHA. Although the demand for fish as n-3 LC-PUFA-containing food has increased, fish catch has reached its limits, and therefore existing marine sources of n-3 LC-PUFA are unlikely to be sufficient to meet current demand and future increases in human needs. As an alternative renewable and sustainable source of n-3 LC-PUFAs, transgenic oilseed plants are being developed. This is achieved by the transfer of genes encoding proteins involved in the EPA and DHA biosynthetic pathways of marine microalgae and microorganisms and fish into oilseed crops. All higher plants have the ability to synthesize major C18-PUFAs, namely, linoleic acid (LA; 18:2) and α-linolenic acid (ALA; 18:3), and some can also synthesize GLA and stearidonic acid (SDA; 18:4 n-3). However, higher plants are unable to further elongate and desaturate these n-3 C18-PUFAs to produce n-3 LC-PUFAs that are characteristic of marine microalgae and are the ultimate source of EPA and DHA found in fish. The synthesis of n-3 LC-PUFAs in plants therefore requires the introduction of genes encoding all of the biosynthetic enzymes required to convert ALA into EPA and DHA. A successful technical example of EPA production was reported in transgenic soybean seeds (Kinney et al., 2004). The embryospecific expression of an EPA pathway constituted from the Δ-6 fatty acid pathway from Mortierella and n-3 desaturases from Arabidopsis and the freshwater mold Saprolegnia diclina increased the EPA content to 10%. Furthermore, the replacement of the Δ-6 desaturase gene from Mortierella with one from Saprolegnia increased the EPA content to 20%. When a Δ-4 desaturase gene from Schizochytrium aggregatum was added to this pathway in soybean embryos, more than 3% DHA was also detected in the embryo oil (Kinney et al., 2004). Another successful example of EPA production was reported in transgenic Brassica juncea seeds expressing lysophosphatidyl acyltransferase from the marine fungus Thraustochytrium (Wu et al., 2005). Furthermore, the addition of an EPA elongase from the fish Oncorhynchus mykiss and a Δ-4 desaturase from Thraustochytrium resulted in the accumulation of DHA (1.5% of total fatty acids).

11.3

IMPROVING THE BIOAVAILABILITY OF MINERALS AND VITAMINS BY GENETIC ENGINEERING

Some minerals (iron, calcium, selenium, and iodine) and vitamins (folate and vitamins E, B6, and A) play a significant role in the maintenance of health. In addition, there is a growing knowledge base indicating that the increased intake of certain vitamins may reduce the risk of diseases such as cancers, cardiovascular diseases, and chronic degenerative diseases associated with aging. Therefore the improvement of dietary patterns has been encouraged. However, nutrient intake associated with optimal health benefits is often not achievable by dietary modification alone. The fortification of foods is an alternative route and genetic engineering is a potentially important route to fortification. Past achievements in and the current status of developments in β-carotene (provitamin A) improvement and iron fortification are presented here.

11.3.1

IMPROVING VITAMIN CONTENTS IN CROP PLANTS BY GENETIC ENGINEERING

Vitamin A deficiency is a global public health challenge, particularly in infants, children, and pregnant and lactating women, because an extra amount is required to support fetal, infant, and child growth. Vitamin A deficiency can result in reduced resistance to infection, impaired cellular differentiation, xerophthalmia, and ultimately blindness and death. It is also associated with anemia. Vitamin A deficiency affects other nutrients as well; for example, iron metabolism is negatively affected and iron is not incorporated effectively into hemoglobin. Vitamin A is taken up either directly from animal foodstuffs or in the form of certain carotenoids with provitamin A activity, such as β-carotene (provitamin A) found in dark green vegetables (such as spinach), palm oil, and © 2010 Taylor and Francis Group, LLC

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orange vegetables (such as carrots). The most abundantly consumed staple crops, rice, maize, and potato, are poor sources of provitamin A. Therefore diets that lack nutritional balance result in a high prevalence of vitamin A deficiency, and the enhancement of β-carotene content in staple crops is of paramount importance to help eradicate this problem. Significant progress has been made in the biofortification of provitamin A in rice (Ye et al., 2000). Although none of the rice cultivars produce carotenoids in the endosperm, rice endosperm is capable of producing the precursor, geranylgeranyl diphosphate, which can be used to produce phytoene by expressing the enzyme phytoene synthase (psy). Because phytoene can be converted into β-carotene by three enzymes (phytoene desaturase, ζ-carotene desaturase, and lycopene β-cyclase), rice plant was transformed by a psy gene from daffodil (Narcissus pseudonarcissus) and a bacterial phytoene desaturase (crtI) gene from Erwinia uredovora under the control of the endosperm-specific promoter and the constitutive CaMV 35S promoter, respectively. The resulting transgenic rice plants accumulated β-carotene, lutein, and zeaxanthin in seeds and served as the prototype for “Golden Rice” (Datta et al., 2003; Al-Babili et al., 2006). Based on the same idea, Golden Rice 2 was created by introducing the maize psy gene and the Erwinia uredovora crtl gene into rice, since psy (the rate-limiting step in carotenoid biosynthesis) from several plant sources has enabled the identification of the psy gene from maize as the most efficient source (Paine et al., 2005; ILSI, 2008b). As expected, Golden Rice 2 accumulated 23-fold more carotene than the prototype plants and contained as much as 37 μg of total carotenoids per gram of dry weight (DW) of grain, of which 31 μg/g was β-carotene (Paine et al., 2005). The β-carotene content in Golden Rice 2 grains is sufficiently high to overcome vitamin A deficiency, although its bioavailability is unknown (ILSI, 2008b). Based on a similar strategy, β-carotene content has also been enhanced in potato tubers (Ducreux et al., 2005; Diretto et al., 2006, 2007) and in maize kernels (Aluru et al., 2008). Golden Potato was created by transforming a bacterial mini-pathway consisting of psy (CrtB), phytoene desaturase (CrtI), and lycopene β-cyclase (CrtY) from Erwinia, under the control of a tuber-specific promoter (Diretto et al., 2007). The resulting transgenic tubers exhibited a deep yellow (golden) phenotype without any adverse leaf phenotypes. Carotenoid content was increased by up to 20-fold (114 μg/g DW) and β-carotene content was increased by as high as 3600-fold (47 μg/g DW) in these tubers. Assuming a β-carotene to retinol conversion of 6:1, 250 g (FW) of Golden Potato is sufficient to provide 50% of the recommended daily allowance (RDA) of vitamin A (Diretto et al., 2007). Transgenic maize with enhanced β-carotene content was created by the overexpression of bacterial genes crtB and crtI from Erwinia under the control of an endosperm-specific promoter (Aluru et al., 2008). Transgenic maize plants exhibited increased total carotenoid content of up to 34-fold with preferential accumulation of β-carotene in the maize endosperm, and the transgenic seeds had high β-carotene contents that were comparable to 50% of the U.S. Institute of Medicine Estimated Average Requirement values (Aluru et al., 2008). β-Carotene-rich tomato and canola were also developed by a similar method (Botella-Pavía and Rodríguez-Concepción, 2006). In addition to the enhancement of β-carotene (provitamin A), vitamin C and vitamin E contents in corn kernels were enhanced (Chen et al., 2003; Cahoon et al., 2003). Recently, an enrichment of the folate content in tomato fruits was also successful (Diaz de la Garza et al., 2007; reviewed in DellaPenna, 2007).

11.3.2

IMPROVING THE MINERAL BIOAVAILABILITY OF CROP PLANTS BY GENETIC ENGINEERING

Iron is the most commonly deficient micronutrient in the human diet and its deficiency leads to anemia. The most widely recognized strategies for reducing micronutrient deficiency are supplementation with pharmaceutical preparations, food fortification, and dietary diversification. Genetic engineering procedures have been added as an alternative approach to enhance iron bioavailability in crop plants. Some examples are (1) the transformation of crop plants with a gene that codes for an iron storage protein (such as ferritin) to increase the iron content in the vegetative parts of the plants, (2) the expression of microbial genes that inactivate iron absorption inhibitors (such as phytate) to

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improve iron absorption, and (3) the introduction of an iron absorption enhancer (such as Cys or Cys-rich peptides) to facilitate iron absorption. The expression of a ferritin gene from common bean (Phaseolus vulgaris) in Japonica rice grains under the control of an endosperm-specific promoter increased the iron content by up to 2-fold (Lucca et al., 2002). Because of this, the iron intake from a daily consumption of about 300 g of rice by an adult would be increased from around 3 mg (normal rice) to about 6 mg (transgenic ferritin-rich rice). This daily 3 mg increase in iron intake represents 20% of the recommended intake for an adult woman of child-bearing age, and it was concluded that the extra iron consumed by eating transgenic ferritin-rich rice grains is of nutritional significance (Lucca et al., 2002). Transgenic ferritin-rich rice plants were developed by other research groups and similar results were reported (Goto et al., 1999; Vasconcelos et al., 2003). The endospermspecific expression of the ferritin gene from soybean (Glycine max L.) in Indica rice increased the iron content of both brown grains and polished white grains (Vasconcelos et al., 2003). Transgenic brown rice grains contained as much as 34.7 μg of iron per gram of total DW of brown seed, with the iron content increasing by up to 2-fold compared with control (17 μg/g DW of iron). Transgenic polished grains contained as much as 37 μg/g DW of iron, with the iron content increasing by up to 3.7-fold compared with control (10 μg/g DW of iron). Based on the U.S. RDA for iron of 18 mg/ day, around 67% of the RDA would be met by ingesting about 300 g of ferritin-rich rice per day. Interestingly, the zinc content of ferritin-rich rice grains before and after polishing was higher than that of the control (Vasconcelos et al., 2003). Zinc deficiency is known to be associated with complications in pregnancy and delivery, as well as with growth retardation and congenital abnormalities. It has also retarded neurobehavioral and immunological development in the fetus. Therefore, it is likely that the GM trait using the soybean ferritin gene is suitable for both iron and zinc biofortification in rice grains. In addition to rice, ferritin-rich lettuce was created by introducing the soybean ferritin gene (Goto et al., 2000). The resultant transgenic lettuce plants had iron contents that were 1.2- to 1.7-fold of those of the control plants. The transgenic lettuce plants had superior photosynthesis rates compared to the controls, and grew larger and faster than the controls during the 3-month period from germination (Goto et al., 2000). Iron bioavailability is also dependent on iron absorption. To prevent iron deficiency anemia, it will be necessary not only to increase iron intake but also to reduce iron absorption inhibitors, such as phytate (inositol hexakisphosphate), which is the major storage form of phosphorus. Ingested phytate chelates divalent ions, such as iron, calcium, and zinc, making them unavailable for uptake. Because nonruminant animals have little or no phytase, which is a phytate-hydrolyzing enzyme, reducing the amount of phytate is expected to improve mineral bioavailability. Based on this idea, a fungal phytase gene was introduced into crop plants to liberate chelated ions and to form phosphate. For example, rice was transformed by the heat- and acid-stable phytase gene from Aspergillus fumigatus under the control of an endosperm-specific glutein promoter (Lucca et al., 2002). Phytase content in the transgenic rice grains was increased by about 130-fold, providing sufficient phytase activity to completely degrade phytate in a simulated digestion experiment. Transgenic rice plants were visually indistinguishable from the nontransgenic plants, indicating that the newly expressed proteins do not affect plant growth and development. In addition, phytate content was not decreased in mature transgenic rice seeds, indicating that the overexpression of foreign phytase did not negatively affect phosphorus content in and germination of rice seeds (Lucca et al., 2002). Various dietary ligands have been shown to enhance trace element absorption. For example, Cys and Cys-rich peptides released from proteins during digestion have a positive effect on iron absorption. Thus, an increase of Cys content in the diet is likely to improve iron absorption. Based on this idea, endogenous Cys-rich metallothionein-like protein (12 Cys residues/72 amino acids) was overexpressed in rice grains (Lucca et al., 2002). The content of Cys residues was increased by about 7-fold in husked rice grains, and varied from 27.4 to 38.9 mg/g protein in the control seeds and from 170.0 to 323.8 mg/g protein in the transgenic seeds. As described above, various GM traits that increase iron content and optimize the contents of iron absorption enhancers and inhibitors were developed, but the synergistic effects have not

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been examined. In the future, use of these GM traits in combination with the optimization of iron bioavailability will create more iron-biofortified crop plants. However there is a potential risk of accumulating unwanted toxic heavy metals, such as cadmium, from contaminated soils. Therefore safety issues must be carefully considered.

11.4

ELIMINATING ALLERGENS BY GENETIC ENGINEERING

Food allergy can be a serious nutritional problem in children and adults, and any food that contains protein has the potential to elicit an allergic reaction in the human population. The allergens in foods are almost always naturally occurring proteins. Recent molecular cloning of genes encoding allergenic proteins offers the possibility of eliminating or reducing undesirable allergenic proteins in crop plants by genetic engineering technology to enhance food safety. For example, soybean storage protein P34 accounts for 85% of IgE responses in soybean-sensitive individuals. Sense-mediated gene silencing of P34 driven by a seed-specific β-conglycinin promoter in soybean resulted in the reduction of P34 content in soybean seeds (Herman et al., 2003). The gene-silenced soybean appeared to be otherwise unchanged by the removal of this allergenic protein; for example, proteomic analysis of extracts from transgenic seeds detected the suppression of P34 expression and no other significant changes in the polypeptide pattern; the transgenic plants matured, flowered, and set seed in the same developmental pattern as the wild-type controls. More importantly, transgenic seed size, shape, and protein and oil contents were also indistinguishable from controls (Herman et al., 2003). Another successful example of allergenic protein reduction was reported in rice (Tada et al., 1996). Rice 14–16 kDa allergenic proteins were identified as multigene products that cross-reacted with IgE of several rice-allergic patients. Antisense-mediated gene silencing of the 14–16 kDa allergenic proteins driven by a rice-seed-specific promoter resulted in marked reduction of the 14–16 kDa allergen content in rice seeds. The allergen content ranged from 60 to 70 μg per transgenic seed, and the values corresponded to an approximately 80% reduction in seed 14–16 kDa allergen content (Tada et al., 1996).

11.5

ELIMINATING TOXINS BY GENETIC ENGINEERING

Plants produce many phytochemicals to protect themselves from predators, and some of these phytochemicals have detrimental effects on human and animal health. For example, phytate, lectins, glycoalkaloids, and cyanogenic glucosides are known as antinutrients or natural toxicants in crop plants. To eliminate such undesirable elements in foodstuffs, genetic engineering is employed to down-regulate genes involved in metabolic pathways for the production, accumulation, and/or activation of antinutrients and toxicants in crop plants. One technical example of toxicant reduction was reported in potato (Solanum tuberosum). Solanaceous species synthesize steroidal glycoalkaloids (SGAs), which are natural toxicants and are believed to serve as a natural defense against insects and other pests. As these compounds can exert toxic effects on humans, they represent a potential source of toxicants, especially in improperly stored or processed potatoes. The major SGAs in potato are α-solanine and α-chaconine, accounting for as high as 95% of the total glycoalkaloid content. Antisense-mediated gene silencing of solanidine glucosyltransferase in potato resulted in a reduction of tuber SGAs (McCue et al., 2006). Another example of toxicant reduction was reported in cassava (Manihot esculenta Crantz). Cassava contains cyanogenic glucosides that are toxic to humans and can lead to serious health disorders, even though cassava is an important food in developing countries. The ingestion of cyanogens (linamarin or acetone cyanohydrin) remaining in incompletely processed cassava roots leads to cyanide poisoning. To reduce linamarin (accounting for 95% of total cyanogens) content, the expression of cytochrome P450 genes (CYP79D1 and CYP79D2) that catalyze the first mediated step in linamarin synthesis in leaves and roots was tissue-selectively inhibited through antisense-mediated gene

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silencing (Siritunga and Sayre, 2003; Siritunga et al., 2004b). When cyanogenic glucoside synthesis in leaves was inhibited tissue-selectively, the resulting transgenic cassava plants exhibited a 60–94% reduction of leaf linamarin content. Interestingly these gene-silenced lines exhibited a 99% reduction of root linamarin content, although the expression of CYP79D1 and CYP79D2 genes in transgenic roots had not been modified. In contrast, when cyanogenic glucoside synthesis in roots was inhibited tissue-selectively, the resulting transgenic cassava root exhibited normal linamarin levels. These results suggest that linamarin is tissue-specifically synthesized in leaves and then transported from leaves (source) to roots (sink). In order to create toxicant-free crop plants by genetic engineering, an understanding of where toxicants are synthesized in the plant body is of great importance. Eliminating toxicants from crop plants has positive effects on human and animal health. However it also means disarming plants of their natural defenses, making them palatable for herbivores and insect feeders under field conditions. Considering such a trade-off caused by GM traits to eliminate toxicants, an alternative GM trait to accelerate detoxification by food processing was proposed in cassava (Siritunga et al., 2004a, 2004b). The overexpression of leaf-specific hydroxynitrile lyase (HNL) in cassava roots increased HNL activity by 13-fold in transgenic cassava roots compared to the wild type, and this has resulted in the acceleration of cyanogenesis by more than 3-fold. Acetone cyanohydrin levels were considerably reduced in processed foods containing HNL which was not inactivated. Transgenic cassava plants that exhibit accelerated cyanogenesis during food processing show linamarin levels that are similar to the steady-state linamarin level of intact plants and retain the beneficial effects of cyanogens, including herbivore deterrence and the promotion of amino acid synthesis in cassava roots.

11.6

PRODUCTION OF AN EDIBLE VACCINE BY GENETIC ENGINEERING

In recent years the development of plant-based therapeutic proteins has created an innovative and exciting opportunity to increase value-added traits in food crops. Using genetic engineering and molecular biology, genes encoding therapeutic proteins are transferred into the nuclear genome of a plant system via genetic transformation, and the transgenic plants become capable of producing therapeutic and diagnostic proteins. Plant expression platforms offer several advantages over conventional microbial fermentation, animal cell cultures, and transgenic animals (Goldstein and Thomas, 2004). The major benefits of plant systems include low production costs, easy and quick scale-up, and overall high product quality. The start-up and running costs of plant systems are lower than those of conventional cell-based production systems because there is no need for fermentation facilities or skilled operators to run them. For example, transgenic tobacco, maize, soybean, and alfalfa plants are expected to yield over 10 kg of therapeutic protein per acre, and this should reduce production costs by approximately 90% compared to other systems as long as adequate yields are achieved (Kusnadi et al., 1997; Larrick et al., 2001; Hood et al., 2002). Furthermore, plant expression platforms have high safety because they lack the endotoxins produced by bacterial cells, they do not harbor human pathogens, and they are not exposed to the possible risks of contamination by unknown mammalian pathogens that may remain undetected in animal cell cultures. If an edible plant is used to produce therapeutic proteins, purification to remove host toxins would not be necessary and screening for mammalian viruses and prions in recombinant proteins produced by mammalian systems would be circumvented. Even though therapeutic proteins produced in plants must be extracted and purified using conventional methods, plant systems would have practical advantages because a simple and economical method can be used. As a result, the extraction and downstream processing steps, whose cost corresponds to more than 85% of the cost associated with recombinant protein production in conventional bacterial and animal expression platforms (Evangelista et al., 1998), can be also circumvented or eliminated in plant expression platforms. Considering these benefits, plant expression platforms are expected to be a good alternative to microbial fermentation and animal cell cultures for the production of therapeutic proteins. They are referred to as molecular farming, Molecular PharmingTM, or molecular biopharming.

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In early work on molecular farming, tobacco was used as the model plant system for the expression of therapeutic proteins. Some examples of successful work are human growth hormone (Barta et al., 1986), serum immunoglobulin G (Hiatt et al., 1989), human serum albumin (Sijmons et al., 1990), Streptococcus mutans surface protein A as a potential dental caries vaccine (Curtiss and Cardineau, 1990), and hepatitis B virus surface antigen (Mason et al., 1992) and Escherichia coli heat-labile enterotoxin subunit B (Haq et al., 1995) as vaccine candidates. Subsequently, many crop species have been investigated as potential hosts for molecular farming; for example, hepatitis B virus antigen was expressed in lupin (Kapusta et al., 1999), lettuce (Kapusta et al., 1999; Marcondes and Hansen, 2008), potato (Richter et al., 2000), cherry tomatillo (Gao et al., 2003), banana (Kumar et al., 2005), tomato (Lou et al., 2007), and rice (Qian et al., 2008). Such plant-based therapeutic proteins have been evaluated in clinical trials involving mice and human volunteers (Haq et al., 1995; Thanavala et al., 1995, 2005; Arakawa et al., 1998; Tacket et al., 1998, 2000, 2004; Mason et al., 1996, 1998; Sandhu et al., 2000; Kapusta et al., 1999; Kong et al., 2001; Gao et al., 2003; Judge et al., 2004; Koya et al., 2005; Arlen et al., 2008; Golovkin et al., 2007; Nochi et al., 2007). Furthermore the productivity of therapeutic proteins by transgenic plants was examined (Valdés et al., 2003; Watson et al., 2004), and some plant-based therapeutic protein products for the treatment of human diseases are rapidly approaching commercialization (reviewed in Ma et al., 2005). The most widely studied plant-based therapeutic protein products include antigens for use as oral vaccines to demonstrate the feasibility of edible plant-based vaccines. As early as 1990, Streptococcus mutans surface protein antigen, the causative epitope of dental caries, was expressed in tobacco as the first step toward achieving a potential plant-based mucosal vaccine (Curtiss and Cardineau, 1990). Since then, many vaccine antigen candidates have been expressed in nuclear transgenic plants. However, contrary to our expectations, there are only a few examples of the high expression of recombinant protein in nuclear transformed transgenic plants. Some examples include hepatitis B virus envelope surface protein (0.01% of total soluble protein (TSP); Mason et al., 1992; Thanavala et al., 1995), Escherichia coli heat-labile enterotoxin subunit B (0.01% of TSP; Haq et al., 1995), and cholera toxin B subunit (CTB) (0.02–0.1% of TSP; Jani et al., 2004), and the expected economic threshold is estimated to be only 1% (Meyers et al., 2008). Therefore new strategies are needed to overcome the limited recombinant protein accumulation before molecular farming can be fully realized. Such strategies include molecular technologies to boost transcription, to direct transcription in tissues suited for protein accumulation, to stabilize transcripts, to optimize translation, to target proteins to subcellular locations and/or tissues optimal for their accumulation, and to engineer proteins to stabilize them (Streatfield, 2007). One successful example was reported in the rice-based cholera vaccine (Nochi et al., 2007). Cereal crop seeds are essentially edible tissues and have the capacity to produce relatively large amounts of recombinant products. In addition, recombinant products accumulating in seeds have been shown to be stable for 6 months, even when stored at room temperature. To achieve high expression and accumulation of inserted vaccine antigen in rice seed, an endosperm-specific expression promoter gene followed by an endoplasmic reticulum retention signal peptide, KDEL, was used for the expression of the codonoptimized CTB gene. The resulting transgenic rice plants stored CTB in protein storage organs PB-I and PB-2 of starchy endosperm cells in seeds. The accumulation level of CTB reached 2.1% of total seed protein, and oral administration of the CTB-expressing rice seed powder to mice induced the production of CTB-specific serum IgG and muscosal IgA antibodies with a neutralizing activity and protective immunity against cholera toxin. In addition, the rice-based CTB vaccine maintained immunogenicity for more than 18 months at room temperature (Nochi et al., 2007). Another example is the soybean-based oral vaccine expressing heat-labile enterotoxin subunit B (LTB) of enterotoxigenic Escherichia coli (Moravec et al., 2007). Soybean seeds have the unique advantage of a high protein content (35–40%) compared to rice and maize (8–10% protein). LTB expression was directed to the endoplasmic reticulum of seed storage parenchyma cells, resulting in LTB accumulation to 2.4% of the total seed protein at maturity and high stability in desiccated seed. LTB-soybean extracts administered orally to mice induced both systemic IgG and IgA and mucosal IgA antibody

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responses, and immunized mice exhibited partial protection against heat-labile enterotoxin challenge (Moravec et al., 2007). These successful examples indicate that cereal crop seed-based oral vaccines are effective and highly practical vaccine regimens against infectious diseases, and offer a new strategy for cold-chain-free and needle-free vaccination against infection. An alternative strategy to improve foreign protein production yield is chloroplast transformation in which recombinant sequences are targeted into the chloroplast genome (Svab et al., 1990, 1993; Maliga, 2004). Examples include candidates for vaccine antigens against cholera, anthrax, tetanus, Lyme disease, plague, amebiasis, rotavirus VP6, canine parvovirus VP2, and human immunodeficiency virus type 1, whose expression levels in chloroplast transgenic plants have reached 4.1%, 14.2%, 18–27%, 10%, 14.8%, 6.3%, 3%, 31.1%, and 40% of TSP, respectively (Daniell et al., 2001, 2004; Koya et al., 2005; Tregoning et al., 2003; Hennig et al., 2007; Chebolu and Daniell, 2007; Birch-Machin et al., 2004; Molina et al., 2004; McCabe et al., 2008). Some chloroplast-derived vaccine antigens have also been evaluated by investigating immune responses, neutralizing antibodies, and pathogen or toxin challenge in animals. The first successful chloroplast transformation was achieved in tobacco (Nicotiana tabacum; Svab et al., 1990). However, the presence of nicotine or other alkaloids in tobacco presents a disadvantage for edible vaccine production. Genetic engineering via chloroplast transformation was recently extended to edible crop plants, including potato (Solanum tuberosum; Sidorov et al., 1999; Nguyen et al., 2005), tomato (Solanum lycopersicum; Ruf et al., 2001; Zhou et al., 2001; Nugent et al., 2005), oilseed Brassicacea (Lesquerella fendleri; Skarjinskaia et al., 2003), carrot (Daucus carota; Kumar et al., 2004a), cotton (Gossypium hirsutum; Kumar et al., 2004b), soybean (Glycine max; Dufourmantel et al., 2004), lettuce (Lactuca sativa L.; Lelivelt et al., 2005; Kanamoto et al., 2006; Ruhlman et al., 2007), rice (Oryza sativa; Lee et al., 2006), cauliflower (Brassica oleracea; Nugent et al., 2006), cabbage (Brassica capitata; Liu et al., 2007), and sugar beet (Beta vulgaris L.; De Marchis et al., 2009). As a result, therapeutic proteins are now produced both in nongreen tissues, such as fruit, root, and tuber, and in edible leafy crops. Among them, lettuce, an edible leafy crop, has attracted the attention of genetic engineers because it not only minimizes downstream protein processing costs but also offers an ideal system for oral intake. Lettuce leaves are consumed without cooking, and the time required to sow seeds and to produce edible biomass is only weeks compared to months for such crops as tomato, potato, and carrot. Furthermore, lettuce is well suited for indoor cultivation by hydroculture systems. The accumulation of proinsulin conjugated to CTB in lettuce chloroplast has been reported (Ruhlman et al., 2007). The extension of chloroplast transformation technology to various species would allow us to improve the nutritive value of crops. One example is the 4-fold enhancement of the provitamin A content in tomato fruit by plastid expression of a bacterial lycopene β-cyclase gene (Wurbs et al., 2007). Chloroplast transformation also allows us to engineer agronomic traits, including insect resistance (McBride et al., 1995; Kota et al., 1999; De Cosa et al., 2001; Hou et al., 2003; Dufourmantel et al., 2005; Chakrabarti et al., 2006; Liu et al., 2008), herbicide resistance (Daniell et al., 1998; Iamtham and Day, 2000; Dufourmantel et al., 2007; Wurbs et al., 2007), disease resistance (DeGray et al., 2001), drought tolerance (Lee et al., 2003a; Zhang et al., 2008), and salt tolerance (Kumar et al., 2004a; Zhang et al., 2008).

11.7 FUTURE PERSPECTIVES This chapter dealt with crop plants which have been nutritionally improved and enhanced by the transgenic approach. Research to improve the nutritive value of crop plants has hitherto been limited by a lack of basic knowledge of plant metabolism and the almost insurmountable challenge of elucidating complex branches of a multitude of metabolic pathways. Recent progress in genomics has revealed ways by which plants produce macronutrients, micronutrients, and undesirable antinutrients and toxicants as a result of a controlled expression of the approximately 30,000 genes that are present in the genome of all plants. Advances in transcriptomics and proteomics are expected to uncover relevant biosynthetic pathways and their related genes (DellaPenna, 2007). With the tools

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harnessed by these “omics” fields, there will be the potential to identify genes of value across species. Metabolomics and nutritional genomics are also expected to contribute to the development of strategies for metabolic engineering (Go et al., 2005; Hall et al., 2008). With these evolving tools, a better understanding of the global effects of metabolic engineering on metabolites, enzyme activities, and fluxes is beginning to be developed. An increase in basic knowledge of plant metabolism will provide the tools necessary to modify more effectively the nutritional contents of crops so they can exert a positive effect on many aspects of human and animal health. An interdisciplinary effort involving human physiology, nutrition science, and plant research will be of considerable importance in this regard. Although a reasonable conclusion would be that the genetic modification of crops, which is an extremely powerful tool, markedly increases the potential of all crop species, the production of nutritionally improved and enhanced crop plants would present a new problem concerning safety assessment. It is estimated that plants produce up to 200,000 phytochemicals across their many and diverse members (Oksman-Caldenty et al., 2004); obviously, a more truncated subset of this number is available on our food palate, with approximately 25,000 different metabolites in general plant foods (Go et al., 2005). Metabolic engineering is generally defined as the redirection of one or more enzymatic reactions to improve the production and accumulation of existing compounds, produce new compounds, or mediate the degradation of compounds. However, there is a risk that modifications of metabolic pathways can produce unexpected effects. In this regard, the regulatory oversight of GM products has been designed to detect such unexpected outcomes in GM crops (ILSI, 2004). As more metabolic modifications are introduced, we must continue to study plant metabolism and the interconnected cellular networks of plant metabolic pathways to increase the likelihood of predicting pleiotropic effects that may occur as a result of the introduced genetic modification. It is, therefore, very encouraging that available analytical techniques, including metabolomics, have been able to detect and assess the safety of these unanticipated effects.

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Engineering of 12 Metabolic Bioactive Phenylpropanoids in Crops Kevin M. Davies CONTENTS 12.1 Introduction .......................................................................................................................... 181 12.2 Phenylpropanoids .................................................................................................................. 183 12.2.1 Lignins and Lignans ................................................................................................. 183 12.2.2 Coumarins, Stilbenes, Chlorogenic Acid, and Related Compounds ........................ 183 12.2.3 Anthocyanins and PAs.............................................................................................. 184 12.2.4 Flavones and Flavonols............................................................................................. 184 12.2.5 Isoflavonoids ............................................................................................................. 184 12.3 Targets for Metabolic Engineering ....................................................................................... 185 12.4 Metabolic Engineering Approaches for Phenylpropanoids .................................................. 186 12.4.1 Reducing Phenylpropanoid Biosynthesis .................................................................. 189 12.4.2 Introducing Novel Biosynthetic Activities................................................................ 190 12.4.3 Increasing Phenylpropanoid Levels .......................................................................... 190 12.5 Concluding Comments ......................................................................................................... 192 References ...................................................................................................................................... 192

12.1

INTRODUCTION

Plants contain a great variety of phenolic compounds, derived from a range of biosynthetic pathways. This review focuses on those from the phenylpropanoid pathway, as these include most of the groups of compounds of current interest for human health studies and is the pathway for which there has been the most extensive plant metabolic engineering to date. Phenylpropanoids are a major group of plant secondary metabolites derived from phenylalanine (Davies and Schwinn, 2006; Boudet, 2007; Yu and Jez, 2008). They are of key importance for plant function both as structural compounds and for responses to the abiotic and biotic environment. It has been estimated that phenylpropanoids account for over 30% of all organic carbon in the biosphere, principally in the form of lignin, and they are therefore a common dietary component (Kyle and Duthie, 2006). Figure 12.1 shows a simplified representation of the proposed biosynthetic pathway to a range of phenylpropanoids of note for this review. There are a great variety of phenylpropanoid structures known. Mostly these are variants of a small group of base compounds found in most plants. Thus, a core section of the biosynthetic pathway is common to most plants, including ferns, in particular to the formation of hydroxycinnamic acids (HCAs), lignins, flavonols, anthocyanins, and proanthocyanidins (PAs, condensed tannins). Variation in individual flavonoid structures is created by modification of the base structure by glycosylation, hydroxylation, or substitution with methyl, sulfur, and aliphatic or aromatic acyl groups. Thus, there are more than 5000 known flavonoid structures, 181 © 2010 Taylor and Francis Group, LLC

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Biotechnology in Functional Foods and Nutraceuticals Phenylalanine PAL HO HO HO O

Cinnamate

Monolignols (coniferyl alcohol)

O

C4H

OH

OCH3 OH

HQT

4-Coumarate

Lignins and lignans

O

O OH HO

4CL

OH

Caffeoyl quinic acids (chlorogenic acid)

4-Coumaroyl-CoA OH

3x Malonyl-CoA OH

CHS + CHR

STS

HO CHS

HO

IFS

3x Malonyl-CoA

Resveratrol OH

3x Malonyl-CoA

Isoflavonoids

OH

O

HO

OH

O Isoflavonoids H CHI OH

FNS

O

Flavone (apigenin)

OH OH

Dihydroflavonols DFR

O

Leucoanthocyanidins

Flavonols (kaempferol)

LAR OH

ANS

Anthocyanidins

ANR

OH HO

OH OH

O OH

F3GT

OH

OH

Proanthocyanidins

O

HO

O

HO

OH OH

OH

F3H FLS

O

IFS

Flavanones

OH HO

O

((–)-Glycinol)

O-Glc OH

Anthocyanins (pelargonidin 3-O-glucoside)

FIGURE 12.1 A section of the phenylpropanoid pathway leading to lignin, lignans, CGA, stilbenes, and various flavonoids. Simplifications include showing only the 4′-hydroxylated flavonoid types (except PAs), no indication of possible secondary modifications (e.g., glycosylation, acylation, and methylation), unlabeled arrows may represent multiple biosynthetic steps, and only one isoflavonoid is shown. Compound names in parenthesis indicate which example is shown from the group of compounds (e.g., pelargonidin 3-O-glucoside is the specific anthocyanin shown). PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; F3GT, uridine diphosphate-glucose (UDPG):flavonoid 3-O-glucosyltransferase; HQT, hydroxycinnamoyl-CoA quinate:hydroxycinnamoyl transferase; CHR, chalcone reductase (also known as polyketide synthase); STS, stilbene synthase; FNS, flavone synthase; FLS, flavonol synthase; IFS, isoflavone synthase (also known as 2-hydroxyisoflavanone synthase); LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase.

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including more than 500 anthocyanins (Andersen and Jordheim, 2006). This diversity of structure has important implications when considering genetic modification (GM) approaches for improving human health characters, as discussed later in the review. There are also sections of the phenylpropanoid pathway that occur only in small ranges of species. For example, the 3-deoxyanthocyanins and 6- or 8-hydroxyanthocyanins have been identified in relatively few species.

12.2 PHENYLPROPANOIDS It is worthwhile outlining the biosynthetic route and functions of the major phenylpropanoid groups of relevance to functional food development, and briefly commenting on the state of knowledge on their importance to human health.

12.2.1

LIGNINS AND LIGNANS

Lignins and lignans are polymeric compounds formed by different biosynthetic routes from monolignol precursors derived from HCAs (Dixon, 2004). Lignins are high-molecular-weight polymers that are one of the major structural components of plant cell walls, notably for xylem vessels. Lignans are dimeric compounds that are abundant in wood resins, although they can occur in a range of tissues, and are linked to plant defense. Food species with significant levels of lignans include grains such as rye, various berry fruit, and green and black tea. However, by far the most abundant source of lignans is flaxseed (Dixon, 2004). Lignins are a major component of dietary fiber, and evidence supporting roles for lignans (or their enteromicrobial metabolites) as phytoestrogens is growing. The biosynthetic pathway to lignins is mostly well defined, although some areas of uncertainty remain. Metabolic engineering of lignin biosynthesis has been carried out, but focused on forage digestibility or benefits for the timber, paper, and biofuel industries [reviewed in Boudet (2007) and Vanholme et al. (2008)] rather than on implications for human diets. Thus, it is not covered in detail in this review. Some of the key biosynthetic steps for lignans have been molecularly characterized, allowing the possibility of metabolic engineering of their production, but with few GM plant studies reported to date (Ayella et al., 2007).

12.2.2 COUMARINS, STILBENES, CHLOROGENIC ACID, AND RELATED COMPOUNDS In addition to monolignols and flavonoids, there are several other compounds of note for human health that are derived from HCAs, in particular coumarins, stilbenes, and caffeoyl quinic acids such as chlorogenic acid (CGA) (Kyle and Duthie, 2006). It is thought that most of these compounds are produced for defense against insect or microbial attack. Coumarins are a varied and widespread group of compounds that can have an adverse effect on human health through photosensitive blistering of the skin; however, they are of limited relevance to functional food development. Of particular interest to functional foods are the stilbenes. Stilbenes are formed by stilbene synthases (STSs), chalcone synthase (CHS)-like enzymes that condense multiple malonyl-CoA units with an HCACoA to form structures with two phenolic rings (Austin and Noel, 2003). Over 300 stilbenoid structures have been reported, but most attention has been given to resveratrol. Resveratrol, which may be common in the diet through grapes and some wines, has generated much interest from experiments looking for compounds that promote life span increases (Knutson and Leeuwenburgh, 2008). Caloric restriction has been shown to significantly increase the life span in mice, Caenorhabditis elegans, Drosophila, and other model species. Data from yeast have suggested that the increased activity of a sirtuin gene (Sir2), encoding a histone deacetylase, is an important part of the mechanism. Resveratrol is a strong activator of Sir2 gene activity, and indeed addition of resveratrol to yeast growth media extended the life span of yeast cells. Supporting results have also been obtained for C. elegans and Drosophila (Griswold et al., 2008), although not all studies support the Sir2 mechanism (Bass et al., 2007). As only one gene needs to be introduced to confer resveratrol production

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(STS), it has been a common metabolic engineering approach across a range of species, for both plant defense and human health goals (reviewed in Delaunois et al., 2008). CGA, an ester of caffeic acid and quinic acid, is abundant in coffee and the major phenolic compound in many Solanaceous food crops. Because it may be abundant in the diet and has high bioavailability, CGA is a potentially important dietary phenolic. Studies have shown a wide range of potential health benefits from CGA in the diet, suggesting a broad-based mechanism of action. The biosynthetic pathway for CGA has been defined and DNA sequences are available for the key steps (Niggeweg et al., 2004).

12.2.3

ANTHOCYANINS AND PAS

Anthocyanins and polymeric PAs are both produced by very well-characterized branches of the flavonoid pathway (Davies and Schwinn, 2006; Tian et al., 2008). Anthocyanins are formed from the base anthocyanidins by glycosylation, and are frequently further modified. PAs are formed from monomers of flavan-3-ols through polymerization. Anthocyanin pigments are widespread in human diets, as they account for many of the red, purple, and blue colors of vegetables and fruit. The role of PAs in plants is less clear, but they are important for seed coat function and are produced as antinutrients for plant defense against herbivory. Like anthocyanins, PAs are common in the diet, notably in beverages such as tea and wine. Both groups of compounds have been the subject of a great many studies to determine whether they can improve human health characters. Numerous in vitro experiments, animal trials, and epidemiological studies have linked the intake of anthocyanins and PAs with a reduced risk of a range of health problems that include heart disease, cancer, diabetes, and degenerative cognitive function conditions such as Alzheimer’s disease (Aron and Kennedy, 2008; Jo and Lee, 2007; Lee et al., 2008). Many studies have looked at general properties such as antioxidant activities, but there has been significant recent progress in defining more specific activities for these compounds in humans. In particular, anthocyanins have been shown to regulate adipocytokine gene expression (Guo et al., 2008; Rie et al., 2007; Tsuda et al., 2006). Other factors in addition to their associated health benefits will likely influence approaches to increasing PA levels in food. Not only do they have potential antinutritive effects, but they also affect important food quality parameters such as increasing astringency and bitterness. DNA sequences have been characterized for almost all of the biosynthetic steps for anthocyanins and PAs, and there have been extensive reports on the metabolic engineering of their production in a range of species.

12.2.4

FLAVONES AND FLAVONOLS

Flavones and flavonols are flavonoids with many functions in plants. They are formed from intermediates common with the biosynthesis of anthocyanins, flavanones, and dihydroflavonols, respectively. Various studies have found that flavones and flavonols are one of the significant groups of flavonoids in average diets (Kyle and Duthie, 2006). As with other flavonoids, flavone and flavonol intake has been linked with beneficial effects on a range of health problems. DNA sequences are available for the enzymes that make the base flavone and flavonol aglycones as well as for some of the secondary modification activities. After anthocyanins, flavonols are the most common phenylpropanoid group for which production has been modified in transgenic plants.

12.2.5

ISOFLAVONOIDS

Isoflavonoids are a large group of flavonoids principally found in legumes (mostly the Papilionoideae subfamily) that are common in the diet from seed crops such as soybeans, peas, and peanuts. They are key plant defense compounds in these species, and there is much interest in the possibility of transferring their production into additional crop species for this purpose. However, there has also

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been much interest in the role of isoflavonoids, or their microbial degradation products, as phytoestrogens with beneficial effects on hormone-dependent breast and prostate cancers, osteoporosis, and a range of other human health characters. The biosynthetic pathway to many different isoflavonoids is now well defined, with DNA sequences available for key biosynthetic enzymes. Extensive reviews of the biosynthesis of isoflavonoids and lignans, and their role as potential phytoestrogens, are available in Dixon (2004) and Tian et al. (2008).

12.3 TARGETS FOR METABOLIC ENGINEERING The initial focus on the antioxidant properties of plant metabolites supported strategies to increase the overall level of bioactive phenolics in plant foods. However, the specific actions of phytoestrogens as hormone mimics have been known for some time, and data are now supporting interactions between other phenolics and particular steps in cellular function, especially gene regulatory pathways, such as for anthocyanins and adipocyte-specific gene expression (Boudet, 2007; Holst and Williamson, 2008). This suggests an argument against the use of overall antioxidant capacity as a measure of beneficial phenolic levels, and raises the question of whether an understanding of the mechanism of action is required before metabolic engineering is undertaken for the development of functional foods. Moreover, although it may be known that a group of phenolic compounds has a beneficial effect, more extensive details are often lacking. These can be numerous and might include the following: Is the bioactivity specific to one structure or common to the whole compound group? (For example, there are more than 500 known anthocyanin structures.) What is the optimum concentration required? What are the key structural features to ensure bioavailability? Are there synergies (or negative consequences) with other food matrix components? Bioavailability is highly variable for phenolics (Manach et al., 2005), and makes the development of Dietary Reference Intake (DRI) values for phenolics, similar to those used for vitamins and minerals, more difficult (Williamson and Holst, 2008). A further challenge for a DRI is the variation between individuals in the uptake and metabolism of phenolics. Moreover, much of the polyphenols and health data are available from in vitro or small-animal trials measuring a few components of a complex physiological response, and may only partially replicate human health consequences. Considering these observations, does the current literature on phenolics and health identify specific compounds as being clear targets for metabolic engineering for functional foods development? Critical reviews of evidence for the beneficial effects of phytoestrogens suggest that, under some circumstances, a high isoflavone diet can reduce the risk of breast cancer, and may also assist with prostate cancer and cardiovascular disease prevention and several postmenopausal conditions. However, benefits may vary depending on the other food matrix components, as enteromicrobial ecology greatly influences the bioactivity. So, even for phytochemicals for which there is strong supporting evidence, such as phytoestrogens, it is difficult to make DRIs. Finley (2005) proposed criteria for assessing the efficacy of plant metabolites in improving human health, and applied these to the literature available at the time. It was considered that the evidence for making recommendations on levels of specific phenolics needed to benefit human health was relatively weak, and supported the conclusion that the ability to engineer plant metabolism was far ahead of the understanding of whether or how the target bioactive metabolites function. The OMICS groups of technologies (genomics, proteomics, metabolomics, etc.) may help overcome some of these limitations. Transcriptomics has been used to examine transcript abundance changes in response to phytochemicals such as isoflavonoids (Dixon, 2004), although frequently only with mammalian cell lines and pure compounds. Of more promise, however, is to apply OMICS to small-animal trials or human dietary trials (if changes can be assayed in blood or urine samples). Metabolomics may assist in defining the levels of all phenolics in the diet, while nutrigenomics offers the prospect of understanding the complex response of the genome, through transcript, metabolite, and proteome profiles, to the dietary components. For example, proteomics has been used to identify specific changes induced in the protein profiles in brain tissues of rats fed

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grape seed extract high in PAs (Kim, 2005; Kim et al., 2006). A particular benefit of such “systems biology” approaches may be to define the effect of specific changes in plant bioactive profiles, such as comparisons of foods derived from near-isogenic lines differing only in levels of one phenolic compound in an otherwise unaltered background. This approach has the advantage of examining phytochemicals in the normal food matrix, rather than as isolated supplements, allowing for synergistic effects to occur. In grape seed extract feeding trials with a tumorigenic-prone rat line, a benefit was identified only when the compounds were included as part of the normal laboratory diet (Kim et al., 2004). Metabolic engineering is ideally placed to develop the material required for these tests. Such plant material is already being used in targeted tests, for example, examining the nutritional benefit of carrots engineered to accumulate twice the usual level of calcium (Morris et al., 2008) and the influence of anthocyanins on the life span of mice (Butelli et al., 2008). An alternative approach for developing functional foods, rather than modifying the production of a single compound and then assaying the effect, is to identify general bioassays for human health benefit, such as life span extension in suitable animal models, and to use these bioassays to drive identification of the most advantageous mix of phytochemicals in the diet. Additionally, OMICS may accelerate the discovery of biomarkers that may contribute assays for measuring beneficial responses to plant metabolites (Kussmann et al., 2006).

12.4 METABOLIC ENGINEERING APPROACHES FOR PHENYLPROPANOIDS In this section some of the main approaches to modifying accumulation of health-related phenylpropanoids are considered, with illustrative examples of application in transgenic plants. The focus is on GM approaches, but it is important to note that many of the phenylpropanoid-related gene sequences, especially those encoding transcription factors, also offer much promise for non-GM approaches, such as marker-assisted breeding. The phenylpropanoid pathway has been at the forefront of the development of metabolic engineering technologies, and there are numerous examples of the successful alteration in transgenic plants of the levels of phenolics with potential human health attributes. Table 12.1 lists some examples from a range of approaches, with an emphasis on more recent literature and on food species. The extent of progress with metabolic engineering technologies for phenylpropanoid biosynthesis can be illustrated by considering the example of tomato (Solanum lycopersicum), to which many of the technologies developed in model systems such as Arabidopsis and petunia have now been applied. The principal phenylpropanoids in tomato fruit, besides lignin, are usually the yellow flavonoid naringenin chalcone and small amounts of flavonols. Introduction of chalcone isomerase (CHI; Schijlen et al., 2006) resulted in the conversion of naringenin chalcone to flavonols (quercetin and kaempferol glycosides). Both flavone production (Schijlen et al., 2006), through a combination of transgenes for CHI and flavone synthase (FNS), and isoflavone production (Shih et al., 2008), by transformation with isoflavone synthase (IFS), have also been achieved in transgenic tomato plants. A transgene for the key enzyme for CGA synthesis, hydroxycinnamoyl-CoA quinate:hydroxycinnamoyl transferase (HQT), resulted in the accumulation of high levels of CGA (Niggeweg et al., 2004). Introduction of either STS or chalcone reductase (CHR) led to the production of resveratrol or 6′-deoxychalcones, respectively, in fruit (Giovinazzo et al., 2005; Morelli et al., 2006; Schijlen et al., 2006). Various combinations of transgenes encoding the regulatory transcription factors that activate the biosynthetic genes (e.g., MYB and bHLH type factors) have been used to increase flavonol levels in fruit (up to 65-fold) or to induce anthocyanin production (Bovy et al., 2002; Butelli et al., 2008; Luo et al., 2008). Alteration of the activity of components of the photomorphogenesis signaling pathway resulted in increases in the levels of both flavonoids and carotenoids. Thus, at least seven different branches of phenylpropanoid biosynthesis have been successfully modified in tomato. Although the results with tomato are the most advanced for a food crop, there are many published studies of successful metabolic engineering of phenylpropanoid biosynthesis in other food species, with Table 12.1 listing more than 12 different food crops.

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TABLE 12.1 Selected Examples of the Metabolic Engineering of Phenylpropanoid Metabolism Target Compound Chlorogenic acid

Species

Approach

Solanum lycopersicum

Constructs to prevent or increase production of hydroxycinnamoyl-CoA quinate:hydroxycinnamoyl transferase Fruit-specific overexpression of AtMYB12 Tuber-specific overexpression of modified version of StMtf1 (MYB) Overexpression of STS

Solanum lycopersicum Solanum tuberosum Resveratrol

Actinidia deliciosa Brassica napus

Carica papaya

Humulus lupulus

Malus domestica Rehmannia glutinosa Solanum lycopersicum

Lignans

Linum usitatissimum

Triticum aestivum Chalcones Flavonols and flavones

Solanum lycopersicum Nicotiana tabacum

Seed-specific overexpression of STS and inhibition of UDP-glucose:sinapate glucosyltransferase gene activity Overexpression of STS under a pathogen-induced promoter Overexpression of STS

Overexpression of STS Overexpression of STS Overexpression of STS

Overexpression of chalcone synthase (CHS), CHI, and dihydroflavonol reductase (DFR) Overexpression of pinoresinol lariciresinol reductase Overexpression of CHS and CHR Overexpression of AtMYB12

Effect on Phenotype (General Comment Only)

Reference

Plants with either reduced or increased (by up to 85%) CGA levels in leaves

Niggeweg et al. (2004)

Increased flavonol (65-fold) and CGA (22-fold) levels Increased CGA, flavonol, and anthocyanin levels

Luo et al. (2008) Rommens et al. (2008)

Resveratrol glucoside (piceid) produced in leaves High levels of resveratrol glycosides produced in seeds and a marked reduction in sinapate ester levels Resveratrol glucoside produced following pathogen inoculation Resveratrol aglycones and glycosides produced in leaves, inflorescences, and hop cones Resveratrol glycosides produced Resveratrol aglycones and glycosides produced Resveratrol aglycones and glycosides produced in fruit, naringenin chalcone levels reduced

Kobayashi et al. (2000) Hüsken et al. (2005)

Increase in total phenolic antioxidant levels, including a slight increase (12–14%) in lignan content Twofold increase in the lignan secoisolariciresinol diglucoside 6′-Deoxychalcones produced in fruit Increased flavonol levels (46-fold for rutin and 83-fold for kaempferol). Reduction in anthocyanins in flowers

Zhu et al. (2004) Schwekendiek et al. (2007)

Szankowski et al. (2003) Lim et al. (2005) Giovinazzo et al. (2005), Morelli et al. (2006), Schijlen et al. (2006) Lorenc-Kukula et al. (2005)

Ayella et al. (2007) Schijlen et al. (2006) Luo et al. (2008)

continued

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TABLE 12.1 (continued) Selected Examples of the Metabolic Engineering of Phenylpropanoid Metabolism Target Compound

Species Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum tuberosum

Anthocyanins

Lactuca sativa

Solanum lycopersicum

Solanum lycopersicum

Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum tuberosum Zea mays Proanthocyanidins

Nicotiana tabacum

Approach

Effect on Phenotype (General Comment Only)

Fruit-specific RNAi downregulation of the DET1 gene Overexpression of the photoreceptor CRYPTOCHROME2 Overexpression of CHI

Increased flavonoid (CGA, chalcone, and flavonol) and carotenoid levels in fruit Increased phenylpropanoid (principally flavonol) and carotenoid levels in fruit Increased flavonol levels

Overexpression of CHI and FNS Fruit-specific overexpression of AtMYB12 Overexpression of maize C1 (MYB) and LC

Flavones produced in fruit

Tuber-specific overexpression of modified version of StMtf1 and RNAi reduction of F3′5′H activity Overexpression of Arabidopsis AtMYB60 (MYB) Overexpression of tomato ANT1 (MYB) or Arabidopsis PAP1 (AtMYB75) Fruit-specific overexpression of Antirrhinum majus DELILA (bHLH transcription factor) and ROSEA1 (MYB) Overexpression of maize LC (bHLH) Overexpression of Perilla frutescens MYC-RP and MYC-GP (both bHLH) Overexpression of maize C1 (MYB) and LC Overexpression of DFR Overexpression of maize P1 (bHLH) Overexpression of ANR and PAP1

Increased flavonol (65-fold) and CGA (22-fold) levels Flavonols increased in fruit; anthocyanins increased in leaves Increased CGA and flavonol levels (100-fold for kaempferol)

Reference Davuluri et al. (2005) Giliberto et al. (2005) Muir et al. (2001) Schijlen et al. (2006) Luo et al. (2008) Bovy et al. (2002) Rommens et al. (2008)

Anthocyanins reduced

Park et al. (2008)

Anthocyanins increased

Mathews et al. (2003), Zuluaga et al. (2008) Butelli et al. (2008)

Anthocyanins greatly increased in fruit

Anthocyanins increased under high light levels Anthocyanins increased

Goldsbrough et al. (1996) Gong et al. (1999)

Flavonols increased in fruit; anthocyanins increased in leaves Increased anthocyanin levels in tubers Increased anthocyanin and flavone levels Epicatechin and gallocatechin monomers, and a series of PA dimers and oligomers produced

Bovy et al. (2002) Stobiecki et al. (2003) Cocciolone et al. (2005) Xie et al. (2006)

continued

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TABLE 12.1 (continued) Selected Examples of the Metabolic Engineering of Phenylpropanoid Metabolism Target Compound

Species

Isoflavonoids

Approach

Malus domestica

Overexpression of LC

Medicago truncatula

Overexpression of ANR

Oryza sativa

Overexpression of ANS

Solanum lycopersicum Nicotiana tabacum

Overexpression of IFS

Nicotiana tabacum Petunia hybrida Lactuca sativa Nicotiana tabacum Lactuca sativa Glycine max

Overexpression of a bifunctional IFS/CHI transgene Overexpression of IFS

Overexpression of IFS with inhibition of F3H (tobacco) or overexpression of PAL (tobacco and lettuce) Overexpression of maize C1 and R (bHLH) with or without inhibition of F3H gene activity

Effect on Phenotype (General Comment Only) Increased levels of anthocyanins (12-fold), epicatechin (14-fold), catechin (41-fold), and dimeric PAs (7–134-fold) in leaves PA oligomers produced in leaf regions producing anthocyanins, associated 50% reduction in anthocyanins Reduction in PA levels and increase in a mixture of flavonoids and anthocyanins Genistein glycosides produced in leaves and fruit Genistein and genistein glycosides produced in petals and young leaves Genistein produced in tobacco petals, petunia leaves and petals, and lettuce leaves

Reference Li et al. (2007)

Xie et al. (2006)

Reddy et al. (2007) Shih et al. (2008) Tian and Dixon (2006) Liu et al. (2007)

Increased genistein levels compared to using only the IFS transgene

Liu et al. (2007)

Ratio of genistein to daidzein reduced. Isoflavonoids levels increased when F3H inhibited

Yu et al. (2003)

Note: Emphasis is given to recent studies on crop species and studies targeting compounds of potential importance to human health.

12.4.1

REDUCING PHENYLPROPANOID BIOSYNTHESIS

Reduction of phenylpropanoid levels may be desired if compounds with adverse effects on health are present (e.g., coumarins), or in order to promote the flow of substrate into other branches of the pathway. Sense suppression, antisense RNA, and RNAi against core biosynthetic gene activity have all been used successfully to reduce the production of various phenylpropanoids. In particular, RNAi has emerged as a powerful and reliable technology, being more effective than sense suppression or antisense RNA for some genes (Nishihara et al., 2005; Nakamura et al., 2006), and allowing allelespecific inhibition (for CHS; Fukusaki et al., 2004), tissue-specific inhibition (CHS; Nakatsuka et al., 2007), and inhibition of all four members of a TF multigene family (Gonzalez et al., 2008). These approaches have been very effective in reducing the levels of specific phenylpropanoids, but in some cases have also led to unexpected pleiotropic phenotypes. In some species inhibiting

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flavonoid production results in male or female sterility (Taylor and Grotewold, 2005), and in tomato results in the production of parthenocarpic fruit (Schijlen et al., 2007). Downregulation of flavanone 3-hydroxylase in the ornamental species carnation increased the levels of methylbenzoate, producing more fragrant flowers (Zuker et al., 2002). Downregulation of flavonol synthase (FLS) in petunia likewise resulted in a redirection of substrate, in this example into anthocyanins, intensifying flower color (Davies et al., 2003). More severe pleiotropic effects may occur when an enzyme early in the phenylpropanoid pathway is targeted, as production of a greater range of downstream compounds will be affected. Thus, attempts to inhibit phenylalanine ammonia-lyase (PAL) activity in model species have generated plants with reduced growth and altered leaf morphology, in addition to reduced phenylpropanoid accumulation (Korth et al., 2001).

12.4.2

INTRODUCING NOVEL BIOSYNTHETIC ACTIVITIES

Although the phenylpropanoid pathway is ubiquitous in plants, there are many variations between species in the range of phenylpropanoids produced. This can be variation of the structures formed within one flavonoid group, for example, different anthocyanin substitution patterns, or the presence or absence of whole groups of compounds. Where associated biosynthetic genes have been identified, the possibility exists of introducing these into species lacking them. This is easier when only one or two biosynthetic activities are required, but more challenging when a whole pathway, for example, for a range of isoflavonoids, is lacking in the target species. As mentioned earlier, stilbenes are of particular interest from a functional food viewpoint, and the key gene for enabling stilbene production (for resveratrol) has been identified from a range of species. STS transgenes have been successfully used to introduce or increase resveratrol production in several species, including apple, hops, oilseed rape, and tomato (Table 12.1). STS in tomato resulted in the production of resveratrol aglycone and two resveratrol glycosides at up to 10 mg kg−1 fresh weight, which compares favorably with the levels reported for red wine (up to 10 mg L −1) (Schijlen et al., 2006). Introduction of the key branch point enzyme IFS has enabled the production of some isoflavonoids in species that normally lack them, but only at low levels. Significant levels were only achieved when IFS expression was combined with approaches to encourage more accumulation of the IFS substrate naringenin (Tian and Dixon, 2006). Other novel compounds introduced to species include 6′-deoxychalcones and flavones (Table 12.1). Commonly, the endogenous glycosyltransferase enzymes are able to act on the novel substrates to produce, for example, genistein, resveratrol, or 6′-deoxychalcone glycosides (Davies et al., 1998; Giovinazzo et al., 2005; Tian et al., 2008). Introduction of novel activities may cause a reduction in the production of other phenylpropanoids, due to competition for common substrates. Thus, CHR caused a reduction in anthocyanins in petunia (Davies et al., 1998), and CHR or STS transgenes reduced naringenin chalcone levels in tomato fruit (Schijlen et al., 2006). Novel biosynthetic activities may also be introduced to increase the structural variation existing within groups of phenylpropanoids already present in the target species. For example, there are numerous examples of changing the type of anthocyanin produced through GM, principally for altering the resulting flower colors. In alfalfa, introduction of an IFS transgene conferred the production of high levels of genistein, which is not one of the isoflavonoids normally accumulated in this legume species (Deavours and Dixon, 2005).

12.4.3

INCREASING PHENYLPROPANOID LEVELS

Two main approaches have proven successful for increasing levels of particular phenylpropanoids in transgenic plants. Introduction of one or more biosynthetic genes that are of rate-determining activity, at least in some tissues or at certain stages of development, has enabled targeted increases in the levels of compounds. For example, overexpressing genes for enzymes early in the flavonoid pathway in flax increased total phenolic levels, with a slight increase in lignan amounts (Lorenc-Kukula et al., 2005).

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This approach has often been effective in increasing the levels of a specific type of flavonoid. An excellent example is the introduction of the HQT transgene into tomato, which increased CGA levels in leaves by up to 85% with no measured reduction in the levels of other phenolics (Niggeweg et al., 2004). If a rate-determining step cannot be identified, or multiple enzymes are rate limiting, then transgenes for multiple biosynthetic steps may be required. In some cases the use of target plant lines with endogenous mutations in the biosynthetic pathway may also be used to aid the effectiveness of transgenes, for example, through promoting the accumulation of specific substrates (Katsumoto et al., 2007). Overexpression of genes encoding the early steps in phenylpropanoid biosynthesis, specifically PAL, cinnamate 4-hydroxylase, and 4-coumarate:CoA ligase, could in theory channel extra substrate into the pathway as a whole. However, mixed results have been obtained with this approach. While the levels of compounds such as CGA have been increased (Shadle et al., 2003), pleiotropic effects on plant growth and development can occur from altering the activity of the entire pathway (Millar et al., 2007). A more promising approach for increasing the overall levels of phenolics is the use of TF transgenes to simultaneously upregulate multiple biosynthetic steps. There are many examples of TF use to dramatically increase anthocyanin levels in plants, and also examples for other flavonoid groups. TF genes are likely to be a cornerstone of future biotechnology approaches for functional foods, either as transgenes or for use in marker-assisted breeding. Two recently published examples for tomato illustrate the power of TF transgenes for modifying the production of human health-related phenolics. Introduction of AtMYB12 into tomato under the control of a fruit-specific promoter activated both theflavonol and caffeoyl quinic acid biosynthetic pathways, resulting in greatly increased levels of both flavonols and CGA (Luo et al., 2008). The total polyphenol content reached 10% dry weight for the fruit. Introduction of a transgene for two anthocyanin pathwayrelated TFs from Antirrhinum majus (DELILA and ROSEA1) into tomato under the control of a fruit-specific promoter resulted in strong anthocyanin accumulation in the fruit, at up to 2.8 mg g−1 fresh weight (Butelli et al., 2008). The transgenic, high anthocyanin tomatoes were used as food components for the diet of a tumorigenic mice line, in parallel tests with nontransgenic tomatoes. Those mice receiving the high anthocyanin tomatoes had a significantly higher average life span than mice receiving nontransgenic tomatoes or no tomato supplement. The results prove the power of TFs to upregulate whole pathways. In other examples, at least eight biosynthetic genes were upregulated by a bHLH transgene in petunia (Bradley et al., 1998), and the AtMYB12 transgene in tomato upregulated at least 14 genes required for flavonol and CGA production (Luo et al., 2008). The use of endogenous TF species also allows for “intragenic/cisgenic” approaches for metabolic engineering, as recently demonstrated for increasing CGA and flavonol levels in potato tubers (Rommens et al., 2008). A limitation for many crop species at present is a lack of understanding of which TFs are important for regulating the production of different groups of phenylpropanoids, and how the various TFs may interact in a regulatory complex for that biosynthetic pathway. Genome sequences of major crop species, expected to be available within a few years for many, will make available all of the TF sequences. The challenge will be to identify rapidly the TFs required for the specific metabolic engineering aim. A recent example of the variations that may occur in the activity of “similar” TFs is the metabolic engineering of anthocyanin biosynthesis in tomato. In maize, LC (bHLH) and C1 (MYB) control anthocyanin production through upregulation of the biosynthetic genes. DELILA (bHLH) and ROSEA1 (MYB) fulfill a similar role in Antirrhinum majus. However, when introduced into tomato, LC + C1 increased flavonol levels in fruit but did not induce anthocyanin production (Bovy et al., 2002), while DELILA + ROSEA1 induced high levels of flavonols and anthocyanins (Butelli et al., 2008). This is probably due to the Antirrhinum majus TF transgenes activating a broader range of phenylpropanoid biosynthetic genes than the maize factors, including CHI and flavonoid 3′,5′-hydroxylase (F3′5′H). F3′5′H is key in tomato for anthocyanin production, due to the substrate selectivity of DFR.

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An alternative approach is to use synthetic TFs designed for the specific purpose. Some TFs may be engineered to recognize and activate the promoter sequences of the target genes, as demonstrated for upregulation of α-tocopherol biosynthesis (Van Eenennaam et al., 2004). Also, the addition of specific protein domains can be used to confer repressive activity on TFs, for example, changing an activator of flavonoid biosynthesis into a repressor of the same pathway (Matsui et al., 2004).

12.5

CONCLUDING COMMENTS

Technologies for the engineering of phenylpropanoid biosynthesis in plants are well advanced. Levels of specific phenylpropanoids, most commonly anthocyanins, flavonols, and resveratrol, have been increased in transgenic plants of a wide range of crop species. These plant lines help define the possibilities for metabolic engineering approaches and also provide material for comparative tests of the health benefits associated with bioactive compounds. Although sufficient data are lacking to allow DRIs for bioactive phenylpropanoids, there is a growing body of evidence supporting a link between increased phenylpropanoid intake and improved health characters. Some food products, such as soybean, are already marketed as having associated health benefits based on their phenylpropanoid content. Thus, the achievement of higher or more consistent levels of active compounds in these crops is a clear current target for metabolic engineering, even in the absence of DRIs. Furthermore, the growing amount of correlative evidence combined with strong public interest in functional foods will likely maintain and increase the drive for the metabolic engineering of food crops, regardless of DRIs for the target compounds.

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Kussmann, M., Raymond, F., and Affolter, M., 2006. OMICS-driven biomarker discovery in nutrition and health. J. Biotechnol. 124: 758–787. Kyle, J.A.M. and Duthie, G.G., 2006. Flavonoids in foods. In: Ø.M. Andersen and K.R. Markham (Eds), The Flavonoids: Chemistry, Biochemistry, and Applications, pp. 219–262. Boca Raton: CRC Press. Lee, Y., Cho, E., and Yokozawa, T., 2008. Effects of proanthocyanidin preparations on hyperlipidemia and other biomarkers in mouse model of type 2 diabetes. J. Agric. Food Chem. 56: 7781–7789. Li, H.H., Flachowsky, H., Fischer, T.C., Hanke, M.V., Forkmann, G., Treutter, D., Schwab, W., Hoffmann, T., and Szankowski, I., 2007. Maize Lc transcription factor enhances biosynthesis of anthocyanins, distinct proanthocyanidins and phenylpropanoids in apple (Malus domestica Borkh.). Planta 226: 1243–1254. Lim, J.D., Yun, S.J., Chung, I.M., and Yu, C.Y., 2005. Resveratrol synthase transgene expression and accumulation of resveratrol glycoside in Rehmannia glutinosa. 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Niggeweg, R., Michael, A.J., and Martin, C., 2004. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat. Biotechnol. 22: 746–754. Nishihara, M., Nakatsuka, T., and Yamamura, S., 2005. Flavonoid components and flower color change in transgenic tobacco plants by suppression of chalcone isomerase gene. FEBS Lett. 579: 6074–6078. Park, J.S., Kim, J.B., Cho, K.J., Cheon, C.I., Sung, M.K., Choung, M.G., and Roh, K.H., 2008. Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa). Plant Cell Rep. 27: 985–994. Reddy, A.M., Reddy, V.S., Scheffler, B.E., Wienand, U., and Reddy, A.R., 2007. Novel transgenic rice overexpressing anthocyanidin synthase accumulates a mixture of flavonoids leading to an increased antioxidant potential. Metab. Eng. 9: 95–111. 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Rommens, C.M., Richael, C.M., Yan, H., Navarre, D.A., Ye, J., Krucker, M., and Swords, K., 2008. Engineered native pathways for high kaempferol and caffeoylquinate production in potato. Plant Biotechnol. J. 6: 870–886. Schijlen, E., de Vos, C.H.R., Jonker, H., van den Broeck, H., Molthoff, J., van Tunen, A., Martens, S., and Bovy, A., 2006. Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit. Plant Biotechnol. J. 4: 433–444. Schijlen, E.G.W.M., de Vos, C.H.R., Martens, S., Jonker, H.H., Rosin, F.M., Molthoff, J.W., Tikunov, Y.M., Angenent, G.C., van Tunen, A.J., and Bovy, A.G., 2007. RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis pathway, leads to parthenocarpic tomato fruits. Plant Physiol. 144: 1520–1530. Schwekendiek, A., Spring, O., Heyerick, A., Pickel, B., Pitsch, N.T., Peschke, F., de Keukeleire, D., and Weber, G., 2007. Constitutive expression of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its derivatives in substantial quantities. J. Agric. Food Chem. 55: 7002–7009. Shadle, G.L., Wesley, S.V., Korth, K.L., Chen, F., Lamb, C., and Dixon, R.A., 2003. Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of l-phenylalanine ammonia-lyase. Phytochemistry 64: 153–161. Shih, C.H., Chen, Y.L., Wang, M.F., Chu, I.K., and Lo, C., 2008. Accumulation of isoflavone genistin in transgenic tomato plants overexpressing a soybean isoflavone synthase gene. J. Agric. Food Chem. 56: 5655–5661. Stobiecki, M., Matysiak-Kata, I., Franski, R., Skala, J., and Szopa, J., 2003. Monitoring changes in anthocyanin and steroid alkaloid glycoside content in lines of transgenic potato plants using liquid chromatography/ mass spectrometry. Phytochemistry 62: 959–969. Szankowski, I., Briviba, K., Fleschhut, J., Schönherr, J., Jacobsen, H.J., and Kiesecker, H., 2003. Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa). Plant Cell Rep. 22: 141–149. Taylor, L.P. and Grotewold, E., 2005. Flavonoids as developmental regulators. Curr. Opin. Plant Biol. 8: 317–323. Tian, L. and Dixon, R.A., 2006. Engineering isoflavone metabolism with an artificial bifunctional enzyme. Planta 224: 496–507. Tian, L., Pang, Y., and Dixon, R., 2008. Biosynthesis and genetic engineering of proanthocyanidins and (iso) flavonoids. Phytochemistry Rev. 7: 445–465. Tsuda, T., Ueno, Y., Yoshikawa, T., Kojo, H., and Osawa, T., 2006. Microarray profiling of gene expression in human adipocytes in response to anthocyanins. Biochemical Pharmacology 71: 1184–1197. Van Eenennaam, A.L., Li, G.F., Venkatramesh, M., Levering, C., Gong, X.S., Jamieson, A.C., Rebar, E.J., Shewmaker, C.K., and Case, C.C., 2004. Elevation of seed alpha-tocopherol levels using plant-based transcription factors targeted to an endogenous locus. Metab. Eng. 6: 101–108. Vanholme, R., Morreel, K., Ralph, J., and Boerjan, W., 2008. Lignin engineering. Curr. Opin. Plant Biol. 11: 278–285. Williamson, G. and Holst, B., 2008. Dietary reference intake (DRI) value for dietary polyphenols: Are we heading in the right direction? Brit. J. of Nutr. 99: S55–S58. Xie, D.Y., Sharma, S.B., Wright, E., Wang, Z.Y., and Dixon, R.A., 2006. Metabolic engineering of proanthocyanidins through co-expression of anthocyanidin reductase and the PAP1 MYB transcription factor. Plant J. 45: 895–907. Yu, O. and Jez, J.M., 2008. Nature’s assembly line: Biosynthesis of simple phenylpropanoids and polyketides. Plant J. 54: 750–762. Yu, O., Shi, J., Hession, A.O., Maxwell, C.A., McGonigle, B., and Odell, J.T., 2003. Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry 63: 753–763. Zhu, Y.J., Agbayani, R., Jackson, M.C., Tang, C.S., and Moore, P.H., 2004. Expression of the grapevine stilbene synthase gene VST1 in papaya provides increased resistance against diseases caused by Phytophthora palmivora. Planta 220: 241–250. Zuker, A., Tzfira, T., Ben-Meir, H., Ovadis, M., Shklarman, E., Itzhaki, H., Forkmann, G., Martens, S., NetaSharir, I., Weiss, D., and Vainstein, A., 2002. Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Mol. Breed. 9: 33–41. Zuluaga, D.L., Gonzali, S., Loreti, E., Pucciariello, C., Degl’Innocenti, E., Guidi, L., Alpi, A., and Perata, P., 2008. Arabidopsis thaliana MYB75/PAP1 transcription factor induces anthocyanin production in transgenic tomato plants. Funct. Plant Biol. 35: 606–618.

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Use of Biotechnology 13 The to Reduce the Dependency of Crop Plants on Fertilizers, Pesticides, and Other Agrochemicals Zeba F. Alam CONTENTS 13.1 Introduction ........................................................................................................................ 198 13.2 Reduced Dependency on Fertilizers ................................................................................... 198 13.3 Biofertilizers .......................................................................................................................200 13.3.1 Symbiotic Nitrogen Fixers ......................................................................................200 13.3.2 Asymbiotic Nitrogen Fixers ....................................................................................200 13.3.3 Phosphate-Solubilizing Bacteria ............................................................................. 201 13.3.4 Organic Fertilizers .................................................................................................. 201 13.4 Herbicide Resistance........................................................................................................... 201 13.5 Glyphosate Resistance ........................................................................................................202 13.5.1 Overexpression of Crop Plant EPSPS Gene............................................................ 203 13.5.2 Use of Mutant EPSPS Genes .................................................................................. 203 13.5.3 Mechanisms to Detoxify Glyphosate ......................................................................203 13.6 Phosphinothricin (Glufosinate) Resistance ......................................................................... 203 13.7 Sulfonylureas and Imidazolinones Resistance ...................................................................204 13.8 Limitations of Herbicide-Tolerant Transgenics In Crop Plants ..........................................204 13.9 Pesticide Resistance ............................................................................................................205 13.9.1 Bacillus thuringiensis (BT) Toxin Gene .................................................................206 13.9.2 Other Microorganism-Derived Resistance Genes ..................................................207 13.9.3 Use and Transfer of Resistance Genes from Higher Plants ....................................207 13.9.3.1 Proteinase Inhibitors ................................................................................207 13.9.3.2 Cowpea Trypsin Inhibitor Gene (CpTI) ...................................................207 13.9.3.3 α-amylase Inhibitor ..................................................................................208 13.9.3.4 Lectins ......................................................................................................208 13.9.4 Resistance Genes from Animals .............................................................................208 13.10 The Commercial Use of Herbicide- and Pesticide-Resistant Plants...................................208 13.11 The Major Impact of Such Transgenic Crops ..................................................................... 210 13.12 Risk Assessment ................................................................................................................. 211 13.13 Future Planning .................................................................................................................. 213 13.14 Conclusion .......................................................................................................................... 214 References ..................................................................................................................................... 214 197 © 2010 Taylor and Francis Group, LLC

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INTRODUCTION

In recent times, we have been faced with the challenges of improving food quality and increasing crop production in order to meet the ever-growing crisis of an increasing world population with the minimum of environmental consequences. Since time immemorial, farmers have used traditional methods to increase their crop production and quality. However, due to recent breakthroughs in science and genetics, more and more farmers have started using biotechnology to increase their agricultural output. Agricultural productivity can be increased by reducing biotic stresses and at the same time using better quality seeds with the optimum use of fertilizers. The challenge is not only to reduce those biotic stresses that reduce the crop yield, such as weeds, pests, and diseases, but also to reduce and minimize the environmental consequences associated with this, such as the consumption of natural resources, conservation of water, and protection of natural habitats and biodiversity. According to a report submitted by the Food and Agricultural Organization of the World Health Organization (2002), the biggest challenge for the agricultural sector will be to deliver twice as much food in 2050 as is produced today; this will become necessary due to the estimated increase in the world population (Conway and Toenniessen, 1999; Cohen, 2003). Regarding the climate changes currently being experienced by our planet and concerns about global warming, this increase in agricultural productivity must be brought about without any further detrimental environmental impact. According to Raven (2008), farmers will have to find the means to reduce environmental impacts by producing more from each unit of land, water, and energy engaged in crop production. There is also a need to adapt cropping systems to suit changes in climate which threaten crop productivity and food reserves at local and global levels (Goklany, 1999). Farmers should be encouraged to adopt and use new technologies like agricultural biotechnology to meet these challenges along with better economic returns. Using biotechnology, attempts have been made to reduce crop dependency on fertilizers, pesticides, and other agrochemicals. This should help to further reduce the impact of these chemicals on the environment without compromising overall crop productivity (Conko, 2003).

13.2 REDUCED DEPENDENCY ON FERTILIZERS Fertilizers are essential for the optimum yield of crop plants. It has been observed that newly cleared lands for agricultural purposes also require the application of all kinds of fertilizers such as nitrogen, potassium, and phosphorus to improve the soil quality and the overall yield. However, we are aware of the adverse effects on the environment resulting from the overuse of nitrogen, potassium, and phosphorus fertilizers and other animal manures. One of these effects is demonstrated in aquatic plants, which show excessive growth due to the runoff from fertilizers and manure into streams, lakes, and other water bodies. Due to this excessive growth, there is depletion in the availability of absorbed oxygen needed by other organisms. Hence one area of biotechnological research focuses on reducing the dependency of crops on fertilizers. Multipronged strategies have been adopted to address this issue. One possibility is to screen crop varieties and select those that have a lower requirement for some mineral nutrients. It has been reported that when soybean tissue samples taken from several varieties are placed on ironor manganese-deficient media, the tissue from some varieties grows better than others (Holt, 1991). The results of such tests are correlated with field tests on deficient soils. One can also try to transfer the capability of fixing atmospheric nitrogen to crop plants to reduce their dependency on chemical nitrogen fertilizers without compromising on the soil fertility (Bruulsema, 2007). Among the major agronomic crops, soybean, alfalfa, and clover can be grown commercially without supplemental nitrogen in the form of fertilizer, manure, or nitrogen carried over from a previous legume crop. The bacteria present in the root nodules of these legume crops convert nitrogen to the required forms and provide it to the host plants. Another strategy involves improving the efficiency of nitrogen-fixing microorganisms which will help provide large amounts of plant-available nitrogen to host crops. The race is also on to

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develop new strains of corn and other nitrogen-fixing crops that have nitrogen-fixing microorganisms. Another more challenging approach is to transfer genes controlling nitrogen fixation from microorganisms to crop plants so that the crop plants themselves will possess the biochemical machinery to fix atmospheric nitrogen, rather than being dependent on microorganisms (Gleason et al., 2006). Further, there has been an intense debate over proposals to relocate the nitrogen-fixing mechanism to the leaves from the root nodules in order to bring them closer to the energy sources (carbohydrates) and to atmospheric nitrogen. However, nitrogen fixing is an anaerobic process and is strongly inhibited by oxygen; therefore, it is important to evaluate how well it will function in an aerobic environment within leaves. If the agricultural nitrogen requirement is met through these strategies, then the amount of energy required to grow crops will be significantly reduced, as well as the degree of dependency on commercially available nitrogen fertilizers. The genetic manipulations for nitrogen fixing involve the following: • Gene alterations to improve nitrogen-fixing efficiency and bacteria–host plant interactions, especially in Rhizobium sp. • Using biotechnology to transfer nitrogen-fixing genes from, for example, Rhizobium sp. to other bacteria such as Agrobacterium tumefaciens, which in turn can infect other crops such as tomato, tobacco, and Petunia so as to fix nitrogen. • Inducing a symbiotic relationship in nonleguminous plants such as wheat, rice, and corn using genetically engineered Rhizobium sp. Using the technique of genetic complementation, the nif genes were isolated from the clone banks of the diazotrophs, Klebsiella pneumonia. This procedure involved the identification and characterization of clones from a wild-type library to restore nitrogen fi xation in the various mutant forms of the original organism. The nif genes of K. pneumonia have been used as hybridization probes for the identification of nif genes from the DNA clone banks of diazotrophic organisms (Buchanan-Wollaston et al., 1981). This approach established the fact that all nitrogen-fixing bacteria possess a similar type of nif gene clusters (MacNeil et al., 1981). Some workers have reported the successful introduction of extra copies of nitrogenase regulatory genes, namely nifA and nifL, into diazotrophs (Hill et al., 1981). The genome or DNA sequence of the rhizobial bacteria of alfalfa which fix nitrogen was reported in 2001 (Galibert et al., 2001). Another bacterium, Gluconacetobacter diazotrophicus, which is found in sugarcane, sweet potato, and pineapples, is an endophyte that lives between the cells of the roots of its host. This organism was reported to be responsible for the low nitrogen requirements of sugarcane and other crop plants and thus acts as a natural fertilizer. The genome of this bacterium has been mapped and sequenced (Pedraza, 2008). There have been several problems associated with the transfer of nif genes into plants. The transfer of 24 kb nif gene cluster becomes ineffective because the normal cellular concentration inactivates the nitrogenase enzyme. Any reduction in O2 causes cell death. Due to the absence of plant promoters (which can respond to nifA regulatory protein), it is impossible to turn on the nif genes in transgenic plants. The plant cells are unable to process nif gene transcripts which are multigenic in nature. Another basic requirement for nitrogen fixation is the nodules on the roots of leguminous plants. Certain genes involved in nodulation called as nod genes have been identified in Rhizobium melitoti. The genetic complementation technique is being used to isolate nod genes and use them for the induction of nodules in crop plants. Successful attempts have been made to induce the bacteria to form nodules in the roots of important crop plants such as rice, wheat, and oilseed rape (Cocking and Davey, 1987). The process of nodulation involves the participation of a large number of nod genes and is influenced by factors such as the concentration of nutrients, soil temperature, light, concentration of CO2, and so on. It has been reported that the release of autoinhibition from calcium/calmodulin-dependent protein kinase (CCaMK) after calmodulin binding is the central

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switch sufficient to activate nodule morphogenesis (Gleason et al., 2006). This finding that a single regulation event is sufficient to induce nodulation has boosted the possibility of transferring the nodulation process to nonlegumes. As reviewed by Good et al. (2004), recent efforts to genetically manipulate the efficiency of nitrogen use in plants involve the overexpression of enzymes in the nitrogen uptake and assimilation pathways. Similarly, many crop plants will grow to full maturity in alkaline soils only in the presence of phosphorus fertilizers. To overcome this problem, around 30 million tones of phosphorus fertilizer are added to farm fields per year around the world. The problem is aggravated because more than two-thirds of global land is naturally acidic or alkaline and phosphorus forms compounds with elemental aluminum, iron, calcium, and magnesium in the soil. If these fertilizers are not used by the plants, then the runoff causes environmental pollution. There have been reports of workers bioengineering corn, tobacco, and papaya plants by introducing a gene from the bacterium Pseudomonas aeruginosa in order to secrete citric acid from their roots (Vijayaraghavan and Yun, 2008). This causes an unbinding of the phosphorus from other elements and makes it available to the plants. The yield in terms of fruit and leaves in these engineered crop plant varieties increased significantly with much smaller requirement of phosphorus fertilizer. There have been successful attempts to create bioengineered rice and corn varieties that show better growth in alkaline soils. Efforts are also underway to modify crop plants such as rice and sorghum to reduce their dependency on phosphorus fertilizers.

13.3

BIOFERTILIZERS

Biofertilizers are nutrient inputs of biological origin used to support plant growth. These are cheap, and environmentally safe alternatives to chemical sources and are fairly easy to produce. They increase the fertility of the soil by increasing its physicochemical properties, texture, and waterholding capacity. The use of biofertilizers helps to reclaim saline and alkaline soils as well as improving the tolerance of plants against abiotic, biotic, and toxic heavy metals. However, they cannot meet the total nutrient needs of plants, and it is therefore necessary to rely on an integrated nutrient supply system using a combination of chemical fertilizers and biofertilizers. Sources which can be used as biofertilizers are as follows.

13.3.1

SYMBIOTIC NITROGEN FIXERS

Diazotrophic microorganisms such as Rhizobium sp. and Bradyrhizobium sp. are used as nitrogen fixers, and have the capacity to fix about 50–150 kg nitrogen/hectare/annum. Using the practice of “green manuring,” leguminous plants with symbiotic nitrogen-fixing bacteria are ploughed into the soil and a nonleguminous crop is grown to take the benefits from the already fixed nitrogen. To further increase this ability to fix nitrogen, a hup+ gene, which plays a role in the uptake of hydrogenase, is used (Eisbrenner and Evans, 1983; Martinez et al., 2008). This helps in the recycling of H2 produced during nitrogen fixation and saves energy required for reduction. Rhizobium strains having a hup+ gene fix more nitrogen (Sekhon et al., 1992). Those strains deficient in nitrate reductase also have an improved nitrogen-fixing ability.

13.3.2

ASYMBIOTIC NITROGEN FIXERS

The asymbiotic nitrogen-fixing bacteria such as Azobacter and Azospirillum directly convert gaseous nitrogen into nitrogen-rich compounds and when they die they enrich the soil with nitrogenous compounds. Blue-green algae not only fixes nitrogen but also improves the biomass. The most efficient strains of Azotobacter fix 30 kg of N2 from 1000 kg of organic matter. Azolla with Anabaena can be used as a biofertilizer because Anabaena azollae maintains a symbiotic relationship by inhabiting the leaf cavities of Azolla.

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TABLE 13.1 Symbiotic Nitrogen-Fixing Bacteria along with the Host Plants Bacterial Species Bradyrhizobium elkani Rhizobium ciceri Bradyrhizobium japonicum Rhizobium leguminosarum Rhizobium meliloti Rhizobium fredi Rhizobium loti Rhizobium elti

Host Plants Soybean Chickpea Soybean Pea, broad bean Alfalfa Soybean Lotus Mung bean, kidney bean

13.3.3 PHOSPHATE-SOLUBILIZING BACTERIA Bacteria such as Thiobacillus and Bacillus can convert the nonavailable inorganic phosphorus present in the soil into usable form of phosphate. These bacteria also produce siderophores which protect the plants from disease by chelating with iron, making it unavailable to pathogenic organisms.

13.3.4

ORGANIC FERTILIZERS

Organic wastes such as animal dung, urine, urban garbage, sewage, oil cakes, and crop residues can be used as organic fertilizers. In countries like India, it is common practice to use animal dung as an organic manure to boost the growth of crop plants (Tables 13.1 and 13.2).

13.4

HERBICIDE RESISTANCE

According to an estimate, the world’s crop yield is reduced by 10–15% due to the damage caused by weeds. A wide range of chemicals are used as weed killers (herbicides) in order to destroy these weeds or unwanted plants. The ideal herbicide is one which has the power to kill weeds without affecting crop plants by quickly distributing within the target plant and which rapidly degrades in the soil. It should not be toxic to animals and other organisms. It has been observed that herbicides are often unable to discriminate between weeds and crop plants, which leads to the destruction of crop plants as well. That is why biotechnologists have focused research on developing herbicideresistant plants. TABLE 13.2 Microorganisms Used as Biofertilizers along with the Crops Category/Microorganism

Crops

Symbiotic Nitrogen Fixers Rhizobium meliloti, Rhizobium ciceri, Rhizobium Legumes, pulses, oilseeds leguminosarum, Bradyrhizobium japonicum Asymbiotic Nitrogen Fixers Azobacter, Azospirillum, blue-green algae (Anabena, Wheat, rice, sugarcane, vegetables Nostic, Plectonemia) Phosphate-solubilizing bacteria Thiobacillus, Bacillus, mycorrhiza (Glomus) Pulses

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The major problems plaguing weed management have been the lack of availability of costeffective herbicides, the limited choice of herbicides with broad-spectrum activity which cause minimum damage and injury to crop plants, and the necessity of multiple applications of herbicides to control the wide range of weed species that affect the overall crop yield. Often the strategy to control weeds has been based on pre-emergence—that is, the herbicide application is decided by the expected weed infestations rather than in response to actual weeds present in a given area. In spite of multiple applications of herbicide, farmers often have to resort to mechanical cultivation and hand weeding to control the spread of weeds in the fields. Genetic engineering has been used to obtain crop plants having tolerance to broad-spectrum herbicides (Botterman and Lemons, 1988). The aim is to increase the crop yield by using methods to improve weed management while minimizing crop injury. Further, the use of herbicide-tolerant crops will not only help to reduce the number of active herbicide ingredients used for weed management but will also reduce the number of herbicide applications per crop season (Fulton and Keyowski, 1992). The other major advantage of using herbicide-tolerant crops is in terms of soil conservation due to low-tillage or no-tillage cropping, which helps to preserve the topsoil. The use of herbicide-tolerant crops enabled soil-conserving low- and no-tillage cropping on more farmland acres and made it more attractive to farmers to use these methods. No-till farming is when weeds are killed with herbicides rather than tilling the soil. With the introduction of biotech herbicide-tolerant crops, no-till crop acreage has increased by nearly 40%. The absence of plowing and tilling leaves a layer of crop and weed residues on the topsoil to protect against wind and rain erosion. The crop seeds are sowed into the soil and the slits are then closed by using rollers. When farmers use these methods, it cuts the soil erosion by 65–95% and helps to build the topsoil. The binding of the topsoil also protects water bodies from sediment pollution. According to an estimate, no-till and conservation tillage farming saves approximately $3.5 billion in water treatment, waterway maintenance, navigation, flooding, and recreation costs. No-till and conservation tillage have also helped to improve soil quality and sustainability by increasing the soil carbon, increased water infiltration, and water-holding capacity. It also produces a 3–6 times increase in the earthworm populations and in-fields wildlife benefits. According to a survey, quail are able to find their food in one-fifth of the time in a no-till field compared to a plowed field because the no-till field has more plant residues and a soil structure with more beetles, insects, and detritovors (Fawcett and Towery, 2002). Another example is the parasitic witchweed which is found in sub-Saharan Africa. This parasitic weed devastates corn and sorghum which are the main food grains of this region. Field trials are in progress to control this weed by planting herbicide-tolerant corn seeds, soaked in a systemic herbicide which can kill the witchweed as it spreads in the crop plants’ roots. This will help to protect and save the food yields of millions of farmers of this region. Herbicide resistance has been easy to achieve as the resistance is controlled by a single gene (Vasil et al., 1992). Several strategies that have been made in this direction are as follows: 1. Overproduction of a herbicide-sensitive biochemical target 2. Reducing herbicide affinity by introducing structural alterations in a biochemical target 3. Introducing mechanisms for detoxification, that is, degradation of the herbicide before it reaches the biochemical target inside the plant cell Transgenic plants against various herbicides such as phosphinothricin (bialaphos), glyphosate, sulfonylurea, glufosinate, imidazolinones, bromoxynil, atrazine, 2,4-d sethoxydim, and so on, have been generated in different food crops, vegetables, and horticultural and ornamental plant species (Benbrook et al., 1996).

13.5 GLYPHOSATE RESISTANCE Glyphosate, a glycine derivative, is a broad-spectrum herbicide which is effective against 76 of the world’s worst 78 weeds. It is marketed as Roundup® by the American chemical company, Monsanto.

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Glyphosate acts as a competitive inhibitor of the enzyme 5-enoyl-pyruvylshikimate 3-phosphate synthase (EPSPS). This enzyme plays a very important role in the shikimic pathway, which results in the formation of aromatic amino acids (tryptophan, phenylalanine, and tyrosine), phenols, and certain secondary metabolites. The enzyme EPSPS catalyses the synthesis of 5-enoylpyruvylshikimate 3-phosphate from shikimate 3-phosphate and phosphoenoylpyruvate. Glyphosate has some structural similarities with the substrate phosphoenol pyruvate. Therefore glyphosate binds more tightly with EPSPS and blocks the normal shikimic acid pathway. Thus, the herbicide glyphosate inhibits the biosynthesis of aromatic amino acids and other important products. The inhibition of protein biosynthesis causes blocking of cell division and plant growth with ultimately the death of the plant. Glyphosate is nontoxic to animals including humans as they do not possess a shikimate pathway. The main strategies applied to provide glyphosate resistance in plants are as follows.

13.5.1

OVEREXPRESSION OF CROP PLANT EPSPS GENE

In Petunia, due to gene amplification the EPSPS gene is overexpressed. Therefore the EPSPS gene from Petunia was isolated and introduced into other plants. There was a 40-fold increase in the synthesis of EPSPS in transgenic plants which provided resistance to glyphosate. These plants can tolerate glyphosate at a dose 2–4 times higher than that required to kill wild-type plants.

13.5.2

USE OF MUTANT EPSPS GENES

An EPSPS mutant gene was first detected in the bacterium Salmonella typhimurium, where a single base substitution (C to T) caused a change in the amino acid from proline to serine. This modified enzyme could not bind to glyphosate and thus provided resistance. Subsequent strategies involved the introduction of mutant gene into tobacco plants using Agrobacterium Ti plasmid vectors. The transgene produced high quantities of the enzyme EPSPS; however, the transformed tobacco plant provided only a limited resistance to glyphosate. The reason for this marginal resistance was that the shikimate pathway occurs in the chloroplasts while the glyphosate-resistant EPSPS was produced in the cytoplasm and was not transported to the chloroplasts. To overcome this problem, the mutant EPSPS gene was tagged with a chloroplast-specific transit peptide sequence which helped the glyphosate-resistant EPSPS enzyme to enter the chloroplasts freely and to confer resistance against the herbicide.

13.5.3

MECHANISMS TO DETOXIFY GLYPHOSATE

The enzyme glyphosate oxidase converts glyphosate to glyoxylate and aminomethylphosphonic acid. This enzyme is present in some soil microorganisms. The gene encoding for glyphosate oxidase was isolated from a soil microorganism Ochrobactrum anthropi and after modifications it was introduced into crop plants, for example, oilseed rape. The transformed plants exhibited very good resistance levels to glyphosate.

13.6 PHOSPHINOTHRICIN (GLUFOSINATE) RESISTANCE Phosphinothricin is a broad-spectrum herbicide and derivative of a natural product called bialaphos. Bialaphos is a tripeptide with a combination of phosphinothricin bound to two alanine residues and is secreted by some species of Streptomyces. Bialaphos is converted to active phosphinothricin by peptidase. Due to a structural similarity between phosphinothricin and glutamate, it acts as a competitive inhibitor of the enzyme glutamine synthase. The inhibition of glutamine synthase leads to the accumulation of ammonia and the inhibition of photosynthesis which eventually leads to the death of the plant. Based on the fact that some Streptomyces sp. possess the natural detoxifying mechanism of phsophinothricin, attempts have been made to develop plants showing resistance against this herbicide. It was observed that the enzyme phosphinothricin acetyl transferase (of Streptomyces sp.) acetylates phosphinothricin, and thus inactivates the herbicide. The gene responsible for coding

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phosphinothricin acetyl transferase (bar gene) was identified in Streptomyces hygroscopicus and was introduced into maize and oilseed rape (Thompson et al., 1987). Transgenic maize and oilseed rape with the bar gene were found to provide resistance to phosphinothricin.

13.7

SULFONYLUREAS AND IMIDAZOLINONES RESISTANCE

The enzyme ALS is a key enzyme in the synthesis of branched chain amino acids, namely, isoleucine, leucine, and valine. Herbicides such as sulfonylureas and imidazolinones are known to inhibit this enzyme. The genes encoding for the mutant forms of this enzyme have been identified, isolated, and characterized. The mutant genes of ALS were transferred to the plants, for example, maize, tomato, sugar beet, and so on, to give resistance to sulfonylureas and imidazolinones. Similarly there are many other cases where a modification of the target led to the induction of herbicide resistance. A gene aroA, isolated from the bacteria Salmonella typhimurium or Escherichia coli, was transferred to tomato and/or tobacco. There have also been reports of workers isolating a gene from Streptomyces hygroscopicus, which encodes an enzyme capable of inactivating the herbicide Basta® (Tsaftaris, 1996). Field tests carried out using the transgenic plants carrying this gene have demonstrated the effectiveness of this gene’s protection against the herbicide. These herbicides can now be sprayed on transgenic crops without inflicting damage on the crops while killing the weeds present in the near vicinity. From 1996 to 2001, herbicide tolerance was the most important trait introduced to many commercially available transgenic crops (Benbrook, 2000, 2001a; Becker et al., 2002). By 2001, herbicide tolerance introduced in soybean, corn, and cotton accounted for 77% of the 626,000 km2 planted with transgenic crops, Bt crops accounted for another 15%, and stacked genes for herbicide tolerance and insect resistance used in both cotton and corn accounted for a further 8% (Benbrook, 2001b) (Tables 13.3 and 13.4).

13.8

LIMITATIONS OF HERBICIDE-TOLERANT TRANSGENICS IN CROP PLANTS

Some of the arguments and limitations cited with regard to herbicide-tolerant transgenics are as follows: 1. One of the main fears of using herbicide-tolerant transgenic crops is the transfer of herbicide tolerance genes to sexually compatible wild relatives or weeds, thus causing a major potential threat to the environment. 2. The creation of “super weeds.”

TABLE 13.3 A List of Herbicide-Resistant Plants with the Transferred Gene Herbicide Glyphosate Glyphosate Sulfonylureas/imidazolinones Phosphinothricin Bromoxynil Cynamide Atrazine Phenocarboxylic acids

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Gene Transfer/Strategy Adopted for Transfer

Transgenic Crops

Inhibition of EPSPS Detoxification by glyphosate oxidase Mutant plant with acetolactate synthase (ALS) bar gene coding phosphinothricin acetyl transferase Nitrilase detoxification Cynamide hydratase gene Mutant plant with chloroplast psb A gene Monooxygenase detoxification

Soybean, tomato Maize, soybean Rice, tomato, maize, sugar beet Maize, rice, wheat, cotton, potato, tomato, sugar beet Cotton, potato, tomato Tobacco Soybean Maize, cotton

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TABLE 13.4 The Mechanisms of Action of Different Herbicides and the Basis of Achieving Resistance against them in Transgenic Plants Herbicide

Pathway Affected

Target Product

Use

Basis of Resistance

Broad spectrum

Overexpression of EPSPS gene; bacterial aro A gene

Broad spectrum

Gene amplification

Glyphosate (Roundup) Amino acid biosynthesis inhibitor

Aromatic amino acid biosynthesis

EPSPS

Phosphinothricin (Basta) Amino acid biosynthesis inhibitor

Glutamine biosynthesis

GS

Sulfonylureas and Imidazolinones Amino acid biosynthesis inhibitor

Branched chain amino acids

ALS

Selected crops

Mutant ALS gene

Selected crops

Mutant PsbA gene, GST gene; detoxification

Selected crops

bxn gene; detoxification

Atrazine (Lasso) Photosynthesis inhibitor

Photosystem II

Photosynthesis inhibitor

Photosynthesis

QB (32 kDa protein) Bromoxynil (Buctril) —

3. Present practices will actually increase the dependence on a few herbicides rather than reduce overall herbicide usage. 4. The problem of weed control can worsen if weeds develop a resistance to such herbicides through the gene flow from transgenic crops. 5. Herbicide tolerance is being sought not only to comparatively environmentally acceptable herbicides but also to conventional, more toxic and persistent products. 6. Some environmentalists still hold the view that conventional and nonchemical methods such as crop rotation, dense plantings, cover cropping, ridge tillage, and others should be preferred over the use of any herbicide, because gene flow is a major risk which should be avoided at all costs.

13.9 PESTICIDE RESISTANCE Among the many issues raised around agricultural chemicals, it is the use of pesticides which is the most hotly debated and delicate. Environmentalists have time and again pointed out the problems caused by the runoff of pesticides into wetlands, streams, and lakes and also their seepage into the groundwater, especially in terms of their damage to biodiversity. These pesticides can kill not only fish but also other aquatic animals, insects, and plants. There are concerns about reduced agricultural productivity because of the destruction of beneficial insects such as bees, butterflies, and pollinating and pest-eating insects due to the overuse of pesticides, which defeats the very purpose of using them. However, a reduction in pesticide use could lower agricultural productivity which in today’s scenario of increasing world population would have worrying consequences. According to an estimate, with the present rate of pesticide use, 20% of the yield in the industrialized world and 40% in Africa and Asia are lost due to insect pests and other pathogens. Biotechnology has been used to exert better control over the insect pests and pathogens by inserting genes which will save the plants from these pests, thereby reducing our dependency on chemical pesticides (Cornejo et al., 2000). Most organisms have a well developed capability and the metabolic machinery to degrade any natural material. Recombinant DNA technology and genetic engineering strategies aim to exploit

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this innate quality present in living organisms in order to bring about the degradation of man-made agrochemicals, chemicals, pollution control, and bioremediation which will further keep the environment clean and sustainable. Another important strategy to keep the environment clean is to find ways to reduce or totally stop the use of chemical pesticides without affecting the overall yield of crop plants (Gianessi et al., 2002). This problem has been solved by creating insect-resistant transgenic crop plants which will help to reduce the consumption of chemical pesticides used in agriculture (Cornejo and McBride, 2002). In addition, chemical pesticides are being replaced by biodegradable bioinsecticides, which are environmentally friendly, nontoxic, and at the same time cost effective. These bioinsecticides are composed of bacterial spores, crystal proteins, and insect fillers and have been successfully tested on insects affecting cotton, maize, cabbage, sunflower, pigeon pea, and safflower. The thrust of major biotechnological research is directed toward improving insect and disease resistance in plants with the aim of developing insect-resistant transgenic crops which will reduce and replace the chemical pesticides without compromising on the yield, safety, and sustainability of crop plants (Phipps and Park, 2002). Some of the approaches relevant to this issue are as follows: 1. There is a need to discover new genes targeted against locally relevant insect pests. 2. Pyramiding the genes to guard against the evolution of resistance in insects. Gene pyramiding is a breeding technique in plants that uses genetic markers to get several resistance genes into a single cultivar of a plant. 3. A proper economic evaluation of biosafety and environmental risk following the release of pesticidal proteins and transgenic crops is a necessity. Much of the progress in inducing insect resistance in transgenic plants has been achieved through the use of the insect-control protein genes of Bacillus thuringiensis (Mujeeb-Kazi and Sitch, 1989). Around 40 different insect-resistant transgenes from bacteria, plants, and other origins have been introduced into plants to increase the level of insect resistance, such as a Bt gene from Bacillus thuringiensis, isopentyl transferase (ipt) gene from Agrobacterium tumefaciens, cholesterol oxidase gene from a Streptomyces fungus, Pht gene from Photorhabdus luminescens, and so on. The resistance genes from higher plants are of two types: (i) proteinase and amylase inhibitors and (ii) lectins, that is, snowdrop lectin (GNA, pea lectin, jacalin, rice lectin, etc.). The resistance genes of animal origin are serine proteinase inhibitors from mammals and tobacco hornworms (Manduca sexta).

13.9.1

BACILLUS THURINGIENSIS (BT) TOXIN GENE

Bacillus thuringiensis is a gram-negative soil bacterium which produces a parasporal crystalline proteinaceous toxin with insecticidal activity. Traditionally, a fermentation process has been used to produce insecticidal spray from these bacteria. These proteins are referred to as insecticidal crystalline protein or ICP. Bt genes code for the Bt toxin, and differ in their spectrum of insecticidal activity with most of them effective against lepidopteran insect larvae and some specifically active against only dipterans and coleopteran insects (Bravo et al., 2007). The insect toxicity of Bt lies in a large protein. The toxins accumulate as crystal proteins inside the bacteria during sporulation. When these parasporal crystals are ingested by the insect, they are converted to active form in the gut of the insect due to the combined action of pH and proteolytic enzymes. This active form of the toxin protein gets inserted into the membrane of the gut epithelial cells of the insect. The insect dies due to disruption to the ion transport across the brush borders/membranes of the insect and the massive loss of cellular adenosine triphosphate (ATP). Several genes encoding lepidopteran-type toxins have been isolated. One such gene from Bacillus thuringiensis subsp. Kurstaki HD-1 contains an open reading frame of 3468 bp encoding a protein of 1156 amino acids. Chimeric Bacillus thuringiensis Kurstaki genes having a CaMV 35S promoter and a sequence coding for an active truncated variant, as well as the full-length gene, have been constructed and expressed in tomato plants (Fischhoff et al., 1987). The level of insecticidal protein

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was enough to kill larvae of Manduca sexta, Heliothis virescens, and Heliothis zea. When progeny of the transgenic plants were analyzed, it was observed that the Bacillus thuringiensis Kurstaki gene segregated as a single dominant Mendelian marker. Another toxin gene has been cloned from Bacillus thuringiensis strain Berlkiner 1715, which produces 1155 amino acid Bt2 protein. When an analysis of the level of expression of a Bt2-based chimeric gene in tobacco plants was carried out (Vaeck et al., 1987), it was observed that there was a correlation between the quality of the toxin and insecticidal activity. The transgenic tobacco plants were found to be protected from the larvae of Manduca sexta. Bt strains have been reported to contain a diverse variety of δ endotoxins-encoding genes. Since the cloning and sequencing of the first insecticidal protein encoding gene in 1981, more than 100 crystal protein gene sequences have been reported. Each crystal protein is unique in having a specific activity spectrum. The Cry 1Ab protein is highly active against the European corn borer so it is used in Bt corn hybrids, whereas the Cry 1 Ac protein is highly toxic to both tobacco budworm and cotton bollworm larvae and is expressed in the Bt cotton varieties. Similarly Cry 3A protein is expressed in Bt potato varieties and provides protection against Colorado potato beetles. Early attempts to express cry 1A and cry 3A proteins under the control of CaMV 35S or Agrobacterium T-DNA promoters resulted in a very low expression in tobacco, tomato, and potato plants (Bravo et al., 2007). When the nucleotide sequence of this gene was modified (alteration of G + C content, removal of some polyadenylation sequences, deletion of ATTTA sequence), there was a 100-fold increase in the Bt toxin product formation. Following this success, the transgenic Bt crops that were found to provide effective protection against insect damage were given approval for commercial planting by the United States in the mid-1990s.

13.9.2

OTHER MICROORGANISM-DERIVED RESISTANCE GENES

Culture filtrates of Streptomyces sp. contain a cholesterol oxidase (CO) protein that showed tremendous toxicity to boll weevil larvae; therefore, this gene was engineered into tobacco. Isopentyl transferase (ipt) gene from Agrobacterium tumefaciens codes for an important enzyme in the cytokinin biosynthetic pathway. Expression of ipt in tobacco and tomato by a wound-inducible promoter has resulted in a decrease in leaf consumption by the tobacco hornworm (Manduca sexta) and reduced survival of the peach potato aphid (Myzus persicae).

13.9.3

USE AND TRANSFER OF RESISTANCE GENES FROM HIGHER PLANTS

Certain genes in higher plants also produce proteins with insecticidal activity and are referred to as non-Bt insecticidal proteins. Common examples are as follows: 13.9.3.1 Proteinase Inhibitors The different proteinases present in the plants are serine, cysteine, aspartic, and metalloproteinases that catalyze the release of amino acids from dietary protein. This provides the nutrients essential for the normal growth and development of insects. Proteinase inhibitors are the proteins that inhibit the activity of proteinase enzymes. Certain plants are known to produce proteinase inhibitors to provide defense against herbivorous insects; when ingested by insects, they interfere with the digestive enzymes of the insect which causes nutrient deprivation and ultimately the death of the insect. Using genetic engineering, this strategy was adopted to introduce proteinase inhibitor genes into crop plants that normally do not produce these proteins. 13.9.3.2 Cowpea Trypsin Inhibitor Gene (CpTI) Certain wild species of cowpea plants found in Africa were found to be resistant to attack by a wide range of insects. These plants had insecticidal proteins which were trypsin inhibitors that actually destroyed the insects attacking the plants. The CpTI gene found in these cowpea plants

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(Vigna unguiculata) is a very effective inhibitor gene that produces antimetabolite substances that provide protection against a number of insect pests such as the Bruchid beetel (Callosobruchus maculates), some of the lepidopteran insects (H. virescens), spodopteran insects (Manduca sexta), coleopteran insects (Callosobruchus, Anthonomus grandis), orthopteran insects (Locusta migratoria), and so on. CpTI gene has been cloned and constructs containing the CaMV 355 promoter and a full-length cDNA clone 550 bp long were used to transform leaf disks of tobacco. The bioassay for the insecticidal activity of transgenic tobacco plants was done with cotton bollworm (H. zea). Insect survival and plant damage were clearly decreased in transgenic plants compared with control. 13.9.3.3 α-Amylase Inhibitor Insect larvae secrete a gut enzyme called α-amylase that digests starch. By blocking the activity of this enzyme by α-amylase inhibitor, larvae can be starved and killed. Genes for three α-amylase inhibitors have been expressed in tobacco with the main emphasis on transferring the gene of α-amylase inhibitor (αAl-Pv) isolated from adzuki bean (Phaseolus vulgaris) which works against Zabrotes subfasciatus and Callosobruchus chinensis. 13.9.3.4 Lectins Lectins are plant glycoproteins and they provide resistance to insects by acting as toxins. The lectin gene (GNA) from snowdrop (Galanthus nivalis) has been transferred and expressed in potato and tomato. Laboratory results with modified potato showed that GNA did not increase the mortality but considerably reduced fecundity. An important feature of this protein is that it also acts as against piercing and sucking insects, especially aphids (Bell et al., 1999). One of the disadvantages is that the protein works well only when it is ingested in large quantities, that is, when insects are exposed to microgram levels in diet incorporation bioassays. A number of lectin-encoding genes (wheat germ agglutinin, jacalin, and rice lectin) have been expressed in transgenic plants, but their insecticidal performance is still very low and therefore far from being effective.

13.9.4

RESISTANCE GENES FROM ANIMALS

Proteinase inhibitor genes from mammals have also been transferred and expressed in plants to provide resistance against insects, but with limited success. The resistance genes involved are primarily serine proteinase inhibitors from mammals and the tobacco hornworm (Manduca sexta). Based on in vitro screening of the inhibition of proteolysis by midgut extracts of a range of lepidopteran larvae, the bovine pancreatic trypsin inhibitor (BPTI), α-antitrypsin (α1AT), and spleen inhibitor (SI) have been identified as promising insect resistance proteins and have been transferred into a range of plants. Manduca sexta-derived proteinase inhibitors, namely antichymotrypsin and antielastase expressed in cotton and chitinase in tobacco, were found to reduce reproduction of Bemisia tabaci and H. virescens, respectively (Tables 13.5 and 13.6).

13.10 THE COMMERCIAL USE OF HERBICIDEAND PESTICIDE-RESISTANT PLANTS According to the International Service for the acquisition of Agri-biotech Applications report, the planting of biotech crops has reached record levels, 114.3 million hectares spread over 23 countries, which is almost a 12.3% increase in acreage over previous years (James, 2007). In 1995, potato varieties expressing Bt crystal protein toxic to Colorado potato beetle were made commercially available. In 1996, cotton varieties expressing the Bt crystal protein toxic to a number of lepidopteran pests, including the tobacco budworm and cotton bollworm, were introduced. Bt corn hybrids with improved resistance to the European corn borer became available to farmers in the United States. Bt cotton varieties carrying the same technology as had been used in the United © 2010 Taylor and Francis Group, LLC

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TABLE 13.5 A Selected List of Plant Insecticidal (Non-Bt) Genes Used for Developing Transgenic Plants with Insect Resistance Plant Gene

Transgenic Plant(s)

Encoded Protein

Protease Inhibitors Potato, apple, rice, Trypsin sunflower, wheat, tomato Tobacco, potato Serine protease Potato, tobacco Serine protease Tobacco, oilseed rape Cysteine protease Tobacco Trypsin α-Amylase Inhibitors

CpTI CII PI-IV OC-1 CMe α-A1-Pv WMAI-1

Pea, tobacco Tobacco

GNA

Potato, rice, sugarcane, sweet potato, tobacco Maize

Resistance to Insect(s) Coleoptera, Lepidoptera Coleoptera, Lepidoptera Lepidoptera Coleoptera, Homoptera Lepidoptera

α-Amylase α-Amylase

Coleoptera Lepidoptera

Lectin

Homoptera, Lepidoptera

Agglutin

Lepidoptera, Coleoptera

Chitinase Tryptophan decarboxylase

Homoptera, Lepidoptera Homoptera

Lectins

WGA

Others BCH TDC

Potato Tomato

States were introduced in Australia. By 2004, biotech crops were grown in 18 countries, covering almost 80 million hectares (James, 2004). Insect-protected crops are the second largest biotech crop, covering 18% of the global biotech total in acreage terms. In the United States alone, biotech Bt crops reduced insecticide use in recent years by nearly 7 million pounds, which has had a major impact on the ecology due to the reduction in environmental pollution. TABLE 13.6 List of Transgenic Plants Showing Insect Resistance Plant Tobacco

Tomato Potato Cotton Pea Rice Maize Sugarcane

Gene Transferred

Insect Resistance

Bt from Bacillus thuringiensis CpTI (cowpea trypsin inhibitor) CpTI Insecticidal protein from Streptomyces Sweet potato trypsin inhibitor gene Bt Cry III from Bacillus thuringiensis Snowdrop lectin (GNA) Bt αAI (α-amylase inhibitor) from bean Bt cryl gene of Bacillus thuringiensis Corn cysteine gene Bt cryII Bt cryI

Manduca sexta Manduca Sexta Heliothis armigera Spodoptera litura (Boll weevil) Spodoptera litura Heliothis armigera Leptinotarsa decenlineata (Colorado potato beetle) Lacanobia oleracea (Tomato moth) Spodoptera Bruchus beetle, Pea weevil Striped stem borer Sitophilus zeamis (Coleoptera) European corn borer Sugarcane borer (Diatracea sachharis)

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TABLE 13.7 A List of Genetically Modified Crop Plants Exhibiting Insect Resistance Crop Plants Alfalfa Cabbage Cotton Egg plant Maize/corn Potato Soybean Sugarcane Sweet potato Tobacco Tomato Rice

Traits Showing Resistance Insect resistance Insect resistance Resistance to bollworms and budworms (Bt toxin) Insect resistance Resistance to corn borer (Bt toxin) Resistance to Colorado potato beetle (Bt toxin); resistance to potato tuber moths Insect resistance Insect resistance Insect resistance Insect resistance Insect resistance Insect resistance (Bt toxin)

Source: Adapted from James, C., 1999. ISAAA Briefs 12: viii.

Almost one-third of biotech crops are now grown in developing countries. Bt corn is grown for food by farmers in South Africa and Philippines, whereas Indian and Chinese farmers are growing large amounts of Bt cotton. Bt cotton is grown in China on almost 7 million of its 12 million acres of cotton. According to official records, Indian farmers are growing only 250,000 acres of Bt crops, which is about 1% of the total Indian cotton area of 22 million acres. From 1996 to 2001, herbicide tolerance and insect resistance were the main traits that were introduced to commercially available transgenic crops (Table 13.7).

13.11

THE MAJOR IMPACT OF SUCH TRANSGENIC CROPS

Since the commercialization of the first genetically modified (GM) crop in 1996, farmers all over the world have planted and grown more than 690 million hectares of transgenic crops without the evidence of a single incidence of health or environmental harm (Food and Agricultural Organization of the World Health Organization, 2004; National Academy of Sciences, 2004). The use of Bt cotton has reduced global insecticide use significantly. According to a survey carried out by the U.S. Department of Agriculture, the total volume of insecticides used to control cotton pests reduced to 2.7 million pounds between the years 1995 and 1999 in six U.S. states where this survey was conducted. An analysis of 1999 harvests of Bt and conventional cotton found an average yield increase of 9% from Bt varieties in that year. The impact of using Bt cotton varieties was further analyzed by the economists of Louisiana State University and Auburn University. They found that farmers saved 3.4 million pounds of raw materials and 1.4 million pounds of fuel oil used in the manufacture and distribution of synthetic insecticides, by using Bt cotton varieties. The farmers also saved 2.4 million gallons of fuel, 93 million gallons of water, and 41,000 10-h days of work which had generally been spent in applying pesticide sprays. Similarly, the use of Bt sweet corn has not only reduced insecticide use by 42–84%, but also saved $1–$2 billion worth of corn each year. The use of Bt potato varieties has cut pesticide applications by about half. In the area of weed management, farmers growing glyphosate-tolerant soybeans have experienced cost savings and a reduction in the number of soybean herbicide treatments; however, there has not been any remarkable increase in the yield. Overall, there has been only moderate success © 2010 Taylor and Francis Group, LLC

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in the net reduction in herbicide use following the planting of Roundup Ready soybeans. However, the adoption of these varieties has induced a shift from relatively more harmful herbicides to glyphosate, which is more “environmentally friendly” as compared to others due to its low toxicity and fast degradation. The use of herbicide-tolerant canola varieties in Canada has led to a 29% reduction in total herbicide use. The use of herbicide-tolerant cotton varieties has led to a shift from more toxic herbicides to glyphosate and other less toxic ones, along with a reduction in the overall herbicide usage of between 20% and 50%. A comprehensive analysis of the global socioeconomic and environmental impact of transgenic crops has been the focus of attention of a number of studies in recent years (Carpenter and Gianessi, 2001). The many benefits associated with the use of transgenic crops include the reduced use of pesticides and insecticides (Brookes and Barfoot, 2007), an increase in safety for nontarget species (Marvier et al., 2007; OECD, 2007), a considerable increase in the adoption of reduced/conservation tillage and soil conservation practices (Fawcett and Towry, 2002), an increase in the crop yield (Brookes and Barfoot, 2007), and a reduction in greenhouse gas emissions. The most exhaustive study by Brookes and Barfoot (2007) showed that the global farm income contributed by GM crops increased by about USD6 billion in 2004. A global 6% reduction in the usage of herbicides and insecticides was observed due to the use of herbicide- and insect-resistant GM soybeans, maize, cotton, and canola (Council for Agriculture Science and Technology, 2002). This had a major positive impact on the environment which, when measured in terms of environmental impact quotient, showed a 14% net gain. Less frequent applications of herbicide and insecticide and a reduction in tillage operations also led to savings in greenhouse gas emissions. An estimated reduction of about 1 billion kg carbon dioxide was brought about by the savings on fuel usage. The use of herbicideresistant GM crops helps to reduce the number of tillage operations necessary for seedbed preparation and weeding which not only helps to reduce tractor fuel usage but also prevents soil erosion. Since the introduction of biotech herbicide-tolerant crops, no-till crop acreage has increased nearly 40%. Two-thirds of soybean growers who have reduced their tillage since 1996 reported the use of herbicide-tolerant crops as the main factor for less need for tillage. This in turn leaves more carbon in the soil, leading to a lower emission of greenhouse gases. The use of high-yielding GM crops, herbicide-, and insecticide-resistant crops has also helped to save millions of acres of wildlife habitat from conversion to agricultural use. In some developing countries, pesticides are sprayed on the crops by hand (Discussion paper, 2003). In China, about 400–500 farmers die every year due to pesticide poisoning. Chinese farmers adopted the planting of Bt cotton varieties in 1997 following which an almost 75% reduction in the quantity of pesticide application was observed. Also, the farmers who planted Bt varieties reported one-sixth as many pesticide poisonings per capita compared to those who planted conventional cotton crops. In Kwa Zulu-Natal province, South Africa, a high rate of esophageal cancer was reported due to the presence of a fungal toxin called Fumonisin. Fumonisin contaminated the corn supply due to inadequate pest protection and environmental conditions. Fumonisin was later found also to be a cause of spina bifida and other birth defects due to its property of blocking the uptake of folic acid. In 2003, 90% of the farmers in the Makhathini Flats were persuaded to grow Bt white corn which not only helped them increase their productivity to 10–80% but also lowered fumonisin levels. This change improved the health of their children and helped the farmers to earn more and have more family time. Hence, the impact made by the use of herbicide-tolerant and insect-resistant crops is quite clear and evident (Table 13.8).

13.12 RISK ASSESSMENT There has been some concern and criticism regarding the safety of using the GM crops (McHughen, 2000; International Life Sciences Institute, 2004). There is still very serious speculation by environmentalists as well as the general public regarding the impact of GM crops (Miller, 1997;

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TABLE 13.8 A List of GM Crops Used Commercially with Insect Resistance and Herbicide Resistance Crop Plant Cotton

Genetically Altered Trait Insect resistance Glyphosate resistance

Corn

Bromoxynil resistance Insect resistance

Glyphosate resistance Glufosinate resistance Insect resistance Soybean Potato Oilseed (canola)

Sweet corn Papaya Alfalfa Apples Lettuce Rice Sugar beet

Glyphosate resistance Insect resistance Glyphosate resistance Glufosinate resistance Herbicide resistance Insect resistance Virus resistance Herbicide resistance Insect resistance Herbicide resistance Herbicide resistance Herbicide resistance

Commercial Name Bollgard® insect-protected cotton Bollgard II® insect-protected cotton Roundup Ready® Flex cotton LibertyLink® cotton BXN Herculex™ I Roundup Ready® corn NK Knockout™ corn NK YieldGard™ hybridcorn Roundup Ready LibertyLink Second-generation YieldGard® cornborer Roundup Ready® soybean Newleaf Roundup Ready Innovator LibertyLink® canola Rogers® brand Rainbow and Sunup Roundup Ready®Alfalfa Bt insect-protected apple Roundup Ready® lettuce LibertyLink® rice Roundup Ready® sugar beet

New Genetics, Food & Agriculture, 2003). In 2000, due to public pressure, McDonalds and the Burger King chain of restaurants stopped the use of GM potatoes for their French fries. Similarly, the report that pollen from Bt corn could kill Monarch butterfly caterpillars created a stir in the scientific community, environmentalists, and the public at large (Losey et al., 1999). This controversy was laid to rest by follow-up studies which clearly showed that while Bt corn pollen could kill nontarget insects such as Monarch butterflies, in actual field conditions the spread of pollen is too insignificant to bring about a substantial change. In fact, it was also reported that since the introduction of Bt corn in the United States in 1996, the population of Monarch butterflies has increased. Further support for this observation came from two years of intensive field studies and research carried out by 29 scientists and published in the Proceedings of the National Academy of Sciences, who reported little or no effect of Bt pollen on Monarchs (Hellmich, 2001). However, the scientific community as well as the general public emphasize the need to establish the food, feed, and environmental safety of these products (IFT Expert Report, 2000). Having acknowledged the potential risks associated with genetic modifications of all kinds in different materials, scientists and various regulatory bodies have established a safety assessment framework exclusively for biotechnology-derived crops. These extensive guidelines and regulatory bodies will help to identify any potential risk to consumers and the environment before allowing commercial use of these new biotech products. The data so far collected has proved that crops developed through modern biotechnology do not pose significant risks over and above those associated with

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conventional plant breeding (National Academy of Sciences, 2004). In fact, the formulation of stringent guidelines and a proper framework for the risk assessment associated with GM crops has made them even safer to use than conventional plants and foods (European Commission, 2001). A number of organizations are involved in this comparative risk assessment, identifying similarities and differences between GM food crops and their conventional counterpart with respect to their safety and nutritional quality (Food and Drug Administration, 1992; Food and Agricultural organization of the World Health Organization, 2002; Codex Alimentarius, 2003; Organisation for Economic Co-operation and Development, 2003; European Food Safety Authority, 2004). Continuous monitoring involving phenotypic, agronomic, morphological, and compositional analyses helps to identify potential harmful effects and assesses product safety (Prakash, 2001).

13.13

FUTURE PLANNING

The first generation of biotech crops was developed with single gene traits of herbicide tolerance and insect resistance by the expression of a given bacterial gene in the crops (Gutterson and Zhang, 2004). In the case of herbicide tolerance, expression of a glyphosate resistant form of the gene CP4EPSPS resulted in plants being tolerant to glyphosate (Padgette et al., 1995). The expression of an insecticidal protein from Bacillus thuringiensis in plants resulted in the protection of plants from damage due to insect feeding (Perlak et al., 1991). Now products are available in the market that induce both herbicide tolerance and insect resistance in the same plant. Farmers are being encouraged to plant crops with “stacked traits” for the management of insects and weeds and “pyramided traits” for the management of insect resistance. This will help them further increase their productivity and profitability. Efforts are being made to develop the next generation of biotech crops which will meet global agricultural challenges much more efficiently, along with providing benefits to farmers as well as consumers. The development of these new second-generation transgenic products will involve the regulation of endogenous plant pathways which will improve quantitative traits such as yield, efficient nitrogen use, and improved tolerance to abiotic stresses. Due to the multigenic nature of these quantitative traits, the mechanism of action underlying these phenotypes will be far more complex. Keeping in mind the consumer benefits, traits like healthier oils and better and enhanced nutritional content will be developed and marketed commercially. Potatoes are the staple food for many populations in developing countries, especially in Asian and African regions. Potato blight is a fungus that destroys potato crops and has been the major cause of potato famine. Rwanda has tripled its potato production since the 1990s, growing 250 pounds per person annually, yet their potato crops remain susceptible to the blight contamination which causes wide-scale hunger and famine. Every year millions of pounds of fungicides are sprayed on the potatoes to protect the crop. The organic farmers spray copper sulfate as fungicide, whereas nonorganic farmers try to use relatively safe and environmentally friendly fungicides to prevent the spread of disease. If one could combine a blight-proof trait with insect and virus resistance, it would be possible to cut global fungicide and insecticide use by tens of millions of pounds per year. In recent years, a collaborative effort between the scientists of Wisconsin, California, and the Netherlands has resulted in the creation of the first “blight-proof” potato. Genetic engineering helped to identify the blight-resistance genes which were inserted into modern, edible, and highyielding potatoes (Wu et al., 1995). These potatoes were found to be resistant to all kinds of blight infections and will be ready for farmers’ fields in the near future. Salt-tolerant tomatoes and canola have been developed by Blumwald and Zhang (2001) by inserting more copies of natural tomato salt pup genes into the genome. These tomatoes were found to grow in nearly 40% seawater. According to Kinney (2006), the use of network maps and metabolic engineering to improve nutrient profiles is going to pave the way for third-generation biotechnology crops and by the second quarter of the century one may expect them to be commercially available.

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CONCLUSION

There is no doubt that agricultural biotechnology is going to play a significant role in the future of the world’s food supply along with traditional agricultural methods and practices. Increased crop yields, greater flexibility in growing environments, and a reduction in the dependency of crop plants on fertilizers, chemical pesticides, and herbicides are going to be the main areas of thrust in the field of agricultural biotechnology in the near future. The development of second- and third-generation biotechnological crops is going to have an immense impact on world food requirement and supply with a minimum negative impact on the environment. However, no one can deny the importance of continuous risk assessment and intensive monitoring at all levels in order to ensure that the environment and consumers are protected against any unforeseen dangers and risks associated with this field. These include increased weed population due to cross-pollination, the development of resistance to Bt in insect populations, and other long-term health concerns provoked by the consumption of biotech products as compared to products obtained by conventional agriculture. However, the data available to date lay to rest the unusually high levels of alarm raised by some environmentalists and other nongovernmental organizations regarding the safety and environmental impact of such products. Stringent laws and legislations are being put in place in order to exercise maximum precautions before releasing these genetically altered plants into a particular environment. The continuous monitoring of the effects of new crops on the environment and the implementation of effective risk management approaches should be an essential component of further research.

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Hellmich, R.L., Siegfried, B.D., Sears, M.K., Stanley-Horn, D.E., Daniels, M.J., Mattila, H.R., Spencer, T., Bidne, K.G., and Lewis, L.C. 2001. Monarch larvae sensitivity to Bacillus thuringiensis-purified proteins and pollen. PNAS. 98: 11925–11930. Hill, S., Kennedy, C., Kavanagh, E., Goldberg, R.B., and Hanau, R., 1981. Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in K. pneumoniae. Nature 290(5805): 424–426. Holt, D., 1991. The impact of biotechnology on the fertilizer and chemical industry. Illinois Fertilizer Conference Proceedings, January 28–30. Institute of Food Technologists, 2000. IFT Expert Report on Biotechnology and Foods (prepared by the Institute of Food Technologists). Chicago, IL: Institute of Food Technologists. Institute of Food Technologists, 2000. IFT Expert Report on Biotechnology and Foods. Chicago, IL: Institute of Food Technologists. International Life Sciences Institute, 2004. Nutritional and safety assessments of foods and feeds nutritionally improved through biotechnology (prepared by a task force of the ILSI International Food Biotechnology Committee). Compr. Rev. Food Sci. Food Saf. 3: 35–104. James, C., 1999. Global review of commercialized transgenic crops. ISAAA Briefs 12: viii. James, C., 2004. Preview: Global Status of Commercialized Biotech/GM Crops: 2007. ISAAA Briefs No. 32. Ithaca, NY: ISAAA. James, C., 2007. Global Status of Commercialized Biotech/GM Crops: 2007. ISAAA Brief No. 37. Ithaca, NY: International Service for the Acquisition of Agri-biotech Applications. Kinney, A.J., 2006. Metabolic engineering in plants for human health and nutrition. Curr. Opin. Biotechnol. 17: 130–138. Losey, J.K., Rayor, L.S., and Carter, M.E., 1999. Transgenic pollen harms monarch larvae. Nature (Lond.) 399: 214. MacNeil, D., Zhu, J., and Brill, W.J., 1981. Regulation of nitrogen fixation in Klebsiella pneumoniae: Isolation and characterization of strains with nif-lac fusions. J. Bacteriol. 145(1): 348–357. Martinez, M., Colombo, M.V., Palacios, J.M., Imperial, J., and Ruiz-Argueso, T., 2008. Novel arrangement of enhancer sequences for NifA-dependent activation of the hydrogenase gene promoter in Rhizobium leguminosarum by viciae. J. Bacteriol. 190: 3185–3191. Marvier, M., McCreedy, C., Regetz, J., and Kareiva, P., 2007. A meta-analysis of effects of Bt cotton and maize on non-target invertebrates. Science 316: 1475–1477. McHughen, A., 2000. Pandora’s Picnic Basket: The Potential and Hazards of Genetically Modified Foods. New York: Oxford University Press. Miller, H.I., 1997. Policy Controversy in Biotechnology: An Insider’s View. Austin, TX: R.G. Landes Company. Mujeeb-Kazi, A. and Sitch, L.A., 1989. Review of advances in plant biotechnology, 1985–88. 2nd International Symposium on Genetic Manipulation in Crops, CIMMYT and IRRI, Mexico, DF Mexico, and Manila, Phillippines, p. 328. National Academy of Sciences, 2004. Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects. Washington, DC: The National Academies Press. National Research Council, 1989. Field Testing Genetically Modified Organisms: Framework for Decisions. Washington, DC. The National Academy Press. New Genetics, Food & Agriculture, 2003. Scientific discoveries–societal dilemmas. The International Council for Science. Organisation for Economic Co-operation and Development, 2003. Considerations for the Safety Assessment of Animal Feedstuffs Derived from Genetically Modified Plants. ENV/JM/MONO. 2003.11. Organisation for Economic Co-operation and Development, Paris. Organisation for Economic Co-operation and Development, 2007. Consensus document on safety information on transgenic plants expressing Bacillus thuringiensis-derived insect control proteins. OECD Environment, Health and Safety Publications Series on Harmonization of Regulatory Oversight in Biotechnology No. 42. Environment Directorate. Organisation for Economic Co-operation and Development, Paris. Padgette, S.R., Kolacz, K.H., Delannay, X., Re, D.B., LaValee, B.J., Tinius, C.N., Rhodes, W.K., Otero, Y.I., Barry, G.F., Eichholtz, D.A., et al., 1995. Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci., 35: 1451–1461. Panda, N. and Khush, G.S., 1995 Host plant resistance to insects. IRRI, CAB International, p. 431. Pedraza, R.O., 2008. Recent advances in nitrogen-fixing acetic acid bacteria. Int. J. Food Microbiol. 125(1): 25–35.

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Perlak, F.J., Fuchs, R.L., Dean, D.A., McPherson, S.L., and Fischhoff, D.A., 1991. Modification of the coding sequence enhances plant expression of insect control protein genes. Proc. Natl. Acad. Sci. USA 88: 3324–3328. Phipps, R.H. and Park, J.R., 2002. Environmental benefits of genetically modified crops: Global and European perspectives on their ability to reduce pesticide use. J. Anim. Feed Sci. 11: 1–18. Prakash, C.S., 2001. The genetically modified crop debate in the context of agricultural evolution. Plant Physiol. 126(1): 8–15. Raven, P., 2008. The environmental challenge: The role of GM crops. In Proceedings of the 53rd Brazilian Congress of Genetics (in press). Sekhon, G.K., Gupta, R.P., Pandher, M.S., and Arora, J.K., 1992. Symbiotic effectiveness of Hup+ Rhizobium, VAM fungi and phosphorus levels in relation to nitrogen fixation and plant growth of Cajanus cajan. Folia Microbiol. 37(3): 210–214. Thompson, C.J., Rao, M.N., Richard, T., Reto, C., Davies, J.E., Lauwereys, M., and Botterman, J. 1987. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J. 6(9): 2519–2523. Tsaftaris, A., 1996. The development of herbicide tolerant transgenic crops. Field Crops Res. 45(1–3): 115–123. Vaeck, N., Reynaerts, A., Hofte, H., Jansens, S., Beukeleer, M.D., Dean, C., Zabeau, M., Montagu, M.C., and Leemans, J., 1987. Transgenic plants protected from insect attack. Nature 328: 33–37. Vasil, V., et al., 1992. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Biotechnology 10: 667–674. Vijayaraghavan, K. and Yun, Y.-S., 2008. Bacterial biosorbents and biosorption. Biotechnology Advances 26(May–June): 266–291. Wu, G., Shott, B.J., Lawrence, E.B., Levine, E.B., Fitzsimmons, K.C., and Shah, D.M., 1995. Disease resistance conferred by expression of a gene encoding H2O2 generating glucose oxidase in transgenic potato plants. Plant Cell 7: 1357–1368.

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Biotechnology 14 Animal Applications and Potential Risks Rama Shanker Verma, Abhilash, Sugapriya M.D., and Chithra R. CONTENTS 14.1 Introduction .......................................................................................................................... 220 14.1.1 The Evolutionary Perspective ................................................................................... 220 14.1.2 The Growth of Animal Cell Culture Techniques ..................................................... 220 14.1.2.1 Aseptic Conditions ..................................................................................... 222 14.1.2.2 Characteristics of Tissue Culture ............................................................... 223 14.1.2.3 Organ Culture ............................................................................................ 223 14.1.2.4 Explant (or Organotypic) Culture .............................................................. 223 14.1.2.5 Applications of Cell Culture ...................................................................... 223 14.1.3 Cell Viability and Cell Toxicity ................................................................................ 223 14.1.3.1 Limitations of Cytotoxicity In Vitro ..........................................................224 14.2 The Technology Involved ..................................................................................................... 225 14.2.1 Cloning ..................................................................................................................... 225 14.2.2 Transgenesis .............................................................................................................. 226 14.2.3 Knockout Technology ............................................................................................... 227 14.3 Transgenic Animals .............................................................................................................. 227 14.3.1 Introduction .............................................................................................................. 227 14.3.2 Transgenic Mice and the Techniques of Transgenesis ............................................. 227 14.3.2.1 Microinjection Method .............................................................................. 228 14.3.2.2 Retroviral Vector Method .......................................................................... 228 14.3.2.3 Embryonic Stem Cell Method ................................................................... 228 14.3.3 Transgenic Cattle (Pig) ............................................................................................. 228 14.3.4 Transgenic Fish ......................................................................................................... 229 14.3.4.1 Transgenic Fish for Human Consumption ................................................. 230 14.3.5 Transgenic Chicken .................................................................................................. 231 14.3.6 Transgenic Animal Bioreactors ................................................................................ 231 14.3.6.1 Recombinant Protein Production in Transgenic Animals ......................... 231 14.4 Applications of Animal Biotechnology ................................................................................ 232 14.4.1 Transgenic Animals for Human Welfare .................................................................. 232 14.4.1.1 Agricultural Applications .......................................................................... 232 14.4.1.2 Medical Applications ................................................................................. 232 14.4.1.3 Industrial Applications............................................................................... 233 14.4.1.4 Animals Used as Models for Human Genetic Diseases ............................ 233 14.4.2 Xenotransplantation .................................................................................................. 233 14.4.2.1 Historical Perspectives ............................................................................... 234 219 © 2010 Taylor and Francis Group, LLC

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14.4.2.2 Potential Animal Organ Donors ................................................................ 235 14.4.2.3 Drawbacks of Using Pigs as Potential Donors........................................... 235 14.4.3 Recombinant Protein Production.............................................................................. 236 14.4.3.1 Uses of Transgenic Animal Models ........................................................... 236 14.4.3.2 The Production of Interferons .................................................................... 236 14.4.3.3 Applications ............................................................................................... 237 14.4.3.4 Production of Erythropoietin ..................................................................... 237 14.4.3.5 Therapeutic Uses of Recombinant EPO .................................................... 237 14.4.4 Vaccine Production ................................................................................................... 238 14.4.4.1 Live or Attenuated Bacteria or Viruses ..................................................... 238 14.4.4.2 Dead or Inactivated Virus .......................................................................... 238 14.4.4.3 Subunit Vaccines ........................................................................................ 238 14.4.4.4 Vaccine Production in Influenza Virus ...................................................... 239 14.4.4.5 Smallpox Vaccine ...................................................................................... 239 14.4.4.6 Rabies Vaccine Production ........................................................................240 14.4.4.7 Equine Herpesvirus Vaccine .....................................................................240 14.4.5 Veterinary Vaccines..................................................................................................240 14.4.5.1 Bacterial Vaccines ..................................................................................... 241 14.4.5.2 Viral Vaccines ............................................................................................ 241 14.4.5.3 Live Vaccines (Complete Life Cycles) ....................................................... 243 14.4.5.4 Killed Vaccines ..........................................................................................244 14.5 Risks and Management......................................................................................................... 245 14.5.1 Potential Risks .......................................................................................................... 245 14.5.1.1 Risks Involved Using Embryonic and Stem Cells in Cloning ................... 245 14.5.1.2 Risks Pertaining to Immunological Disorders .......................................... 245 14.5.1.3 Risks Pertaining to the Susceptibility to Diseases ....................................246 14.6 Conclusion ............................................................................................................................246 Acknowledgments.......................................................................................................................... 247 References ...................................................................................................................................... 247

14.1 INTRODUCTION 14.1.1

THE EVOLUTIONARY PERSPECTIVE

Animal biotechnology is the use of science and engineering to modify living organisms. Its aim is to enhance products by improving farm and domestic animals. Examples of animal biotechnology include creating transgenic animals (animals with one or more genes introduced by human intervention), using gene knockout technology to produce animals with a specific inactivated and/ or overproducing gene for human consumption. Animal biotechnology can also be used to produce nearly identical animals by somatic cell nuclear transfer (SCNT) (or cloning) for superior characteristics and also as living factories. Table 14.1 illustrates key events in the advances in animal biotechnology.

14.1.2 THE GROWTH OF ANIMAL CELL CULTURE TECHNIQUES The history of animal tissue culture goes back to 1880 when Arnold showed that leucocytes could divide outside the body. Later, in 1903, Jolly studied the behavior of animal tissue explants in serum, lymph, or ascitic fluid after being grown ex vivo. The early twentieth century is considered a turning point in cell culture history as it led Harrison in 1907 to culture frog tadpole spinal chord in a lymph drop. Later, in 1913, Carrel developed the methodology for culturing cells free of contamination;

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TABLE 14.1 Key Events in the Development of Animal Biotechnology Date

Event

1665

Robert Hooke discovered the cross-sections of cork “cells” Leeuwenhoek discovered the first protozoa and bacteria, called animalcule Schleiden and Schwann proposed their “cell theory” Rudolf Virchow formulated the theory that all cells are derived from cells Wilhelm Roux cultivated the first cells from chick embryos in salt solution Harrison isolated pieces of frog embryonic tissue known to give rise to nerve fibers animation for years HeLa cells were derived from the cancerous growth of Henrietta Lacks, who died in of cervical cancer Somatic cell transfer technology Fibroblast growth factor, monoclonal antibodies Mcdb-selective serum-free media; matrix interactions Production of recombinant tissue-type plasminogen activator in mammalian cells Industrial-scale culture of transfected cells for the production of biopharmaceuticals Culture of human adult mesenchymal stem cells Tissue engineered cartilage Culture of human embryonic stem cells Induced pluripotent stem cell development

1692 1838–1839 1855 1885 1907 1995 1971 1975 1978 1984 1990 1991 1998 1998 2006–2007

References Alberts et al. (2007) Alberts et al. (2007) Alberts et al. (2007) Alberts et al. (2007) Alberts et al. (2007) Alberts et al. (2007) Alberts et al. (2007) Wilmut et al. (1997) Gospodarowicz et al. (1978b) Gospodarowicz et al. (1978b), Ham and McKeehan (1979) Collen et al. (1984) Butler (1991) Caplan (1991) Aigner et al. (1998) Thomson et al. (1998) Okita et al. (2007)

subsequently, other suitable culture methods using a variety of media and aseptic techniques were developed. Animal cell culture is a technique which involves the in vitro maintenance and propagation of animal cells in a suitable nutrient medium under strict laboratory conditions and in a controlled environment. The media composition of animal cell culture consists of nine essential amino acids— histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. A few cells such as liver cells can make tyrosine from phenylalanine. Liver and kidney cells both have the property of producing glutamine, but others cannot and have to be provided with this ability from an external source. The other important constituent of the medium is vitamins, which cells cannot make at all or cannot make in adequate amounts. Various salts, glucose, serum as well as the noncellular parts of blood and growth factors are also added to make the medium desirable for cell culture. Certain physiological properties should also be taken into consideration for media formulation, such as pH, CO2, O2, temperature, and osmomolality. • pH: The appropriate value is 7.0–7.4 but it may differ according to the type of cells. • Carbon dioxide: This generally exists in the form of carbonic acid and its concentration in the dissolved state depends on the temperature and atmospheric tension. The presence of CO2, HCO3, and pH are interrelated, as an increase in one leads to a decrease in the other. Recently, a new bicarbonate buffer named 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) has been used, which is considered more efficient and suitable; however, it

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has some constraints, such as cost effectiveness and toxicity. In consideration of these, the bicarbonate buffer is still a common buffer for cell culture. • Oxygen: This is the most important component for animal cell culture. Cells require dissolved O2 to be present in the medium but higher concentrations of O2 can be toxic due to the generation of free radicals. Selenium, glutathione, and mercaptoethanol have the tendency or ability to counteract free radicals generated by O2. • Temperature: Temperature has a direct influence on the growth of cells. The optimal temperature for animal cell culture is ±37°C, although it may also depend on the body temperature of the source animal from which the cells are derived. A typical medium may or may not contain serum. The latter is called a serum-free medium. Serum-free media are used to obtain a desired and defined composition in accordance with cell requirements so as to achieve quality control and the proper regulation of differentiation. The major advantage of using serum-free media is that there is less chance of contamination. Media with serum are less frequently used because it is difficult to obtain serum that is totally free of contaminants like bacteria and viruses. Media can also be defined on the basis of their mode of derivation: 1. Natural media are media derived from biological sources and have constituents derived from animals, for example, plasma, serum, lymph, amniotic fluid, and so on. 2. Artificial media are media containing partly defined components specified for a cell requirement; this ensures that the pH and toxicity of the components are maintained (Freshney, 2005). 14.1.2.1 Aseptic Conditions The maintenance of aseptic conditions is a very important factor before starting the cell culture. This includes environmental control, personal hygiene, equipment and media sterilization, and other associated quality control measures (including media, glassware, and instruments, as well as the environment to which the cultures are briefly exposed during transfer procedures). The most basic requirement of successful cell culture is personal hygiene. The human skin harbors naturally occurring bacterial and fungal inhabitants that shed microscopically and ubiquitously. Measures include: a. Workers should take a shower before entering the lab; hair should be clean and properly tied up b. Hands and arms should be cleaned thoroughly with antibacterial soap c. Clean and cuffed laboratory coats and latex gloves should be worn as protection against hazardous biological agents and chemicals d. Gloves should be frequently disinfected with 70% ethanol to complete sterility e. Gloves should be disposed off after use and laboratory coats should be washed within the laboratory facility or sent out for cleaning f. Hands should be washed after removing protective gloves Any items that come into contact with the culture must be sterile. This includes direct contact (e.g., a pipette used to transfer cells) and indirect contact (e.g., flasks used to temporarily hold a sterile reagent before liquating the solution into sterile media). Sterile disposable plastic items such as culture flasks and containers, test tubes, pipettes, and filters are now readily available and are reliable alternatives to the laborious cleaning and sterilization methods that must be adopted for the reuse of glass items in the laboratory. Ideally, all aseptic work should be conducted in a laminar hood and flame sterilization is also used as a direct and localized method of decontamination.

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All these procedures are employed in order to set up a complete sterilized condition. This is likely to eliminate problems such as potential contamination during the transfer of media in culture flasks, test tubes, or bottles in an exposed area and the sterilization of small instruments such as forceps or wire and inoculating loops and needles before and after transfers. 14.1.2.2 Characteristics of Tissue Culture Tissue culture deals with the in vitro cultivation of organs, tissues, and cells. This technique is not only limited to animal cells, but also includes the in vitro cultivation of plant cells. Tissue culture can be subdivided into three major categories: organ culture, explants culture, and cell culture. 14.1.2.3 Organ Culture Organ culture is a three-dimensional culture technique, which retains the histological features of the tissue in vivo and thus maintains its structure and differentiation capacity. This is accomplished by culturing the tissue at the liquid–gas interface on a grid or gel. However, there are a few disadvantages associated with this technique. Each piece of tissue has to be propagated individually, making it a difficult to assess the reproducibility of a response. • The cells of interest may be small in number and thus it is difficult to detect and quantify the cells • Sometimes there is an inadequate supply of oxygen and nutrients throughout the tissue because of the absence of a functioning vascular system, leading to necrosis • The use of stirred cultures or roller bottles for culturing cells could be an alternative method of providing air and soluble nutrients 14.1.2.4 Explant (or Organotypic) Culture In explant culture, tissue is cultured in a rich medium containing serum, which is allowed to attach to an appropriate substrate coated with collagen. Previously, explants were maintained in Maximov chambers, where the cells were grown on cover slips sealed over a depression in a thick glass slide. Although this technique is still used, regular culture dishes are much more convenient for the purpose as they need not be disassembled and reassembled for each feeding. 14.1.2.5 Applications of Cell Culture • Cell culture has been used extensively in the understanding of cell metabolism and the cell cycle • It has been used for drug testing, toxicity, and efficiency on cell lines, as the test drug generally passes through many phases after which it gets approved and marketed, preventing harmful and fatal effects on animal species • It has significance in studying cell-to-cell interaction and intracellular flux • It has simplified industrial production of therapeutically significant biological compounds (hormones and proteins) both in terms of efficiency and speed • Since the cells behave differently in different conditions and different environments, animal cell culture can be exploited to understand the deeper concepts of regenerative medicine

14.1.3 CELL VIABILITY AND CELL TOXICITY In recent advancements, there has been an emphasis on the use of cell culture rather than animal models. This is becoming the preferred way of screening pharmaceutical drugs, because it satisfies ethical questions raised by animal activists as well as the regulatory board. As cells are explanted from a living environment (in vivo) there are fundamental issues with cell viability, especially when

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the cells are subjected to experimental manipulations. Many experiments are currently carried out on cells rather than by using animals as models in order to avoid harmful effects on animals. One such example is the study of the potential cytotoxicity of compounds used for pharmaceutical, cosmetic, and food additive purposes before they become available to the general public. 14.1.3.1 Limitations of Cytotoxicity In Vitro Toxicity is a complex process occurring inside the cells, leading to direct cellular damage, neurotoxicity, and other inflammatory effects. Tissue and systemic responses: The major concern here is with in vivo tissue responses, including fibrosis, kidney failure, inflammatory reaction, or systemic reactions such as pyrexia and vascular dilation. Pharmacokinetics: A number of events take place inside a body when it is subjected to a drug including tissue reactions, clearance, and metabolism. It is not possible for all these parameters to be stimulated in isolated cells although studies related to multicultural tumor spheroids for drug penetration could be done. Metabolism: Body metabolism is complex, causing toxic substances to become nontoxic and nontoxic to become toxic. All these processes are carried out by the liver in vivo, and thus special care should be taken to expose cells to the same type of compounds in vitro (Freshney, 1994). The following section will describe various techniques used in the study or development of the effects and screening of drugs and drug interactions. 14.1.3.1.1 Cell Viability and Cell Cytotoxicity Assay These assays developed for cell viability and cell cytotoxicity are based on membrane integrity, cellular respiration, radioisotope incorporation, calorimetric assays, and luminescence test. 14.1.3.1.1.1 Membrane Integrity-Based Assay These are common viability assays. Here, membrane damage could be caused by cell separation, freezing, and thawing as well as cell disaggregation, and this can be determined with the help of dyes (naphthalene black, erythrosine, and trypan blue). Viable cells are impermeable to these dyes. Neutral red or diacetyl fluorescein can also be used in place of the abovementioned dyes. The other assays used are labeled chromium enzymes and fluorescent probes. These have limitations in that they are not able to predict final survival, although they are faster than using dyes. 14.1.3.1.1.2 Dye Exclusion Assay This is the method of choice for suspension cultures rather than for monolayer cultures, as dead cells tend to detach from them. Dead cells in suspension will take up dyes such as naphthalene black and trypan blue, leaving viable cells unstained. However, a limitation of using this method is that sometimes reproductively dead cells remain unstained, giving them a viable appearance when viewed under microscope. 14.1.3.1.1.3 Dye Uptake Assay In this method the dye used is diacetyl fluorescein, which is taken up by the viable cells rendering it impermeable to the membrane. Live cells therefore emit fluorescent green light which is not the case with dead cells. 14.1.3.1.1.4 Labeled Chromium Uptake Assay This method uses 51Cr to determine the cytotoxic activity of cells. Labeled chromium is used to bind to the intracellular protein which leaks out through the membrane in cases of cell membrane damage. This can be determined easily since the leakage is proportional to the damage. 14.1.3.1.2 Survival Assays Survival assays measure viability. Survival is defined as the capacity for the development of a cell after cytotoxic agent exposure, its ability to regenerate or its proliferative capacity.

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14.1.3.1.2.1 Clonogenic Assay Clonogenic assay is also known as colony formation assay or cell survival assay and this technique is based on the in vitro growth of a cell into a complete colony of cells (minimum 50 cells). This technique deals with studying cell reproductive death as well as the effectiveness of cytotoxic lethal agents when treated with ionizing radiation. The cells are seeded in appropriate dilutions to form colonies in 1–3 weeks. Usually only a small proportion of the seeded cells have the capability to develop into colonies. These are fi xed with glutraldehyde solution 6.0%v/v, stained with crystal violet 0.5%w/v, and counted using a stereomicroscope. 14.1.3.1.2.2 MTT Assay The [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] MTT assay is also known as the [3-(4-5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfonyl)-2-tetrazolium] MTS assay and is a rapid versatile and quantitative technique, which uses standard colorimetric assays that measure color change. It is used to determine the cytotoxicity of potential medicinal agents and toxic materials. The MTT calorimetric assay determines the ability of viable cells to convert a soluble tetrazolium salt MTT into an insoluble formazan precipitate, giving a purple color. Cells in the growth phase (log phase) are exposed to a cytotoxic drug. The drug is then removed, allowing cells to proliferate (minimum 2 to 3 population doubling time). The surviving cells are then detected indirectly using the MTT dye. The concentration is measured spectrophotometrically. 14.1.3.1.3 Metabolic Assay A metabolic assay employs the metabolic measurements (DNA, RNA, and protein synthesis) and is usually carried out once the cells have been exposed to a cytotoxic drug. However, there are certain limitations because the outcome of metabolic estimation is not always the cell number, since these assays cannot differentiate between the metabolic and proliferative activity of cells. 14.1.3.1.4 Mutagenic Sister Chromatin Assay The sister chromatin method employs the radioactive nucleotides in the replication process of DNA to detect sister chromatin exchange, as these mutagenic sister chromatins (SCEs) are sensitive to mutation and chromosomal breaks. The method employed is fluorescence plus Giemsa technique. 14.1.3.1.5 Inflammation Assay Inflammation assays are used for testing allergic reactions caused by the administration of synthetic compounds both externally and internally, such as pharmaceuticals, cosmetics, and so on. For example, the Draize test is done to test shampoos by administering them to rabbit eyes, and ELISA is used for the measurement of paracrine responses in cases when allergens are administered epidermally.

14.2 THE TECHNOLOGY INVOLVED 14.2.1 CLONING Scientists use reproductive cloning techniques to produce multiple copies of mammals which are nearly identical copies of other animals, including transgenic animals, genetically superior animals, and animals that produce high quantities of milk or have some other desirable trait. To date, cattle, sheep, pigs, goats, horses, mules, cats, rats, and mice have been cloned using cloning technology. The first cloned animal was a sheep named Dolly in 1996 (Gospodarowicz et al., 1978b). Reproductive cloning uses SCNT technology. In SCNT, the nucleus from an egg cell (oocyte) is removed and a nucleus from a donor adult somatic cell is introduced, which may be from any cell except a germ cell. After the transfer of this nucleus, the resultant embryo is implanted into the uterus of a surrogate female and develops into an adult animal (Figure 14.1).

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Biotechnology in Functional Foods and Nutraceuticals The cloning of Dolly Sheep 1 Cells are removed from the udder and cultured

Sheep 2 Eggs are removed

Udder cell

Egg cell

The nucleus of the udder cell is placed next to the egg cell fused with electric current

Nucleus is removed from the egg cell

The egg cell develops into an embryo…

… This is implanted into surrogate mother

FIGURE 14.1

The offspring clone of Sheep 1

A representation of the first animal cloning.

14.2.2 TRANSGENESIS Transgenic technology is a technique which produces considerable alterations in the sequences of nucleotides in DNA segments of the gametes of early embryos. These alterations are mainly substitutions, insertions, deletions, or the rearrangement of nucleotides in DNA segments of these gametes (Boyd and Samid, 1993), leading to mutations. These mutations can lead to the death of cells and few of them can be repaired; other mutations cause genetic variations which result in genetic diseases. The overwhelming majority of these alterations in DNA do not have any appreciable effect in larger animals, particularly farm animals, because the dead cells are not used for such purposes. Most natural mutations occurring in early embryos have a deleterious effect, leading to a failure in fertilization, death of embryos, and congenital abnormalities. However, sometimes these natural mutations are beneficial. The same principle applies to transgenic alterations that have both detrimental and favorable effects (Margawati, 2003). This technology is used with the intention of producing animals carrying desired molecules for sociological needs and animals with traits which will improve the quality of livestock. These procedures involve: 1. The isolation of a gene which codes for a product of potential interest 2. The preparation of a DNA construct that allows the gene to be expressed in the tissue of choice 3. The transfer of the DNA construct in vitro to a recently fertilized 1-cell ovum 4. The reimplantation of the treated ovum into a suitable recipient 5. Analysis of the offspring

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Definition: Transgenesis is a phenomenon in which an exogenous DNA construct is integrated into the chromosome of an animal to maintain a stable inheritable character. As the injected DNA is integrated into the genome of the animal it is expected to be transmitted in the germline to the next generation. In this way significant changes in the characteristics of the animal can be achieved. The first successful experiment using the above-mentioned technique has been performed in mice and in recent years has been extended to other animals. This work is motivated by the need to: • Increase the efficiency of food conversion in domestic animals • Generate animals with improved characteristics • Use animals as production units for compounds of therapeutic interest

14.2.3 KNOCKOUT TECHNOLOGY In addition to the use of transgenics and cloning, researchers can use gene knockout technology to inactivate, or “knock out,” a specific gene. With this technology they can create a possible source of replacement organs or mass of tissues. The replacement process of transplanting cells, tissues, or organs to another species is referred to as xenotransplantation. Currently, the pig is the major animal being considered as a viable organ donor to humans. Unfortunately, pig cells and human cells are not immunologically compatible. Pigs, like almost all mammals, have markers on their cells that enable the human immune system to recognize them as foreign and to reject them. Genetic engineering is used to knock out the pig gene responsible for the protein that forms the marker to the pig cells.

14.3 TRANSGENIC ANIMALS 14.3.1 INTRODUCTION Humans have been dependent on animals such as cow, buffalo, goat, sheep, poultry, pig, and fish for milk, meat, egg, and wool. Thus there have been constant efforts to improve the quality and productivity of the stock. Originally this was important for survival; now it is more for commercial purposes. In the early days, the selective breeding method was adopted for this purpose. This technique mainly involved a combination of mating and the selection of animals with improved genetic traits. Although costly and time-consuming, this method was preferred then due to the nonavailability of other techniques. It took years to obtain a desired trait in animals with a long gestation period. Current economic and consumer demands suggest that quantum changes in the productivity and quality of domestic animals are essential. Methods developed in the early 1980s mean that dramatic changes in an animal phenotype can now be achieved within a single generation.

14.3.2 TRANSGENIC MICE AND THE TECHNIQUES OF TRANSGENESIS The technology that enables stable and transmissible integration of cloned DNA segments into the mammalian genome was first established for the mouse. Mouse is the animal of choice for transgenic experiments because it produces a number of eggs. A mouse normally produces 5–10 eggs, while a super ovulated mouse can produce up to 40 eggs, which is not the case with larger animals. The generation of the super mouse was the combination of many years of research effort in technologies such as: • Microinjection method • Retroviral vector method • Embryonic stem cell method

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14.3.2.1 Microinjection Method At present this is the most effective procedure for the introduction of foreign DNA. It consists of the direct microinjection of several hundred linear DNA molecules into the large (male) pronucleus of a fertilized one-cell egg (Gordon et al., 1980). The successful generation of transgenesis demands that at least one of these become integrated in the genome. Usually, several DNA copies are integrated in a regular head-to-tail fashion but other types of arrangements also occur (Palmiter and Brinster, 1985). The injected eggs are transferred into the oviducts of pseudopregnant foster mothers and are allowed to develop. Some 10–20% of the eggs survive the microinjection and reimplantation procedure. This injected segment is capable of producing tissue-specific expression for a functional protein (E.T.S., 1997). Although proven to be a very efficient technique, it is consuming, costly, and labor intensive. There are also chances that a number of pieces of DNA might get incorporated at a single site and these transgenes may not lead to expression, overexpression or underexpression (E.T.S., 1997) (Figure 14.2). 14.3.2.2 Retroviral Vector Method This technique has generally been used to produce transgenic mice where DNA fragments of small size (8 kb) could effectively be transferred. However, a drawback to this technique lies in retroviral contamination. This can interfere in the signal that determines expression of the inserted gene. There is also a risk of losing regulatory sequences. It is therefore not a commonly used method (FELASA, 1982). 14.3.2.3 Embryonic Stem Cell Method In the embryonic stem cell method, cells at the blastocyst stage are proliferated in a cell culture and have the capability to differentiate into different type of cells when transferred into a host blastocyst embryo. Here the foreign DNA is inserted into the embryonic stem cells by the electroporation or microinjection method as mentioned earlier, and the desired segment is identified with the help of Pcr and marker genes (Gossler et al., 1986). Next, the gene construct of interest is fused to blastula cells and the modified blastula is implanted into the host mother. The offspring obtained from this are mated with normal animals to obtain transgenic species. The advantage of this technique is that the DNA of interest can be inserted to the exact locus. However, the first offspring produced from such animals are chimeras (Molecular Therapeutics, 2008). Thus, to obtain true transgenic animals we have to wait until the second generation. The reproductive tissue obtained from first-generation chimeras is derived from blastula rather than stem cells (Figure 14.3).

14.3.3 TRANSGENIC CATTLE (PIG) Hemoglobin, the oxygen carrying protein of red blood cells (RBC), is used as a substitute in experiments on blood transfusion. This is due to the fact that hemoglobin can be stored for a few months, whereas whole blood can only be stored for a week. There is also no risk of contamination such as HIV in the case of RBC. Thus, transgenic pigs that produce human hemoglobin have been developed. Pigs can be used as potential human organ donors (Lee et al., 1997). Transgenic pigs with omega-3-fatty acid rather than the omega-6-fatty acid commonly found in meat could potentially cause pork sausages to become a healthy food choice, effective in reducing the occurrence of heart disease, cancer, and diabetes. However, there are certain limitations: • The free hemoglobin is naked so it cannot transport oxygen effectively • It is easily degraded and the breakdown product may cause kidney damage • There is always a risk of contamination by pig virus

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229 The steps in the production of transgenic mice

Eggs harvested after super ovulation

Newly fertilized egg “New” DNA inserted by microinjection

Several embryos are implanted into a pseudopregnant foster mother

Offsprings

DNA from tissue samples is analyzed for presence of new DNA

Mice with required expression of gene selected

FIGURE 14.2 A schematic representation of the steps used in the production of transgenic animals.

Transgenic hemoglobin can cause allergic reactions and is thus not ideal for use in major surgery, but it can be used in minor surgery.

14.3.4 TRANSGENIC FISH Transgenic fish production offers many potential economic advantages for commercial aquaculture. The major advantage of this technique is the introduction of novel genes. Several transgenic fish (catfish, salmon, trout, flounders, and oysters) have been developed using this technique. These animals are now being engineered for traits that make them better suited for aquaculture such as faster growth, disease resistance, and temperature tolerance. The genes engineered into these fish come from a variety of organisms including other fish, coral, mice, and humans. One of the most

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Donor blastocyst ES cells Transgene

Transfection

ES cells with transgene

Microinjection into blastocyst

Selection of ES cells with transgene

Implant Foster mother

Transgenic mice

Embryonic stem cell method for producing transgenic mice

FIGURE 14.3

A schematic representation of the production of transgenic mice using stem cells.

interesting areas in transgenic fish production is the introduction of growth hormones. It is generally observed that the growth rate of many fish species normally used for aquaculture is slow but currently this is being enhanced by creating a transgenic fish by incorporating a gene construct encoding growth hormone (Rahman et al., 2005). By doing so there is a 3- to 11-fold gain in weight. These techniques have been specifically studied in wild salmonid species, relative to the growth achieved in domesticated strains (Devlin et al., 2001). It has been observed that the growth response is strongly influenced by the intrinsic growth rate and genetic background of the host strain. Intrinsic growth rate hormone transgenes engineered into highly domesticated fish will not necessarily lead to further growth enhancement (Devlin et al., 2001). It was observed in Oncorhyncus mykins eggs when a very slow growing wild strain was microinjected with rainbow trout in salmon gene construct overexpressing growth hormone (construct on MTGH1). Like Coho salmon the transgenic trout produced a great difference in weight by 14 months postfertilization. The methodologies used for transgenic fish production are same as transgenic mice production. Transgenic fish were first produced by this methodology in 1990 (Chen and Powers, 1990). 14.3.4.1 Transgenic Fish for Human Consumption A few studies have shown that the use of transgenic fish as food has led to negative health effects, because the transgenic fish contain new proteins that could induce severe allergic reactions. The new proteins could also interrupt or amplify other functions in the fish, potentially making the fish

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less nutritious and even toxic. Such unforeseen consequences of gene insertion are called secondary effects. The Food and Drug Administration (FDA) recognizes that a transgene cannot be turned off once it is inserted into an organism and this may lead to uncontrolled expression. Depending on where the transgenes are inserted, they can also effect the expression of other genes. Further, a foreign growth hormone genetically inserted into a fish variety such as salmon may increase the production of other compounds such as insulin in the fish. Besides actually engineering genes for growth hormones to be injected into fish, scientists are also experimenting with injecting Tilapia cichlid (fish) with a genetically engineered growth hormone (rBGH) derived using cow genes. rBGH is currently used in dairy cows in the United States to increase milk production, but its use has been criticized and banned by many other countries due to concerns that the hormone has a detrimental effect on cows and causes milk to have increased levels of IGF-1, a substance linked to cancer in humans.

14.3.5 TRANSGENIC CHICKEN The production of transgenic chicken is a very complicated process. The reason behind this is that, while one sperm fertilizes one ovum in higher animals, in the case of chickens several sperm fertilize one ovum, making the identification of the fertilizing male pronuclei difficult. There is also no evidence so far to suggest that embryonic stem cells have been found in chickens. Despite these difficulties, transgenic chicken lines have been established and offer a number of advantages over both plant and animal systems as listed below: • • • •

The purification of protein is comparatively simple Good manufacturing practices have been established for vaccine production Antibodies produced have enhanced cell killing properties Eggs having more protein and less fat have been developed

Transgenic chicken production: Blastoderm cells are removed and transfected. Now the transfected blastoderm cells are reintroduced into the subgermplasm space of the irradiated blastoderm of freshly laid eggs. Thus the transgenic lines of chicken are well established.

14.3.6 TRANSGENIC ANIMAL BIOREACTORS 14.3.6.1 Recombinant Protein Production in Transgenic Animals Dr David Bullock’s laboratory was the first to develop transgenic mice at Baylor College of Medicine using β-casein driven constructs. The sequences required to target gene expression were localized to approximately 1 kb of the promoter and the first exon and intron of the gene. Matrix attachment regions (MARs) were shown to participate in the insulation of the transcription elements from the surrounding chromatin in tissue culture cells and transgenic mice (McKnight et al., 1996). This study concluded that mammary gland specific gene expression requires operative interaction between several factors and is not controlled by a single transcription factor. The main focus of these studies was: • To design a construct that led to efficient expression of foreign proteins in the mammary gland • To develop the proper post-translational processing of these proteins • To develop an effect on lactation and transgene overexpression in the mammary gland In recent years a number of genes encoding medically important proteins have been isolated and characterized. These include insulin, plasminogen activator-1 antitrypsin, and coagulation factors viii and ix. The purification of these proteins from blood and tissue is an expensive and

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time-consuming process which carries the risk of transmitting infectious diseases such as AIDS. The emergence of genetic engineering techniques has stimulated attempts to prepare such human proteins from recombinant microorganisms; however, the expression of genes encoding human blood proteins in microbial hosts has often proved unsatisfactory because the foreign proteins were not correctly processed. The expression of cloned genes in mammalian tissue culture has proved a viable strategy, although batch fermentation of animal cells is both expensive and technically demanding. Techniques have recently been developed for introducing cloned DNA segments into the germ lines of higher animals where they may be expressed and propagated as new Mendelian loci. A combination of large daily protein output, excellent post-translational processing abilities, ease of access to the recombinant protein, and low production costs when compared to industrial high-volume fermentors has made the transgenic mammary gland an excellent candidate for the production of recombinant protein.

14.4

APPLICATIONS OF ANIMAL BIOTECHNOLOGY

14.4.1 TRANSGENIC ANIMALS FOR HUMAN WELFARE The benefits of these animals for human welfare are divided into various areas: • Agriculture • Medicine • Industry 14.4.1.1 Agricultural Applications These can be further classified as a. Breeding: Selective breeding is a year-round process aiming to achieve a desired trait for increased milk production and growth rate, and is time-consuming and difficult compared to traditional breeding methods. Using developments in molecular biology, animals are being developed in shorter times and with more precision. This has made it easier to increase the yield (Moore and Mepham, 1995). b. Quality: With the advent of transgenic technology, transgenic cows have been developed that produce more milk which contains less lactose or cholesterol 12; cattle with more meat and wool have also been developed adopting this technology. c. Disease resistance: Although this is a recent development, attempts have been made to develop influenza-resistant pigs. However, the number of genes in farm animals for disease resistance is limited. 14.4.1.2 Medical Applications a. Xenotransplantation: There are a huge number of people dying every year due to lack of replacement organs, particularly in cases of heart, liver, and kidney diseases. Transgenic pigs have the potential to alleviate this shortfall but currently this is hampered by a pig protein (α-1,3-galactosyl-transferase), which is not found in primates. b. Pharmaceuticals: Animal commodities such as insulin, blood anticlotting factors, and growth hormone have been obtained from transgenic cows, sheep, and goats (Chemical and Engineering News, 2009). Currently, much research has focused on developing milk from these cattle for the treatment of diseases such as phenylketonuria, hereditary emphysema, and cystic fibrosis.

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• In 1997 the first transgenic cow developed was named “Hamed Rosie.” The milk produced from this cow was protein rich, containing the human gene α-lactalbumin. c. Human gene therapy: The potential here for treating the 5000 named genetic diseases is huge and transgenic animals play a major role. Human gene therapy involves the addition of a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the same gene. • The A.I Vietanen Institute in Finland has produced a calf with a gene that has the ability to produce a substance that promotes the growth of red cells in humans. 14.4.1.3 Industrial Applications There are a number of industrial applications. These include: • In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats began to manufacture silk along with their milk and secreted tiny silk strands from their bodies. These strands were used as thread for weaving purposes. • Toxicity-sensitive transgenic animals have been produced for chemical safety testing. • Microorganisms have been engineered to produce a wide variety of proteins that synthesize enzymes to enhance chemical reactions (FELASA, 1982). 14.4.1.4 Animals Used as Models for Human Genetic Diseases Genetically modified mice have proved to be excellent models for the study of specific human genetic diseases and the development of possible therapies (Wagner et al., 1995). For example, trials conducted on mouse models for Huntington’s disease have identified a number of potential therapies that are suitable subjects for clinical trials (Sathasivam et al., 1999). Transgenic mouse models for Alzheimer’s disease have been developed where efforts have been made for therapeutic intervention. However, there are certain limitations in using mouse as a model for human diseases, because murine physiology, anatomy, and lifespan differ significantly from those of humans, rendering it an inappropriate model (Miller et al., 1996). Therefore it is more advantageous to produce animal models in suitable livestock species because the physiology of some species is very similar to that of humans. Thus modified sheep with mutations in the gene for cystic fibrosis have been developed as a successful model because there is enough evidence to show close anatomical, functional, and electrophysiological similarities between a sheep lung and human lung. A further advantage of a sheep model in comparison to mice is their longer lifespan which enables the assessment of longer term therapeutic procedures. The rabbit is also a promising model for cystic fibrosis, atherosclerosis, and cardiovascular diseases, since it is phylogenetically closer to primates than rodents and it is a larger animal to facilitates the monitoring of physiological changes. A pig model for the rare human eye disease retinitis pigmentosa has been developed by the expression of a mutated rhodopsin gene in transgenic pigs, providing a large animal model with similar physiology. The pig eye is similar in size to the human eye enabling the evaluation of applicable surgical procedures.

14.4.2 XENOTRANSPLANTATION Xenotransplantation of animals to humans is defined as the transplantation of living cells, tissues, organs, and human body fluids that constitute an alternative to material of human origin. This technique bridges the shortfall in human material needed for transplantation. This concept was pioneered a century ago, when transplanting organs was considered ethically controversial and the grafts were rejected because of unknown forces later identified to be immune responses (Figure 14.4).

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Diagram: Type I diabetes treatment from insulin producing cells derived from pig’s pancreas

Animal cell therapy

Insulin producing cells from pancreas Transplanting to recipient’s body

Insulin derived from pig cells

Diagram: The pig liver cells passed through external device for the treatment of liver failure

Animal external therapy

Liver Purified patient’s blood Bilized patient waiting for liver donation Cells passed through external chambers Diagram: Human failed kidney replaced by pig’s kidney

Animal organ transplant

Kidney Kidney replacement

Patient with restored kidney function

FIGURE 14.4 A diagram illustrating three different types of animal-to-human transplantation. The first shows animal cell therapy, where insulin-producing cells from the pancreas of a pig are transplanted into a human, so that the human can produce his own insulin. The second type is animal external therapy, where cells from a pig’s liver are transferred into an external device, used to clean the blood of a person who has experienced liver failure, and who is waiting for a transplant. The third type is animal organ transplantation, where a whole organ (e.g., a kidney) is transplanted into a human to replace a failed kidney. The patient thus experiences restored kidney function.

14.4.2.1 Historical Perspectives Alexis Carrel is the father of experimental organ transplantation because of his pioneering work using vascular techniques. Carrel and Guthrie’s work contributed a lot to the science of transplantation from 1904 to 1906 (Wrighton et al., 1995). The main work performed by them is as follows: 1. 2. 3. 4.

Performed autogenous vein grafts Performed leg transplantation in dogs Developed the famous patch graft technique for widening the narrowed vessels Performed heterotrophic experimental transplantation, for example, parts of a small dog were transplanted into the neck of a larger dog 5. Developed a buttonhole technique for the anastamosis of donor and a recipient vessels in kidney transplantation to prevent thrombus formation

In 1906 Jaboulay transplanted kidneys from goats, sheep, and monkeys into humans; this attempt failed. Later in 1910 Urger transplanted a nonhuman kidney into a man dying of renal failure (Michon et al., 1953). The man died after one day, resulting in a failure of the technique. In 1932 Neuhof transplanted a lamb kidney into a patient with mercury poisoning who survived for nine days. In 1950 Demikhov transplanted a heterotopic heart and lung; the animal survived for 9 h (Demikhov, 1950). No similar significant outcomes of other experiments were observed until the 1950s. Organ transplant failure was attributed to powerful unknown forces which were eventually to be identified as the body’s immune system. The first successful transplant was that of a kidney from identical twins. Michon and Hamburger successfully performed donor kidney transplantation in Paris in

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1952. In 1956 Harrison et al. performed the first kidney transplantation between monozygotic twins using no immunosuppression (Harrison et al., 1956). At that time an understanding of transplant immunobiology and immunosuppressive drugs had just started to develop. Starzl and colleagues were trying xenotransplantation using chimpanzees or baboons. The momentum of xenotransplantation accelerated in the 1990s with the discovery of porcine retrovirus. Concerns about the risk of cross-species infections resulted in clinical trials. 14.4.2.2 Potential Animal Organ Donors Since primates are the closest relatives to humans, nonhuman primates were the first to be considered as a potential organ source for xenotransplantation into humans. Chimpanzees were originally thought to be the best option since their organs are of similar size and they have blood type compatibility with humans. However, since chimpanzees are listed as an endangered species, other potential donors were sought. Although baboons are more readily available, they are not practical as potential donors due to their smaller body size, lower frequency of blood group O (the universal donor), long gestation period, and typically low number of offspring. In addition, a major problem with the use of human primates is the increased risk of disease transmission, since they are so closely related to humans. Xenotransplantation between baboons and humans raises the issues of xenozoonoses (Nishitai et al., 2005). The organisms of greatest concern are the herpes virus and retroviruses which can be screened for and eliminated from the donor pool (Ashton-Chess et al., 2003). Others include Toxoplasma gondii, Mycobacterium tuberculosis, and the encephalomyocarditis virus. Pigs are currently thought to be the best animals for organ donation (Figure 14.4). The risk of cross-species disease transmission is decreased because of their increased phylogenetic distance from humans (Dooldeniya and Warrens, 2003). They are readily available; their organs are anatomically comparable in size. Also, new infectious agents are less likely since they have been in close contact with humans through domestication for many years (Lawson Handley et al., 2007). Current experiments in xenotransplantation most often use pigs as the donor and baboons as human models (Cozzi and White, 1995). 14.4.2.3 Drawbacks of Using Pigs as Potential Donors It has been discovered that the rejection of pig organs by primates is due to the presence of a special group of disaccharides (Gal-α1,3-Gal) in pigs, which is absent in primates. This codes for a 1,3-galactotransferase, present in pigs and not in primates (Miyagawa et al., 2005). Scientists are optimistic that knockout pigs lacking the gene encoding the enzyme a 1,3-galactosyl-transferase can be developed in the next few years. Another approach is to introduce genes in primates that can degrade or modify Gal-α1,3-Gal disaccharide groups, leading to reduced immunogenicity. Besides the above there are other strategies to avoid hyperactive organ rejection by the hosts in xenotransplantation (Fishman and Patience, 2004): 1. The expression of antibodies against pig disaccharides 2. The expression of compliment-inactivating protein on cell surfaces The above approaches may overcome the immediate hyperactive rejection of organs. The next problem is the delayed rejection which involves the macrophages and natural killer cells of the host. Another concern of xenotransplantation is that the endogenous pig retrovirus could become activated after organ transplantation. This may lead to new genetic changes with unknown consequences. This strategy is still limited to laboratory experiments (Bach et al., 1998). A lot of effort is needed to make it a reality. There is much debate concerning the ethics of xenotransplantation and the majority of it is negative (Correa and Sgreccia, 2001).

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14.4.3 RECOMBINANT PROTEIN PRODUCTION 14.4.3.1 Uses of Transgenic Animal Models 14.4.3.1.1 Pharmaceutical Products Banting first discovered the hormone insulin in 1921. At that time, abattoir animals were the source of human insulin. This method was the primary solution for type 1 diabetes mellitus. Although bovine and porcine insulin are similar to human insulin, their composition differs in one amino acid. Alanine is present in place of threonine at the C terminal end and B terminal chain of human insulin. As a consequence the immune system produces antibodies against it, neutralizing its actions and resulting in inflammatory responses at injection sites. Added to this, complications ensuing from regular injection of foreign substance have led to a decline in the production of animal derived insulin (http://www.gene.com/gene/news/press-releases/display. do?method=detail&id=4160). Attempts to produce insulin by recombinant DNA technology only started in the late 1970s. The basic technique involved the insertion of human insulin gene and the promoter gene of lac operon onto the plasmids of Escherichia coli. Human insulin is the only animal protein which has been made in bacteria in such a way that its structure is absolutely the same as that of the natural molecule. This reduces any complications of antibody production. However, one problem has been the contamination of the final product by the host cells, resulting in the contamination of the fermentation broth. A purification process has been used to remove the contamination. Today, this procedure is performed by using yeast cells as the growth medium since they secrete an almost complete human insulin molecule, a perfect three-dimensional structure. This minimizes the need for complex and costly purification procedures (Walsh, 2005). 14.4.3.1.2 Production of Recombinant Insulin (Humulin) The first step involves the chemical synthesis of DNA chains that carry the specific nucleotide sequences characterizing the A and B polypeptide chains of insulin. The required DNA sequence is known because the amino acid compositions of both chains have been mapped. Sixty-three and 90 nucleotides are required for synthesizing the A and B chains, respectively. A codon at the end of each chain terminates the protein synthesis by a signaling process. An anticodon incorporating the amino acid methionine is then replaced at the beginning of each chain to remove the insulin protein from the amino acids of the bacterial cells. The synthetic genes of A and B chains are then separately inserted into the gene of the bacterial enzyme β-galactosidase, which is carried in the vectors plasmid. At this stage it is necessary to ensure that the codons of the synthetic gene are compatible with those of the β-galactosidase. 14.4.3.1.3 Interferons Alick Isaacs and Jean Lindemann discovered interferon in 1957. It is an antiviral substance which interferes with the viral replication process. Interferons are proteinaceous substances of molecular weight 20,000–30,000. There are three types, categorized as follows: • Interferon-α • Interferon-β • Interferon-γ 14.4.3.2 The Production of Interferons Mammalian cells produce interferons when they are infected by viruses. The nucleic acid of viral origin stimulates the host DNA to produce proteinaceous substances called interferons. These interferons have a role in inhibiting the replication process of viruses. The replication process differs from species to species. It is a vector-mediated process which requires cDNA synthesized from mRNA of a specific interferon. The cDNA is then inserted into a vector. Earlier, E. coli was used,

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but now it is replaced by yeast Saccharomyces cerevisiae. This is because yeast has the property of glycosylation as in mammalian cells whereas a bacterium does not. Attempts are in progress to make hybrid interferons having a range of antiviral activities, where the researchers are trying to bypass the glycosylation step which would enable the use of bacteria as the vectors. According to the latest research, hybrid interferon tan α has been produced in which a fusion polypeptide comprising an N-terminal segment is directly joined to a C-terminal segment. And the N-terminal segment corresponding to amino acid residue 1 to about residue 28 of a mature IFN-γ and said C-terminal segment corresponding to residue 29 of the C-terminal residue of mature IFN-α. 14.4.3.3 Applications Interferon-α, -β, and -γ are used for the treatment of a number of diseases including cancers, myelomas, AIDS, multiple sclerosis, herpes zoster, hepatitis C, and genital warts. 14.4.3.4 Production of Erythropoietin Kidneys synthesize erythropoietin (EPO); human EPO has a molecular weight of 34,000. It acts on the bone marrow to increase the production of RBC. Stimuli such as bleeding or the scarcity of oxygen at high altitudes trigger the release of EPO. The approval for therapeutic use of EPO was given in 1989. People suffering from kidney failure can be kept alive by dialysis, but dialysis cleans only the waste blood. Without a source of EPO, these patients suffer from anemia. With the help of recombinant DNA technology it is possible to produce recombinant human EPO to treat these patients. EPO level measured the amount of hormone also called as erythroid hormone erythropoietin (EPO) in the blood. The hormone acts on stem cells in the bone marrow to increase the production of RBC. It is made by the cells in the kidney, which release the hormone when oxygen levels are low. 14.4.3.5 Therapeutic Uses of Recombinant EPO • Some of the drugs used to treat AIDS (e.g., zidovudine (AZT)) cause anemia as a side effect. Recombinant EPO helps AIDS patients cope with this problem • Recombinant EPO improves anemia which is frequent side effect of cancer chemotherapy • EPO has been shown to be beneficial in certain neurodegenerative diseases like schizophrenia • Severe blood loss in Jehovah’s witnesses, whose religion forbids them to receive blood transfusions, can also be helped with recombinant EPO • EPO plays an important role in the brain’s response to neuronal injury. EPO is also involved in the wound healing process EPO increases the hematocrit, thus enabling more oxygen to flow to the skeletal muscles. Some cyclists (and distance runners) have used recombinant EPO to enhance their performance. The kidney cells that make EPO are specialized and are sensitive to low oxygen levels in the blood. Although recombinant EPO has exactly the same sequence of amino acids as the natural hormone, the sugars attached by the cells used in the pharmaceutical industry differ from those attached by the cells of the human kidney. This difference can be detected by testing the athlete’s urine. Over two dozen young professional cyclists have died unexpectedly (usually during the night) after using recombinant EPO. Researchers suspect that the EPO induced the increase in their hematocrit levels leading to a reduction in heart rate and causing death. Prolonged exposure to reduced oxygen levels (e.g., living at high altitude) leads to increased synthesis of EPO. In mice, and perhaps in humans, the skin mediates this effect. Mouse skin cells can detect low levels of oxygen (hypoxia) and if this persists, blood flow to the kidneys diminishes, leading to increased synthesis of EPO by them. Recently it has been found that EPO is also synthesized in the brain when oxygen becomes scarce (e.g., following a stroke), and helps protect neurons from damage. Perhaps recombinant human EPO

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will turn out to be useful for stroke victims as well. The EPO gene has been found on human chromosome 7 (in band 7q21). EPO is produced not only in the kidney but also, to a lesser extent, in the liver. Different DNA sequences flanking the EPO gene act to control kidney versus liver production of EPO. Early treatment with EPO has been shown to significantly increase the risk of retinopathy of in premature infants and is not recommended.

14.4.4 VACCINE PRODUCTION The prevention of diseases by vaccination is the most significant medical achievement of mankind. Vaccines currently prevent more than three million deaths per year. Use of animal biotechnology in the field of vaccine production goes back to the eleventh century. In those days it was used in a crude form. The dried scabs of smallpox patients were ground and prepared as a powder which was then sprayed in the noses of normal people. It was found that in general these people were less susceptible in the case of sudden outbreaks. Smallpox was a highly infectious disease with a high death rate; survivors frequently developed deformities like blindness and mental retardation, and so on. It was only in 1796 that Jenner experimented on a small boy with exudates from a cowpox lesion. He repeated it twice with a gap of week and inoculated some human volunteers. Those volunteers did not develop smallpox. Thus smallpox has been eradicated with vaccination; polio is expected to be eradicated in the coming era in a similar fashion using vaccines. New vaccines for pneumococcal disease and human papillomavirus (HPV) infection are also being produced. It is because of the development of various vaccines during the twentieth century that the average human life span has increased by approximately 30 years. Vaccines are basically of three types: 1. Live or attenuated bacteria or viruses 2. Dead or inactivated virus 3. Subunit vaccines 14.4.4.1 Live or Attenuated Bacteria or Viruses The best example of this is the smallpox vaccine, also the first vaccine developed for various purposes. It consists of a live bovine poxvirus that is similar to the smallpox virus, causing the disease in humans but not in cows. This concept has been adopted for vaccines against several bacterial and viral pathogens, including Mycobacterium tuberculosis, Vibrio cholera Salmonella typhi, yellow fever virus, measles virus, mumps virus, polio virus, rubella virus, varicella-zoster virus, adenovirus, and rotavirus. 14.4.4.2 Dead or Inactivated Virus This is a method of vaccine production in which the inactivated or killed pathogenic organism is used for vaccine production. All the antigens of the organism are exposed to the immune system, but the organism itself is rendered harmless. A few examples are the influenza virus, hepatitis A virus, rabies virus, and whole-cell Bordetella pertussis. They do not possess the ability to replicate in vivo and thus are less immunogenic than live vaccines. 14.4.4.3 Subunit Vaccines Most bacteria such as Clostridium tetani and Corynebacterium diphtheriae produce toxins that lead to pathogenic conditions. It is a well known fact that antibody-mediated neutralization of such toxins can prevent disease; thus the vaccines are based on detoxified models of these toxins, known as toxoids. There are certain antibodies which are directed against the bacterial polysaccharides, for example, Neisseria meningitidis and Streptococcus pneumoniae. It is surprising that these polysaccharide vaccines are most effective in adults and only poorly effective in children (Johri et al., 2006).

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14.4.4.4 Vaccine Production in Influenza Virus Influenza is a widespread disease affecting 10% of the population every year, including both humans and animals (pig, horses, birds, and chickens). These viruses are very clever in that they frequently change their antigenic properties. Researchers worldwide are trying to formulate vaccines against them, so as to reduce their widespread affect. The surveillance rate of the vaccine includes protection against all three strains recognized so far (see http://www.cdc.gov/flu). Vaccine production includes the following steps 1. Strain selection: Strain selection comes under the jurisdiction of World Health Organization (WHO) officials to analyze and identify the dominant circulating strains. This information is submitted to the FDA for recommendations as to which strains are to be selected for vaccine production. The strains selected should be the most prevalent and damaging strains, having similar antigenic properties. The FDA authorities for vaccine production distribute the selected strain seeds. Forecasting or future prediction is also carried out for the coming year’s strain to begin vaccine production prior to FDA approval. 2. Manufacturing and production: Manufacturing and production include the separate production of each strain, which are later combined for the vaccine production. Specially prepared chicken eggs are used for this purpose. The eggs were collected and cleaned with disinfectants for seven months. They are injected with one strain each and incubated so as to allow for virus multiplication. Then the virus-loaded fluid is collected for purification. 3. Testing and purification: The fluid collected is passed through several purification steps along with special chemical treatments so as to ensure the viruses are killed or inactivated. The killed viruses are then chemically disrupted and the fragments collected from different batches are combined after the completion of quality control tests. FDA and manufacturers then test the vaccine concentrate so as to determine the amount and yield to ensure it is adequate for immunization purposes. 4. Vaccination: The vaccine, after undergoing FDA approval and licensing, is then ready for marketing and commercial use for immunization. 14.4.4.5 Smallpox Vaccine Smallpox is highly contagious, caused by the variola virus, a double-stranded DNA virus belonging to the genus orthopoxvirus which includes cowpox, monkeypox, and vaccinia. Poxviruses mainly affect the skin and cause disease in humans (smallpox) and animals (e.g., swinepox, camelpox, sheeppox, goatpox, and fowlpox). Vaccine production for these pox viruses goes back to the Chinese era several centuries BC, when they would expose a healthy person to biological matter from the smallpox lesions of an infected person in order to confer immunity. Chinese doctors also dried and ground up the crusts of smallpox scabs and used tubes to blow the material into the noses of healthy persons. In Africa, Asia Minor, and parts of Europe, people swallowed smallpox scabs or had doctors scratch smallpox lymph into their skin (variolation). By the end of the seventeenth century it was common practice in England and America to scrape the lesions of a person infected with smallpox and then introduce the matter onto the skin of healthy children and adults to cause a mild form of smallpox, which would then confer immunity. The above process is known as variolation. This had some positive action but many more side effects, leading to permanent scars or even blindness from the intervention. Many people were unknowingly infected with syphilis, tuberculosis, or hepatitis because the biological matter obtained from the lesions was taken from persons who were also suffering from serious diseases. This also contributed to the spread of smallpox throughout populations and to Edward Jenner’s discovery in 1796. He infected an eight-year-old boy with cowpox by scraping pus from the lesions of a child infected with cowpox onto the skin of the boy. He then checked the boy’s immunity twice by scraping pus from the lesions of a person with smallpox onto the boy’s skin. The boy never came down with smallpox.

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There are currently known to be multiple strains of vaccinia virus with varying degrees of virulence for humans and animals. Scientists around the world, working on new vaccines for diseases such as HIV, have created recombinant vaccinia viruses from several strains of vaccinia virus. 14.4.4.6 Rabies Vaccine Production Rabies, a viral disease affecting all warm-blooded animals, is widespread in many regions of the world. Rabies vaccines is manufactured in primary mammalian cell cultures such as hamster kidney (Fenje and Pinteric, 1966), dog kidney (Van Steenis et al., 1985), and fetal calf kidney (Atanasiu et al., 1974), or in avian cell cultures such as chicken embryo (Barth et al., 1984) and quail embryo (Bektimirova et al., 1983), following Kissling’s methodology. A second group of vaccines is produced in diploid cells of regular cariogenicity and duplication, mainly of human origin (WI38 MRC5). Some vaccines of animal origin, such as rhesus monkey fetus (Burgoyne et al., 1985), likewise pertain to this group. A third group includes vaccines developed in heteroploid cell culture such as the Vero line 24, BHK21 (Erbolato et al., 1989) and NIL2 (Diamond, 1967) cell lines, where rabies virus replicates with very good yields reaching a high viral titer (106 –LD50 in mice), have been accepted as substrate. These cell types allow an easy industrial scale-up, producing cultures through different systems (roller, spinner flask, stirred bioreactors, airlift, and hollow fiber), and thus increasing the yield. Vaccines manufactured with live attenuated virus are permitted for animal immunization (Abelseth, 1964), allowing oral vaccination of wildlife, employing rabies virus strain SAD, or SAD-B19 clone. These cells are very sensitive. The addition of certain adjuvants is permitted (Fenje and Pinteric, 1966). Vaccines for human use are usually employed for treatment after exposure to rabies virus (five or more doses, depending on the type of vaccine) and have been more highly developed, as such vaccines must be fully inactivated. Normal diploid karyotype cells are employed as substrate for rabies virus replication, containing either oncogenes or transformed genes. 14.4.4.7 Equine Herpesvirus Vaccine This is the vaccine developed for protecting horses with EHV-1 and/or EHV-4. The vaccine commonly includes inactivated EHV-1 (e.g., chemically inactivated EHV-1 KyA virus) and an adjuvant. The adjuvant is composed of cross-linked olefinically unsaturated carboxylic acid polymer having bioadhesive properties. The vaccine may also include antigens against other equine pathogens such as inactivated EHV-4 and inactivated A1 and/or A2 strains of equine influenza virus. Methods for protecting horses against EHV-1 and/or EHV-4 and associated diseases and methods of producing the equine herpesvirus vaccine are also provided.

14.4.5 VETERINARY VACCINES Veterinary vaccines currently available in Europe and other parts of the world have been developed by the veterinary pharmaceutical industry. The development of a vaccine for veterinary use is an economic endeavor that takes many years. There are many obstacles along the path to the successful development and launch of a vaccine. The industrial development of a vaccine for veterinary use usually starts after the proof of a concept that is based on robust academic research. The major goals of veterinary vaccines are to improve the health and welfare of companion animals or increase the production of livestock in a cost-effective manner, and prevent animal-to-human transmission from both domestic animals and wildlife. These diverse aims have led to different approaches in the development of veterinary vaccines from crude but effective whole-pathogen preparations to molecularly defined subunit vaccines, genetically engineered organisms or chimeras, vectored antigen formulations, and naked DNA injections. Recent developments in bacterial, viral, and parasite vaccine research and development are also addressed and the developments of novel adjuvants that use the expanding knowledge of immunology and disease pathology are described.

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14.4.5.1 Bacterial Vaccines 14.4.5.1.1 Inactivated Vaccine Bacterial vaccines are mainly inactivated whole-cell cultures (or bacterins), inactivated culture supernatants (or toxoids), crude extracts of the cell surface, or attenuated live preparations. Advances in our knowledge and biological technologies provide the opportunity to develop improved vaccines. Inactivated crude preparations had the advantage that knowledge of the molecular basis of pathogenesis was not a prerequisite to vaccine development, since there was a good chance they contained the antigens necessary to stimulate protective immunity, lipopolysaccharides, teichoic acid, or other cell wall constituents. These components led to the heavy stimulation of innate immune systems so that undesirable adverse effects were likely. Nonetheless, the traditional approach remains a perfectly acceptable solution for certain diseases (Andre-Fontaine et al., 2003). 14.4.5.1.2 Live Vaccines Early live vaccines had the advantage of inactivated antigens combined with the potential for in vivo dependent gene expression to provide a more protective immune stimulus (Feberwee et al., 2001). 14.4.5.1.3 DNA Recombinant Vaccine DNA vaccination is a relatively recent development where genes encoding protective immunogens are inserted into nonreplicating elements. These elements can be taken up by host cells; the foreign antigen are initially presented intracellularly and then are presented on the cell surface in association with major histocompatibility complex (MHC) antigens to T cells. This offers the advantages (1) that the Th1/Th2 balance of immune responses can be modulated to achieve more effective immunity, and (2) that the DNA is highly stable at room temperature and easily quality assurance (QA) controlled during production (Jechlinger, 2006). Consequently, not only are new adjuvant strategies needed to make recombinant subunit or DNA vaccines work, but also bacteria may themselves provide the solution. The most intensively investigated adjuvants are cholera and E. coli heat labile ADP-ribosylating enterotoxins (Lycke, 2004), the zonula occludens toxin of Vibrio cholerae (De Magistris, 2006), Mycobacterium tuberculosis heat shock protein 70 (Bulut et al., 2005), Salmonella typhimurium FljB (Simon and Samuel, 2007), synthetic analogs of bacterial lipoproteins (Ghielmetti et al., 2005), synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides, and monophosphoryl lipid A (Jiang et al., 2007). 14.4.5.2 Viral Vaccines 14.4.5.2.1 Rabies Vaccines The production of rabies vaccines for animal use, whether live or inactivated, has developed during the past two decades with the increasing use of continuous cell lines as a substrate and the adoption of fermentor technology for antigen production. These vaccines are produced for administration to domestic animals or wild species by parenteral or oral routes according to the vaccine characteristics. Highly immunogenic inactivated cell culture vaccines for the immunization of dogs via the parenteral route are now widely available. More recently, a third generation of live veterinary rabies vaccine has been developed using recombinant technology. Depending upon the expression system, these vaccines can be used either parenterally or orally. The development of recombinant DNA technology has initiated a new era in rabies control. Recombinant vaccines cannot exhibit the residual pathogenicity caused by rabies because they contain only single nonvirulent gene products. The majority of the safety requirements for modified live-virus vaccines are also applicable to recombinant vaccines. Various recombinant constructs (e.g., animal poxviruses or human or canine adenovirus as vector) expressing the rabies glycoprotein were tested in different target and nontarget wildlife species by the oral route. The only recombinant that is now produced and used in large quantities is a vaccinia-based recombinant product expressing the rabies glycoprotein (VRG). © 2010 Taylor and Francis Group, LLC

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14.4.5.2.2 Combined Vaccines Combined vaccines are already used for the immunization of dogs and cats. Several different antigens are incorporated in canine rabies vaccine, such as canine distemper, canine hepatitis, leptospirosis, and canine parvovirus. Combined rabies vaccines for cats may include various other antigens such as feline panleukopenia virus, feline calicivirus, and feline parvoviruses. A combined rabies and foot-and-mouth disease vaccine is available for use in cattle, sheep, and goats. 14.4.5.2.3 α Herpesviruses The deletion mutants’ biotech approach has been particularly successful for pseudorabies virus (PRV) and bovine herpesvirus-1 (BHV-1). In contrast, for equine herpesvirus-1 and -4 (EHV-1; EHV-4), deletion of nonessential virus glycoprotein and enzyme genes with the aim of deriving attenuated live virus vaccine candidates similar to PRV and BHV-1, have been fruitless (Patel and Heldens, 2005). 14.4.5.2.4 Circoviruses Porcine circovirus-2 (PCV-2) is a cause of multiorgan disease and significant economic loss in domestic pigs (Allan and Ellis, 2000; Krakowa et al., 2002). Immunoprophylaxis against PCV-2 infection of domestic pigs is with conventional and biotech vaccines, such as a baculovirus expressed PCV-ORF2 protein. Additionally, many biotech experimental PCV-2 vaccines have been investigated. Examples of such vaccines are PCV-2 ORF 2 gene expressed in PRV vector, an apathogenic PCV-1/PCV-2 chimera vaccine, and DNA plasmid vaccines (Fenaux et al., 2004), 14.4.5.2.5 Flaviviruses Several biotech approaches have been investigated for classical swain fever vaccine (CSFV) vaccines. These include baculovirus expressed E2 glycoprotein (Ahrens et al., 2000), chimeric bovine virus diarrhoea virus (BVDV)-CSFV vaccine (Van Gennip et al., 2000), and recombinant PRV expressing CSFV-E2 glycoprotein, protecting against diseases due to PRV and CSFV (Van Zijl et al., 1991). 14.4.5.2.6 Influenza and Paramyxoviruses Influenza A viruses cause greater problems in birds than mammals. In particular, some strains of H5 and H7 subtypes are highly pathogenic, causing high mortality and posing a zoonotic risk to man along with some H9 subtypes. A likely approach for the development of vaccines would be, firstly, cloning and site-directed mutagenesis to turn the HA-gene into a nonpathogenic form and, secondly, the production of so-called high-growth reassortants producing considerable amounts of the new HA protein, which is, among others, the protective antigen in influenza virus. The latter can be achieved by reverse genetics and transfection techniques (Wood and Robertson, 2006). This has been the approach for the currently circulating and potentially pandemic H5N1 avian influenza virus, which has caused more than 250 human deaths so far. 14.4.5.2.7 Iridoviruses and Orbiviruses African horse sickness (AHS) is a highly fatal, insect transmitted disease of equidae caused by the African horse sickness virus (AHSV), an orbivirus. Currently, conventional mouse brain or tissue culture grown vaccines are in use but future vaccines may use AHSV VP2 protein as a subunit biotech vaccine (Ranz et al., 1992). For bluetongue virus (BTV), while there are egg-adapted polyvalent vaccines to prevalent serotypes in use, biotech approaches have been investigated which hold much promise. Thus BTV-like and virus core-like structures have been constructed using major (VP3, VP7) and minor (VP1, VP4, VP5) capsid proteins. The VP proteins were produced in baculovirus multiple expression vectors. These virus-like single- and double-shelled particles emulsified in Freund’s incomplete adjuvant or Montanide ISA-50 adjuvants were highly immunogenic and protective for naïve sheep (Roy, 1992). © 2010 Taylor and Francis Group, LLC

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14.4.5.2.8 Retroviruses and Viruses without Vaccines Monovalent and subunit feline leukemia virus (FeLV) glycoprotein expressed in E. coli is marketed in the European Union (EU). The immunogenicity of an experimental vaccinia virus recombinant that expresses bovine leukemia virus envelope protein (gp51) has given promising results in bovine calves and rabbits (Valikhov et al., 1997). It is important and relevant to point out that there are animal diseases for which there are still no vaccines at present. The γ herpesviruses, namely alcephaline herpesvirus-1 (AHV-1) and ovine herpesvirus-2 (OHV-2), cause fatal excessive lymphoid proliferation in ruminant hosts such as cattle and deer species (Nettleton et al., 1998; Hussey et al., 2000). AHV-1 and OHV-2 are innocuous in reservoir hosts, wildebeest and sheep, respectively. Two new fatal zoonotic infections due to related paramyxoviruses, called Hendra and Nipah viruses, affect horses and pigs, respectively, and also man. The reservoirs for both viruses are petropid bats found in Australia and the Far East (Haplin et al., 2000). Currently no vaccines are available for these viruses. 14.4.5.2.9 Parasite Vaccines Classically, the most important parasitic diseases in humans and animals are treated and/or controlled by using chemotherapeutics (Cornelissen and Schetters, 1996). A series of developments (including drug resistance) has given impetus to research into parasite vaccines. As a result, a number of vaccines against parasitic diseases are now commercially available. It is envisioned that more parasite vaccines will come to market and will aid in the control of parasitic diseases (Vercruysse et al., 2004). 14.4.5.3 Live Vaccines (Complete Life Cycles) 14.4.5.3.1 Transient Infections The most obvious examples of such a vaccine are the live vaccines against coccidiosis in chickens (Williams, 2002). As this type of infection is transient (the parasite “passes” through the chicken) the infection is self-limiting and no chemotherapeutic treatment is necessary to cure the infection. 14.4.5.3.2 Selection for Less Virulent Strains The virulence of parasite strains derived from a single isolate can be variable. For example, using Babesia bovis isolates, passage through splenectomized animals can select for strains of reduced virulence. The infection that develops is less virulent and the animals develop immunity against subsequent challenge infections (De Waal and Combrink, 2006). Similarly, strains with reduced virulence can be selected from Eimeria isolates. Some commercially available coccidiosis vaccines for broilers contain strains that are selected after repeated passage through chickens. These socalled precocious strains require less time to develop into oocysts, and the numbers of progeny are reduced compared to the wild-type parent population (Williams, 1994). 14.4.5.3.3 Temperature-Sensitive Strains Some parasite strains have been selected that differ from the wild-type strains in that they cause a self-limiting infection. One such example is the temperature-sensitive strain of Toxoplasma gondii. This strain, which resulted from chemically induced attenuation, can be propagated successfully in vitro at relatively low temperatures, but will not propagate successfully at the body temperature of the target animal. As a result the infection will self-cure (Lindsay et al., 1993). This vaccine has not been commercialized. 14.4.5.3.4 Infection Treatment In case the parasite has a tendency to survive in the host for longer periods of time, chemotherapeutic cure of the infection is also required. An example of this approach is the live vaccine against

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Theileria parva infection. The vaccine is based on isolates of virulent Theileria parva strains, which are used to infect cattle that are simultaneously treated with a long-acting tetracycline preparation to control the infection (Boulter and Hall, 1999). 14.4.5.3.5 Live Vaccines (Incomplete Life Cycles) The early life cycle stages are sufficiently immunogenic to induce protective immunity; selection for parasite strains with truncated life cycles is another strategy to develop vaccines. A good example is the Toxoplasma gondii S48 strain. This strain has lost the capacity to develop from the tachyzoite into the bradyzoite stage, and thus does not form tissue cysts. The tachyzoites induce a transient infection in the host, while triggering protective immune reactions (Buxton, 1993). Irradiation of parasites has also been used as a mechanism to truncate the life cycle. The live vaccine against lungworm infection in cattle contains L3 larvae of Dictyocaulus viviparus that do not develop further than the L4 stage. Vaccinated cattle are immune to challenge with L3 larvae (Urquhart, 1985). Virulent parasite strains could be genetically modified to reduce their virulence. This principle has been investigated using Toxoplasma gondii in which a tetracycline-dependent regulatory element was cloned in front of an essential gene. The parasite may be propagated during the vaccine production phase in the presence of tetracycline. Once injected into the target animal, the parasite will not be able to continue propagation in the absence of tetracycline, as this will lead to blocking of the expression of the essential gene (Van Poppel et al., 2006). 14.4.5.4 Killed Vaccines 14.4.5.4.1 Whole Organisms If no live vaccine strains are available, or the use of live vaccines is undesirable, one may want to inactivate the parasites prior to the formulation of a vaccine. Such preparations by themselves do not induce protective immunity and an appropriate adjuvant and formulation must be developed. Examples of such vaccines are the vaccine against abortion in cattle due to Neospora caninum infection (Schetters and Gravendyck, 2006) and a vaccine against giardiasis in dogs (Olson et al., 2000). 14.4.5.4.2 Subunit Vaccines In some cases the antigens are produced using recombinant DNA technology. The best example is a vaccine against Taenia ovis in sheep, which is based on recombinantly produced parasite antigens that induce antibodies which block the attachment of oncospheres to the gut epithelium (Harrison et al., 1999). Saponin adjuvant was shown to be most efficacious. Another example is the vaccine against the cattle tick Boophilus microplus (Willadsen, 2004). The vaccine contains recombinantly produced gut wall antigens of the tick. Upon vaccination of cattle, high levels of antibodies to the gut wall of ticks are produced. During feeding of the tick on the vaccinated animal these antibodies are ingested and destroy the gut epithelium of the tick, thus killing the parasite. 14.4.5.4.3 Vaccine Immunopotentiators or Adjuvants Vaccine adjuvants may also increase the overall magnitude of the antigen-specific response in order to reach a minimal level of protective antibody concentration or effector T cell population. In addition, adjuvants can prolong the duration of vaccine effector immune responses, allowing for fewer or no booster immunizations, which becomes critical during mass vaccinations. Moreover, certain vaccine adjuvants are able to positively affect the quality of the immunophenotype, for example, induction of cell-mediated T cell immunity, considered necessary for the control of many types of intracellular pathogens. Antigen dose (cost) sparing is enabled by certain adjuvants, which is relevant in the case of expensive or cumbersome antigen production systems.

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RISKS AND MANAGEMENT

14.5.1 POTENTIAL RISKS 14.5.1.1 Risks Involved Using Embryonic and Stem Cells in Cloning How do stem cells decide which tissues to become and how many to make of each type of tissue? To date we understand that turning genes on and off is central to the process of human development, but we do not know much about how and who makes these decisions during the process of human development. We also do not know exactly how stem cells are turned on or off. Some of our most serious medical conditions, such as cancer and birth defects, are due to abnormal cell specialization and cell division. A clear molecular understanding of normal cell development is necessary for scientists to know exactly what goes wrong with cells when cancer or birth defects occur. This will one day be understood and we will able to correct the birth defect or reverse the cancer onset. Once we delineate these events, we will eventually be able to use these embryonic stem cells for better cloning to produce disease-free animals and eventually a disease-free human race. 14.5.1.2 Risks Pertaining to Immunological Disorders Animal biotechnology faces a variety of uncertainties, safety issues, and potential risks as it moves away from classical genetic breeding and towards recombinant technology used in the development of specific livestock for the production of vaccines and other smaller molecules such as growth factor. For example, concerns have been raised regarding issues such as the use of unnecessary genes in constructs designed to generate transgenic animals, the use of vectors with the potential to be transferred to or contribute sequences to other organisms, the potential effects of genetically modified animals on the environment, the effects of biotechnology on the welfare of animals, and potential human health and food safety concerns about meat or animal products derived from animal biotechnology (see http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/ GuidanceDocuments/Biotechnology/default.htm). Before animal biotechnology can be used widely by animal agriculture production systems, additional research will be necessary to determine if the benefits of animal biotechnology outweigh these potential risks. The USDA Biotechnology Risk Assessment Grants program supports environmental risk assessment research projects on genetically engineered animals. In addition, the NRI Animal Protection program supports research projects to determine the effects of genetic modification on the health and wellbeing of animals. Advances in animal biotechnology have been facilitated by recent progress in sequencing and analyzing animal genomes, the identification of molecular markers (microsatellites, expressed sequence tags [ESTs], quantitative trait loci [QTLs], etc.), and a better understanding of the mechanisms that regulate gene expression. The potential for genetically engineered animals, particularly fish, insects, shellfish, and other animals which easily escape and become feral to disrupt ecosystems or to introduce novel genes to a population was one of the top concerns of the committee. Another concern raised by the committee was whether food products from genetically altered animals would be safe for human consumption. Although the committee found no evidence of a threat, it was noted that more information was necessary. Other potential risks identified are as follows: • The creation or spread of novel zoonotic diseases through xenotransplantation • Diminished welfare among genetically altered animals or animals undergoing certain techniques • Social, ethical, or cultural conflicts • Insufficient regulatory framework to adequately address concerns related to biotechnology (see http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/Guidance Documents/Biotechnology/default.htm)

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Taking in account of animal cloning technology: Reproductive cloning is expensive and highly inefficient. More than 90% of the cloning attempts fail to produce a viable offspring. Also, more than 100 nuclear transfer procedures could be required to produce a viable clone. The animals cloned in this manner have compromised immune function and higher rates of infection, tumor growth, and other disorders. This has been proved by Japanese and Australian studies in which animals that were born healthy died within days. Studies from the White Head Institute of Biomedical Research in 2002 reported that the genomes of cloned mice are compromised and 4% of their genes function abnormally. The cause for these changes is not the mutation but abnormal activation or expression of certain genes. 14.5.1.3 Risks Pertaining to the Susceptibility to Diseases 14.5.1.3.1 Transmissible Spongiform Encephalopathies The transmission of bovine spongiform encephalopathy (BSE) from cattle to humans is called Creutzfeld–Jacob disease. Data published so far suggest that BSE resulted from the transmission of scrapie from sheep to cattle. Although it is not a very common disease, it is caused by a prion agent that causes modification in an endogenous protein, a product of the PrP gene. The gene encoding PrP was knocked out in mice using homologous recombination and the mice homozygous for this knockout were found to be resistant to scrapie. Although the resistance of PrP-mutant sheep and cows to scrapie and BSE, respectively, has yet to be demonstrated, this approach might be a route towards the eradication of transmissible spongiform encephalopathies (TSEs) in livestock species. 14.5.1.3.2 Avian Influenza Virus Avian influenza virus is another potential threat to human health and is a source of a new flu virus. The natural reservoirs of these flu viruses are aquatic birds, reared pigs, and poultry and these reservoirs are supposed to be involved in the transfer of this disease to human beings. These hosts are likely to enable both reassortment between different strains of avian flu and between avian influenza and human influenza viruses. A case study of the H5N1 strain of avian influenza conducted in 2004 was the largest on record and resulted in 32 fatal human cases. The use of transgenic technology to produce flu-resistant chickens by using lentiviral vectors to introduce sequences that express small RNAs would be a significant development.

14.6 CONCLUSION The potential benefits of animal biotechnology are numerous, and include: the enhanced nutritional and micronutrient content of food for human consumption for a healthy and disease free life style; a more abundant, cheaper, and varied food supply; agricultural land-use savings; a decrease in the number of animals needed for the food supply; improved health of animals and humans; the development of new, low-cost disease treatments for humans; and an increased understanding of human disease. It has been reported recently that phenotypic abnormalities have been observed in aged cloned mice from embryonic stem cells when these mice have been kept long term. The aged cloned mice showed multiple abnormalities such as an increase in body weight, some phenotypic abnormalities in the kidneys, testes and thymus, and lower urea nitrogen in their serum biochemical values (Rudenko and Matheson, 2007). However, after comprehensively reviewing the production of cattle, swine, sheep, or goats by SCNT and comparing it with the current assisted reproductive technology, the FDA did not find any adverse reactions. Therefore the FDA has concluded that food from cattle, swine, sheep, and goat are as safe to eat as food from the same species derived from conventional methods (Shimozawa et al., 2006). Yet despite these potential benefits, several areas of concern exist around the use of biotechnology in animals. To date, the majority of the world population is uncomfortable with genetic

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modifications to animals. This may be repaired by more dialogue between society and scientists. Public concern regarding the leaching of these genetically engineering organisms into the environment or their spread into existing species is a major concern. More data may be required to prove the safety and eco-friendly nature of these technologies.

ACKNOWLEDGMENTS Part of this work is supported by a grant from the Department of Biotechnology (BT/PR7951/ MED/14/1193/2006). Authors are indebted to Usha Nagarajan and Sreejit P for their assistance in the preparation of the manuscript.

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of Micro-RNA in 15 Application Regenerative Nutraceuticals and Functional Foods Ji Wu, Huacheng Luo, Li Zhou, and Jie Xiang CONTENTS 15.1 Introduction .......................................................................................................................... 251 15.2 Functional Foods and Nutraceuticals ................................................................................... 251 15.2.1 miRNA in Functional Foods and Nutraceuticals ..................................................... 252 15.2.2 miRNA and Diseases................................................................................................ 253 15.2.3 miRNA and Regenerative Medicine......................................................................... 254 15.3 Diseases Associated with Functional Foods and Nutraceuticals ......................................... 256 15.4 Perspective ............................................................................................................................ 258 References ...................................................................................................................................... 259

15.1 INTRODUCTION The concept of physiologically relevant functional food was first introduced in Nature with the headline Japan Explores the Boundary between Food and Medicine (Swinbanks and O’Brien, 1993). The news item reported on some novel biotechnology-produced foods, and gave special emphasis to their medical value. References to new concepts in nutrition outlined in the article implied that Functional Food Science fuels the research and development into functional foods, and that a strategy for functional foods development has been defined (Gibson and Williams, 2000; Figure 15.1). At present, there are several critical criteria involved in the evaluation of a particular functional food. As well as its value as a functional food, and based on science and technology, this evaluation should focus on biochemical, physiological, molecular and cell biological data, and take into account other modern biosciences. Functional foods and nutraceuticals have been identified which play an significant role in human health and disease. An important approach to the development of a functional food is the identification and validation of relevant markers, including biomarkers, which can predict potential benefits related to a target function in vivo. Moreover, micro-RNAs (miRNAs) as a kind of biomarker have been confirmed and are closely correlated not only with human health, but also with human disease research. Therefore, miRNAs are considered as useful biomarkers and are widely applied in functional foods and nutraceuticals.

15.2 FUNCTIONAL FOODS AND NUTRACEUTICALS Functional foods are defined as the foods that contain potentially healthful products, including “any modified food or food ingredient that may provide a health benefit beyond that of the traditional nutrients it contains.” They have attracted widespread interest because of the possibility that these selected foods could actually promote health (Gibson and Williams, 2000). With the development 251 © 2010 Taylor and Francis Group, LLC

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Function Mechanism

Functional test and effect

Hypothesis-driven nutrition studies

Enhanced function

FIGURE 15.1

Disease prevention

The strategy for functional food development.

of functional foods and nutraceuticals, nutrition science has entered into the era of nutrigenomics, the aim of which is to establish personal nutrition. The study of the interaction between nutrition and an individual’s genome or the response of an individual to different diets is coined as “nutrigenomics” or “nutritional genomics.” The research purpose of the discipline is to find out how the nutriment and other chemical substances regulate the expression of the gene and the mechanism of metabolism (Manuela et al., 2006). Recently, biotechnology, which used to be applied mainly in modern molecular genetics research, has also been widely employed in nutritional genomics. DNA chip and proteomics are common methods of elucidating the relationship between a nutriment and the genes. In addition, the complementary DNA (cDNA) chip plays an important role in researching the gene expression map in absence of nutriment, proper nutriment, or redundant nutriment conditions, which will help to find more biomarkers to estimate the nutritional status. Currently, nutritional requirements are set only partly according to the biochemical index rather than according to gene expression. Thus, several sensitive, reliable biomarkers will probably be needed to adequately assess the benefits and risks associated with the increased consumption of specific foods and their bioactive components through different levels, including DNA, RNA, protein, and to establish more precise and reasonable nutritional requirements.

15.2.1

MIRNA IN

FUNCTIONAL FOODS AND NUTRACEUTICALS

Dietary supplements are also commonly known as nutraceuticals, a term derived from the combination of “nutrition” and “pharmaceutical.” This word denotes extracts from foods identified to have a medicinal effect on human health, including vitamins, minerals, botanicals, and others. The metabolic pathway is comprised of carbohydrates, fatty acids, cholesterol, amino acids, and so on, and when a metabolic syndrome occurs, it often leads to a series of health problems or diseases, such as diabetes, hyperlipidemia, hypertension, or arteriosclerotic cardiovascular diseases. A stable metabolism is currently considered to be of great importance for a healthy life. Some important genes are regulated by nutraceuticals, such as carbohydrates, fatty acids, cholesterol, and amino acids. More obviously, it is implied that a nutraceutical will contain an extract or food confirmed to be of physiological benefit or to provide an effective protection against certain chronic diseases. Research on liver and adipose tissue of rats showed that a high-carbohydrate diet could promote the expression of these genes, but that gene expression could be suppressed by starvation or a high-fat diet (Foufelle et al., 1996). Takanabe and colleagues also reported upregulated expression of miR-143 in the mesenteric fat of high-fat diet induced obese mice; this finding implies that miR-143 might regulate expression of the adipocyte genes involved in the pathophysiology of © 2010 Taylor and Francis Group, LLC

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obesity (Takanabe et al., 2008). Moreover, these findings have demonstrated that glucose is a primary inducer of in vitro gene expression (Takanabe et al., 2008). Glucose that can induce the transcription of the insulin gene has also been characterized (Docherty and Clark, 1994). This feature is obviously associated with the diabetic syndrome. In addition, in vitro studies have shown that the expression of a small number of genes of adipose cells can be increased by the presence of fatty acids (Schoonjans et al., 1996). Researchers also found that fatty acids could inhibit gene expression in rats. If this same inhibition happens in vivo, it stands to reason that nutrition could regulate the number of adipocytes and will be a crucial determinant of a possible expansion of this tissue, associated with the obesity syndrome (Schoonjans et al., 1996). Some researchers have revealed that gene regulation induced by nutraceuticals, foods of specific composition, includes a very large number of mechanisms involved in the regulation of crucial pathways as well as cell differentiation and proliferation. Therefore, these researchers imply that these are of great importance at certain periods. As for other components of cell functions, it is very likely that gene regulation by nutraceuticals is subject to variations linked to genetic polymorphisms among individuals, especially combined with the application of miRNA biotechnology in nutraceuticals and functional foods. Similarly, miRNAs, which are the most subtle reporters for cells exposed to the nutritional environment and the molecular basis of the body’s nutritional requirements and differentiation to response, have strong potential to become the new biomarkers in nutrigenomics, and this assumption will become more likely in the near future after deeper, richer acquaintance with miRNAs. Thus, miRNAs provide a novel way for searching for new biomarkers, and might well be applied in the function research for functional foods. At present, the study of functional food is not thorough enough. Up to now, we still have no idea exactly how the effectual elements work and interact with genetic material or other metabolites. The expression profi le of miRNA or the diversification of miRNA expression could be used as new clues in the comprehension of the effect of function foods. Biological systems are extremely complex and exhibit properties that extend far beyond observations associated with individual independent cellular processes. As a result, it is vital to evaluate the entire biological system from the nutrigenomics perspective involving the critical evaluation of DNA polymorphisms or single nucleotide polymorphisms (SNP) of a gene, the expression of that specific gene, the expression of specific processed messenger RNA (mRNA) (alternative splicing), the expression of miRNA, protein production from that mRNA, post-translational modification of the resultant protein, and the formation of respective metabolites (Berezikov et al., 2005). At the present time, nutrigenetics, epigenomics, transcriptomics, proteomics, and metabolomics drive a broad application of this kind of approach so that a comprehensive picture emerges from not only a single cell but tissues or even whole organisms. Studies on miRNAs in functional food can help to unveil the mechanisms of molecular events accompanying carcinogenesis and perturbations during cell death processes. Besides, researches on these novel small RNAs will improve our understanding of impact that individual’s response to diet makes to these processes, which is also one of the research aim of the nutrigenomics.

15.2.2

MIRNA AND

DISEASES

miRNAs are short noncoding RNAs that can regulate specific gene expression by binding to target mRNAs, in general, which can negatively regulate specific gene expression by translational repression, degradation, or destabilization of target genes (Zhuo et al., 1999; Lukiw, 2007). Researchers confirmed the vital roles of miRNAs in controlling fundamental biological processes, such as various kinds of metabolism pathway, development of organism, cell differentiation, proliferation, and apoptosis (Chen et al., 2006). Furthermore, accumulating evidence validated that miRNAs relate to a wide variety of human diseases, such as significant infections, cardiovascular disease, neurological and muscular disorders, and repression and progression of cancers (Xu et al., 2007; Yang et al.,

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2007). Therefore, downregulation and upregulation of miRNA expression are often associated with series of developments of human tissues and organs, and diseases including cancers, neuronal diseases, and vascular diseases (Table 15.1). The indispensable roles of miR-17-92 and miR-21 in cancer cell development have been verified using experimentations. miR-17-92 and miR-21 can intriguingly enhance lung and hepatocellular cancer growth and proliferation, respectively (Hayashita et al., 2005; He et al., 2005; O’Donnell et al., 2005; Meng et al., 2007). Besides, miR-155, miR-221 and miR-222 are also reported to promote cancer cell growth and proliferation (Eis et al., 2005; Galardi et al., 2007; Le Sage et al., 2007). Moreover, miR-106a and miR-106b-25 cluster are upregulated in colon cancer and human gastric cancers, respectively (Volinia et al., 2006; Datta et al., 2008; Figure 15.2). A research group found that increasing the expression of miR-146a in cervical cancer will promote cell growth and proliferation (Wang et al., 2008). Another report of oncogenic miRNAs is from Huang (2008) and colleagues, who elucidated that the miRNAs mir-373 and mir-520c can obviously promote breast cancer cell migration and invasion in vitro and in vivo (Huang et al., 2008; Figure 15.2). In contrast, some miRNAs have been confirmed to suppress the growth and proliferation of cancer cells or even promote their apoptosis. Overexpression of let-7 was reported to repress the growth and proliferation of lung cancer cells (Takamizawa et al., 2004; Johnson et al., 2007). In leukemic cells, miR-15 and miR-16 are found to be able to promote cancer cell apoptosis by suppressing a protein named antiapoptotic B cell lymphoma 2 (Bcl2) (Cimmino et al., 2005). Bommer et al. (2007) and Tazawa et al. (2007) had found that the overexpression of miR-34 family could inhibit growth of nonsmall cell lung cancer cells by downregulating a series of genes increase cell cycle progress. Also miR-34a acted as a tumor suppressor, could induce senescence-like growth arrest in human colon cancer cells (Bommer et al., 2007; Tazawa et al., 2007). MiR-181c has a distinct negative effect on glioma cells growth (Liu et al., 2007). Liu et al. (2009) reported that miR-34c, miR-145, and miR-142-5p were repressed in transgenic lung cancers. miRNA-223 and miR-1 have a distinct effect on inhibiting the proliferation of hepatocellular carcinoma (HCC) cell (Volinia et al., 2006; Wong et al., 2008). As Li et al. reported that overexpression of miR125b could obviously suppress the cell growth and phosphorylation of HCC cell line (Scott et al., 2007). Sohail et al. (2008) elucidated that miR-335 and miR-126 had been identified as metastasis suppressor miRNAs in human breast cancer. Yang et al. (2009) had showed that miR-373 and miR-520c had exerted their distinct downregulating effect in PCa. Wang (2008) had demonstrated that reducing the expression of miR-143 and miR-145 in cervical lesions and cervical cancer was inhibited in cervical cancer cell growth and proliferation (Wang et al., 2008). And Liu (2008) had showed that suppressing the expression of miR-27a was able to repress gastric cancer cell growth (Liu et al., 2008). With more and more miRNAs will be identified, a large number of therapeutic targets of cancer would spring up. Therefore, understanding of these cancer-associated miRNAs will give a deeper insight into developing effective therapies in the future.

15.2.3

MIRNA AND

REGENERATIVE MEDICINE

As studies have shown that the expression activities of miRNAs associates with activities of some oncogenes or tumor suppressors, interference by artificial miRNAs implies a potential therapy to conquer cancer. They could even be considered as excellent diagnostic or prognostic medicines. Moreover, some miRNAs function as tumor suppressors are useful in developing in vivo expression systems. Because miRNAs have most of identical function to siRNAs performing efforts to anticancer therapies, if successful, will provide effective tools for the delivery of miRNAs (Bumcrot et al., 2006).

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TABLE 15.1 Diverse Functions of miRNAs in Some Kinds of Diseases, Cancer, Metabolism Pathway, and Development of Tissues Categories Brain

Diseases Type Alzheimer’s Parkinson’s diseases Schizophrenia

Muscle

Skeletal muscle impairment

Cardiovascular

Cardiomyocyte apoptosis Coronary artery disease Cardiac hypertrophy and heart failure Proliferative vascular diseases Obesity

Immunity Metabolism

Immunity injury Diabetes Lipid metabolism

Cancer

Lung cancer Liver carcinomas

Breast tumor B-cell lymphomas B-cell leukemia

Prostate cancer (PCa) Testicular germ cell tumors Brain cancer Neuroblastoma Colorectal neoplasia PCa Thyroid anaplastic carcinomas

Cervical carcinogenesis

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miRNA miR-131 (Zhuo et al., 1999), miR-9, miR-124a, miR-125b, miR-128, miR-132, miR-219 (Lukiw, 2007) miR133b (Kim et al., 2007) miR-134 (Schratt et al., 2006), miR-130b (Shifman et al., 2002), let-7 (Perkins et al., 2007), miR-26b, -30e, -92, -24 (Burmistrova et al., 2007), miR-212, miR-106b (Nelson et al., 2006) miR-206 (Anderson et al., 2006), miR-181 (Chen et al., 2006), miR-1 (Chen et al., 2006; Zhao et al., 2005; Lim et al., 2005), miR-133 (Chen et al., 2006; Zhao et al., 2005) miR-1, miR-133 (Chen et al., 2006; Xu et al., 2007; Yang et al., 2007) miR-1 (Yang et al., 2007) miR-195 (Van Rooij et al., 2006) miR-21 (Ji et al., 2007) miR-14 (Xu et al., 2003), miR-122 (Kru Tzfeldt et al., 2005; Esau et al., 2006), miR-278 (Teleman et al., 2006a) miR-146a/b, miR-132, and miR-155 (Taganov et al., 2006) miR-375 (Poy et al., 2004), miR-9 (Plaisance et al., 2006) miR-14 (Xu et al., 2003), miR-278 (Teleman et al., 2006b), miR-122 (Esau et al., 2006) miR-17-92 (Hayashita et al., 2005), Let-7 (Takamizawa et al., 2004), miR-34a-c (Bommer et al., 2007) miR-122 (Kutay et al., 2006; Gramantieri et al., 2007), miR-21 (Meng et al., 2007), let-7c (Shah et al., 2007), miR-199a, miR-195, miR-200a, miR-125a (Murakami et al., 2006) miR-125b, miR-145, miR-155 (Iorio et al., 2005), miR-21 (Si et al., 2007), miR373, miR-520c (Huang et al., 2008) miR-155 (Eis et al., 2005), miR-17-19b (He et al., 2005) miR-142 (Lagos-Quintana et al., 2002), miR-17-92 (Hayashita et al., 2005; He et al., 2005; O’Donnell et al., 2005), miR-155 (Eis et al., 2005), miR-15, miR-16 (Cimmino et al., 2005), miR-221, miR-222 (Felli et al., 2005) miR-221, miR-222 (Galardi et al., 2007) miR-372, miR-373 (Voorhoeve et al., 2007) miR-181a-c,miR-181b (Ciafre et al., 2005) miR-34a (Welch et al.,,2007), miR-9, miR-125a, miR-125b (Laneve et al., 2007) miR-143, miR-145 (Micheael et al., 2003) miR-221, miR-222 (Galardi et al., 2007) miR-181a, miR-181c (He et al., 2005), miR-30d, miR-125b, miR-26a, miR-30a-5p (Visone et al., 2007), miR-222, miR-220, miR-213 miR-143, miR-145, miR-146a (Wang et al., 2008)

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let-7, miR-15, miR-16, miR-181c miR-34 family, miR-34c, miR-145, miR-143, miR-142-5p, miR-223, miR-1, miR125b

miR-17-92, miR-21, miR-155, miR-221, miR-222, miR-106a, miR-106b, miR373, miR-520c. Cancer cells

FIGURE 15.2

miR-335, miR-126, miR-373, miR-520c, miR-27a.

Downregulative (⊢) or upregulative (→) function of miRNAs in cancer cells.

Therefore, if miRNA expression data can be effectively applied to construct signature with clinical value, miRNAs have obvious advantages: they can be long-lived in vivo and very stable in vitro (Lim et al., 2005). Nowadays, miRNA biotechnology has been widely used in research on muscle development and function, neuropsychiatry, schizophrenia, chronic myeloid leukemia and cardiovascular diseases, and so on. According to their characteristics, Haseltine (2003) classified regenerative medicines into four categories: human substances (proteins and genes), cells and tissues (a form of tissue engineering), embryonic stem (ES) cells, and novel materials (Haseltine, 2003). However, very few of them have been functionally analyzed, especially functional foods and regenerative nutraceuticals. Growing needs in understanding functions of miRNAs and accumulating benefits in scientific researches and therapeutic applications using RNAi aroses dramatic development of artificial and natural miRNAs. However, further studies are still in need to elucidate whether miRNA-mediated therapy is an effective approach for the chemoprevention of human cancers. Several studies have established the potential application of miRNA-based therapy in diseases or even cancer. Jan Stenvang (2008) reported that locked nucleic acid (LNA)-mediated targeting of miR122 in mice effectively repressed the liver disease (Stenvang et al., 2008). The induction of cellular apoptosis by miR-15a and miR-16-1 in chronic lymphocytic leukemia (Cimmino et al., 2005), suppression of proliferation of cancer cells by let-7 (Takamizawa et al., 2004), and the reduced migration and invasion induced by miR-125 in breast cancer cells (Scott et al., 2007). Obviously, the utilization of the anti-miR-21 oligonucleotides to reduce the colangiocarcinoma cells with gemcitabine manifests that miRNA-based therapy can be effectively connected with chemotherapy (Galardi et al., 2007). Therefore, the above results show that there will be a promising prospect in disease or cancer therapy.

15.3

DISEASES ASSOCIATED WITH FUNCTIONAL FOODS AND NUTRACEUTICALS

There are many studies confirmed that the dietary agents could induce cell apoptosis, which is considered as a natural phenomenon that plays critical role in organisms development (Ahmad et al., 1997; Khan et al., 2007). Many diseases were associated with aberrantly regulated programmed cell death. This aberration will ultimately lead to inhibition of apoptosis and propagation of diseases such as cancer. By now, what we have known about apoptosis is that it occurs primarily through two well-recognized pathways in cells, one is the intrinsic, mitochondrial-mediated pathway and the other is the extrinsic, death receptor-mediated pathway (Khan et al., 2008). Many natural dietary components could induce apoptosis through different ways, but the exact mechanism is still poorly known. We infer that the dietary components might modulate apoptosis through impacts on protein expression and function, mRNA expression, and on the human genome, either directly or indirectly (Table 15.2).

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TABLE 15.2 Different Dietary Agents Play Roles in Apoptosis Dietary Agents Death receptor-mediated pathway

Luteolin Epigallocatechin-3-gallate (EGCG) Lupeol

Curcumin

3,30-Diindolylmethane Resveratrol Sanguinarine Mitochondrial-mediated pathway

Genistein Fisetin

Pomegranate Ginger constituents Delphinidin Capsaicin Allyl and benzyl isothiocyanates Lycopene Ellagitannins of pomegranate fruit Silibinin

Roles Sensitize both TRAIL-sensitive as well as TRAIL-resistant cancer cells to TRAIL-induced apoptosis (Shi et al., 2005) Inducing the expression of death-associated protein kinase 2, Fas ligand, Fas, and caspase-4 (Pan et al., 2007) Will cause a significant increase in the expression of Fas receptor with a significant increase in the expression of Fas-associated protein with death domain (FADD) protein (Saleem et al., 2005) Will induce apoptosis in human melanoma cells through a Fas receptor/caspase-8 pathway independent of p53 (Bush et al., 2001) Significant downregulation of the c-FLIP (Zhang et al., 2005) Can induce the clustering of Fas (Delmas et al., 2003) Can reduce protein levels of procaspase-3, Bcl-2, cIAP2 (Kim et al., 2008) Induce apoptosis through activation of caspase-9 and -3 and cytosolic release of cytochrome c (Ng et al., 2007) Induce poly(ADP-ribose) polymerase (PARP) cleavage, increase in Bax, decrease in Bcl-2 and modulation in the expressions of Bcl-2-family proteins (Khan et al., 2008) Cause an change in the protein expression of Bax and Bcl-2 (Malik et al., 2005) Induction of mitochondrial transmembrane potential alteration, cytochrome c release (Mori et al., 2006) Cause nuclear condensation and fragmentation, PARP cleavage, and loss of mitochondrial membrane (Lazze et al., 2004) Induce apoptosis involving reactive oxygen species generation, and activation of caspase-3 (Sanchez et al., 2006) Cause disruption of mitochondrial membrane potential, activation of multiple caspases (Zhang et al., 2003) Reduce mitochondrial transmembrane potential, induce apoptosis (Hantz et al., 2005) Cause induction of apoptosis via intrinsic pathway through Bcl-XL and caspase-9 and effector caspase-3 (Larrosa et al., 2006) Cause downregulation of survivin and an increase in p53 expression together with enhanced apoptosis in human bladder tumors (Schmidt et al., 2008)

To our astonishment, the dietary agents are highly selective to their targets. They could induce cell to death or to proliferation, according to the cell’s situation. Bickford P. C. (2006) had demonstrated that many matural compounds in blueberry, green tea, catechin, carnosine, and vitamin D3 could promote the development of human bone marrow more obviously when compared with other stimulating factor, such as human granulocyte macrophage-colony simulating factor (hGM-CSF), and the combination of these nutrients showed effect on proliferating human hematopoietic progenitors (Bickford et al., 2006). Although the mechanism of the process has not clarified, these components have showed great potential as regenerative medicine to stimulate endogenous stem cell to promote

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Biotechnology in Functional Foods and Nutraceuticals Nutritional genomics and proteomics Genomic DNA

Epigenomic CH3-DNA

Post-translational mRNA Proteins

Bioactive food component

Enzymes, structural, transport, signaling caused by phosphorylation, glycosylation, etc.

FIGURE 15.3 Influence of bioactive food components on genetic, epigenetic, and proteomic events. A host of bioactive food components are known to influence one or more stages of this process (Milner, 2004).

healing. Thus, elucidation of events associated with both apoptosis and carcinogenesis provides the opportunity for dietary intervention with the plethora of bioactive components in the diet. In short, we could refer that dietary components must be possibly play roles in cell through modulating the expression of miRNA (Figures 15.3 and 15.4).

15.4 PERSPECTIVE Functional foods have been considered as those whole, fortified, enriched, or enhanced foods containing some healthy elements (e.g., vitamins and minerals). When consumed at an efficient level as part of a varied diet on a regular basis, these foods could improve health. Also, nutraceutical, which is any substance that is a food or a part of a food, provides medical or health benefits, including maintenance of the stability of metabolism, the prevention and treatment of disease. As some reports showed that with the change of metabolism, several alterations in some nutrient-related genes would follow, thus the expression of associated miRNAs would be regulated (Chen et al., 2006; Rist, 2006). Abnormal upregulation or downregulation of miRNAs contributes more or less to some serious diseases or cancer, such as significant infections, cardiovascular disease, and neurological and muscular disorders, as well as liver or lung cancers, and so on. Therefore, reasonable and scientific application of functional foods and nutraceutical will keep our body in a homeostasis state physiologically and psychologically, thus reduce the incidence of some diseases or cancer. In general, fully integrating the advantages of both miRNA biotechnology and functional foods have a bright application prospect in the future.

Nutrigenetics Nutritional epigenomics Bioactive food components

DNA

mRNA

Nutritional transcriptomics Proteomics

Metabolomics

miRNA

Protein

Metabolite

FIGURE 15.4 Nutrigenetics, nutritional transcriptomics, nutritional epigenomics, proteomics, and metabolomics are necessary to understand the role of nutrition in carcinogenesis.

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Functional foods

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Nutraceutical In vitro

Absorption

Metabolism pathway Cytoplasm

Homeostasis Lipid

Amino acid

Glucose

Nutrient genes miRNA Homeostasis

Nucleus Diseases

FIGURE 15.5

Health

The relationship among functional foods, metabolism, miRNA, diseases, and health.

In 1984, a proposal was put forward for discriminating functional foods from ordinary foods and medicines or pharmaceuticals, and confirming the role of functional foods in keeping the homeostasis of our bodies. We absorb nutrients from foods every day to maintain our health; even completely healthy boys and girls must do that, since otherwise they are unable to lead healthy lives. Generally speaking, if some reason make us become unhealthy, and abnormal, or even sick, it is necessary to take medicines for the purpose of treating the disease. However, a recent medical concept suggests that there is a semihealthy state between healthy and sick (Figure 15.5). Therefore, we can reasonably choose a number of nutritional products (functional foods or nutraceuticals) to adjust the homeostasis of our bodies, and to maintain health (Figure 15.5).

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Production 16 Microbial of Organic Acids and Its Improvement by Genome Shuffling Takashi Yamada CONTENTS 16.1 Introduction .......................................................................................................................... 265 16.2 Major Organic Acids Produced by Fermentation .................................................................266 16.2.1 Citric Acid.................................................................................................................266 16.2.2 Gluconic Acid ...........................................................................................................266 16.2.3 Acetic Acid ............................................................................................................... 268 16.2.4 Propionic Acid .......................................................................................................... 268 16.3 Improved Acid Tolerance by Genome Shuffling .................................................................. 268 16.3.1 Principle of Genome Shuffling ................................................................................. 268 16.3.2 Genome Shuffling of Lactobacillus for Improved Acid Tolerance .......................... 269 16.4 Microbial Production of Hydroxycitric Acid (HCA) ............................................................ 270 16.4.1 HCA: Plant-occurring Organic Acid ........................................................................ 270 16.4.2 Bioactivities of Natural HCA.................................................................................... 271 16.4.3 Screening of Microorganisms That Produce HCA .................................................. 272 16.4.4 Improved HCA Production by Genome Shuffling ................................................... 272 16.5 Future Perspectives ............................................................................................................... 274 Acknowledgments.......................................................................................................................... 274 References ...................................................................................................................................... 274

16.1

INTRODUCTION

Acetic acid and lactic acid were produced and used in food preparation processes, prior to the knowledge that microorganisms are involved in the production of these acids. When sugar sources were left to ferment into alcohol by nonindustrialized methods, acidification occurred easily. Moreover, the generation of lactic acid was already known through the acidification of milk, and the technology of lactic acid fermentation had been applied to pickling vegetables in the Orient. Techniques for the culture of microorganisms advanced through the nineteenth century up to the beginning of the twentieth century, when microbiological research accelerated. As a result of decades of research, the number of identified microbial organic acids has increased, and organic acid producers have been characterized. The industrial fermentation processes for several organic acids, including citric acid, gluconic acid, and lactic acid, have been firmly established. Moreover, the biosynthetic mechanisms for these acids have been elucidated. In the twenty-first century, innovative research is expected to advance 265 © 2010 Taylor and Francis Group, LLC

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biotechnological production processes. The interest in functional elements in foods has increased in recent years. This chapter includes a summary of the major organic acids that are produced by fermentation, and discusses the biosynthesis and application of organic acids and the microorganisms that produce them. A pioneering study on genome shuffling for the improvement of acid tolerance is introduced, a technology which is potentially powerful for the rapid improvement of complex phenotypes such as acid tolerance. Mutant acid-tolerant strains reveal improved acid production. The author also demonstrates the discovery of microorganisms that produce an organic acid with unique physiological properties, occurring in plants, which are promising food additives. The production of this organic acid has been improved by genome shuffling. Finally, the future perspectives for improved microbial production of organic acids are discussed.

16.2 MAJOR ORGANIC ACIDS PRODUCED BY FERMENTATION 16.2.1

CITRIC ACID

Citric acid is an intermediate of the tricarboxylic acid (TCA) cycle. Citric acid is enriched in citrus fruits, such as lemon and oranges, and used to be extracted from them. Several fungi accumulate citric acid. Citric acid fermentation was discovered in a culture of Penicillium glaucum on sugar medium in 1893 (Wehmer, 1893). Subsequently, numerous strains of Aspergillus were found to accumulate citric acid at highly acidic conditions (pH 2.5–3.5) (Currie, 1917). This discovery overcame contamination problems and enabled the industrial production of citric acid, and subsequently citric acid producers with higher yield were screened. The improvement of the strains has been achieved by mutagenesis, followed by the selection of an improved mutant known as classical strain improvement (CSI) (Grewal and Kalra, 1995; Haq, 2001; Soccol et al., 2006). There have been many other attempts to optimize citric acid production with respect to substrates and the fermentation processes (Mattey, 1992; Soccol et al., 2006; Papagianni, 2007). Commercial production of citric acid utilizes Aspergillus niger in an industrial-scale submerged fermentation process (Mattey, 1992). Bulk production of citric acid by fermentation exceeded 1.4 million tons per year worldwide in 2004 and continues to increase each year. Because of the general recognition of its safety to humans, a pleasant acid taste, high water solubility, and chelating and buffering properties, citric acid is frequently added to beverages (soft drinks, syrups, wines, and ciders), to foods (jellies, jams, preservatives, ice creams, processed cheese, and candies), to pharmaceuticals as an effervescent agent in powders and tablets, and to cosmetics and toiletries as a buffering agent (Milsom and Meers, 1985a; Soccol et al., 2006). Citric acid is generated via the condensation reaction of citrate synthase between oxaloacetate and acetyl-CoA, both of which are converted from pyruvic acid (Figure 16.1a). Although many studies have examined the mechanism of citric acid accumulation in large quantities with regard to carbon utilization, the biosynthesis of citric acid, its excretion from mitochondria, its transport from cytosol to the external medium, and its metabolic regulation, many questions remain unanswered (Karaffa and Kubicek, 2003). Genome sequence analysis demonstrates that Aspergillus niger contains several genes encoding oxaloacetate biosynthesis, two pyruvate carboxylases and four malate dehydrogenases. Moreover, Aspergillus niger possesses one cytosolic citrate synthase and three putative mitochondrial citrate synthases. Similar redundancy is found in aconitases. Gene duplications are thought to be important for the efficient production of citric acid by Aspergillus niger (Pel et al., 2007). Historically, the development of a submerged process for citric acid production was important because it was the first aerobic fermentation. This technology has been applied to the fermentation of antibiotics, including penicillin.

16.2.2 GLUCONIC ACID Gluconic acid is found in honey, fruit juice, and wine. It exists in equilibrium with the cyclic ester glucono-δ-lactone in aqueous solution. Gluconic acid and its lactone have been utilized as naturally

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Glucose Glycolytic pathway Pyruvate decarboxylase

O H3C

C

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Pyruvic acid

COOH

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COOH H

COOH

COOH

Citric acid

Isocitric acid

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(b) H

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Glucose dehydrogenase

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Alcohol dehydrogenase

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Acetic acid

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O

COOH

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COOH

CH2

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Succinyl-CoA

FIGURE 16.1 Schematic representation of metabolic pathways of microbial organic acids. (a) Citric acid, (b) gluconic acid, (c) acetic acid, (d) propionic acid. Solid arrow represents one enzyme reaction and dashed arrow indicates multiple reactions catalyzed by several different enzymes. Organic acids are anion form in solution at physiological pH.

occurring food additives (Ramachandran et al., 2006), and they are identified as generally recognized as safe (GRAS) substances by the U.S. Food and Drug Administration (2004). About 60,000 tons of gluconic acid and its salts are produced worldwide annually. Glucono-δ-lactone is used in baking powders for use in dry cake. Calcium gluconate is used to treat calcium deficiency in humans due to its chelating properties. Sodium gluconate is used in the cleansing of glassware © 2010 Taylor and Francis Group, LLC

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and in metal cleaning to remove rust (Ramachandran et al., 2006). Interestingly, gluconic acid was found to increase the number of Bifidobacterium in human feces, suggesting that it is a promising prebiotic (Asano et al., 1994). Industrial fermentation has been performed mainly with Aspergillus niger in a submerged process and the yield is close to the theoretical one (Milsom and Meers, 1985b). Acetic acid-producing bacteria (e.g., Gluconobacter oxydans) are also effective producers of gluconic acid (Gupta et al., 2001; Ramachandran et al., 2006). The Aspergillus niger genome contains one intracellular and three secreted glucose oxidase genes. Moreover, it has 11 catalase genes catalyzing hydrogen peroxide generated by glucose oxidase to water and O2. Two gluconic acid-specific kinases are present, suggesting that the catabolism of gluconic acid proceeds via the phosphorylation of 6-phosphogluconic acid (Pel et al., 2007). Gluconobacter oxydans incompletely oxidizes carbohydrates, including sugar, in a process that is referred to as oxidative fermentation (Gupta et al., 2001). Gluconic acid is produced from glucose by glucose dehydrogenase bound to its cytoplasmic membrane (Figure 16.1b; Goodwin and Anthony, 1998). It is excreted and subsequently accumulates in the extracellular space, leading to a decrease in pH, thereby preventing the propagation of many other microorganisms. The genome sequence indicates that this bacterium has a simple respiratory chain. Because of the limited proton-translocating abilities, the incomplete oxidation process is thought to be accelerated in this bacterium (Prust et al., 2005).

16.2.3

ACETIC ACID

Acetic acid is produced mainly by chemical synthesis or dry distillation; however, vinegar is produced through aerobic fermentation by acetic acid bacteria, the genera of Acetobacter and Gluconobacter (Matsushita et al., 1994). In the fermentation process, acetic acid is converted from ethanol as a substrate by cytoplasmic membrane-bound pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase and quinine-dependent aldehyde dehydrogenase (Figure 16.1c; Gupta et al., 2001; Matsushita et al., 2002). Acetobacter is different from Gluconobacter in its ability to further oxidize acetic acid to form carbon dioxide; Acetobacter has a complete citric acid cycle but Gluconobacter does not (Matsushita et al., 1994; Prust et al., 2005). Moreover, Acetobacter species is more tolerant to acetic acid than Gluconobacter species, explaining why the productivity of acetic acid is higher with Acetobacter species than Gluconobacter species (Matsushita et al., 1994).

16.2.4

PROPIONIC ACID

Propionic acid and its salts are used as antibiotic food additives and precursors of flavor (Huitson, 1968; Herting et al., 1974; Leuck, 1980; Boyaval and Corre, 1995). Propionibacterium produces propionic acid, and the fermentation is important for giving flavor to fermented foods. When Emmental cheese is produced, Propionibacterium ferments lactate to form acetate, propionate, and carbon dioxide, and this fermentation is necessary for forming the scent and holes (termed as cheese eyes) by carbon dioxide (Figure 16.1d; Hettinga and Reinbold, 1972; Playne, 1985). The growth of Propionibacterium is slow, and it usually takes 1–2 weeks for the batch culture. The yield of propionic acid is low because of its inhibitory effect on bacterial growth. Moreover, the purification of propionic acid is expensive and requires its separation from by-products, including acetic acid. Hence, the chemical synthesis is superior to fermentation, but the natural process is desired for food use (Playne, 1985; Boyaval and Corre, 1995).

16.3 IMPROVED ACID TOLERANCE BY GENOME SHUFFLING 16.3.1

PRINCIPLE OF GENOME SHUFFLING

First-generation microbial strains were screened from natural isolates in a laboratory that falls short of the commercial performance of industrial-scale fermentation. Common limitations include low

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yields and poor tolerance to process conditions. Hence, efforts towards strain improvement were made to meet commercial requirements. With respect to organic acid fermentation, the biosynthesis and excretion of organic acids, tolerance to low pH, and cell viability are thought to influence the yield of organic acids. However, information on the detailed mechanism is still limited, as discussed for citric acid fermentation. Sophisticated metabolic engineering is not practical to improve a poorly understood and complex phenotype such as the overproduction of organic acids in genetically uncharacterized strains. Thus, the improvement of industrial microorganisms that produce organic acids has been traditionally accomplished by CSI, sequential random mutagenesis, and screening. In this process, the wild-type strain is treated with mutagens, and one subtly improved mutant is selected among progenies with genetic diversity. This mutant is subsequently subjected to the next round of mutagenesis and screening. Thus, CSI is a sequential process of accumulating individual mutations. CSI is robust but, unfortunately, very time and resource intensive. Additionally, deleterious mutations are also accumulated (Yamada et al., 2007a, 2007b). Genome shuffling is a novel approach for addressing this issue (Zhang et al., 2002). In this process, improved mutants are obtained by mutagenesis or chemostat-mediated adaptation. The pooled population with genetic diversity is shuffled by homologous recombination. Among various methods for recombination between bacterial cells, protoplast fusion is broadly applicable. Since the population of the improved mutants includes a pool of useful genetic diversity, the shuffling of the population generates a new combination of mutant progenies with greater performance. Improved progenies are selected and repeatedly subjected to the next round of shuffling. Genome shuffling thus accelerates directed evolution through recursive recombination of improved progeny (Figure 16.2a and b). In the pioneering study of genome shuffling, a single fusion resulted in the efficient production of two-, three-, and four-marker progenies, whereas recursive fusion markedly improved the generation of the progenies in complex progeny in the model actinomycete, Streptomyces coelicolor. Moreover, the increased production of the polyketide antibiotic tylosin was achieved in Streptomyces fradiae by genome shuffling, where two rounds of genome shuffling (1 year, 24,000 assays) were sufficient to achieve results that had previously required 20 rounds of CSI (20 years, 1,000,000 assays) (Zhang et al., 2002). Given that genome shuffling relies on homologous recombination, the obtained mutant population is not considered a genetically modified organism.

16.3.2

GENOME SHUFFLING OF LACTOBACILLUS FOR IMPROVED ACID TOLERANCE

Genome shuffling was applied to a poorly characterized industrial strain of Lactobacillus in order to improve acid tolerance. The improved acid-tolerant strains successfully exhibited improved lactic acid production (Patnaik et al., 2002). Lactic acid is added to beverages and foods as a pH-adjusting ingredient and a preservative (Vickroy, 1985). Its calcium salt is used to treat calcium deficiencies in humans (Allen, 1982). Lactic acid fermentation occurs at a minimum pH of 5.0–5.5. Thus, fermentation at or below the pKa of lactic acid (~3.8) is preferred. At this low pH, the free lactic acid is predominant, facilitating a purification of lactic acid by direct extraction with organic solvent. Therefore, acid tolerance is an industrially important phenotype to reduce the waste and cost of product purification. In the genome shuffling study, two classical methods were used to generate populations of acid-tolerant variations of wild-type Lactobacillus (LB-WT). The first population (pop-adap) was obtained by chemostat-mediated adaptation of LB-WT. It was achieved by slowly decreasing the fermentation pH from 6.0 to 4.1 over 1200 h. The second population (pop-NTG) was generated by using nitrosoguanidine (NTG) and was enriched on pH gradient plates. Pop-adap and pop-NTG populations colonized a more acidic region of the plate than that survived by LB-WT on pH gradient plates. Two subpopulations, pop1 and pop2, were isolated from pop-adap and popNTG cultures on pH gradient plates. Subsequently, the two pH-tolerant populations were shuffled by means of five rounds of recursive pool-wise protoplast fusion. Strains after the fifth rounds of shuffling grew at lower pH than LB-WT and showed 4-fold higher density and produced 3-fold more lactic acid than the wild-type strain at pH 4.0 (Patnaik et al., 2002). This approach has the

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Fusion

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Improved Protoplasts mutants obtained by mutagen or chemostat adaptation (b) Classical strain improvement

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FIGURE 16.2 Schematic representation of genome shuffling. (a) Homologous recombination of improved mutants by protoplast fusion, (b) classical strain improvement versus genome shuffling.

potential to facilitate metabolic engineering and to accord a nonrecombinant alternative to the rapid production of improved organisms.

16.4 MICROBIAL PRODUCTION OF HYDROXYCITRIC ACID (HCA) 16.4.1 HCA: PLANT-OCCURRING ORGANIC ACID Interest in functional food additives and dietary supplements for disease prevention has increased. To respond to this need, an expansion of the chemical library, evaluation of biological activities,

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and efficient production of chemicals are required. Tropical plants are a rich source of valuable secondary metabolites. Some alkaloids, terpenoids, and polyphenols are useful as medicines and food additives. HCA is an organic acid that is found in limited numbers of semitropical and tropical plant species and which possesses unique physiological properties (Jena et al., 2002; Yamada et al., 2007a). HCA has a hydroxyl group at the second carbon of citric acid. As shown in Figure 16.3, it has four stereoisomers, (2S, 3S), (2R, 3R), (2S, 3R), and (2R, 3S), and their lactone forms. Each of the stereoisomers can form a lactone ring and, in general solution, HCA is a mixture of nonlactone and lactone forms (Yamada et al., 2007a). Of these, (2S, 3R)-HCA is concentrated at the calyxes of Hibiscus subdariffa and Hibiscus rosa-sinensis, which are cultivated in several tropical and semitropical countries. The dried flowers of Hibiscus subdariffa are used to make herbal tea (Hibiscus, Roselle, Jamaica, Karkade) in many areas. Roselle juice is a popular beverage and herbal medicine (Griebel, 1939), whereas (2S, 3S)-HCA is a major acid component in the fruit rinds of Garcinia species, including Garcinia cambogia, Garcinia indica, and Garcinia atroviridis, which grow prolifically on the Indian subcontinent and in western Sri Lanka (Lewis and Neelakantan, 1965; Jena et al., 2002). The rinds are traditionally used in combination with salt for curing fish in south and southeastern Asia. Extracts of Garcinia fruits have become popular worldwide as an ingredient in diet supplements. The other stereoisomers, (2R, 3R) and (2R, 3S)-HCA, have not been isolated from natural sources (Yamada et al., 2007a).

16.4.2

BIOACTIVITIES OF NATURAL HCA

Hibiscus-type (2S, 3R)-HCA lactone inhibits porcine pancreatic α-amylase and small intestine α-glucosidase (Hansawasdi et al., 2000). In a human cell model system, it reduced carbohydrate digestion (Hansawasdi et al., 2001). Administration of these α-amylase and α-glucosidase inhibitors suppresses increases in blood glucose and insulin levels (Jenkins et al., 1981; Tattersall, 1993; Bischoff, 1994). Acarbose, an α-glucosidase inhibitor, is widely used as a therapeutic agent for diabetes treatment; however, it must be used with care because of its side effects. Therefore, natural Hibiscus HCA may be a possible safe food additive for helping Garcinia-type diastereomer COOH HO HO H

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COOH

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HOOC O

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(Hibiscus) (Streptomyces sp. U121)

FIGURE 16.3 Structure of HCA stereoisomers. HCA has four stereoisomers, (2S, 3S), (2R, 3R), (2S, 3R), and (2R, 3S), and their lactone forms. Garcinia cambogia produces (2S, 3S)-HCA. Hibiscus subdariffa and Streptomyces sp. U121 produce (2S, 3R)-HCA.

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prevent diabetes and obesity. Garcinia-type (2S, 3S)-HCA is a competitive inhibitor of mammalian adenosine-5′-triphosphate (ATP) citrate lyase, which catalyses the extramitochondrial fission of citrate to oxaloacetate and acetyl-CoA. The subsequent decrease in acetyl-CoA represses fatty acid synthesis lipogenesis (Watson et al., 1969; Lowenstein, 1971; Sullivan et al., 1974, 1977; Lowenstein and Brunengraber, 1981; Rao and Sakariah, 1988). A novel calcium– potassium salt of Garcinia HCA (HCA-SX) with improved bioavailability was shown to cause body weight reduction in rats but no significant toxicological changes under 90 days repeated dose toxicity test, suggesting HCA-SX is a safe dietary supplement (Ohia et al., 2002; Shara et al., 2003, 2004; Preuss et al., 2004; Roy et al., 2004).

16.4.3

SCREENING OF MICROORGANISMS THAT PRODUCE HCA

The availability of HCA is limited by the restricted habitat of its source plants and the difficulty of stereoselective organic synthesis due to the presence of two asymmetric carbons in the molecule. Additionally, the biosynthetic mechanism remains unknown in plants. Hence, the author attempted to screen microorganisms to find an alternative source of bulk optically pure HCA (Hida et al., 2005). The bulk production of citric acid by a fungus, Aspergillus niger, has been established as described above. Additionally, tartaric acid can be fermented by Gluconobacter suboxydans (Kotera et al., 1972). The success of microbial production of structural analogs of HCA gave us the idea of producing natural HCA from microorganisms. As a result of culture tube assays of more than 2000 microbial strains, two strains, Streptomyces sp. U121 and Bacillus megaterium G45C, had metabolites with retention times similar to those of authentic HCA on high performance liquid chromatography (HPLC). These metabolites were purified from their culture broths after transformation to methyl esters, followed by column chromatography on silica gel. Purified products from both strains were found to be the Hibiscus-type HCA diastereomer by electrospray ionization mass spectrometry (ESI-MS) and 1H-nuclear magnetic resonance (NMR) studies, but the absolute configuration could not be identified (Hida et al., 2005). Thus, optical resolution for synthetic (2S, 3R)- and (2R, 3S)-HCA methyl ester enantiomers was developed by using an HPLC system with a chiral column. The results clearly indicated that both strains of microbes synthesize optically pure Hibiscus-type (2S, 3R)-HCA (Hida, et al., 2006).

16.4.4 IMPROVED HCA PRODUCTION BY GENOME SHUFFLING Streptomyces sp. U121 yielded only 30 mg/L (2S, 3R)-HCA, and all attempts to improve the yield by fermentation technologies, including altering the media composition, were unsuccessful. Hence, genome shuffling was applied to this strain to achieve a rapid improvement in HCA production (Hida et al., 2007), because this method can be applied to improving the yield of most metabolic pathways for particular chemicals and for modifying other complex phenotypes in uncharacterized bacteria such as Streptomyces sp. U121, in which the details of the HCA biosynthetic pathway and its regulatory mechanisms remain unclear. First, spores were treated with mutagens; however, the initial mutagenesis and subsequent genome shuffling were quite inefficient. Hence, trans-epoxyaconitic acid (EAA, Figure 16.4a) was utilized. EAA is an analog of HCA, and it is more antibiotic than HCA at a given concentration because it contains an epoxide moiety. The use of EAA for screening agar medium yielded mutant strains with slightly higher levels of HCA production than the wild-type strain. Furthermore, the higher concentration of EAA inhibited the regeneration of nonfused protoplasts. When protoplast fusion was performed among the improved population for genome shuffling, colonies appeared even in the presence of EAA. These results suggest that mutant protoplasts with enhanced EAA resistance were formed by fusion and then selectively regenerated (Figure 16.4a). Mutant population with enhanced EAA resistance produced significantly higher levels of HCA. Selected mutants after three rounds of shuffling (totally 500 assays) showed 5–6-fold increased HCA production as well as increased cell growth. Mutant

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(a) Parent Streptomyces sp. U121 (Spores) NTG Fusion Subtle improved mutant population

(Protoplasts)

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No colony (Non-fused protoplast eliminated)

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EAA-resistant Streptomyces sp. U121 cell EAA

HCA

Biosynthesis Excretion? Putative transporter EAA

EAA HCA

HCA

HCA

HCA

FIGURE 16.4 Genome shuffling of Streptomyces sp. U121 for improved production of HCA. (a) Efficient screening of a mutant library using trans-epoxyacetic acid (EAA), an antibiotic analog of HCA in genome shuffling. (b) Possible mechanism of improved HCA production in EAA-resistant Streptomyces sp. U121 cells.

EAA-resistant strains did not have EAA hydrolytic activity, so EAA-resistant strains may have had enhanced EAA-excretion activity, leading to HCA resistance. Enhanced HCA resistance contributed to enhanced cell growth in the presence of HCA, and the overall improved production of HCA was achieved in the culture system (Figure 16.4b). Consequently, the isolation of mutants resistant to the antibiotic analog EAA contributed to the achievement of an improvement in HCA production in this bacterium in a reasonably short period of time. These results suggest that an efficient screening system is critical to obtaining an improved mutant population by genome shuffling (Hida et al., 2007; Yamada et al., 2007b).

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16.5 FUTURE PERSPECTIVES This chapter presents an overview of organic acid fermentation, genome shuffling of bacteria with improved acid tolerance, discovery of a novel microbial organic acid, and a subsequent improvement in its production by genome shuffling. Organic acids are contained in fermented foods, and human beings have a long history of ingesting them via these foods. In general, organic acids prevent foods from decomposition and are safe for human consumption. The usage of organic acids will increase if their beneficial physiological properties for human health are better understood. It is not surprising that two bacterial strains that produce HCA were newly discovered. As described above, previous research has focused on organic acids that accumulate in large amounts in culture media. If analytical technology with higher sensitivity is applied, still unknown organic acids could be discovered from microorganisms as organic acids are potential sources of nutraceuticals. Industrial organic acid fermentation of citric acid and gluconic acid has a high yield that surpasses the chemical syntheses. The industrial processes were established after successive efforts for several decades. Hence, genome shuffling might be useful to improve the production of a newly discovered organic acid in the near future. The establishment of an efficient screening system that selects improved mutants is important. Acid tolerance can be used to improve organic acid productivity; however, the specific mutations that have been introduced in the improved mutants are only poorly understood. The organic acid transporter is one possible target. However, mutations probably occur throughout the genome. Recent advances in genome-wide studies on microorganisms could enable us to identify the critical mutations. Such information could provide new insights into the metabolic flow and cellular physiology of microorganisms. Additionally, fermentative production of organic acids from renewable resources has recently been advanced. It is expected that organic acids will become promising building-block chemicals to produce materials such as polymers in the chemical industry (Tsao et al., 1999; Lee et al., 2004; Song and Lee, 2006). Likewise, organic acids could become precursors of important chemicals in the food and pharmaceutical industries. In summary, the microbial production of a unique organic acid and its rapid improvement is a promising method for developing functional foods and nutraceuticals.

ACKNOWLEDGMENTS Screening and genome shuffling of HCA producers were performed with Drs H. Hida, and Y. Yamada. This work was in part supported by Iwata Chemical Co., Ltd., and the High-Tech Research Center Project for Private Universities on the Evolution of Green Science for Quality Improvement of Environmental and Health, matching a fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science, and Technology), 2004–2008.

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Prust, C., Hoffmeister, M., Liesegang, H., Wiezer, A., Fricke, W.F., Ehrenreich, A., Gottschalk, G., and Deppenmeier, U., 2005. Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. Nat. Biotechnol. 23: 195–200. Ramachandran, S., Fontanille, P., Pandey, A., and Larroche, C., 2006. Gluconic acid: Properties, applications and microbial production. Food Technol. Biotechnol. 44: 185–195. Rao, R.N. and Sakariah, K.K., 1988. Lipid-lowering and antiobesity effect of (−)-hydroxycitric acid. Nutr. Res. 8: 209–212. Roy, S., Rink, C., Khanna, S., Phillips, C., Bagchi, D., Bagchi, M., and Sen, C.K., 2004. Body weight and abdominal fat gene expression profile in response to a novel hydroxycitric acid-based dietary supplement. Gene Expr. 11: 251–262. Shara, M., Ohia, S.E., Schmidt, R.E., Yasmin, T., Zardetto-Smith, A., Kincaid, A., Bagchi, M., Chatterjee, A., Bagchi, D., and Stohs, S.J., 2004. Physico-chemical properties of a novel (−)-hydroxycitric acid extract and its effect on body weight, selected organ weights, hepatic lipid peroxidation and DNA fragmentation, hematology and clinical chemistry, and histopathological changes over a period of 90 days. Mol. Cell. Biochem. 260: 171–186. Shara, M., Ohia, S.E., Yasmin, T., Zardetto-Smith, A., Kincaid, A., Bagchi, M., Chatterjee, A., Bagchi, D., and Stohs, S.J., 2003. Dose- and time-dependent effects of a novel (−)-hydroxycitric acid extract on body weight, hepatic and testicular lipid peroxidation, DNA fragmentation and histopathological data over a period of 90 days. Mol. Cell. Biochem. 254: 339–346. Soccol, C.R., Vandenberghe, L.P.S., Rodrigues, C., and Pandey, A., 2006. New perspectives for citric acid production and application. Food Technol. Biotechnol. 44: 141–149. Song, H. and Lee, S.Y., 2006. Production of succinic acid by bacterial fermentation. Enzyme Microb. Technol. 39: 352–361. Sullivan, A.C., Singh, M., Srere, P.A., and Glusker, J.P., 1977. Reactivity and inhibitor potential of hydroxycitrate isomers with citrate synthase, citrate lyase, and ATP citrate lyase. J. Biol. Chem. 252: 7583–7590. Sullivan, A.C., Triscari, J., Hamilton, J.G., and Miller, O.N., 1974. Effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat: II. Appetite. Lipids 9: 129–134. Tattersall, R., 1993. α-Glucosidase inhibition as an adjunct to the treatment of type 1 diabetes. Diabetic Med. 10: 688–693. Tsao, G.T., Cao, N.J., Du, J., and Gong, C.S., 1999. Production of multifunctional organic acids from renewable resources. Adv. Biochem. Eng. Biotechnol. 65: 243–280. U.S. Food and Drug Administration., 2004. Agency Response Letter GRAS Notice No. GRN 000136. Available at http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/ GRASListings/ucm153958.htm Vickroy, T.B., 1985. Lactic acid. In: M. Moo-Young (Ed.), Comprehensive Biotechnology, Vol. 3, pp. 761–776. Oxford: Pergamon Press. Watson, J.A., Fang, M., and Lowenstein, J.M., 1969. Tricarballylate and hydroxycitrate: Substrate and inhibitor of ATP: Citrate oxaloacetate lyase. Arch. Biochem. Biophys. 135: 209–217. Wehmer, C., 1893. Note sur la fermentation Citrique. Bull. Soc. Chem. Fr. 9: 728. Yamada, T., Hida, H., and Yamada, Y., 2007a. Chemistry, physiological properties, and microbial production of citric acid. Appl. Microbiol. Biotechnol. 75: 977–982. Yamada, T., Hida, H., and Yamada, Y., 2007b. Isolation of hydroxycitric acid-producing Streptomyces sp. U121 and generation of improved mutants by genome shuffling. Actinomycetologica 21: 40–45. Zhang, Y.X., Perry, K., Vinci, V.A., Powell, K., Stemmer, W.P.C., and del Cardayré, S.B., 2002. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415: 644–646.

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Part III New Frontier in Food Manufacturing Process

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Biotechnology 17 Microalgal in the Production of Nutraceuticals Niels-Henrik Norsker, Maria Barbosa, and René Wijffels CONTENTS 17.1 Microalgae in the Human Food Chain .................................................................................280 17.1.1 Scale of Microalgal Nutraceutical Production ......................................................... 281 17.1.2 Production Costs of Microalgal Biomass ................................................................. 283 17.1.3 Microbiological Food Quality Assurance ................................................................ 283 17.1.4 Health Concerns with Microalgal Products ............................................................. 285 17.1.5 Quality by Self-Regulation ....................................................................................... 285 17.2 Lipid Production with Microalgae ........................................................................................ 286 17.2.1 Lipid Contents........................................................................................................... 286 17.2.2 Lipid Classes ............................................................................................................. 287 17.2.3 Modification of Lipid Contents by Growth Conditions ............................................ 287 17.2.4 Yield of Lipids in Autotrophic Microalgal Productions ........................................... 287 17.3 Carotenoid Production with Microalgae .............................................................................. 287 17.3.1 Chemical Nature and Biological Function of Carotenoids ....................................... 288 17.3.2 Biological Function of Carotenoids .......................................................................... 290 17.3.3 Secondary Carotenoids ............................................................................................. 290 17.3.4 β-Carotene ................................................................................................................ 291 17.3.5 Induction of β-Carotene Formation .......................................................................... 291 17.3.6 Astaxanthin............................................................................................................... 291 17.3.7 Induction of Astaxanthin Formation ........................................................................ 292 17.3.8 Biomass Productivity in Natural Daylight ................................................................ 293 17.3.9 Astaxanthin Formation and Irradiation .................................................................... 293 17.3.10 Industrial Haematococcus Production .................................................................... 294 17.4 Carbohydrates ....................................................................................................................... 294 17.4.1 Chlorella Growth Factor........................................................................................... 294 17.5 Microalgal Production Methods: Autotrophic, Heterotrophic, Mixotrophic ....................... 294 17.5.1 Autotrophic Growth .................................................................................................. 295 17.5.2 Efficiency of the Photosynthesis ............................................................................... 297 17.5.3 Growth Rate and Light Intensity .............................................................................. 297 17.5.4 Heterotrophic Microalgal Productivity..................................................................... 299 17.5.5 Biomass Production Systems .................................................................................... 299 17.5.6 Open Ponds ...............................................................................................................300 17.5.7 Photobioreactors ....................................................................................................... 301 17.5.8 Tubular Reactors ....................................................................................................... 302 17.5.9 Flat-Panel Reactors ................................................................................................... 305 17.5.10 Bubble Column and Air-Lift Reactors .................................................................... 305 279 © 2010 Taylor and Francis Group, LLC

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17.5.11 Other Types of Photobioreactors ............................................................................306 17.5.12 Fermenters ..............................................................................................................306 17.6 Future Development Prospects in Microalgal Nutraceuticals Production ...........................307 References ......................................................................................................................................307

17.1

MICROALGAE IN THE HUMAN FOOD CHAIN

The subject of this chapter is research and applied aspects of microalgal biotechnology in the production of nutraceuticals. Nutraceuticals, defined as food supplements and functional foods, currently produced and marketed from microalgal biomass, comprise a rather short range of carotenoids, fatty acids, and wholecell products. A number of substances of microalgal and cyanobacterial origin are currently under development as novel drugs, but these compounds lie outside the definition of nutraceuticals. Whereas terrestrial plants have a long history of use in natural medicine and health products, the use of microalgae as food or food supplements is almost entirely new. Only a few examples of the traditional use of microalgae are known, including the use of natural sources of the cyanobacteria Spirulina and Nostoc in China as dietary supplements in times of food scarcity. The modern use of microalgae as food supplements started with the production of Chlorella in Taiwan and Japan. Some sources refer to Japanese discoveries of the use of microalgae as vitamin sources during the Second World War as the beginning of the development of microalgal nutraceuticals, whereas the first industrial production of Chlorella started in Taiwan in 1965. Algal biomass was produced in shallow, circular ponds, typically 15 cm deep, and stirred by a rotating central arm that functions as a scraper to keep the cells suspended. The force on the arm limits the diameter of these tanks to about 50 m. Circular ponds are still widely in use (Lee, 2001). In the 1960s, much emphasis was on microalgal cultivation for the production of protein for human dietary purposes out of fear of a global food shortage and exaggerated projections of microalgal yield and production costs. Chlorella factories were projected and seen as a supply of plant protein for a starving world. The global food shortage fear was called off with the help of the green revolution, and microalgae failed to materialize as cheap, mass-produced protein sources; however, a nutraceutical niche production eventually emerged. In 1976, Spirulina production for human food supplements in large raceway ponds was started in California by Earthrise Corporation and shortly after also in Hawaii (Oswald, 2003). Although the number of algal plants is several hundred, the largest algal plants are still smaller than most conventional farms—the average U.S. farm size in 2000—was 449 acres (182 ha) compared with the largest microalgal plant, Earthrise Farms, with its mere 44 ha (Spolaore et al., 2006). It is noteworthy that development of the Spirulina industry in a very short time rose out of a very fruitful academic environment at the University of California/Berkely in the early 1960s, where algal wastewater treatment technology was also being developed and investigated by Oswald, Gouleke, and coworkers (Oswald, 2003; Oswald, 1995; Sheehan et al., 1998). The development today is highly dependent on several life science disciplines, including bioprocess engineering, traditional plant physiology and biochemistry, genetics and taxonomy, and molecular biology. However, with ever-increasing public focus on food safety, quality assurance is becoming important and many microalgal food supplement producers are ISO or GMP certified. Regulatory matters will undoubtedly play an increasing role, with particularly the European Union (EU) moving toward a strict regulation of the food supplements area. The recent possibility of having generic health claims approved by EFSA (European Food Safety Agency) will without doubt also affect the microalgal nutraceuticals business. Currently, a total of more than 2000 claims have been submitted to EFSA for approval, indicating the extent of industrial interest in serious nutraceutical product marketing.

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Molecular biology is gradually becoming a tool in microalgal biotechnology for the investigation and engineering of metabolic pathways. Also, the genetic improvement of strains to increase product formation would in principle be an obvious advantage from the production point of view, but there is little indication that consumer skepticism with genetically modified organisms for food production is decreasing. Molecular biology in the field of microalgae is very young, and the basic transformation methods are still under development. A good review of current microalgal molecular technologies can be found in the monograph of León and Fernández (2007) and will not be discussed here. Improvement of strains, however, may also be carried out using traditional random mutagenesis and phenotype selection methods and has, for example, been applied to improve fatty acid production in microalgae (Baldwin, 2001). Intellectual property development of product applications is an important cost factor in the nutraceuticals industry and constitutes a significant overhead on the sales costs. Product quality similarly is an important cost factor. The manufacturing process is important for the quality of the product, in terms of both nutritional quality and public health matters. The manufacturing process for microalgal nutraceuticals is traditionally divided into three parts: biomass production, product formation, and downstream processing, which also includes packaging. Whereas downstream processing is little different from conventional industrial food processing and is regulated accordingly, microalgal biomass production is very different from other types of raw materials production and quality control is difficult to establish. Nevertheless, microbial quality standards typically have been transferred from other types of plant products such as for instance the U.S. standards for contents of insect fragments and rodent hairs, copied from grain quality standards. Biomass production takes place in ponds, photobioreactors, or fermenters. There are very few examples of harvesting microalgae from the wild; one example is the production of Aphanizomenon flos-aqua from Klamath Lake in Oregon. There are also a number of examples where natural Spirulina blooms are used for food by local people, such as in Chad and in Myanmar in a naturally alkaline volcanic crater lake (Tredici, 2004). In contrast, almost all macroalgal production (kelp, seaweeds) is either harvested directly from natural resources or cultured in the open sea. The definition is of considerable interest in relation to nutraceuticals production. Ponds are open to contamination from microorganisms, insects, and foreign matter. Photobioreactors are closed, largely preventing contamination; however, bacteria-free operation cannot usually be accomplished. Fermenters, on the other hand, can be completely sterilized at setup and operated axenically (“bacteria free”) for extended periods of time. Light, however, is difficult to introduce, and fermenters therefore require that algae can grow heterotrophically. Product formation denotes a certain process step where biomass is induced to produce a particular compound at an increased rate. The production of so-called secondary metabolites in microalgae may be induced by simply maintaining photosynthesis under conditions of nutrient deprivation and the contents of, for example, carotenoids may be increased by orders of magnitude. In this manner, photosynthetic organisms offer quite a unique processing opportunity.

17.1.1

SCALE OF MICROALGAL NUTRACEUTICAL PRODUCTION

Nutraceuticals constitute a very large part of the industrial production of microalgae (the remaining part is mainly aquaculture feed and fine chemicals for cosmetic and pharmaceutical uses). The production is dominated by Spirulina and Chlorella, about 3000 ton of each species. Spirulina is marketed in 70 countries (Gantar and Svircev, 2008). Chlorella is produced by more than 70 companies, with 400 ton/year being the largest production by a single company (Spolaore et al., 2006). The Dunaliella production is the third largest, 1500 ton/year, followed by that of Haematococcus. A strongly growing market is microalgal oils, rich in polyunsaturated fatty acids, produced heterotrophically from the thraustochytrid Schizochytrium (a simple algal form, related to phycomycetes) and the dinophyceans Crypthecodinium.

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Docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-rich oils from Schizochytrium and Ulkenia are being used to increasing extents in fortified foods, including dairy products, bread, and cereals, excluding milk-based drinks. Lately, the Standing Committee on the Food Chain and Animal Health, EFSA recommended permission to extend the range of products to which addition of microalgal oils is allowed to include bakery products (energy bars) and milk-based beverages. In March 2009, Martek, which supplies Schizochytrium oil to which the ruling applied, published sales of US$87 million for the first quarter of the year, which represents an increase of 5% compared to the same period the year before. In 2003, the total annual sales were US$111 million (Martek, 2003). The present markets for microalgal nutraceutical products are shown in Table 17.1. The figures are compiled from various sources of scientific and market information, and should probably be taken with some caution; it appears that much of the information dates back from 2000 to 2001. Also the DHA–EPA market may be underestimated, with quarterly Martek sales now reaching US$87 million. Chlorella is produced predominantly in Japan and Taiwan (traditionally in shallow circular ponds), but about half the production is now heterotrophic, based on glucose. Spirulina can be produced relatively easily in open raceway systems because of its ability to grow at very high alkalinity, which excludes most other organisms. For practical purposes, Spirulina, Dunaliella, and Chlorella are currently the only algal species that can be produced industrially in open systems (Lee, 2001). These three species have a very distinct competitive advantage that makes them amenable for open system production. Dunaliella thrives at very high salinities and Spirulina thrives at high alkalinities; nevertheless, contamination with other cyanobacteria does happen (Shimamatsu, 2004). Chlorella grows very fast, and by fast dilution of open Chlorella ponds, competitive advantage over other algae can be obtained. Even then, contaminations with other algae occur, for example, Oscillatoria and Chlorella in Spirulina cultures (Grobbelaar, 2003). Except for these examples of “competitiveness engineering,”

TABLE 17.1 Overview of Existing Markets for Microalgal Nutraceuticals

Compound

Volume of Total Market Product Size (US$ × 106) (ton/year)

Astaxanthin β-Carotene EPA, refined DHA, refined Water-soluble food pigments (phycobiliproteins) Aphanizomenon (as dry biomass) Nostoc Chlorella (as dry biomass) Spirulina (as dry biomass)

250 200 100 200

100 300 300

Microalgal Part of Volume (%) Approx. 1 25a 0 (?)

Product Prize (US$/kg) Volume of Nonalgal Microalgal Algal Product Product (ton/year) Product 0.3–0.5

800

30

2000 600

>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.

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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.

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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.

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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

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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)

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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

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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

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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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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).

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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.

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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)

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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.)

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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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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: <1 kW/ha (Sheehan et al., 1998). For this reason, the raceway pond is still the most cost-effective cultivation system for Spirulina.

FIGURE 17.4 Cyanotech’s microalgae production facility in Kona, Hawaii. Raceway ponds with cultures of Spirulina and Haematococcus. (Courtesy of Cyanotech Corporation.)

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FIGURE 17.5 Hutt Lagoon, West Australia. The ponds are red from carotenoid-rich Dunaliella salina. The two lakes in the foreground of the picture are 125 ha each. (Courtesy of Cognis GmbH.)

There are drawbacks and limitations of the system. Rain can be a serious problem and production is not always possible during the rainy season. In Thailand, where DIC (Dainippon Ink and Chemicals Inc.) has operated a Spirulina production site near Bangkok since 1978, the mean annual precipitation is 1400 mm. Rain dilutes the medium, which may result in culture infection by green algae, protozoa, and/or insects (Shimamatsu, 2004). The open system also inevitably results in various types of foreign matter in the product. Dunaliella, which is produced for β-carotene production, can similarly grow in open ponds because of its ability to grow at salinities near saturation (5.5 M) (Shaish et al., 1992), which almost excludes all other organisms. Haematococcus (used for astaxanthin production) can be grown in open raceways for the product formation step, where the cysts are robust and not susceptible to bacterial infections or grazing by protozoans. Part of the biomass production can even be accomplished in open ponds, provided that sufficiently large, pure starter cultures are used. Under the Aquatic Species program, a range of species was tested in outdoor raceway systems, including Phaeodactylum tricornutum, Tetraselmis suecica, Chaetoceros gracilis, Cyclotella sp., Synechococcus sp., Navicula sp., and Cyclotella cryptic. For short periods of time several of these strains showed high areal productivities (Sheehan et al., 1998), and Nannochloropsis has been produced in small outdoor raceway ponds in Israel (Lubzens et al., 1997). However, owing to the occurrence of both grazers and competing autotrophic organisms, the ponds must be frequently restocked. The largest pond facilities in operation for microalgal nutraceuticals production are the Australian ponds belonging to Cognis, which produces Dunaliella for β-carotene. The ponds are naturally mixed and the largest, Hutt Lagoon, is 250 ha, with a volume of about 106 m3 (Borowitzka, 1999) and a depth up to 50 cm with an average of 30 cm (Figure 17.5). The obtainable densities are very low and harvesting costs are therefore significant. Little information is available about Japanese open pond Chlorella production. It is believed to be gradually replaced by the heterotrophic production of Chlorella in fermenters, which, in terms of quality, gives a more predictable production.

17.5.7

PHOTOBIOREACTORS

Photobioreactors are optimized for light absorption and are therefore transparent with a large surface:volume ratio. Photobioreactors may result in 10–100 times higher volumetric productivities

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TABLE 17.4 Examples of Productivity in Outdoor Photobioreactors Light Path Bioreactor Type (cm) Location Tubular Tubular Tubular Tubular Tubular Tubular Tubular Tubular (inclined) Undular tub. Bubble column Flat plate (inclined) Flat plate (inclined) Flat plate (inclined) Flat plate

Areal Volumetric Productivity Productivity Algal Species (g DW/m2/day) (g DW/L/day)

Reference

Italy Spain Israel

Spirulina Spirulina Spirulina Phaeodactylum Porphyridium Phaeodactylum Phaeodactylum Chlorella sorokiniana Spirulina Phaeodactylum Spirulina

33

2.7 0.69 0.30

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Israel

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51

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Carlozzi (2003) Miron et al. (1999) Miron et al. (1999), Qiang Hu (1996) Qiang Hu (1996)

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24

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Tredici et al. (1991)

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Cheng-Wu et al. (2001)

13 2.6 2.5 3 6 6 6

Italy Italy Israel Spain Spain Spain Spain

20 10.4

10

25 27.8 27

20

47.7

0.25 1.6 2.76 1.50 1.9 1.2 1.47

Torzillo et al. (1986) Giuseppe Torzillo (1993) Richmond et al. (1993) Molina Grima et al. (1996) Camacho et al. (1999) Molina et al. (2001) Acién Fernández et al. (2001) Ugwu (2002)

(biomass productivity per unit reactor volume) than open ponds, which have obvious advantages in terms of downstream processing costs and more controlled product quality. The volumetric productivities of outdoor, autotrophic photobioreactors are typically 1–2 g DW/L2/day (see Table 17.4). Using artificial illumination, the volumetric productivity of Spirulina, however, in Richmond (2000) of 28.8 g DW/L/day was reached. With photobioreactors, areal productivity may be a better basis for the comparison of systems performance than volumetric productivity, but this parameter is ambiguous because of the different physical shapes of the photobioreactors. The area may be taken as the entire footprint of the solar collector or it may be the direct surface of the reactor or the horizontal projection of the area solar collector. Furthermore, as light and temperature conditions vary during test periods, it is difficult to compare the productivity of the systems. Very rarely are systems compared in parallel at similar times and geographical location. In large reactor systems, peak values of about 50 g DW/m 2/day in ideal climatic situations have been recorded (Table 17.4). It is difficult to conclude much from a comparison of literature values as the data encompass variability in latitude, local irradiation levels, reactor types, and data collection periods. Generally, for a good geographical location, average summer productivity values tend to be around 25 g DW/m 2/day. There are three fundamentally different designs in enclosed photobioreactors in microalgal nutraceuticals production: the tubular photobioreactor (Figure 17.6), the flat-plate reactor (Figure 17.7), and the bubble column (Figure 17.8).

17.5.8

TUBULAR REACTORS

One of the first studies of tubular bioreactors was by S.J. Pirt in 1983 (Pirt et al., 1983), and this bioreactor is still the most widely applied closed photobioreactor. It is rather easy to manufacture, but must be carefully engineered and operated in order to perform well. A tubular reactor is based

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FIGURE 17.6 Tubular pilot plant in a greenhouse, Foundation Cajamar facilities, Almería (under construction). A white reflecting surface under the tubes increases the irradiation taken up by the algal culture. (Courtesy of E. Molina Grima.)

on recirculation of the algal culture in a tubular solar collector, where photosynthesis (and hence oxygen accumulation) takes place. With regular intervals, oxygen is stripped in the degasser tank and carbon dioxide is added to the culture. In the solar collector, cells are recycled over a light gradient between the dark center of the tube and the periphery. Cycle periods are determined by the turbulence in the tube. Large tubular plants are, for example, the Israeli Haematococcus producing

FIGURE 17.7 Flat-panel reactor. The panels in the photograph are built for artificial illumination between panels, for which reason they are placed close to each other. (Courtesy of Otto Pulz.)

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FIGURE 17.8 Fifty-liter bubble column reactors for algae cultivation at the Energy Research Centre of the Netherlands, Petten, the Netherlands. (Courtesy of Hans Reith.)

plant from Alga Technologies Ltd., the German Chlorella producing plant in Klötze near Berlin, now belonging to the French company Roquette, and the Mera Pharmaceuticals Haematococcus producing plant in Hawaii. Other tubular plants include Biofence (http://www.variconaqua.com/), a commercially available vertically stacked tubular plant, sized for aquaculture purposes, and Biocoil (Hall et al., 2002), a cylindrically “wound” tubular plant. Optimizing the productivity in tubular reactors involves both fluid mechanics and algal growth physiology. Parameters influencing the light regime include fluid velocity, light path, irradiation, biomass density, and tube length between the degassers, and biological characteristics of the algal strain that are important for the suitability of the strain to cultivation in a given tubular reactor system include shear sensitivity, light integration capacity, and sensitivity to oxygen supersaturation. At normal operating biomass densities, algal culture in the tubes is so dense that light does not penetrate all the way through the culture but, as a result of turbulence in the tubes, algae are shifting between inner, dark zones and outer, illuminated zones (light–dark cycling). Light–dark cycling depends on fluid velocity and is necessary at a certain level for maintaining high productivity, as established in several empirical studies (Acién Fernández et al., 2003; Hall et al., 2002). However, providing high fluid velocities is not a simple solution to the problem for two reasons: energy costs from maintaining high turbulence are considerable and some algae may be damaged by high shear stress with accompanying loss of productivity and product quality (shear damage may result in bacterial growth). Keeping a fluid regime in the transition zone between laminar and fully turbulent flow appears to be sufficient to secure optimum productivity under high irradiation in tubular reactors. With a diameter of 3–6 cm, this is equivalent to fluid velocities from 0.3 to 0.6 m/s (Giuseppe Torzillo, 1993; Acién Fernández et al., 1998). Under a dynamic light environment (irradiation can vary by two to three orders of magnitude as a consequence of cloud cover) and considering species variability in sensitivity to oxygen supersaturation and light acclimation, it is obvious that optimization is complex and that there is good reason to assume that average summer productivities may be doubled from the current standard of about 25 g DW/m2/day.

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FLAT-PANEL REACTORS

Flat panels are vertical or inclined 1–20 cm thick rectangular vessels (Figure 17.7). Degassing and mixing in the panels is accomplished by air sparging and is one of the critical parameters of the system because productivity strongly depends on gas flow rate in combination at high biomass densities and irradiation at least for Spirulina, for which these relations have been documented (Richmond, 1996, 2000; Qiang and Richmond, 1996). The use of compressed air for sparging is an important cost factor and rather high gas flow levels are applied to obtain sufficient mixing. Flat-panel reactors are, at this point, still in the experimental stage and more detailed studies of the economy of operation are required to fully develop the design of flat-plate reactor systems. Several large pilot plants are reportedly under construction at the moment, and this may represent a change of industrial preference for flat-plate reactors as opposed to tubular systems.

17.5.10

BUBBLE COLUMN AND AIR-LIFT REACTORS

The bubble column (Figure 17.8) is a transparent cylindrical vessel, mixed with gas sparging. Most aquaculture microalgal production takes place in such bubble columns, designed as semiopen polyethylene bags either supported by a metal mesh (volume 150–500 L) or hanging (volume 0–150 L) from a metal framework. Such bag cultures are in effect bubble columns. Nondisposable versions are also applied in aquaculture, for example, transparent fiberglass reinforced polyester tanks. Closed, acrylic bubble columns have been suggested and investigated for commercial nutraceuticals production (see Figure 17.9). High volumetric productivities can be obtained with bubble columns, and the construction principle is currently being applied by a German company NOVAgreen (Fahrendorf, 2009) that produces microalgal compounds for pharmaceutical use. The air-lift reactor resembles the bubble column, but has a structured mixing pattern. When comparing areal productivity, it is important to realize that the areal productivity of inclined systems is given as the productivity of the reactor relative to the horizontal area it occupies.

FIGURE 17.9

A 200 L steam sterilizable reactor with internal illumination.

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Comparing systems on the basis of volumetric productivity alone is insufficient because the liquid volume per unit area may vary. For a small (2 m3), commercially available glass flat-plate photobioreactor system, construction costs of US$4 per liter were calculated (Cheng-Wu et al., 2001; Richmond and Cheng-Wu, 2000). The system was intended for the small-scale production of microalgae for aquaculture purposes where total costs of US$50–100 per kg DW actually are economically competitive.

17.5.11

OTHER TYPES OF PHOTOBIOREACTORS

The Czech Institute of Microalgal Biotechnology in Trebon (49°N latitude) developed a very different system for microalgal production in 1963, known as the inclined plate or cascading system. In this system, the algal culture is recirculated over open-air, inclined glass plates (1.6–1.7%), on which it flows at 6–8 mm depths with a velocity of 66 cm/s. The average summer net (i.e., less night respiration losses) productivity values achieved were 38 g DW/m2/day for Chlorella with photosynthetic efficiencies of 7.1% (PAR) and peak values of 50 DW/m 2/day. Annual productions at favorable sites were projected at 80–100 ton DW. Production costs were claimed to be only a fraction of those of raceway ponds (Doucha and Lívansky, 2006, 2009). At the present time, the system is only operated for experimental purposes, but a similar system has been applied for commercial Dunaliella production in Australia (Borowitzka, 1999). Another reactor with potential application in small-scale nutraceuticals production is the dome reactor, which is a hemispherical flat-plate reactor applied by Fuji Chemical Corporation in Hawaii for the production of Haematococcus-based astaxanthin.

17.5.12

FERMENTERS

For heterotrophic production of microalgae, conventional fermenters are applied. Many algae can grow heterotrophically (using, for instance, glucose or acetate as the carbon source) and reach volumetric productivities of 20 g DW/L/day (Lee, 2001). For comparison, the volumetric productivity of some industrial bacterial or yeast fermentations is still higher. For example, Candida utilis can reach 174 g DW/L/day (Lawford et al., 1979) in oxidative fermentations. With the use of organic substrates in the media, it is imperative that the culture is free of any foreign organisms (axenic). Fermenters are stainless steel tanks, typically with a volume of 100–300 m3. Fermenters are steam sterilized and kept axenic throughout the duration of the culture by protecting all inlets to the fermenter (including sterile filters at gas and substrate inlets) and steam sealing all entry points of contaminants to the reactor (such as ports and stirrer shaft bearings). Steaming refers to filling and flushing the reactor with steam at a pressure of 1.3 bar and ensuring that temperature is maintained at 121°C for a desired period of time, usually 30 min. At present, only glass, and only as small-diameter tubes (<25 cm) or as small-sized slates, is approved for pressurized steaming. The only sterilizable, illuminated reactor design appears to be a steel vessel with inserted glass tubes from which the reactor may be illuminated from within. Small surface:volume ratios may be obtained in this way; the steel tank used for mixotrophic inoculum production of microalgae, shown in Figure 17.9, has a surface:volume ratio of 0.3. Photobioreactors, on the other hand, are optimized for light entry. This means that they have a large surface:volume ratio in the range of 25–80 m 2/m3 (Pulz and Pulz, 2001), which in turn implies that the reactors can sustain only moderate pressure. The presently largest, steam-sterilizable photobioreactor produced industrially is presumably the thin-tube Biostat PBR from Sartorius, with a maximum volume of 100 L. In contrast, industrial fermenters used for the production of Schizochytrium or Crypthecodinium are conventional industrial fermenters, and typically range in volume from 100 to 200 m3. The process applied is a fed batch, meaning that growth medium is gradually fed to the reactor under the cultivation process. One problem with traditional stainless steel fermenters used with marine

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organisms is that warm seawater is highly aggressive to steel. Martek has, for example, addressed this problem by developing a cultivation process with reduced chloride in the medium.

17.6 FUTURE DEVELOPMENT PROSPECTS IN MICROALGAL NUTRACEUTICALS PRODUCTION With the recently introduced novel foods regulations in the EU and U.S. nutraceutical market, any modified or novel production process for a nutraceutical is subject to large preapproval costs. While undoubtedly beneficial for public health concerns, this constitutes an additional market barrier for small- and medium-sized companies. At the same time, public research funding institutions have grown accustomed to substantial private industry cofunding of research in areas with applied potential, and private participation is typically a prerequisite in applied public research projects. A serious concern is therefore that growing market barriers may serve to prevent development within this field and reduce the introduction of new nutraceutical products with health benefits.

REFERENCES Abe, K., Hattori, H., and Hirano, M., 2007. Accumulation and antioxidant activity of secondary carotenoids in the aerial microalga Coelastrella striolata var. multistriata. Food Chem. 100: 656–661. Acién Fernández, F.G., Fernández Sevilla, J.M., Sánchez Pérez, J.A., Molina Grima, E., and Chisti, Y., 2001. Airlift-driven external-loop tubular photobioreactors for outdoor production of microalgae: Assessment of design and performance. Chem. Eng. Sci. 56: 2721–2732. Acién Fernández, F., García Camacho, F., Sánchez Pérez, J., Fernández Sevilla, J., and Molina Grima, E., 1998. Modeling of biomass productivity in tubular photobioreactors for microalgal cultures: Effects of dilution rate, tube diameter, and solar irradiance. Biotechnol. Bioeng. 58: 605–616. Acién Fernández, F.G., Hall, D.O., Cañizares Guerrero, E., Krishna Rao, K., and Molina Grima, E., 2003. Outdoor production of Phaeodactylum tricornutum biomass in a helical reactor. J. Biotechnol. 103: 137–152. Apt, K.E. and Behrens, P.W., 1999. Commercial developments in microalgal biotechnology. J. Phycol. 35: 215–226. Balachandran, P., Pugh, N.D., Ma, G., and Pasco, D.S., 2006. Toll-like receptor 2-dependent activation of monocytes by Spirulina polysaccharide and its immune enhancing action in mice. Int. Immunopharmacol. 6: 1808–1814. Baldwin, N., 2001. Application for the Approval of DHA-rich Oil. EU. Barber, J. and Nield, J., 2002. Organization of transmembrane helices in photosystem II: Comparison of plants and cyanobacteria. Phil. Trans. R. Soc. Lond. B. 357: 1329–1335. Barclay, W., Meager, K., and Abril, J., 1994. Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms. J. Appl. Phycol. 6: 123–129. Becker, W., 2004. Microalgae in human and animal nutrition. In: A. Richmond (Ed.), Handbook of Microalgal Culture. Oxford: Blackwell. Behrens, P.W. and Kyle, D.J., 1996. Microalgae as source of fatty acids. J. Food Lipids 3(4): 259–272. Belay, A., 1997. Mass culture of Spirulina outdoors—the earthrise farms experience. In: A. Vohnshak (Ed.), Spirulina (Arthrospira) plantensis. Physiology, Cell-biology and Biotechnology. Bristol, PA: Taylor & Francis. Belay, A., 2007. Spirulina (Arthrospira): Production and qualtiy assurance. In: D.M.E. Gershwin and A. Belay (Eds), Spirulina in Human Nutrition and Health. Boca Raton, FL: CRC Press. Belay, A., McCalmont, M., and Kitto, G., 1997. Development of an immunoassay to detect insect contamination of microalgal products. J. Appl. Phycol. 9: 431–436. Ben-Amotz, A. and Avron, M., 1990. The biotechnology of cultivating the halotolerant alga Dunaliella. Trends Biotechnol. 8: 121–126. Ben-Amotz, A., Shaish, A., and Avron, M., 1989. Mode of action of the massively accumulated β-carotene of Duniella bardawil in protecting the alga against damage by excess irradiation. Plant Physiol. 91: 1040–1043.

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Innovation of Technology 18 The for Microalgae Cultivation and Its Application in Functional Foods and the Nutraceutical Industry Akira Satoh, Masaharu Ishikura, Nagisa Murakami, Kai Zhang, and Daisuke Sasaki CONTENTS 18.1 Introduction .......................................................................................................................... 313 18.1.1 About Yamaha Motor Co. Ltd .................................................................................. 313 18.1.2 Some Background on the Research and Development into Microalgal Biotechnology by Yamaha Motor ............................................................................. 314 18.2 An Overview of Microalgae Mass Culture in Functional Foods and the Nutraceutical Industry .......................................................................................................... 314 18.3 Research and Development into the Commercial Production of Microalgae at Yamaha Motor .................................................................................................................. 316 18.4 Astaxanthin Raw Material Manufacturing Process ............................................................. 318 18.4.1 Outline of the Factory and Manufacturing Process ................................................. 318 18.4.2 Quality Assurance System Under Good Manufacturing Practice Conditions ......... 320 18.5 Astaxanthin and its Source, Hematococcus Alga ................................................................ 321 18.5.1 Astaxanthin............................................................................................................... 321 18.5.2 Structure and Source of Astaxanthin ....................................................................... 322 18.5.3 Antioxidative Activity of Astaxanthin ..................................................................... 322 18.5.4 Haematococcus Algae .............................................................................................. 322 18.6 Benefits of Astaxanthin for Human Health Management .................................................... 323 18.6.1 Astaxanthin and Atopic Dermatitis ......................................................................... 323 18.6.2 Astaxanthin and Brain Health .................................................................................. 323 18.6.3 Astaxanthin and Metabolic Syndrome ..................................................................... 324 18.7 Concluding Remarks ............................................................................................................ 326 Acknowledgment ........................................................................................................................... 326 References ...................................................................................................................................... 326

18.1 18.1.1

INTRODUCTION ABOUT YAMAHA MOTOR CO. LTD

Yamaha Motor Co. Ltd. was a spin-off from its parent musical instrument company, Nippon Gakki Co. Ltd. (now Yamaha Co. Ltd.) and became an independent company in 1955, supplying the latest 313 © 2010 Taylor and Francis Group, LLC

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motorcycles in Shizuoka, Japan. At the time more than 100 motorcycle companies existed in Japan; now only four companies exist, including Yamaha Motor. Yamaha Motor is mainly concerned with manufacturing and supplying motorcycles, marine products, and many other types of power products worldwide. Approximately 90% of its sales come from motorcycles, marine products, and power products: only 10% of all their sales are generated in Japan, the remaining 90% being overseas orders. In 2006, Yamaha Motor Co. Ltd. pioneered a new business concerned with biotechnology, which Yamaha Motor originally developed to provide bulk astaxanthin as a functional food ingredient.

18.1.2

SOME BACKGROUND ON THE RESEARCH AND DEVELOPMENT INTO MICROALGAL BIOTECHNOLOGY BY YAMAHA MOTOR

In 1997, a meeting was held in Kyoto, Japan, to discuss the international convention on the stabilization of greenhouse gas concentration in the atmosphere. At the meeting, many advanced countries agreed to reduce the emissions of six greenhouse gases, including CO2. In Japan, the Kyoto meeting was considered very important for many people and companies, alerting them to the effects of global warming. Environmental issues concerning global warming set Yamaha Motor on the road to researching and developing carbon dioxide reduction and fixation technology. In addition, they began to develop environmentally friendly power products, such as lower CO2 emission engines and fuel cells. Yamaha Motor became interested in the biofixation of CO2 using algal photosynthesis, which has a CO2-fixing capability far superior to that of higher plants. In the creation of an efficient biofixation system based on microalgae mass culture, Yamaha Motor began developing new conceptual photobioreactors using fluid dynamics technology that was previously utilized in the field of marine products (see Section 18.3 for details). Throughout the research, they accumulated specific technology for microalgae mass cultivation, including several photobioreactors in which each microalgal cell could be supplied with adequate light photons, dissolved CO2, and the nutrients required for maximum photosynthesis. The successful development of high-density mass cultivation technology led them to a further challenge in microalgal biomass production, producing astaxanthin on a commercial scale as a functional foods for the nutraceutical industry.

18.2 AN OVERVIEW OF MICROALGAE MASS CULTURE IN FUNCTIONAL FOODS AND THE NUTRACEUTICAL INDUSTRY Microalgae, including cyanobacteria, are microscopic photoautotrophs in which inorganic compounds and sunlight energy are converted into biomass. Applied research on algal culture and biomass began in the late 1940s (Burlew, 1953). This research included alternative and unconventional protein sources, photosynthetic gas exchange for space travel, aquaculture feed, wastewater treatment, renewable energy sources, biological fixation of greenhouse CO2, and production of recombinant biopharmaceuticals (Borowitzka, 1995; Becker, 2004; Spolare et al., 2006; Eriksen, 2008). It was discovered that microalgae synthesize a variety of valuable substances, including carbohydrates, lipids, vitamins, pigments, and other biological active compounds (Borowitzka, 1995; Becker, 2004; Pulz and Gross, 2004; Spolare et al., 2006; Cardozo et al., 2007). Microalgal cultivation technology and its subsequent biomass and products have received much research attention in the last 5–6 decades due to their potential as possible commodities and other industrial applications. However, only a limited number of microalgae have been found suitable to produce competitively priced products for use in functional foods and the nutraceutical industry (Vonshak, 1990; Borowitzka, 1999; Ben-Amotz, 2004; Cysewski and Lorenz, 2004; Hu, 2004; Iwamoto, 2004; Pulz and Gross, 2004; Spolaore et al., 2006). These include: Chlorella, mainly cultured in Japan, Taiwan, and Germany, with an annual production of 2000 tons dry weight; Arthrospira (Spirulina) and Aphanizomenon flos-aquae, mainly cultured in China, India, USA, and Japan, with an annual production of 3000

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and 500 tons dry weight, respectively; Dunaliella, mainly cultured in Australia, Israel, and the United States, with an annual production of 1200 tons dry weight and which is used as a source of β-carotene; Haematococcus, mainly cultured in the United States, Israel, Sweden, and Japan, with an annual production of 300 tons dry weight, used as a source of astaxanthin; Crypthecodinium and Schizochytrium, mainly cultured heterotrophically in the United States, with an annual production of 240 and 10 tons, respectively, used as a source of docosahexaenoic acid (DHA). The bottleneck of the microalgal business is price competitiveness, and this depends on difficulties with microalgal cultivation. These problems are derived from the growth and metabolic characteristics of a selected strain (e.g., growth rate, cellular content of a target product, optimum conditions, and stress tolerance), the culture system used (ponds and photobioreactors), and the sustainability of the culture and hence target product (e.g., contamination risks, productivity, quality, and seasonal effects). Before discussing the commercial production of microalgae at Yamaha Motor, details of the technology and problems are briefly mentioned. For further details on microalgae, culture systems, and their advantages and problems, readers are referred to the book edited by Richmond (2004) and reviews by Borowitzka (1999), Lee (2001), Pulz (2001), and Tredici (2004). Commercial cultivation of microalgae is mainly performed under outdoor conditions using open-air systems (circular and raceway ponds) and natural sunlight, due simply to the economics of production. One of the major problems with open-air systems of outdoor cultures is contamination risk, that is, difficulties in maintaining a monoalgal culture (species control) and prevention of bacterial and/or protozoal overgrowth (sterility). An extreme culture environment is therefore necessary to reduce the contamination risk, and a major reason why only a limited number of microalgae have been successfully mass cultured under outdoor conditions using open-air systems and marketed commercially. Examples include, Dunaliella, which requires high saline culture conditions, Spirulina, which requires high alkaline conditions, and Chlorella, which grows well in nutrient-rich media. Another major technical problem affecting sustainable productivity in microalgal cultures is the difficulty of supplying an adequate amount of light to each algal cell. Light irradiation reaching the surface of the culture is decreased by increasing either the distance from the surface or the cell density, due to mutual shading, leading to limited light energy for algal growth. In open-air systems, therefore, pond depth must be less than 50 cm and cell density at harvest is low (less than 0.6 g dry weight/L) and therefore a very large culture area is required for commercial-scale production of algal biomass—hundreds of hectares, provided by a number of ponds, each stretching to 1000 m2. Mixing, temperature and gas transfer are also important factors for algal growth, but these factors are difficult to control in open-air systems. Microalgal culture technology has developed a closed system using a photobioreactor to overcome the problems discussed above, especially contamination risk and light limitation, and consequently to achieve higher cell density. In typical photobioreactors, a series of transparent tubes, flat plate chambers, cylinders, or sleeves are positioned vertically, horizontally, coiled, or at a desired angle. Mixing and temperature control are also improved in comparison with open-air systems. An optical path in the photobioreactor is a precise parameter which can be altered through the diameter of tube or thickness of the chamber, to control algal growth and productivity in photoautotropic cultures. In general, a shorter optical path produces a higher cell density, while culture volume per reactor decreases, resulting in a larger surface area-to-volume ratio. The high cost of bioreactors and difficulties in scale-up are the two bottlenecks of photobioreactors, which limits successful use of commercial scale photobioreactors in the microalgal business (Tredici, 2004). Both ponds and outdoor photobioreactors have been used for autotrophic cultivation of Haematococcus pluvialis, used in astaxanthin production (Olaizola and Huntley, 2003; Cysewski and Lorenz, 2004; Del Campo et al., 2007). Constant irradiation of sufficiently high light and temperature control are required, since light intensity and temperature are the most critical factors affecting the growth of H. pluvialis and astaxanthin accumulation (Margalith, 1999). In addition,

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sterility is a severe problem in the cultivation of Haematococcus, compared with other successfully mass-cultured algae (Dunaliella, Spirulina, and Chlorella), since no selective culture environment to prevent bacterial and/or protozoal overgrowth is currently available for this alga (Margalith, 1999). A culturing technology which enables both increased astaxanthin production and sterile culture conditions is required. An indoor cultivation system has recently been developed to manufacture an astaxanthincontaining H. pluvialis algal biomass, namely the “YAMAHA High-efficiency Photobioreactor.” The development of this high-efficiency photobioreactor is described in the next section.

18.3 RESEARCH AND DEVELOPMENT INTO THE COMMERCIAL PRODUCTION OF MICROALGAE AT YAMAHA MOTOR As described in Section 18.1.2, Yamaha Motor became engaged in the development of outdoor photobioreactors for the biofixation of CO2 using algal photosynthesis. In order to achieve high-density algal cultures with high photosynthetic activity, we at Yamaha Motor focused on the “flashing light effect,” also called “intermittent illumination,” which had been reported to be a means of utilizing a larger fraction of the sunlight shining on a given area (Emerson and Arnold, 1932; Weller and Franck, 1941; Rieke and Gaffron, 1942; Tamiya and Chiba, 1949; Kok, 1953). The “flashing light effect” is a situation in which cells in a high-density culture are exposed to light and dark periods in turn at high frequencies by mixing. Cell mixing in a reactor was found to be very important but it was suggested that mixing performance would depend on the shape of the reactor. As a novel approach to the design and screening of photobioreactors, we applied computational fluid dynamics to analyze the mixing performance of various shapes of photobioreactor (Sato et al., 2002, 2005; Tomita et al., 2005a, 2005b). In these studies, we made several types of photobioreactor including dome-, parabola-, pipe-, and diamond-type. Side views of the dome- and pipe-type photobioreactors are shown in Figure 18.1. These photobioreactors were then evaluated in terms of light reception, global mixing, and local mixing, followed by an evaluation of algal biomass productivity using Chlorococcum littorale, which is regarded as an extremely high-CO2 tolerant algal strain, suitable for high-density culture (Kodama et al., 1993). A computational fluid dynamics analysis of the pipe-type photobioreactor is shown in Figure 18.2. This shape of bioreactor was found to be the best at light reception and biomass productivity: 20.5 g/m2/day in dry weight was observed during winter days in Japan. This pipe-type photobioreactor was then applied to the outdoor cultivation of Chaetoceros calcitrans,

FIGURE 18.1

The side view of a dome-type (a) and pipe-type (b) photobioreactor at Yamaha Motor.

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FIGURE 18.2 Computational fluid dynamics analysis of the pipe-type photobioreactor.

a valuable algal strain widely used as a feed in marine hatcheries. A biomass productivity of 37.3 g/m2/day was observed for Chaetoceros calcitrans. The culture volume of the pipe-type photobioreactor could be increased up to approximately 200 L. In addition, the simultaneous use of artificial light and sunlight to increase algal productivity was made possible by inserting fluorescent lamps into the inner cavity, since the space could be sealed completely after the insertion of lamps to prevent rainwater invasion. However, further enlargement of the reactors was not possible due to the strength of the structural material and difficulties in cleanup and sterilization. The pipetype photobioreactor is therefore applicable for small-scale on-site production of live microalgae for aquaculture feeds and we have indeed marketed live Chaetoceros calcitrans cells since 2002 (Yamauchi, 2003). Our next R&D project was mass cultivation of H. pluvialis for production of a more valuable compound, astaxanthin. Due to the scale-up problems of the pipe-type photobioreactor, we decided to use a vertical flat-plate-type reactor for this project, since previous research had shown this type of reactor to have excellent light illumination efficiency, leading to superior algal growth (Zhang et al., 2001). Our first efforts involved outdoor cultivation of H. pluvialis (Figure 18.3), however, as described in the previous section, bacterial and/or protozoal overgrowth was a severe problem due to the lack of a selective culture environment. In addition, biomass productivity and astaxanthin yield were very unstable and depended on weather conditions and seasonal differences, since algal growth and astaxanthin accumulation are greatly influenced by light availability and temperature (see Section 18.2). In order to establish a stable industrial manufacturing process, we moved to indoor cultivation using artificial lights, and this resulted in the development of the “YAMAHA High-efficiency Photobioreactor.” High-quality and high-quantity astaxanthin-containing algal oil have thus been produced since October 2006, and the manufacturing process was given a health food raw material Good Manufacturing Practice (GMP) certification. Details of our manufacturing process (GMP approved) are described in the next section.

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FIGURE 18.3 An outdoor vertical flat-plate-type photobioreactor at Yamaha Motor, used in the cultivation of H. pluvialis.

18.4 18.4.1

ASTAXANTHIN RAW MATERIAL MANUFACTURING PROCESS OUTLINE OF THE FACTORY AND MANUFACTURING PROCESS

A factory dedicated to the manufacture of astaxanthin raw material (astaxanthin-containing dried algal powder of H. pluvialis), namely the “Fukuroi Factory II,” was completed in October 2006 as the first and only factory capable of producing the raw material in Japan. The proprietary indoor cultivation system, demonstrating practical use of a number of the “YAMAHA High-efficiency Photobioreactors” is the main production facility (Figure 18.4). This facility is operated in an

FIGURE 18.4 The indoor cultivation system known as the “YAMAHA High-efficiency Photobioreactor” in the Fukuroi Factory II.

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unattended manner at night, enabling highly controlled batch cultures under continuous illumination, with lower labor costs. The factory is located on a site of approximately 37,000 m2 in the Kuno Industrial Park of Fukuroi city, Shizuoka prefecture. The building area is approximately 1800 m2, with 3300 m2 total floor space. At present, the production capability is approximately 20 tons of dried algal powder per year, however, the factory is designed as a unit of standardized equipment, making it possible to increase the production capability easily and quickly in response to market expansion by increasing the number of units required. The manufacturing process consists of several steps before shipment: cultivation, separation, drying, packaging, and quality inspection (Figure 18.5a). The process is entirely optimized under the concept of “manufacturing high-quality and safe products.” Briefly, at the cultivation step, the bioreactors are continuously illuminated with optimized synthetic light during a culture period and maintained at the desired temperature to maximize astaxanthin accumulation in algal cells. The quality of water is also carefully considered for optimum astaxanthin production. In order to minimize the risk of contamination, the reactors are isolated in a class 100,000 clean room (Figure 18.4); the cultivation and all operations are performed in the same or a more strictly controlled environment under sanitary quality control. Great care is also taken in the system design and operations at

Astaxanthin oil

Shipping inspection

Packaging (b) Concentration

Extraction

Shipping inspection

Packaging

(a)

Drying

Separation

Cultivation

FIGURE 18.5 A production flow scheme for astaxanthin raw material production (a) and subsequent astaxanthin-containing H. pluvialis oil (b).

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subsequent steps of harvesting the algal cell culture up to packaging the dried algal powder, both to protect the astaxanthin from degradation or deterioration by bacteria, excess heat, oxygen, and light, and to prevent contamination from foreign bodies. In our manufacturing process, raw material with an astaxanthin content of over 5% is consistently produced (Zhang, 2005, 2007), which is much higher than that obtained from outdoor cultivation, where it reduces to less than 2% in the winter season (personal communication). This stable supply of highly sterilized biomass with a high astaxanthin content is advantageous, not only to maintain low extraction costs during downstream processing, but also as a quality and safety guarantee for further downstream business partners and end-users.

18.4.2 QUALITY ASSURANCE SYSTEM UNDER GOOD MANUFACTURING PRACTICE CONDITIONS GMP is the rule employed to produce high-quality and safe products, by which a manufacturing system is guaranteed constant quality throughout the whole process, from the raw material stock to product shipment. Although the GMP was originally imposed as a legal duty for medicine manufacturers, similar GMP conditions have been applied to manufacturers of cosmetics, food additives, and health foods in recent years. In February 2005, the Japanese Ministry of Health, Labor and Welfare published a voluntary inspection guideline on GMP in the manufacturing of health foods and the safety of raw materials (No. 0201003). We designed and improved our factory to meet the GMP requirements and in July 2007 the Fukuroi factory II was given a health food raw material GMP certification by the Japanese Health Food Standards Association (JIHFS). Our quality assurance system had thus established a reliable GMP system, certified by an outside organization. Our GMP is composed of three basic principles: keeping mistakes to a minimum level; preventing pollution and product loss; and designing a management system for guaranteed high-quality products. The indispensable requirements to achieve this GMP are listed below, where (1) through (4) are germane to management side while (5) through (8) refer to equipment. 1. The complete preparation of various standard operation procedures (SOPs), operation manuals and operational records; correct operation of equipment strictly observing these SOPs and manuals. In addition, correct storage of the operation records, for easy access. 2. A traceability system using serial numbers for each product lot. 3. An education and training system, and skills improvement for all staff and workers at the plant. 4. Execution of a rigorous plant self-check system, based on our GMP, at the end of every run. 5. Clean-room installation, complete control of air-conditioning facilities and purification control in all production processes. 6. Manufacturing environment maintenance to prevent cross-contamination and foreign body mixing. 7. Installation of appropriate inspection equipment for both the process control and product standard test. 8. Running the automatic production monitoring system over 24 h. Astaxanthin raw material manufactured under GMP conditions in the Fukuroi factory II is shipped to a medicine manufacturer for further extraction to prepare astaxanthin-containing H. pluvialis oil (Figure 18.5b). The extraction process to produce astaxanthin oil also fulfills both the production system and quality assurance system in accordance with the principles of GMP. Our GMP-grade astaxanthin oil product (Figure 18.6) is prepared using an advanced quality assurance system equal to that used in orally administered medicine.

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FIGURE 18.6 Yamaha Motor’s GMP-grade astaxanthin oil product.

18.5 ASTAXANTHIN AND ITS SOURCE, HEMATOCOCCUS ALGA 18.5.1

ASTAXANTHIN

Astaxanthin, a natural lipophilic tetra terpenoid with a deep red color, is a carotenoid like β-carotene and lycopene and is widely distributed in nature, especially in marine organisms including salmon, salmon roe, shrimp, crab, and microalgae (Hussein et al., 2006). It is a xanthophyll from the carotenoid group, with oxygen-containing functional groups, and also possesses hydroxyl and oxo functional groups. In nature, astaxanthin is found in its esterified form or binding form, bound to proteins, because these forms are more stable than the dialcohol form. Plants, algae, and microorganisms can synthesize de novo carotenoids; however, animals lack the ability to synthesize these compounds and so must acquire them from their diet. Many carotenoids are known as provitamin A as they can be cleaved at the central C15=C15′ double bond and converted into vitamin A in vivo (Goodman and Huang, 1965; Olson and Hayashi, 1965). In fish, many xanthophylls, including astaxanthin, are reported to be converted reductively to retinol in vivo (Katsuyama and Matsuno, 1988). In contrast, human astaxanthin is reported to be cleaved asymmetrically at the C9 position and is therefore regarded as a non-provitamin A carotenoid (Kistler et al., 2002). Xanthophyll esters are thought to be hydrolyzed prior to absorption (Zaripheh, 2002). Only nonesterified astaxanthin is detected in serum after the ingestion of esterified astaxanthins (CoralHinostroza et al., 2004; Odeberg et al., 2003). Xanthophylls are mixed with bile acid to make a micelle, and are absorbed as a micellar solution by the intestinum tenue after intake. The absorbed xanthophylls are then incorporated by intestinal mucosal cells into chylomicra and released into the lymph. In the lymph, chylomicra-containing xanthophylls are digested by lipoprotein lipase, reducing their size, and xanthophylls reach the liver as chylomicra remnants. In the liver, they are incorporated into lipoprotein, which is synthesized in the liver, and translocated to each tissue. It was recently reported in humans that blood xanthophylls translocate more easily to red blood cells than to plasma when compared with hydrocarbon carotenoids (Nakagawa et al., 2008). It is suggested that the difference in the chemical nature of xanthophylls and hydrocarbon carotenoids causes the difference in their in vivo behavior.

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Due to their high antioxidant properties and other functions (see Section 18.6), many astaxanthincontaining nutraceuticals with potent effects on human health are coming onto the market.

18.5.2

STRUCTURE AND SOURCE OF ASTAXANTHIN

The astaxanthin molecule has a symmetric configuration and two chiral centers; two carbon atoms adjacent to hydroxyl functional groups are chiral. There are three enantiomeric isomers of astaxanthin, (3S, 3′S), (3R, 3′R), and (3R, 3′S). Chemically synthesized astaxanthin is a mixture of 1:2:1 of (3S, 3′S), (3R, 3′S), and (3R, 3′R) enantiomer, respectively. This is mainly used in the field of aquaculture as a reviver and is not used as an ingredient in human neutraceuticals. A green microalga (H. pluvialis), a red yeast (Phaffia rhodozyma), and crustacean by-products are commercially available as natural sources of the astaxanthin pigment. These sources are often used in the nutraceutical industry because of recent natural food trends and safety concerns. The forms of astaxanthin in these natural sources are slightly different from each other. Astaxanthin from Haematococcus is the (3S, 3′S) isomer (Renstrom et al., 1981) and is almost esterified with fatty acid to form mono- or diesters (Johnson and An, 1991). In contrast, Phaffia rhodozyma is reported to synthesize the (3R, 3′R) isomer (Torissen et al., 1989), which is mainly unesterified (Andrewes and Starr, 1976). Haematococcus alga is considered to be the most efficient natural source of astaxanthin (Hussein et al., 2006) and is presently used as the main source of natural astaxanthin (see Section 18.5.4).

18.5.3

ANTIOXIDATIVE ACTIVITY OF ASTAXANTHIN

Carotenoids are generally known to possess powerful antioxidative activity, thought to be because of their long conjugated polyene system (Nishida et al., 2007). The stable structure and strong antioxidative activity of astaxanthin is considered to be due to the conjugation of the oxo group to the polyene system. Astaxanthin is reported to show a strong quenching effect against singlet oxygen, with potency more than 100-fold higher than that of α-tocopherol (Miki, 1991). This same study also reports that astaxanthin shows strong activity against lipid peroxidation. In addition, astaxanthin is reported to have no pro-oxidative properties: other carotenoids, such as β-carotene, lycopene, and zeaxanthin, under certain conditions, are considered to possess pro-oxidative properties (Martin et al., 1999).

18.5.4

HEMATOCOCCUS ALGAE

H. pluvialis, Flotow, Volvocales, Chlorophyceae, is a unicellular freshwater green microalga. In response to environmental conditions, the green flagellated cells (vegetative cells) gradually transform into cyst cells without flagellae (the aplanospores), accompanied by a marked accumulation of astaxanthin, resulting in the formation of red-colored cells (Margalith, 1999). The size of a vegetative cell is less than 10 μm in diameter, although it gradually increases to over 40–50 μm after transforming into cyst cells. In oxygenic photosynthetic organisms, carotenoids play important roles in the light-harvesting complex and in the protection of photosynthetic machinery, by dissipating excess light energy (Frank and Cogdell, 1996). These types of carotenoids are referred to as primary carotenoids and are essential in metabolism (Krishna and Mohanty, 1998). These carotenoids are localized in thylakoid membranes of the chloroplast. In contrast, secondary carotenoids such as astaxanthin are not functionally obligatory for photosynthesis. Astaxanthin in H. pluvialis accumulates in cytoplasmic lipid globules of the cell: accumulation occurs in response to environmental stimuli, such as high light intensity and oligotrophic conditions. In H. pluvialis, it is considered that astaxanthin acts as a sunshade (Hagen et al., 1994), to provide protection from photodamage (Hagen et al., 1993), or to minimize oxidation of storage lipids (Sun et al., 1998).

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323

BENEFITS OF ASTAXANTHIN FOR HUMAN HEALTH MANAGEMENT

As a company which supplies functional food ingredients, it is important that we investigate the effectiveness of the ingredient we produce. It is generally accepted that oxidative stress is involved not only in the normal aging process but also in the pathogenesis of many acute, chronic, and agerelated diseases, including inflammation, cancer, cardiovascular disease, neurodegenerative disease, lung disease, and eye disease. Natural antioxidants are scavengers of reactive oxygen species including free radicals, and therefore have broad implications in antiaging and human health management. As described in the previous section, astaxanthin has a strong antioxidative activity and hence various physiological efficacies. In the next section, we present our clinical evidence for the efficacy of our astaxanthin-rich H. pluvialis oil on oxidative stress-related diseases/aging, especially atopic dermatitis, brain health, and metabolic syndrome.

18.6.1

ASTAXANTHIN AND ATOPIC DERMATITIS

There are many lines of evidence suggesting that allergic disorders, such as atopic dermatitis, asthma, and rhinitis, are mediated by oxidative stress (Bowler and Crapo, 2002; Okayama, 2005). We observed that the administration of astaxanthin significantly suppressed ear swelling in NC/Nga mice which had been previously sensitized by intradermal injections of mite antigen into the ear pinnae, inducing atopic dermatitis-like lesions (Iio et al., 2009). We further investigated the effect of astaxanthin intake on atopic dermatitis by a double-blind, randomized, placebo-controlled clinical trial (Satoh et al., 2009a). In this study, patients (aged 19–51 years) with mild to moderate atopic dermatitis ingested astaxanthin-rich H. pluvialis oil, equivalent to 12 mg of astaxanthin dialcohol (Ax group: six men and eight women) or corn oil (placebo group: six men and seven women) contained in soft capsules, once daily for 4 weeks. Before and after administration, evaluations were made on severity (Scoring atopic dermatitis; SCORAD), pruritus (visual analog scale; VAS), quality of life (Skindex-16 and state trait anxiety inventory; STAI), immune function (Th1/Th2 and blood catecholamines), and antioxidative status (urine 8-OHdG and isoprostane). No significant difference in severity and pruritus was apparent between the Ax and placebo groups. However, the Ax group showed significant amelioration of symptoms evaluated by Skindex-16 and on anxiety state evaluated by STAI. The ameliorating effect on anxiety by astaxanthin was supported by the level of the stress hormone, dopamine, which was significantly decreased in the Ax group. The level of urine 8-OHdG, which is reported to be high in patients with atopic dermatitis (Tsuboi et al., 1998), decreased significantly in the Ax group, indicating that astaxanthin intake has an antioxidative efficacy. Furthermore, the study revealed that there was a statistically significant shift in the Th1/Th2 balance toward Th1 in the Ax group. Atopic dermatitis is characterized by Th2-dominated allergic skin inflammation. It is known that higher anxiety in patients enhances the Th2-type response, due to dysregulation of the neuro immune system, leading to worsening of the allergic symptoms (Hashizume and Takigawa, 2006). The findings of this study, which showed that patients in the Ax group showed reduced anxiety accompanied by a shift in the Th1/Th2 balance toward Th1, strongly suggests that astaxanthin is a promising food factor in the management of atopic dermatitis.

18.6.2

ASTAXANTHIN AND BRAIN HEALTH

Brain aging, either natural or in neurodegenerative disorders, is also closely associated with oxidative stress, subsequent damage to cellular components (DNA oxidation/mutation, protein modification/aggregation, and lipid peroxidation), inflammation, and other factors, including excess calorie intake, insulin resistance, and mitochondrial dysfunction leading to neuron death (Matison et al., 2002; Schon and Manfredi, 2003). In connection with the effect of astaxanthin on the factors involved in brain aging, we have observed the following findings: (1) astaxanthin protects neuroblastoma

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cells from oxidative-stress induced apoptosis in vitro (Ikeda et al., 2008; Tsuji et al., 2008); (2) in dopaminergic neuron-specific manganese superoxide dismutase (MnSOD)-deficient mice which display Parkinsonian symptoms, oral administration of astaxanthin resulted in improvement of body weight loss and behavior function and hence an increase in life span (Tsuji et al., 2007); (3) astaxanthin improves mitochondrial function by maintaining a high mitochondrial membrane potential, stimulating respiration, maintaining mitochondria in a reduced state even under oxidative challenge (Wolf et al., 2009). In addition, a memory-improving effect of astaxanthin in mice has been reported by other research groups (Hussein et al., 2005; Zhang, 2007). However, there had been no studies investigating the effect of astaxanthin on the higher cognitive function of the human brain. We therefore conducted a preliminary clinical evaluation, an open-label trial using 10 otherwise healthy male subjects (50–69 years of age) who complained of age-related forgetfulness (Satoh et al., 2009b). Age-associated memory impairment is a common condition characterized by very mild symptoms of cognitive decline that occur as part of the normal aging process (NYU Medical Center/NYU School of Medicine, 2005, http://www.med.nyu.edu/adc/forpatients/memory.html). In our study, subjects ingested astaxanthin-rich H. pluvialis oil, equivalent to 12 mg of astaxanthin dialcohol, contained in soft capsules, once daily for 12 weeks. Cognitive function was evaluated before administration (at baseline) and again every six weeks during the study, using either the CogHealth tool (CogState; Melbourne, Australia) or the event-related P300 recognition response elicited by an auditory task. CogHealth is a cognitive function test specifically designed to detect changes in healthy or mildly impaired subjects at an early date (Collie et al., 2003). The trial revealed significant reduction in response time on all tasks in CogHealth: psychomotor speed, impulse control, working memory, episodic learning, and attention, after 12 weeks intake of astaxanthin. The accuracy on the “working memory” task in CogHealth was also significantly improved after 12 weeks of treatment. The P300 peak amplitude tended to increase after 12 weeks astaxanthin administration, indicating that astaxanthin might increase patient information processing capacity and selective attention. All tasks investigated by CogHealth and P300 are indispensable for a skilful performance in daily life activities such as driving, which is a complex form of activity involving specific cognitive and psychomotor functions. Age-related decreases in these tasks are therefore a serious problem, causing an increased number of traffic accidents and deaths in older people. Double-blind, randomized, placebo-controlled clinical trials investigating the effect of astaxanthin on CogHealth tasks and driving performance are currently in progress.

18.6.3

ASTAXANTHIN AND METABOLIC SYNDROME

Metabolic syndrome is characterized by a clustering of metabolic risk factors for cardiovascular disease in one person. The risk factors include abdominal obesity, insulin resistance, elevated blood glucose, atherogenic dyslipidemia (high tryglycerides, low HDL cholesterol, and high LDL cholesterol), and hypertension (Cornier et al., 2008; Grundy, 2008). Metabolic syndrome is closely associated with oxidative stress and adipose tissue inflammation, and the improving effects of astaxanthin on metabolic syndrome have been suggested in several animal models (Uchiyama et al., 2002; Naito et al., 2004, Hussein et al., 2005; Ikeuchi et al., 2007; Watanabe et al., 2007; Akagiri et al., 2008) and in a clinical study (Satoh et al., 2009b). We attempted to confirm the clinical efficacy of astaxanthin in an open-label uncontrolled study using volunteers at risk of metabolic syndrome (Uchiyama and Okada, 2008). In this trial, a total of 17 volunteers between 22 and 65 years of age (13 men and 4 women) at risk of developing metabolic syndrome were administered with astaxanthin-rich H. pluvialis oil, equivalent to 8 mg of astaxanthin dialcohol, twice daily for 12 weeks. A significant increase in adiponectin levels and a significant decrease in tumor necrosis factor (TNF)-α levels were observed. In addition, a significant decrease in glycohemoglobin (HbA1c), a diagnostic marker of diabetes, was observed. Adiponectin has an antidiabetic effect; it decreases blood glucose and is therefore regarded as a good indicator of metabolic syndrome (Matsushita et al., 2006), while TNF-α increases insulin resistance. It is generally

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accepted that the reduced secretion of adiponectin and increased secretion of TNF-α in dysfunctional adipose tissue (inflamed adipose tissue with enlarged adipocytes and impaired preadipocyte differentiation) increase insulin resistance in the pathogenesis of metabolic syndrome (Gustafson et al., 2007). Our results showed that astaxanthin intake increased adiponectin levels and decreased TNF-α levels, which strongly suggests that astaxanthin has a positive effect on the proinflammatory state of dysfunctional adipose tissue and hence on elevated blood glucose. This is confirmed by a significant decrease in glycohemoglobin (HbA1c) in humans at risk of metabolic syndrome. While the clinical efficacy was in the process of evaluation by administrating astaxanthin as an encapsulated oil-form in the studies above, we investigated the effect of astaxanthin on body fat in human subjects by using an astaxanthin-containing beverage as a test material. We prepared the test beverage using water-mixable astaxanthin oil. The study was a randomized, double-blind, placebo-controlled, parallel-group comparison trial, performed to conform with the Helsinki declaration. A total of 36 volunteers, aged between 20 and 65 years (41.0 ± 7.3 on average), with a high body mass index (BMI) of 25 or more, were administered 50 mL of the beverage containing 0 mg (placebo group) or 6 mg of astaxanthin dialcohol (astaxanthin group), once daily for 12 weeks. Body fat was evaluated by measuring abdominal visceral fat areas (VFA), subcutaneous fat areas (SFA), and total fat areas (TFA) using a computerized tomography (CT) scan, before (baseline) and after 8 and 12 weeks of dosing. The statistical differences in VFA, SFA, and TFA between baseline and postdosing values were determined by paired t-tests, while those between the placebo group and astaxanthin group were tested by unpaired t-tests, using SPSS for Windows (ver. 13.0J; SPSS Inc., Chicago, Illinois, USA). Differences with p < 0.05 were considered significant. The timecourse changes in VFA, SFA, and TFA are shown in Figure 18.7. The astaxanthin group showed a marked reduction in TFA in comparison with the placebo group, with significant differences after eight weeks dosing. VFA in the astaxanthin group was significantly reduced after eight weeks when compared with the placebo; no significant difference was observed after 12 weeks, possibly due to fat homeostasis. A marked reduction in SFA was also observed in the astaxanthin group after 8 and 12 weeks when compared with the placebo. In addition, no adverse effects, laboratory abnormalities, or adverse events attributed to administration of the test material were apparent among the vital signs, laboratory analysis (hematology, hepatic, and renal function tests), and subject interviews.

(a)

(b)

(c)

6 3

Δcm2

0 –3 –6 –9 –12

0

4

8

12

0

4 8 12 Evaluation period (weeks)

0

4

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FIGURE 18.7 Time course changes in TFA (a), VFA (b), and SFA (c) in the astaxanthin group (closed squares) and placebo group (open triangles). The differences in TFA, VFA, and SFA between respective baseline values and after treatment values are expressed as Δcm2. The number of subjects was 26 and 10 in the astaxanthin and placebo group, respectively. *p < 0.05.

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These results suggest astaxanthin could be safely administered as a beverage for use in body fat management. Further clinical studies remain to be performed to confirm our findings.

18.7 CONCLUDING REMARKS In this chapter, we discussed our indoor photobioreactor, a highly controlled microalgal cultivation technology, with high sterility, light illumination efficiency, and therefore high and stable biomass productivity on a commercial scale. Although microalgae have been recognized as promising organisms to provide sources of natural products in functional foods in the nutraceutical and pharmaceutical industry, only a few algal strains and their products have been successfully marketed so far, as described in Section 18.2. Our microalgal cultivation technology, using an indoor photobioreactor, is considered to be competitive in the production of high-value bioactive metabolites with low cellular content. The use of sterilized, high-density algal suspensions can be advantageous in downstream processing such as concentration and extraction processes, particularly for neutraceuticals and pharmaceuticals obtained from microalgae which are difficult to culture outdoors. Another possible application of our technology seems to be the cultivation of genetically engineered microalgae that will most likely be banned from outdoor cultivation systems. Transgenic algal strains that express good concentrations of several recombinant therapeutic proteins have already been successfully generated (Mayfield et al., 2007; Fletcher et al., 2007; Siripornadulsil et al., 2007). Further improvements of our cultivation technology, as well as innovations of other important technologies such as solar electricity and low-cost reactor materials, are needed to develop more competitive and economically feasible algal biomass production systems. Together with finding useful algal strains and their metabolites, the innovations of our cultivation technology and other cultivation-related technologies will bring advancement and expansion to microalgal business opportunities in the human health industry.

ACKNOWLEDGMENT The authors wish to express their sincere gratitude to Dr. Shigetoh Miyachi, Professor Emeritus, University of Tokyo, for his kind supervision and contributions to the Yamaha Motor Life Science Institute.

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of Nattokinase 19 Production as a Fibrinolytic Enzyme by an Ingenious Fermentation Technology Safety and Efficacy Studies Shinsaku Takaoka, Kazuya Ogasawara, and Hiroyoshi Moriyama CONTENTS 19.1 19.2 19.3 19.4

19.5 19.6

19.7

19.8

Introduction .......................................................................................................................... 332 NK as a Fibrinolytic Enzyme ............................................................................................... 333 Production of NK.................................................................................................................. 335 Assay Method ....................................................................................................................... 337 19.4.1 Definition of Activity Unit ........................................................................................ 337 19.4.2 Assay Method of NK ................................................................................................ 337 Bioavailability of NK............................................................................................................ 338 Safety .................................................................................................................................... 338 19.6.1 Animal Studies ......................................................................................................... 338 19.6.1.1 Single Oral Dose Toxicity Study ............................................................... 338 19.6.1.2 Oral Repeated-Dose 28-Day Toxicity Study ............................................. 338 19.6.1.3 A 13-Week Oral Toxicity Study ................................................................. 338 19.6.2 Pathogenicity Study of NSK-SD Producing Bacteria ............................................... 339 19.6.3 Mutagenicity ............................................................................................................. 339 19.6.3.1 Reverse-Mutation Assay ............................................................................ 339 19.6.3.2 Chromosomal Aberration Test ................................................................... 339 19.6.4 Human Studies.......................................................................................................... 339 Efficacy .................................................................................................................................340 19.7.1 In Vitro Assessment .................................................................................................. 341 19.7.2 Animal Studies ......................................................................................................... 341 19.7.3 Human Clinical Studies ............................................................................................ 342 19.7.3.1 Fibrinolytic Effect ...................................................................................... 342 19.7.3.2 Reduction of Hypertension ........................................................................ 343 19.7.3.3 Decrease of Blood Viscosity ......................................................................344 19.7.3.4 Antiplatelet Activity ...................................................................................344 Future Prospectives...............................................................................................................344 19.8.1 Vitamin K2 ................................................................................................................ 345 19.8.2 Poly-γ-Glutamic Acid ............................................................................................... 345 19.8.3 Food with Specified Health Claim ........................................................................... 345 331

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19.9 Conclusion ............................................................................................................................346 Acknowledgment ...........................................................................................................................346 References ......................................................................................................................................346

19.1

INTRODUCTION

Fermentation technology has played an indispensable role in the development and evolution of Japanese traditional foods, such as “natto” which is prepared by fermenting boiled or steamed soybeans in the presence of Bacillus subtilis var. natto, or miso which is also prepared by fermenting soybeans and rice. These fermented products are commonly known to provide health benefits as well as taste and flavor enhancements. Miso paste, in particular, is often used in soup for daily meals and can also be used as an additive. It plays an important role in providing the unique, sophisticated taste and flavor to a wide variety of Japanese foods. Even today such traditional fermented foods are a popular part of the Japanese dietary culture. Natto has a long dietary history, eaten as a Japanese traditional food for 2000 years (National Federation of Cooperatives on Natto, 1977; Sumi et al., 1987). Natto has a unique aroma, flavor, and texture, and many Japanese people like to include it in their daily meals. In fact, natto is commercially available in almost every supermarket in the country under numerous brand names and at a reasonable price (Figure 19.1). Furthermore, natto was a deep-rooted ingredient in the Japanese diet and was perceived by consumers as an excellent source of protein, fiber, and other essential nutrients; the other primary source of protein was traditionally obtained from marine animals such

FIGURE 19.1 A picture of commercially available natto procured from a supermarket. Brown fermented soybeans show stick-threads.

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as fish before Japan became westernized. Thus, as a functional food, natto has been perceived as attractive to consumers who expect it to help them maintain a healthy body and to prevent various diseases. Until recently, scientific evidence to substantiate the functions of natto has not been available, for example, its use as a folk remedy for heart and vascular diseases, to relieve fatigue, and as an antiberiberi agent (National Federation of Cooperatives on Natto, 1977; Sumi et al., 1987). In 1987 an enzyme contained in natto was found to have distinct fibrinolytic activity and was named nattokinase (NK) (Sumi et al., 1987). Fibrinolysis is the process by which fibrin clots or coagulated blood products are broken down. Although the molecular mechanism of fibrinolysis is not expanded in this chapter, details can be found elsewhere (Cesarman-Maus and Hajjar, 2005). Such a process plays a crucial role in response to the formation of thrombus after an injury to a blood vessel. Thrombus in the blood vessel probably causes vascular occlusions, leading to vascular diseases. The intake of natto has been known to have a role in the prevention of such diseases for many years. The finding of fibrinolytic activity in natto has accelerated studies on NK’s mechanistic action as well as on the safety and efficacy of the enzyme being supplemented for its health benefits. In this chapter, we describe the evolution of this fibrinolytic enzyme known as NK into a dietary supplement or so-called healthy food (SCHF) thought to contribute to the prevention of thrombotic diseases such as cardiovascular and cerebrovascular diseases. The term SCHF is used to define a scientifically substantiated ingredient with proven safety and efficacy (Ohama et al., 2006). Furthermore, we attempt to update the latest studies on NK, providing new insight about its biological activities in addition to the fibrinolytic activity of NK.

19.2

NK AS A FIBRINOLYTIC ENZYME

NK is scientifically known as Subtilisin NAT. NK was produced by fermentation of B. subtilis var. natto. NK is a serine protease, classified as a member of the Subtilisin family. It has a molecular weight of 27,728 which is composed of 275 amino acid residues (Fujita et al., 1993) and its primary structure is shown in Figure 19.2. The nucleotide sequence of the NK (Subtilisin NAT) gene exhibits a high homology with other Subtilisin enzymes: it is 99.5% homologous to Subtilisin E and 99.3% homologous to Subtilisin Amylosacchariticus (Nakamura, 1992). H2N – Ala –Gln – Ser – Val – Pro – Tyr – Gly – Ile – Ser – Gln – Ile – Lys– Ala – Pro – Ala – Leu – His – Ser – Gln – Gly – Tyr – Thr – Gly – Ser – Asn – Val – Lys – Val – Ala – Val – Ile – Asp* – Ser – Gly – Ile – Asp – Ser – Ser – His – Pro – Asp – Leu – Asn –Val – Arg – Gly – Gly – Ala – Ser – Phe – Val – Pro – Ser – Glu – Thr – Asn – Pro – Tyr – Gln – Asp – Gly – Ser – Ser – His* – Gly – Thr – His – Val – Ala – Gly – Thr – Ile – Ala – Ala – Leu – Asn – Asn – Ser – Ile – Gly – Val – Leu – Gly – Val – Ala – Pro – Ser –Ala – Ser – Leu – Tyr – Ala – Val – Lys – Val – Leu – Asp – Ser – Thr – Gly – Ser – Gly – Gln – Tyr – Ser – Trp – Ile – Ile – Asn – Gly – Ile – Glu – Trp – Ala – Ile – Ser – Asn – Asn – Met – Asp – Val – Ile – Asn – Met – Ser – Leu – Gly – Gly – Pro – Th r – Gly – Ser – Thr – Ala – Leu – Lys – Thr – Val – Val – Asp – Lys – Ala – Val – Ser – Ser – Gly – Ile – Val – Val – Ala – Ala – Ala – Ala– Gly – Asn – Glu – Gly – Ser – Ser – Gly – Ser – Thr – Ser – Thr – Val – Gly – Thr – Pro – Ala – Lys – Tyr – Pro – Ser – Thr – Ile – Ala – Val – Gly – Ala – Val – Asn – Ser – Ser – Asn – Gln – Arg – Ala – Ser – Phe – Ser – Ser – Val – Gly – Ser – Glu – Leu – Asp – Val – Met – Ala – Pro – Gly – Val – Ser – Ile – Gln – Ser – Th r – Leu – Pro – Gly – Gly – Thr – Tyr – Gly – Ala – Tyr – Asn – Gly – Thr – Ser* – Met – Ala – Thr – Pro – His – Val – Ala – Gly – Ala – Ala – Ala – Leu – Ile – Leu – Ser – Lys – His – Pro – Thr – Trp – Thr – Asn – Ala – Gln – Val – Arg – Asp – Arg – Leu – Glu – Ser – Thr – Ala – Thr – Tyr – Leu – Gly – Asn – Ser – Phe – Tyr – Tyr – Gly – Lys – Gly – Leu – Ile – Asn – Val – Gln – Ala – Ala – Ala – Gln – COOH

FIGURE 19.2

The primary structure of NK.

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*Active site

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Biotechnology in Functional Foods and Nutraceuticals (a)

(b)

0 min

(c)

30 min

(d)

(e)

1h

2h

5h

FIGURE 19.3 Changes in fibrinolytic activity of NK with time as indicated using fibrin plates (a−e) to simulate artificial thrombus.

A potent fibrinolytic activity of NK is demonstrated using fibrin plates in Figure 19.3. The activity of NK was confirmed in an in vitro study (Sumi et al., 1987). A subsequent study using dogs with experimentally induced thrombosis further confirmed its fibrinolytic activity, when a mild enhancement of activity was observed after oral administration of NK (Sumi et al., 1990). Purified NK has been shown to degrade fibrin clots in two different ways. One way is to act directly on the substrate or fibrin with a potent fibrinolytic activity which exhibits a much stronger activity than that of plasmin in clot lysis assays (Fujita et al., 1993, 1995b). The other mechanism is to degrade fibrin indirectly by affecting plasminogen activator inhibitor type 1 (PAI-1), which is the primary inhibitor of tissue-type plasminogen activator (Urano et al., 2001). The elucidated activities of NK thus far are summarized in Figure 19.4. Importantly, NK does not suppress the formation of fibrin from fibrinogen; therefore, NK does not inhibit the formation of blood clots. These NK

Nattokinase 3

1 Attack Degradation

Attack

Fibrin degradation products (FDP)

Plasmin Fibrinogen Increase

Plasminogen activator inhibitor type 1 (PAI-1)

Tissue plasminogen activator (t-PA)

2 Activation (in vitro)

Thrombin

Degradation

Fibrin thrombus

Urokinase Plasminogen Pro-urokinase

FIGURE 19.4 The proposed mechanism of NK on fibrinolysis. Arrows show the sites where NK works. NK as a serine protease exhibits multifunctional activities.

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activities differ from other fibrinolytic enzymes such as streptokinase and urokinase. Moreover, NK is orally ingestible and still maintains its enzymatic activities, while the other fibrinolytic enzymes must be used intravenously in order to be active.

19.3 PRODUCTION OF NK Natto is prepared by fermenting boiled soybeans with B. subtilis var. natto. The soybeans are usually fermented at 40°C for 14–18 h until they convert into dark brown beans with the presence of sticky, viscous, string-like material. In addition, natto possesses a notorious sour aroma and nutty flavor. In this fermentation process, B. subtilis var. natto produces nutrients such as vitamin B2, amino acids, and dietary fiber as well as functional materials including NK, vitamin K2, poly-γglutamic acid (PGA), isoflavone, and polyamine. Japan Bio Science Laboratory Co. Ltd. (JBSL) has a proprietary product containing a selected and patented strain of B. subtilis var. natto. These bacteria were isolated from commercialized microorganism B. subtilis var. natto by JBSL. The NK production method was found to yield NK in a culture medium primarily containing corn starch as the carbon source, defatted soybean as the nitrogen source, and inorganic salts such as calcium carbonate. All culture medium nutrients are food or food additives approved by the Japanese government for human consumption. NSK-SD is a raw material of the dietary supplements produced by JBSL. The production methods of NSK-SD are as follows. The first process of NSK-SD production is fermentation. Providing proper aeration and agitation is an important process in large-scale fermentation. NK is produced under an optimal temperature of around 35°C for the culture period of 2–3 days. This cultured medium usually contains NK activity of about 1000–2000 FU/mL; a definition of the NK activity unit is given in the next section. Additionally, vitamin K2 with concentrations ranging from 10 to 100 μg/g dry weight is contained in this cultured medium. Although vitamin K2 is useful as a blood coagulant, it is an unfavorable by-product for the supplementation of NK since one of the major health benefits of NK is its antithrombotic effect; thus, vitamin K2 is eliminated subsequently. The second process of NSK-SD production is filtration and removal of the vitamin K 2. Chitosan dissolved in acetic acid solution as the high molecular weight aggregator and diatom earth as the filter aid are added to the cultured medium. Filtration is performed under pressure to give a clear filtrate. The chitosan–acetic acid solution absorbs the solids, bacterial cells, and hydrophobic materials including vitamin K2 in the cultured medium. Consequently, after chitosan treatment 99.0– 99.9% of the vitamin K2 in the filtrate is removed. This solution is then filtered by 0.2 μm pore size membrane. The third process of NSK-SD production is the concentration and removal of natto flavor. The filtrate is further concentrated by an ultrafiltration membrane unit. The majority of compounds having a molecular weight of 10,000 (MW) or less can be removed by this procedure. The obtained concentrate of the culture medium is then filtered by 0.2 μm pore size membrane filters. At this point, the concentration of vitamin K2 in the solution is less than 0.01 μg/g dry weight. The final process of NSK-SD production is powdering. Appropriate additives such as watersoluble dietary fiber are added to the concentrate to produce powder upon spray-drying. The production flow chart of NK is outlined in Figure 19.5. The finished product of NSK-SD is a white (milk-white) colored powder with little or no odor, having an enzymatic activity unit of more than 20,000 FU/g. The recommended dosage is 2000 FU/ day. All vitamin K2, which potentially increases blood coagulation, has been removed. NSK-SD is produced from nongenetically modified soybeans and a selected, patented strain of B. subtilis var. natto. NSK-SD is stable in a pH of 5.5–10.0 at 25°C for 24 h. NSK-SD is stable at 50°C for 1 h. Optimal fibrinolytic activity takes place at around 65°C at pH 10.5. Also, NSK-SD is under pressure up to 2000 kg/cm2 and can therefore be pressed to prepare tablets. NSK-II, the brand name for JBSL’s soft gel capsule product, retains 75–85% of the activity at a pH of 2.0, mimicking gastric fluid, for 30 min at 37°C.

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Biotechnology in Functional Foods and Nutraceuticals (a)

(b)

(c)

(d) U F

(e)

(f )

(g)

(h) Additive

FIGURE 19.5 The production flow of NSK-SD using B. subtilis var. natto. (a) Main culture, (b) filtration, (c) sterile filtration, (d) ultra filtration (UF), (e) sterile filtration, (f) membrane filtration, (g) standardizing, and (h) spray drying.

As dietary supplement, NK is generally used in soft gel dosage form. Employing the assay method described below, the activity of commercially available natto products are compared and shown in Figure 19.6. Vitamin K2 concentration in NK is assessed by high-performance liquid chromatography (HPLC). NSK-SD is found to contain no vitamin K2, while the contents of vitamin K2 in other NK products ranged from 0.49 to 2.64 μg/g (Table 19.1).

A B C D E F G H I J K L M N O P Q R S T U Ave.

1590 1000 1610 930 1770 1550 3290 1270 2050 1170 1150 1480 2240 1670 1130 2040 620 2250 1180 1230 2100 1590 0

FIGURE 19.6

500

1000

1500

2000

2500

3000

A comparison of the NK activities of commercially available natto per pack.

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TABLE 19.1 A Comparison of Vitamin K2 Concentrations in NK Products in the Marketplace in Japan and the United States Product Name NattoGoldTM Nattokinase NSP-2TM Ultra Nattokinase NSP-2TM NSK-SDTM

Activity (FU/g)

Vitamin K2 (μg/g)

15,600 11,400 19,000 24,900

0.49 1.93 2.64 Not detected

19.4 ASSAY METHOD It is essential to establish an assay method for the measurement of NK activity in order to ensure the presence of the expected enzymatic unit, as well as to guarantee the quality and safety of the product. NSK-SD as a SCHF can be formulated in a wide variety of dosage forms such as soft gel capsule, hard shell capsule, tablet, paste, granule, and so on (Ohama et al., 2006). An accurate and standardized measurement of NK activity is crucial for determining the appropriate dose for daily oral ingestion. The present assay method is officially validated by the Japan Health Food and Nutrition Association (JHFA) in Tokyo, Japan and the Japan Natto Kinase Association (JNKA) in Tokyo, Japan for any NK activities in SCHF products.

19.4.1

DEFINITION OF ACTIVITY UNIT

NK activity is determined by optically measuring the quantity of acid-soluble low-molecular products, which increases with the hydrolysis of the peptide linkages when NK acts on fibrin as a substrate. One unit (1 FU) is defined as the amount of the enzyme that increases the absorbance of the sample at 275 nm by 0.01 per min under the conditions described below.

19.4.2 ASSAY METHOD OF NK A mixture of 1.4 mL of 50 mmol/L borate buffer (pH 8.5, containing NaCl) and 0.4 mL of 0.72% fibrinogen in buffer is preincubated in a water bath at 37.0 ± 0.3°C for 5 min. 0.1 mL of thrombin, dissolved in buffer and adjusted to 20 U/mL, is added to the mixture and agitated by a vortextype mixer. After reacting for 10 min, 0.1 mL of the sample solution is added and mixed for 5 s at 37.0 ± 0.3°C. After initiating the reaction, agitations at 20 or 40 min are performed for 5 s. After reacting for 60 min, 2.0 mL of trichloroacetic acid (0.2 mol/L) solution is added to terminate the reaction. The sample solution is further mixed and incubated for 20 min at 37.0 ± 0.3°C. The mixture is transferred into a micro test tube and centrifugation is performed at 15,000 × g for 5 min. Finally, 1.0 mL of supernatant is delivered into a cuvette using a Pasteur pipette and the absorbance (AT) at 275 nm is recorded. The following equation was used to calculate the NK activity per weight (FU/g): (AT − AB) ___ 1 × D, NK activity (FU/g) = __________ × 1 × ___ 0.01 60 0.1 where AT is the absorbance of the sample, AB is the absorbance of blank (same as the sample except that it was added to the mixture after terminating the reaction), and D is the dilution rate of the sample. Standardized NK 1.000 g is accurately weighed and diluent is added to prepare NK activity per volume to be 1.00 FU/mL. The standard sample should be assayed simultaneously at every test batch to confirm the accuracy of the test result.

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BIOAVAILABILITY OF NK

The bioavailability of NK was demonstrated in a study using rats by measuring the transportability of NK across the intestinal tract. A dose of 80 mg NK/kg was delivered intraduodenally to the rats. Blood samples were drawn at predetermined intervals. NK activity was detected in the plasma at 3 and 5 h after administration. Furthermore, NK activity at 30 min after administration was measured by monitoring the presence of degraded products of fibrinogen in the plasma. Coagulation time, determined as plasma recalcification time, was prolonged when compared to baseline at 3 and 5 h following the administration of NK (Fujita et al., 1995a).

19.6 SAFETY NK is an enzyme which has been present in a commonly consumed Japanese food, natto, for centuries without any adverse effects (National Federation of Cooperatives on Natto, 1977; Sumi et al., 1987). The safety of NSK-FD, NSK-SD, or NSK II has been evaluated by JBSL in a wide range of studies using both animals and humans. NSK-FD and NSK-SD are raw dietary supplement materials produced by JBSL. NSK-FD is a freeze-dried powder with a natto flavor. NSK II is a soft gel capsule product produced by JBSL.

19.6.1

ANIMAL STUDIES

Safety studies using animals include (1) a single oral dose toxicity study, (2) an oral repeated-dose 28-day toxicity study, and (3) a 13-week oral toxicity study. 19.6.1.1 Single Oral Dose Toxicity Study NSK-FD with an activity of about 13,000 FU/g was tested orally by the administration of a single dose to Sprague-Dawley rats. A group of 10 rats (five males and five females) received 2000 mg (26,000 FU)/kg body weight, and the other group received the placebo. The animals were observed for 14 days and subjected to a necropsy at the end of the observation period. The study was performed in compliance with the Ministry of Health and Welfare (MHW) Guidelines (1997). The trial of a single oral dose of the NSK-FD showed no deaths for the study periods while diarrhea was observed in two male rats and soft stools in three male rats and all female rats tested at one day after dosing. The body weight gain of treated rats was not found to be adversely affected. No gross pathological alterations were evident at terminal necropsy in any of the rats. Based on these results, the minimal lethal dose (LD50) of NKS-FD after single oral administration in male and female Sprague-Dawley rats was found to be greater than the limit dose level of 2000 mg/kg body weight (BILIS, 1999). 19.6.1.2 Oral Repeated-Dose 28-Day Toxicity Study A repeat-dose study for 28 days was conducted with Sprague-Dawley rats. A dose of 167 mg/kg/day NSK-SD (3700 FU/kg body weight/day) was orally administered to six male and six female rats. The other group with the same number of animals was given a placebo. The potency of NSK-SD was calculated as being equivalent to 100 times the common intake from commercially available packs of natto (50 g) by a 60 kg human. Rats were examined for clinical signs, body weight, food consumption, urinalysis, and ophthalmological health. The study methodology was in accordance with MHW guidelines (1993). At the end of 28 days the rats were bled for hematological and chemical analysis of blood and then euthanized for necropsy and histopathological examination. The study results demonstrated that no toxic effects were found in the oral administration of NSK-SD (Kobuchisawa Laboratories, 2002). 19.6.1.3 A 13-Week Oral Toxicity Study A repeat dose of 90 days (13 weeks) was also conducted using Sprague-Dawley rats to assess the toxicity of doses at 100, 300, and 1000 mg/kg/day NSK-SD (21,900 FU/g). A placebo group was

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included for comparison. Four groups of animals were tested; each group consisted of 24 animals, 12 males and 12 females. The study methodology was in accordance with the guidelines established by the MHW (1997). The results of the study showed no deaths of tested animals, no NK-related alterations in clinical signs, no changes in body weight or food consumption, no ophthalmological abnormalities, and no adverse effects on urinalysis, including water consumption. Results also indicated that hematology as well as blood chemistry and pathology were normal at the end of the study (Bozo Research Center, 2004).

19.6.2 PATHOGENICITY STUDY OF NSK-SD PRODUCING BACTERIA The safety of the NSK-SD producing bacteria, isolated from commercially available natto bacteria (B. subtilis var. natto) was tested using 5-week-old mice (ICR-strain). A single oral inoculation of control or 7.55 × 108 colony forming unit (CFU) was given to a group of 10 mice. Each group consisted of five males and five females. The mice were observed for 14 days after inoculation. Results demonstrated no mortality and no abnormalities in general health or body weight. No treatment-related abnormalities were observed in the histopathology examinations during autopsy and no bacteria were found in any of the tissues examined during autopsy. It was concluded that the bacteria used in the production of NSK-SD had no potential for infectivity, pathogenicity, or toxicity (Gifu Research Laboratories, 2003).

19.6.3 MUTAGENICITY It was demonstrated that NSK-SD was nonmutagenic in the reverse-mutation assay (Ames test) against five strains of bacteria and also in a chromosomal aberration study conducted in CHL/IU cells. 19.6.3.1 Reverse-Mutation Assay The mutagenicity of NSK-SD (20,000 FU/g) was tested in five strains of bacteria: Salmonella typhimurium TA98, TA1537, TA100, TA1535, and Escherichia coli WP2uvrA. NSK-SD was tested at six dose levels ranging from 15.5 to 5000 mcg/plate. Negative and positive controls were included. Positive controls included those without metabolic activation. The results of a dose study as revealed by revertant colony counts of the test substance groups with and without metabolic activation were less than 2 times the negative control values for all strains tested. NSK-SD is not considered to be mutagenic (Kobuchizawa Laboratories, 2003a). 19.6.3.2 Chromosomal Aberration Test Tests for cell growth inhibition and chromosomal aberration were conducted with CHL/IU cells from the lung of a female Chinese hamster. NSK-SD (20,000 FU/g) inhibited cell growth sharply at 0.156 mg/mL and higher concentrations. The chromosomal aberration test was performed at concentrations lower than those which caused cell growth inhibition. NSK-SD was incubated with the cells for 6 h (short term) or 25 h (long term) with and without metabolic activation. The period of 25 h was selected as it was 1.5 times the cell cycle for CHL/IU cells. The short-term chromosomal aberration test was conducted without metabolic activation at three doses (0.110, 0.078, 0.055 mg/mL). The long-term test was conducted using the latter concentrations. Both positive and negative controls were included. The results of the experiments indicated that chromosomal aberration occurred at less than 5% in all doses tested without exhibiting dose-dependency. It was concluded that NK did not produce chromosomal aberrations in CHL/IU cells at any concentration (Kobuchizawa Laboratories, 2003b).

19.6.4 HUMAN STUDIES The safety of NSK II was performed in a randomized, double-blind human clinical study with 31 healthy male and female subjects (20–64 years old; body mass index (BMI) between 18

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and 28). Nine subjects (five males and four females) took a placebo and 22 subjects (10 males and 12 females) took three NSK II soft gel capsules (2000 FU/3 capsules) per day for 4 weeks, followed by a 2-week observation period. All subjects visited the clinic at the beginning of the study and then 4 and 6 weeks after initial treatment. During visits to the clinic, interviews were conducted along with measurements of body weight, blood pressure, and pulse rate. In addition, blood was taken and urine collected. Subjective symptoms were recorded daily. No significant adverse effects were reported for either group. Mild adverse events reported for the placebo group were diarrhea (four cases) and back pain (one case). Mild adverse effects found in the treatment group included diarrhea (three cases) and common cold (two cases). The other effects observed were constipation, pimples, stomach pains, menstrual cramps, and headache. Body weight slightly increased in both the placebo and treatment groups, which was considered to be clinically not significant. There were also minor changes in hematological profi les in both groups that were not deemed clinically significant. No effect was noted on blood pressure or pulse rate. No significant changes in urinary analysis were observed. It was confi rmed that oral daily administration of three capsules (630 mg) of NSK II for 4 weeks was safe (Ogasawara et al., 2006). In recent years, consumers taking medications such as warfarin as an anticoagulant have asked questions about drug–food interactions. Hence, another study was performed to assess the safety of the administration of NSK II to patients taking physician-prescribed warfarin. This was a double-blind, placebo-controlled study with 60 adult cardiovascular patients (male and female). Treatment groups were administered two capsules of NSK II (1700 FU) as low dose and four capsules of NSK II (3400 FU) as high dose per day after breakfast for 26 weeks. No adverse effects were observed for either group as a result of taking a combination of the two agents; therefore, concomitant intake of NSK II and warfarin may not cause any food–drug interaction (Ninomiya et al., 2008). Furthermore, an open label study evaluated the safety of NSK-FD as a concomitant oral fibrinolytic agent for those who had experienced strokes. The study included 12 patients with an average age of 53.3 years who were in hospital in a conscious state with acute mild to moderate ischemic stroke of noncardiac origin. All patients were administered heparin s.c. (7600 IU/day) and antiplatelet drugs (low-dose aspirin 150 mg to 325 mg/day Clopidogrel) along with NSK-FD (6000 FU/day, three doses for 2000 FU) for 7 days. The patients were then monitored for 3 months (90 days). The results exhibited no mortality during the course of the study. No incidents of hemorrhagic transformation of the infarct as confi rmed by CT scan were reported in any of the patients. The conditions of the subjects were evaluated using three internationally recognized scales: National Institute of Health Stroke Scale, Modified Rankin Scale, and Barthel Index. According to these scales, five patients had an overall favorable response. Coagulation and fibrinolytic assays were performed on days 1, 2, and 7. Significant changes compared to day 1 were as follows: bleeding time increased on day 7, clotting time increased on days 2 and 7, and D-dimer levels decreased on days 2 and 7. There were three adverse events that might have been attributed to NSK-FD: (1) prolonged activated partial thromboplastin time, (2) moderate hematemesis, and (3) an abnormal liver function test. All of these events were temporary. Thus, the fi ndings in the study suggest that NK may be safely administered to patients with stroke as an adjunct to standard medical treatments (Japan Bio Science Laboratory, 2004; Shah et al., 2004).

19.7 EFFICACY In vitro experiments as well as animal and human studies prove that NK does indeed have fibrinolytic activity. Results of such studies also suggest that serine protease may control high blood pressure, reduce red blood cell (RBC) rouleaux formation, decrease blood viscosity, and inhibit platelet aggregation.

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IN VITRO ASSESSMENT

Initially, fibrinolytic activity was observed when the vegetable cheese natto was applied directly to fibrin. The fibrinolytic activity was about 40 CU (plasma units)/g wet weight and the isolated protease was named NK (Sumi et al., 1987). The fibrin degradation unit is defined as fibrinolytic activity of 40 CU (equivalent to 30 FU). Experiments using a clot lysis assay (cross-linked fibrin) revealed that purified NK was 4- to 5-fold more potent than the fibrinolytic activity of plasmin. NK cleaved fibrinogen and fibrin, producing degraded products similar to those of plasmin. When the kinetics of NK and plasmin were measured, NK was 3-fold less active in the cleavage of fibrinogen as a substrate compared to plasmin (Kcat/Km) but 6-fold more efficient in the cleavage of crosslinked fibrin (Sumi et al., 1990). Preparations of pure NK and NSK-SD (bulk powder plus capsule contents) were tested in a series of in vitro experiments in human plasma. Test concentrations (0.2–1.6 FU/mL) were calculated as twice the plasma concentration of the highest recommended dose (4000 FU) assuming 100% bioavailability in a 5-L average blood volume. In this system, the functional activity of fibrinogen to form fibrin in response to thrombin was not altered by concentrations of 0.2–0.8 FU/mL of NK. Only at the highest concentrations of 0.8 and 1.6 FU/mL did NK reduce the activity of fibrinogen. This finding suggested that NK should not affect the body’s ability to respond to tissue wounding, when taken at recommended doses (Ero and Lewis, 2008). Unlike urokinase, NK does not promote fibrinolysis by directly stimulating plasminogen activator activity (Sumi et al., 1990). Instead, it was reported to degrade the PAI-1. PAI-1 is the primary inhibitor of the tissue-type plasminogen activator (t-PA). NK cleaves active recombinant PAI-1 into low-molecular-weight fragments at concentrations of 0.02–1.00 nmol/L. NK reduces the activity of the inhibitor while enhancing t-PA-induced lysis of fibrin clots in a dose-dependent manner (0.06–1 nmol/L) (Fujita et al., 1995). In contrast to the above study, an in vitro test in human plasma reported that NK (0.8 and 1.6 FU/mL) slightly increased the presence of PAI-1 (Ero and Lewis, 2008). The effects of NK on RBC aggregation and blood viscosity were also measured in an in vitro experiment. Blood samples incubated with NK at final concentrations of 15.6, 31.3, 62.5, and 125.0 activity units/mL resulted in 21.9%, 25.9%, 49.7%, and 62.0% inhibition of RBC aggregation respectively as compared to the control. NK reduced blood viscosity at lower shear rates but no changes in viscosity at high shear rates were observed (Pais et al., 2006).

19.7.2 ANIMAL STUDIES The fibrinolytic activity of NK was tested in dogs using an experimental thrombosis model by infusing bovine fibrinogen and thrombin into the animals. Three dogs were treated with NK and six dogs were given a placebo as control. Four capsules of NK (250 mg/capsule: 2.13 CU/mg equivalent to 1600 FU) or placebo were orally administered. Angiograms were obtained before the induction of the thrombus and from 2.5 to 24.0 h afterwards. In the control group, there was no sign of lysis 18 h after induction of thrombosis. In contrast, the dogs treated with NK had a complete restoration of blood circulation within 5 h (Fujita et al., 1993). The fibrinolytic activity of NK was also examined in a rat model, in which thrombus was formed in the common carotid artery by damaging the endothelial cells of the vessel wall with acetic acid. In this model, urokinase or t-PA given intravenously at a constant rate of 20 min restored blood flow (45%) over 60 min. There was no restoration of blood flow with saline. NK was tested in this model in doses of 0.02, 0.04, and 0.12 μmol/kg (intravenous) and its activity was compared to plasmin and elastase. NK caused a dose-related recovery of blood flow (18%, 42%, and 62%) after 60 min. When the activity of NK and plasmin was compared on a molar basis, NK was 4-fold more efficient than plasmin. There was no recovery with elastase. Degradation of cross-linked fibrin was determined by the presence of D-dimer γ–γ chain remnants in the plasma. D-dimer remnants were detected in the blood after treatment with NK as well as urokinase and t-PA. The feasibility of using NK

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therapeutically for fibrinolysis would depend on its ability to digest fibrin without acting on fibrinogen. Values for residual plasma fibrinogen following administration of a dose of NK were reduced by one-third to the approximate activity level of plasmin and the residual fibrinogen level was 53%. This is a greater amount of residual fibrinogen than the 33% remaining after treatment with plasmin at a comparative level. The results imply that NK may be safer than plasmin at an appropriate dose level (Sumi et al., 1990). Thickening of vascular intima is thought to be part of the progression of arteriosclerotic plaques which can lead to heart attack and stroke. The ability of NK to inhibit the progression of intimal thickening was tested in a rat model. In this model, endothelial damage to the femoral artery was induced by intravenous injection of Rose Bengal followed by irradiation with transluminal green light. Twenty-one days after endothelial injury, significant intimal thickening was observed. Administration of NK (50 or 100 CU/animal, equivalent to 38 and 75 FU/animal) began 3 weeks before the endothelial injury and then continued for another 3 weeks. NK reduced the development of intimal thickening from an area of 1.28 ± 1.14 mm2 in the control group to 0.79 ± 0.60 mm2 and 0.71 ± 0.27 mm2 in the low- and high-dose groups. The difference between the thickening in the control group and the high-dose group was significant (p < 0.05). When the intima/media ratios were compared for the three groups, both treatment groups were significantly different from the control group (p < 0.05). There was no difference between the control and treatment animals in the time taken to develop occlusion following injury. However, differences were observed in the morphology of the mural thrombi. In the control group the center of the vessel reopened with mural thrombi attached to the vessel walls. In the NK groups, thrombi near the vessel walls showed lysis and most thrombi were detached from the vessel wall surface. The control group had thrombi attachment lengths measuring 858 ± 430 mm at 8 h after injury. NK reduced the attachment length in a dose-dependent manner with the measurement of 173 ± 105 mm for the high-dose group, a significant difference (p < 0.05) as compared to that of the control. Bleeding times for the three groups did not differ (Suzuki et al., 2003). NK was previously demonstrated to decrease blood pressure in animal models using Wistar male rats weighing 400–450 g. The rats were intraperitoneally injected with 0.5 mL of a lyophilized natto extract dissolved in 80% ethanol which was equivalent to 25 mg natto or about 0.8 FU total or 2 FU/ kg body weight. Blood pressure was measured using the tail artery. The average systolic blood pressure of six rats significantly (p < 0.05) decreased 2 and 3 h after administration of the natto extract by 12.6% and 13.2%, respectively. The systolic blood pressure decreased from 166 ± 14 mm Hg at baseline to 144 ± 27 mm Hg after 3 h (Maruyama and Sumi, 1998).

19.7.3 HUMAN CLINICAL STUDIES Various clinical studies demonstrated that NK or NSK-II possessed activities such as (1) fibrinolysis, (2) reduction of hypertension, (3) decrease of blood viscosity, and (4) platelet-aggregatory inhibition. 19.7.3.1 Fibrinolytic Effect Preliminary evidence that NK would have a fibrinolytic effect on human subjects was reported in 1990 (Maruyama and Sumi, 1998). Twelve health volunteers (21–55 years old) were given a single dose of 200 g natto (equivalent to about 6000 FU) or a control of boiled soybeans in a crossover single-dose designed study at a 2-week interval. Blood was collected 2–24 h after ingestion. Euglobin (clot), lysis time (ELT) significantly decreased 2, 4, and 8 h after intake of natto compared to the soybean control. In another experiment the volunteers were given two enteric-coated capsules containing NK (650 mg/capsule; 2.13 CU/mg) 3 times a day following meals (equivalent to 3000 FU/ day) for 8 days. Blood was collected each day. Euglobulin fibrinolytic activity (EFA) gradually but not significantly increased over the course of 8 days. The degraded products from fibrin and fibrinogen (FDP) in the serum were not measured. The FDP levels in the serum spiked on the first day and

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then slowly decreased over the 8-day period. The levels were significantly different from baseline on days 1 through 4 (Fujita et al., 1993). In a different study, a single oral dose of 30 g lyophilized natto (equivalent to 200 g original wet weight which is equivalent to 6000 FU) was given to five volunteers (51–86 years old). Blood samples were taken 2–24 h after intake. Fibrinolysis was evaluated 4–8 h after intake. EFA significantly increased after 5 h and FDP measurements remarkably increased 6 and 8 h after administration. EFA increased from 1.9 ± 2.7 mm2 at baseline to 4.5 ± 3.3 mm2 after 2 h, 1.33 ± 7.2 mm2 after 4 h, and 8.7 ± 7.4 mm2 after 8 h. The FDP levels at baseline, 6 h, and 8 h were 0.75 ± 0.52, 5.50 ± 2.74, and 2.75 ± 1.37 mcg/mL, respectively. The FDP was further decreased following additional intakes on days 2 and 4 (Sumi et al., 1996; Sumi and Maruyama, 1998). A double-blind, placebo-controlled study with 30 human subjects (male and female; average age 59) explored the administration of NK to patients taking prescribed warfarin for possible food–drug interaction. The theory behind the combination of the two agents was that the addition of NK might help stabilize the fibrinolytic effect of warfarin. The treatment group was given two capsules of NSK II (1700 FU) per day after breakfast for 26 weeks. As a result, there were significantly decreased rates of change in prothrombin and prothrombin-INR compared to placebo (p < 0.05). Treatment was particularly effective for those over 60 years of age. Activated partial thromboplastin time and prothrombin time were closed to reference values compared to the placebo group after 4 months (p < 0.05). In addition, lower rates of change were noted for activated partial thromboplastin and prothrombin, and prothrombin-INR times (Ninomiya and Yamada, 2008). 19.7.3.2 Reduction of Hypertension Hypertension is a global public health concern, affecting about 50 million individuals in the United States and one billion individuals worldwide (Kearney et al., 2005; Chobanian et al., 2003). High blood pressure is postulated as the most crucial risk factor for cardiovascular disease. In Japan, the disease is the third highest mortality cause of the 2000 s (Ohama et al., 2006). Moreover, hypertension is an important predictor of stroke, acute myocardial infarction, and end-stage renal disease (Iseki et al., 2000). It has been reported that the occurrence of cardiovascular disease in Asia is relatively low, attributable to traditional food from Asia (Artaud-Wild et al., 1993; Keys et al., 1984). In fact, it is traditionally known that natto in the diet tends to lower blood pressure (National Federation of Cooperatives on Natto, 1977). A previous study of natto and NK for the control of high blood pressure suggested that such foods might exert their beneficial effect through the inhibition of the angiotensin converting enzyme (ACE) (Kim and Yamamoto, 1992). However a recent clinical study found no difference in blood levels of ACE following treatment with NK but did report a decrease in rennin activity (Kim et al., 2008). This study suggested that the mechanism whereby NK decreased blood pressure might be attributable to the inhibition of rennin activity as further described below. Briefly, a randomized, double-blind, placebo-controlled study was conducted with 73 hypertensive subjects (20–80 years old) with initial systolic blood pressures between 130–159 mm Hg. The subjects received one capsule of NSK-II (2000 FU/capsule) per day or a placebo for 8 weeks. After 8 weeks of treatment there were significant decreases in systolic and diastolic blood pressure compared to placebo. Both treatment and placebo groups had some reduction in blood pressure, with the net decreases for the treatment group being 5.5 mm Hg in systolic blood pressure and 2.8 mm Hg in diastolic blood pressure. There was also a net decrease in plasma rennin activity (1.17 ng/mL/h) in the treatment group compared to the control (p < 0.05). However, there was no significant difference in ACE levels between the two groups. In an open label clinical study, 30 g of lyophilized extract (80% ethanol; equivalent to 200 g natto, about 6400 FU) were orally administered for four consecutive days to human subjects who had high blood pressure. In four of the five subjects the systolic and diastolic blood pressure decreased when measured in the supine position. The systolic average values decreased by 10.9%

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from 173.8 ± 20.5 to 154.8 ± 12.6 mm Hg. The diastolic blood pressure decreased by 9.9% from 101.0 ± 11.4 to 91.2 ± 6.6 mm Hg (Maruyama and Sumi, 1998). A randomized, placebo-controlled, crossover study was conducted with 20 male and female subjects (18–75 years of age) with a variety of disease states such as essential hypertension, hypercoagulable states, autoimmune diseases, and diabetes. Half of the subjects in the study received 4000 FU (2000 FU twice a day of NSK) and the other half received placebo. After 4 weeks the groups crossed over and received the alternate intervention. There was a significant decrease in systolic blood pressure compared to baseline for the NSK group (p = 0.039) and no significant change in diastolic blood pressure as compared to baseline. The placebo treatment did not cause any change in systolic or diastolic pressure (Krishman Medical Association S.C, 2003). 19.7.3.3 Decrease of Blood Viscosity The effects of NK on blood flow were studied in a placebo-controlled crossover study with 15 healthy subjects aged 30–49 years (seven men and eight women). The volunteers were given three capsules of NSK (2000 FU/capsule or total of 6000 FU) in a single dose (Group A) or a placebo (Group P). There was a 2-week wash-out period before switching treatments. Blood flow was measured using the PeriScan PIM II method. In Group A, there was a significant increase in blood flow in the right and left middle fingers 80, 120, and 180 min after intake of NK (p < 0.01). Compared to Group P (placebo) there was a significant effect 180 min after intake (Group P 0.10 ± 011 V compared to Group A 0.42 ± 0.08 V; p = 0.034). Group A also had an increase in blood flow in the back of the right and left hands at 40, 80, 120, and 180 min compared to baseline (p < 0.01). When the volunteers were subdivided according to the BMI, those with a BMI over 23 treated with NK had a statistically significant increase blood flow compared those on placebo (p = 0.046) (Pais et al., 2006). 19.7.3.4 Antiplatelet Activity A series of experiments to assess the antiplatelet effects of NK was performed in human studies. An open label pilot study was first conducted by supplementing NK in soft gel capsules (4000 FU, equivalent to the NK activity of two packs of commercially available natto) to healthy human volunteers (Takaoka et al., 2003). The assay used was an aggregometer with a light-scattering method (Moriyama et al., 2003; Iizuka et al., 2007; Moriyama et al., 2009), which helped to determine the antiplatelet activity of NSK II based on the extent of platelet aggregatory inhibition and also the distribution of platelet aggregates based on the developmental formation of platelet aggregates with time and three sizes. An additional study using NK demonstrated that administration of NSK II could inhibit spontaneously occurring platelet aggregation (Takaoka et al., 2004). A further study led to the establishment of a possible mechanistic action of NK on platelet-aggregatory inhibition, suggesting that fibrin degradation products might have played a pivotal role in this inhibition (Takaoka et al., 2005). A previous study reported that urokinase could inhibit platelet aggregation through mechanisms independent of the generation of plasmin but rather through mechanisms which might be associated with the fibrinogen of a fragment as a responsible inhibitor of platelet aggregation (Torr-Brown and Sobel, 1996). These studies appear to demonstrate the antiplatelet effects of NSK II and provide support for the observation that NK prevents the generation of platelet microaggregates in the early phase of platelet activation and the formation of thrombi which lead to vascular occlusion. Thus, oral administration of NK soft gel is a good supplement for lifestyle-related diseases such as cardiovascular diseases.

19.8

FUTURE PROSPECTIVES

During the production process of NK useful by-products are also yielded in the culture media, including vitamin K2 and PGA. Recently, vitamin K2 has been successfully recovered from the cultured media.

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19.8.1

345

VITAMIN K2

Vitamin K2 is a fat-soluble vitamin which acts as a coenzyme and participates in the synthesis of a number of proteins involved in blood clotting and bone metabolism. It is possible to recover vitamin K2, previously removed by extraction from the filtration residue in the chitosan treatment, with an organic solvent such as ethanol or less polar solvents and then to concentrate the extract. Molecular distillation or steam distillation is employed. The recovered vitamin K2 can be used in medical treatments or in the prevention of osteoporosis. Although additional refinements could reduce the recovery cost of this vitamin K2, such products are already available in the marketplace. Vitamin K2 is an ingredient in the foods with specified health claim (FOSHU) category in Japan, which allows the claim that it “helps absorb calcium into the bone” (Yamaguchi et al., 1999).

19.8.2 POLY-γ-GLUTAMIC ACID PGA is a water-soluble and biodegradable substance that comprises d- and l-glutamic acid polymerized through γ-glutamyl bonds. Furthermore, PGA (consisting of 30–5000 glutamic acids) is responsible in part for the slippery characteristics of natto (Figure 19.7). In 2003, PGA was approved as an active ingredient of functional food under the FOSHU system with a health claim that it “helps absorption of calcium.” In light of its FOSHU approval, a largescale fermentation of PGA was attempted in order to search for suitable conditions for commercial PGA production (Ogawa et al., 1997). Incidentally, during the production of NK at its present industrial scale, a substantial amount of PGA has been detected in the culture media, which is eliminated as a by-product. Thus, the introduction of an efficient recovery methodology is important to reclaim PGA during NK production.

19.8.3 FOOD WITH SPECIFIED HEALTH CLAIM In 1991, the MHW, now the Ministry of Health, Labor and Welfare (MHLW), introduced the FOSHU system. Details are discussed by Ohama et al. (2006). FOSHU was based on the concept of “functional foods” which benefit the structure and function of the human body, as a result of research studies on the health benefits of foods since 1984. The Ministry of Education organized national research and development projects to evaluate the functionalities of various foods. Researchers from diverse scientific fields defined new functions of foods by incorporating previously recognized functions of nutrition, sensory/satisfaction, and the physiological effects of ingredients in foods. A representative FOSHU product based on material derived from traditional Japanese food is the fermented bonito or “Katsuobushi” peptide which has a health claim of “lowering blood pressure” (Fujita et al., 2001). Natto and its NK ingredient is another possible candidate for FOSHU, also categorized under the health claim of “lowering blood pressure.”

CO CH2 CH2 NH

CH

–

COO

n

FIGURE 19.7

The structure of PGA which is found in the stick-threads of natto.

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19.9 CONCLUSION Natto, a traditional Japanese food, is a source of functional ingredients such as the fibrinolytic enzyme known as NK, and vitamin K2. The large-scale production of functional foods has made it possible to commercialize them as ingredients for dietary supplements. NK, in particular, plays an important role in antithrombosis, which may potentially help to prevent vascular disorders such as cardiovascular disease. The present NK production methodology successfully eliminates vitamin K2 as a coagulation agent, which is considered as a contaminant in the final NK product. NK has been demonstrated to be safe, and proven to show not only fibrinolytic activity but also antihypertensive, antiplatelet, and other blood circulatory beneficial activities to help prevent vascular diseases. NK could thus be used as a dietary supplement replacement for aspirin or nonsteroidal anti-inflammatory drugs for the prevention of thrombi formation without any side effects. The incorporation of additional steps in the NK production process can isolate vitamin K2 and possibly PGA for their utilization in other health benefits.

ACKNOWLEDGMENT One of the authors (Hiroyoshi Moriyama) is supported by funds from MTI, Inc. (Tokyo, Japan) and the Japan Bio Science Laboratory Co., Ltd. (Osaka, Japan).

REFERENCES Artaud-Wild, S.M., Connor, S.L., Sexton, G., and Connor, W.E., 1993. Differences in coronary mortality can be explained by differences in cholesterol and saturated fat intakes in 40 countries but not in France and Finland. A paradox. Circulation 88: 2771–2779. BILIS, Environmental Biological Life Science Research Center, Inc., Shiga, Japan. Final report: Singe oral dose toxicity study of BIOZYME NSK-FD POWDER in rats. February 26, 1999. Bozo Research Center, Inc., Tokyo, Japan. Final report: A 13-week oral toxicity study of nattokinase DS (NSK-SD) in rats. B-5170, June 16, 2004. Cesarman-Maus, G. and Hajjar, K.A., 2005. Molecular mechanisms of fibrinolysis. Brit. J. Haematol. 129: 307–321. Chobanian, A.V., Bakris, G.L., Black, H.R., et al., 2003. National heart, lung, and blood institute joint national committee on prevention, detection, evaluation, and treatment of high blood pressure education program coordinating committee: The seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure: The JNC 7 report. JAMA 289: 2560–2572. Ero, M.P. and Lewis, B.H., Coagulation profile of nattokase. A study report by Machaon Diagnostics, Inc., 3023 Summit Street Oakland, CA, USA, February 20, 2008. Fujita, M., Hong, K., Ito, Y., Misawa, S., Takeuchi, N., Kariya, K., and Nishimuro, S., 1995a. Transport of nattokinase across the rat intestinal tract. Biol. Pharm. Bull. 18: 1194–1196. Fujita, M., Ito, Y., Hong, K., and Nishimuro, S., 1995. Characterization of nattokinase-degraded products human fibrinogen or cross-linked fibrin. Fibrinolysis 9: 147–164. Fujita, M., Nomura, K., Hong, K., Ito, Y., Asada, A., and Nishimuro, S, 1993. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem. Biophys. Res. Commun. 197: 1340–1347. Fujita, H., Yamagami, T., and Oshshima, K., 2001. Effects of an ace-inhibitory agent, katsuobushi oligopeptide in the spontaneously hypertensive rat and in borderline and mildly hypertensive subjects. Nutr. Res. 21: 1149–1158. Gifu Research Laboratories, JBS Co., Ltd., Gifu, Japan. Final report: Pathogenicity study of nattokinase DS (NSK-SD) producing bacteria in mice. Study No. JBS-03-MOPG-549, June 9, 2003. Iizuka, T., Nagai, M., Taniguchi, A., Moriyama, H., and Hoshi, K., 2007. Inhibitory effects of methyl brevifolincarboxilate isolated from Phyllanthus niruri L. on platelet aggregation. Biol. Pharm. Bull. 30: 382–384. Iseki, K., Kimura, Y., Wakugami, K., et al., 2000. Comparison of the effect of blood pressure on the development of stroke, acute myocardial infarction, and end-stage renal disease. Hypertens. Res. 23: 143–149.

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Japan Bio Science Laboratory Co., Ltd. Clinical study report: An open clinical pilot study to evaluate the safety and efficacy of natural super kinase as an add-on oral fibrinolytic agents to low molecular weight heparin & anti-platelet in acute ischaemic stroke. DS-NK007, February 28, 2004. Kearney, P.M., Whelton, M., Reynolds, K., Muntner, P., Whelton, P.K., and He, J., 2005. Global burden of hypertension: Analysis of worldwide data. Lancet 365: 217–223. Keys, A., Menotte, A., Aravanis, C., et al., 1984. The seven countries study: 2289 deaths in 15 years. Prev. Med. 13: 141. Kim, J.-Y., Gum, S.-N., Paik, J.-K., Lim, H.-H., Kim, K.-C., Ogasawara, K., Inoue, K., Park, S., Jang, Y., and Lee, J.-H., 2008. Effects of nattokinase on blood pressure: A randomized, controlled trial. Hypertens. Res. 31: 1583–1588. Kim, S. and Yamamoto, K., 1992. The in vivo role of rennin-agiotensin system. Cell Sci. 8: 146–151. Kobuchisawa Laboratories, Fuji Biomedix Co., Ltd., Yamanashi, Japan. Final report: Oral repeated-dose 28-day toxicity study of Nattokinase DS in rats. FBM 02-2704, August 8, 2002. Kobuchizawa Laboratories, Fuji Biomedix Co., Ltd, Yamanashi, Japan. Final report: Reverse-mutation assay of nattokinase DS in bacteria. FBM 02-8766, February 28, 2003a. Kobuchizawa Laboratories, Fuji Biomedix Co., Ltd, Yamanashi, Japan. Final report: Chromosomal aberration test of nattokinase DS in cultured mammalian cells. FBM 02-8767, February 28, 2003b. Krishman Medical Association S.C. Evergreen Park, IL 60805, USA, Effect of NSK-SD on blood pressure by oral administration. July 12, 2003. Maruyama, M. and Sumi, H., 1998. Effect of Natto Diet on Blood Pressure. pp. 1–3. Japan Technology Transfer Association (JTTAS). Moriyama, H., Hosoe, T., Wakana, D., Itabashi, T., Kawai, K., Iizuka, T., Hoshi, K., Fukushima, K., and Lau, F.C., 2009. Assay-guided informatory screening method for antiplatelet effect of adenosine isolated Malbranchea filamentosa IFM 41300: Inhibitory behaviors of adenosine in different solvent. J. Health Sci. 55: 103–108. Moriyama, H., Iizuka, T., Nagai, M., and Hoshi, K., 2003. Adenine, an inhibitor of platelet aggregation from the leaves of Cassia alata. Biol. Pharm. Bull., 26: 1361–1364. Nakamura, T., Yamagata, Y., and Ichishima, E., 1992. Nucleotide sequence of the Subtilisin NAT gene, aprN, of Bacillus subtilis (natto). Biosci. Biotechnol. Biochem. 56: 1869–1871. National Federation of Cooperatives on Natto, 1977. In: A Historical Record of Natto, Foods Pioneer. pp. 1–367. Tokyo: Natto Research Center (in Japanese). Ninomiya, J. and Yamada, S., 2008. Clinical study of effect and safety of nattokinase administration on cardiovascular patients with warfarin. Jpn. Pharmcol. Ther., 36: 453–464, (in Japanese). Ogasawara, K., Iuchi, S., and Shionoya, K., 2006. An oral safety study of nattokinase containing food, natural super kinase II: A randomized, placebo controlled, double-blind study. Prog. Med. 26: 1137–1148 (in Japanese). Ogawa, Y., Yamaguchi, F., Yuasa, K., and Tahara, Y., 1997. Efficient production of γ-polyglutamic acid by Bacillus subtilis (natto) in jar fermenters. Biosci. Biotechnol. Biochem. 61: 1684–1697. Ohama, H., Ikeda, H., and Moriyama, H., 2006. Health foods and foods with health claims in Japan, Toxicology 221: 95–111. Pais, E., Alexy, T., Holsworth, R.E., and Meislman, H.J., 2006. Effects of nattokinase, a pro-fibrinolytic enzyme, on red blood cell aggregation and whole blood viscosity. Clin. Hemorheol. Microcirc. 35: 139–142. Sumi, H. and Banba, T., and Kishimoto, N., 1996. Strong pro-urokinase activators proved in Japanese soybean cheese natto. Nippon Shokuhin Kagaku Kaishi 43: 1124–1127 (in Japanese). Sumi, H., Hamada, H., Nakanishi, K., and Hiratani, H., 1990. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematol. 84: 129–143. Sumi, H., Hamada, H., Tsushima, H., Mihara, H., and Muraki, H., 1987. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese natto; a typical and popular soybean food in the Japanese diet. Experientia 43: 1110–1111. Sumi, H. and Maruyama, M., 1998. Enhancement of Fibrinolytic System and Thrombolytic Effect by Natto Diet, pp. 5–10. Japan Technology Transfer Association (JTTAS) 5–10. Shah, A.B., Rawat, S., Mehta, S., Takaoka, S., Sato, K., and Ogasawara, K., 2004. An open clinical pilot study to evaluate the safety and efficacy of natto kinases an add-on oral fibrinolytic agent to low molecular weight heparin &anti-platelets in acute ischeamic stroke. Jpn. Pharmacol. Ther. 32: 437–445 (in Japanese). Suzuki, Y., Kondo, K., Matsumoto, Y., Zhao, B.Q., Outsuguro, K., Maeda, T., Tsukamoto, Y., Urano, T., Umemura, K., 2003. Dietary supplementation of fermented soybean, natto, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial injury in rat femoral artery. Life Sci. 73: 1289–1298.

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Takaoka, S., Murata, Y., Taniguchi, A., Moriyama, H., and Hoshi, K., 2003. The 123rd Annual Meeting of the Pharmaceutical Society of Japan, Nagasaki, Japan. Antiplatelet effects of nattokinase (NSK-SD), p. 134 (abstract in Japanese). Takaoka, S., Sato, K., Taniguchi, A., Moriyama, H., and Hoshi, K., 2005. The 125th Annual Meeting of the Pharmaceutical Society of Japan, Tokyo, Japan. Antiplatelet effects of fibrin degradation products produced by nattokinase (NSK-SD), 30-0460 (abstract in Japanese). Takaoka, S., Taniguchi, A., Moriyama, H., and Hoshi, K., 2004. The 124th Annual Meeting of the Pharmaceutical Society of Japan, Osaka, Japan. Effects of nattokinase (NSK-SD) on spontaneous platelet aggregation. 29[P2]III-468 (abstract in Japanese). Torr-Brown, S.R. and Sobel, B.E., 1996. Antiplatelet effects of uronikanse and their clinical implications. Therapy and Prevention 7: 63–68. Urano, T., Ihara, H., Suzuki, Y., Oike, M., Akita, S., Tsukamoto, Y., Suzuki, I., and Takada, A., 2001. The profibrinolytic enzyme Subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor. J. Bio. Chem. 276: 24690–24696. Yamaguchi, M., Taguchi, H., Gao, Y.H., Igarashi, A., and Tsukamoto, Y., 1999. Effect of vitamin K2 (menaquinone-7) in fermented soybean (natto) on bone loss in ovariectomized rats. J. Bone Miner. Metab. 17: 23–29.

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of Antihypertensive 20 Synthesis GABA-Enriched Dairy Products Using Lactic Acid Bacteria Kazuhito Hayakawa CONTENTS 20.1 Introduction .......................................................................................................................... 349 20.2 GABA and Hypertension ...................................................................................................... 350 20.3 Fermented Milk and Hypertension ....................................................................................... 351 20.4 Production of GABA Using Two Strains of LAB ................................................................ 351 20.5 Antihypertensive Effect of Fermented Milk Containing GABA ......................................... 354 20.6 Mechanism of the Antihypertensive Effect of GABA ......................................................... 356 20.7 Conclusions ........................................................................................................................... 358 References ...................................................................................................................................... 358

20.1

INTRODUCTION

High blood pressure is the leading risk factor for cardiovascular disease, which remains one of the world’s most important public health problems (World Health Organization, International Society of Hypertension Writing Group, 2003; European Society of Hypertension and European Society of Cardiology, 2007). Hypertension affects approximately one billion individuals worldwide. Hypertension develops because of a number of factors, including those related to diet and lifestyle. It is well known that nutritional factors influence blood pressure; for example, excessive salt and alcohol intake may lead to an increase in blood pressure (Suter et al., 2002). On the other hand, some foods decrease blood pressure and reduce the risk of hypertension. For example, a study of 5000 people found that those with normal blood pressure reported a higher consumption of milk than did hypertensive subjects (Ackley et al., 1983). The authors of that study suggested that some component of milk, probably calcium, exerts a protective effect against hypertension. A large-scale intervention trial on dietary approaches to stop hypertension (DASH) demonstrated that a diet rich in fruits, vegetables, low-fat dairy products, fiber, and minerals (calcium, potassium, and magnesium) significantly lowers blood pressure and is effective as a fi rst-line therapy (Moore et al., 2001; National High Blood Pressure Education Program Coordinating Committee, 2002; DASH-Sodium Trial Collaborative Research Group, 2003; Appel et al., 1997; Miller et al., 2000). Many studies have addressed the effect of food components on blood pressure, but only a few have assessed the influence of small quantities of

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an amino acid as a dietary supplement. This chapter focuses on γ-aminobutyric acid (GABA), found in a variety of fruit and vegetables.

20.2

GABA AND HYPERTENSION

GABA is an amino acid that is present in various vegetables and fruits but not in meat or dairy products (Table 20.1). We usually ingest small amounts of GABA from vegetables and fruit. GABA is one of the major inhibitory neurotransmitters in the central nervous system and is also found in the peripheral tissues; it has been proposed to play an important role in the modulation of cardiovascular function (Gillis et al., 1980) by acting not only within the central nervous system but also within these peripheral tissues (DeFeudis et al., 1981; DeFeudis et al., 1982). Indeed, GABA has been reported to reduce blood pressure in experimental animals (Takahashi et al., 1955) and humans (Elliott and Hobbiger, 1959) following its systemic or central administration, and it has been suggested that the depressor effect induced by systemic administration of GABA (Takahashi et al., 1958; Stanton, 1963) is due to blockade of the sympathetic ganglia (Stanton and Woodhouse, 1960). The blood–brain barrier is impermeable to GABA (Kuriyama and Sze, 1971), and the concentration of GABA in the brain is not changed following intravenous GABA injection (Roberts et al., 1958; Tsukada et al., 1960; Gelder van, 1958). Thus, the antihypertensive effects seen following intraperitoneal or intravenous administration of GABA are due to its actions within the peripheral tissues (presumably, blood vessels or the autonomic nervous system). GABA is able to modulate vascular tone by suppressing noradrenaline release in isolated rabbit ear arteries and rat kidneys (Manzini et al., 1985; Monasterolo et al., 1996; Fujimura et al., 1999). However, the effect of dietary GABA has attracted little attention as a factor influencing blood pressure, and it is not known whether low-dose oral administration of GABA affects blood pressure.

TABLE 20.1 Characteristics of γ-Aminobutyric Acid (GABA) Structural formula

NH2–CH2–CH2–CH2–COOH; MW 103

Biosynthesis

H

COOH H

C

NH2

CO2

H

COOH Existence in animals Physiology

Pharmacological effect (central action)

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NH2

CH2

CH2 CH2

C

Glutamate decarboxylase (EC 4.1.1.15 )

CH2 COOH

l-Glutamic acid GABA Brain, small intestine, fallopian tubes, islets of Langerhans Inhibitory neurotransmitter GABA receptor Type A; Cl− channel Type B; Ca2+, K+ channel Antidepressant, increases growth hormone, reduces blood pressure

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20.3

351

FERMENTED MILK AND HYPERTENSION

The history of fermented milk goes back a long time. Fermented milk was recognized as an important food as early as 4000 b.c. At that time, fermentation was used for the biopreservation of milk. However, since the publication of The Prolongation of Life by Nobel laureate E. Metchnikoff (1908) at the beginning of the twentieth century, the focus has been directed towards the physiological functions of fermented milk. It has been suggested that the use of lactic acid bacteria (LAB) and fermented milk containing these microbes and metabolic products may improve human health (Shortt et al., 2004). Fermentation of milk by LAB generates new functions that are not associated with the milk itself. For example, lactic acid produced by fermentation of lactose not only enhances the taste of milk products, but also induces physiological actions such as promoting digestive tract movement and improving intestinal flora owing to the suppression of pathogen growth. In addition, there are other benefits such as the increased digestibility of proteins and lactose following biodegradation with LAB. Furthermore, interest has recently been directed to a variety of benefits through the activation of the immune system, reduction of serum cholesterol, and antitumor efficacy (De Roos and Katan, 2000). Viable microbes conferring beneficial effects on their hosts are called “probiotics” (Salminen et al., 1998). Some studies have reported that, in both single-dose and long-term regimens, various fermented milk or cheese products have stronger hypotensive effects than milk itself (Nakamura et al., 1995; Yamamoto et al., 1999; Saito et al., 2000; Seppo et al., 2003). Some LAB release peptides from the protein of the milk by proteolysis; these peptides can inhibit angiotensin-converting enzyme (ACE), which is instrumental in inducing hypertension (Nakamura et al., 1995; Yamamoto et al., 1999). The effects of antihypertensive peptides (Val-Pro-Pro and Ile-Pro-Pro) in fermented sour milk have been demonstrated in spontaneously hypertensive rats (SHR) (Nakamura et al., 1995) and in mildly hypertensive humans (Hata et al., 1996). A placebo-controlled human study showed that the blood pressure of hypertensive subjects decreased significantly between 4 and 8 weeks after daily intake of this sour milk (Hata et al., 1996). From a different perspective, we used two types of LAB to develop a fermented nonfat milk product that contains GABA. Although not normally detectable in milk, GABA was produced from the protein of the milk by the metabolic actions of the two LAB.

20.4 PRODUCTION OF GABA USING TWO STRAINS OF LAB We tried to establish a novel fermented milk product containing GABA produced by LAB. At first, we searched for LAB strains that produce GABA. Many kinds of LAB were cultured in Rogosa medium with added 1% sodium l-glutamic acid. A few strains of Lactococcus lactis and Streptococcus thermophilus produced GABA in the culture broth, but most LAB did not (Table 20.2). Lactococcus lactis YIT 2027 was an especially good strain for GABA production: the conversion ratio from l-glutamic acid to GABA by glutamate decarboxylase (GAD) (EC 4.1.1.15) was near 100%. The GAD activity in Lc. lactis YIT 2027 was highest at 30–45°C and pH 5.0–5.3 (Figure 20.1). pH-stat culture (pH kept at 7.0 with NaOH) of Lc. lactis YIT 2027 resulted in markedly higher cell growth than non-pH-stat culture, but GABA was not produced. Therefore, from the point of view of GABA production, acidic fermented milk is beneficial, because the GAD activity of Lc. lactis YIT 2027 is optimal under acidic conditions, and GABA accumulates during the fermentation process. To create a novel fermented milk product containing GABA, we examined the GABA productivity of Lc. lactis YIT 2027 in fat-free milk. GABA productivity was shown to be very low, because the levels of free l-glutamic acid, the precursor of GABA, are low in nonfat milk. However, plenty of l-glutamic acid is included as a constituent amino acid in milk protein (approximately 20%). Therefore, we attempted to make use of another LAB to release the l-glutamic acid from

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TABLE 20.2 GABA Productivity of Various Lactic Acid Bacteria (Examples from 110 Strains) GABA Productivity

Strain Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus casei Lactobacillus casei Lactobacillus casei Lactobacillus casei Lactobacillus delbrueckii Lactobacillus delbrueckii Lactobacillus delbrueckii Lactobacillus delbrueckii Lactobacillus delbrueckii Lactobacillus delbrueckii Lactobacillus delbrueckii Lactobacillus fermentum Lactobacillus gasseri Lactobacillus gasseri Lactobacillus gasseri Lactobacillus gasseri Lactobacillus helveticus Lactobacillus helveticus Lactobacillus helveticus Lactobacillus helveticus Lactobacillus johnsonii Lactobacillus johnsonii Lactobacillus johnsonii Lactobacillus kefir Lactobacillus pentosus Lactobacillus plantarum Lactobacillus plantarum

YIT 0070 YIT 0165 YIT 0191 YIT 0198 YIT 0281 YIT 0282 YIT 0003 YIT 0180 YIT 0209 YIT 9029 YIT 0058 YIT 0086 YIT 0162 YIT 0181 YIT 0182 YIT 0183 YIT 0206 YIT 0081 YIT 0164 YIT 0168 YIT 0192 YIT 0279 YIT 0083 YIT 0085 YIT 0113 YIT 0177 YIT 0202 YIT 0219 YIT 0284 YIT 0222 YIT 0238 YIT 0101 YIT 0102

− − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

GABA Productivity

Strain Lactobacillus plantarum Lactobacillus rhamnosus Lactobacillus reuteri Lactobacillus rhamnosus Lactobacillus sakei Lactobacillus salivarius Lactobacillus zeae Lactococcus lactis Lactococcus lactis Lactococcus lactis Lactococcus lactis Lactococcus lactis Leuconostoc lactis Leuconostoc lactis Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Bifidobacterium adolescentis Bifidobacterium adolescentis Bifidobacterium bifidum Bifidobacterium bifidum Bifidobacterium breve Bifidobacterium breve Bifidobacterium catenulatum Bifidobacterium catenulatum Bifidobacterium infantis Bifidobacterium infantis Bifidobacterium lactis Bifidobacterium longum Bifidobacterium longum Bifidobacterium pseudocatenulatum

YIT 0132 YIT 0105 YIT 0197 YIT 0125 YIT 0259 YIT 0104 YIT 0078 YIT 2008 YIT 2013 YIT 2027 YIT 2052 YIT 2105 YIT 3004 YIT 3005 YIT 2001 YIT 2021 YIT 2037 YIT 2046 YIT 2047 YIT 4011 YIT 4087 YIT 4007 YIT 4013 YIT 4014 YIT 4065 YIT 4016 YIT 4118 YIT 4018 YIT 4019 YIT 4121 YIT 4021 YIT 4037 YIT 4072

− − − − − − − − ++ +++ + +++ − − − − − ++ ++ − − − − − − − − − − − − − −

GABA productivity was estimated by the ratio of conversion from l-glutamic acid to GABA in modified Rogosa medium with 1% glutamate: −, 0%; +, 1–10%; ++, 11–80%; +++, >80%.

the milk protein. We cultured many kinds of LAB in nonfat milk and determined the amounts of free l-glutamic acid present after cultivation (Table 20.3). Of the strains we examined, we found that Lactobacillus casei strain Shirota released the highest amounts of l-glutamic acid from milk protein during the fermentation process. On the basis of these results, we anticipated that GABA productivity would be enhanced by coculturing Lc. lactis YIT 2027 with Lb. casei strain Shirota. Glutamic acid is dissociated from the protein of the milk by the protease of Lb. casei strain Shirota, and the glutamic acid is converted to

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Synthesis of Antihypertensive GABA-Enriched Dairy Products (a)

(b)

100

100 Relative activity (%)

Relative activity (%)

353

80 60 40 20 0

80 60 40 20

0

20

40

60

0 4.0

4.5

Temperature (°C)

5.0 pH

5.5

6.0

FIGURE 20.1 The effects of temperature and pH on the activity of crude GAD from Lc. lactis YIT 2027. (a) Enzyme activity was assayed in 100 mM sodium phosphate-citrate buffer (pH 5.3) at different temperatures. (b) Enzyme activity was assayed in 100 mM sodium phosphate-citrate buffer at 35°C. Relative activity is shown in proportion to activity at pH 5.3, 35°C.

TABLE 20.3 L-Glutamic Acid-Releasing Activity of Various Lactic Acid Bacteria (Examples From 110 Strains) Glutamic Acid Release

Strain Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus casei Lactobacillus casei Lactobacillus casei Lactobacillus casei Lactobacillus casei Lactobacillus delbrueckii Lactobacillus delbrueckii Lactobacillus delbrueckii Lactobacillus fermentum Lactobacillus gasseri Lactobacillus gasseri Lactobacillus gasseri Lactobacillus helveticus Lactobacillus helveticus Lactobacillus johnsonii Lactobacillus johnsonii Lactobacillus kefir

YIT 0070 YIT 0191 YIT 0165 YIT 0003 YIT 0078 YIT 0180 YIT 0209 Shirota strain YIT 0086 YIT 0181 YIT 0183 YIT 0081 YIT 0164 YIT 0168 YIT 0192 YIT 0083 YIT 0113 YIT 0219 YIT 0284 YIT 0222

++ ++ + + + + + +++ ++ + ++ + ++ + + ++ + ++ +++ +

Glutamic Acid Release

Strain Lactobacillus pentosus Lactobacillus plantarum Lactobacillus reuteri Lactobacillus rhamnosus Lactobacillus rhamnosus Lactobacillus sakei Lactobacillus salivarius Lactococcus cremoris Lactococcus lactis Leuconostoc lactis Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus Streptococcus thermophilus

YIT 0238 YIT 0102 YIT 0197 YIT 0105 YIT 0125 YIT 0259 YIT 0104 YIT 2007 YIT 2027 YIT 3005 YIT 2001 YIT 2021 YIT 2037 YIT 2046 YIT 2047 YIT 2090 YIT 2103 YIT 2104 YIT 2105 YIT 2109

+ + + + + + + ++ + + + + + + + + + + + +

l-Glutamic acid-releasing activity was estimated by the amount of l-glutamic acid after cultivation in a 20% skim milk aqueous solution: +, 0–10 mg/100 mL; ++, 11–20 mg/100 mL; +++, >20 mg/100 mL.

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30

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20 15 10

15 10 5

5 0

20

0 0

1 2 Culture time (day)

3

0

1 2 Culture time (day)

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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.

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185 180 175

115 110 105

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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

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8 Age (weeks)

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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).

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0

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16

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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.

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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.

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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

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Biotechnology in Functional Foods and Nutraceuticals

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

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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

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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 <0.01%

Lb. casei

Mixture of fatty acids

Pediococcus acidilactici Mixture of fatty acids

Saccharomyces cerevisiae Vegetable wax a b

c

d

1.6 × 1011

Control 8% Gastricb Control <0.01%

7.5 × 1010

1.45 × 1010

Coated cells 15% Thermalc Coated cells 69% Coated cells 25% Powdered milkd

Control 15% Not tested

Coated cells 69%

Recovery rates in percentage after stability tests. Control consists of uncoated freeze-dried cells. Gastric stability: 1 g of freeze-dried powder or coated particles is placed in 100 mL of 0.1 NHCl solution (pH 1.2) and incubated at 37°C for 1 h. Thermal stability: 10 g of freeze-dried or coated particles are introduced in sealed tubes, kept in a water bath at 50°C for 24 h. Stability in powdered milk: freeze-dried or coated particles are mixed into powdered milk at a rate of 1% (w/w). The mixtures were divided into samples in welded polythene pouches, kept at 30°C for four months.

(Table 21.5). Improved stability of spray-coated cells to simulating gastric conditions, high temperature and during storage in powdered milk compared to uncoated freeze-dried cells was also reported (Table 21.5) as well as a better resistance of cells to compression (Durand and Panes, 2002).

21.5.3 PRODUCTION OF WATER-INSOLUBLE FOOD-GRADE MICROCAPSULES BY EMULSION AND SPRAY-DRYING This technique consists of encapsulating milk fat droplets containing dried probiotic bacteria in whey protein-based water-insoluble microcapsules, using emulsification and spray-drying in a twostep continuous process (Picot and Lacroix, 2003a, 2003b). Soluble whey protein polymers were used as coating material and formed, upon rehydration, a stabilizing film around milk fat globules containing micronized powder of freeze-dried bacteria. This low-cost technique permits production of large quantities of low-diameter (<100 μm) microcapsules. Control of size distribution of the different elements constituting the capsules is a critical step in the method. Freeze-dried powder of bacteria can be micronized to reduce particle size. However, micronization affects thermotolerance of probiotics notably for heat-sensitive strains, leading to high cell loss (>99%) during microencapsulation, and especially, the spray-drying step (Picot and Lacroix, 2003c, 2004). The use of fresh cells in heat-treated whey protein suspension followed by spray-drying was a less destructive method, with survival rates after spray-drying of 26% for B. breve and 1.4% for the more heat sensitive B. longum (Picot and Lacroix, 2004). Cell viability of B. breve entrapped in whey protein

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microcapsules using this method was significantly higher than those of free cells after 28 days in yoghurt stored at 4°C (+2.6 log), and after sequential exposure to simulated gastric and intestinal juices (+2.7 log) (Picot and Lacroix, 2004). However, no protective effect of encapsulation was observed for the tested B. longum strain. These findings emphasize the need to take the technological properties of probiotic strains into consideration with regard to processing, especially heat stability, when using spray-dry encapsulation.

21.6 CONCLUDING REMARKS Technological development of probiotics have until recently focused on achieving highest cell numbers after cell production and, in products in an economical way. Throughout this chapter, we have shown that the life of probiotics is riddled with many hurdles, during production, addition to products and digestion until reaching the target site of the gastrointestinal tract. Adaptation mechanisms of probiotic cells to adverse environments are based on overexpression of specific or general stress-response proteins and physiological modifications. Our knowledge about mechanisms and responses of probiotic cells to stressing conditions has considerably developed over the last few years, especially thanks to the progress in molecular approaches and this has largely contributed to the development of new technologies based on the exploitation of cellular stress response to efficiently stabilize and protect sensitive probiotic cells. However application of this basic knowledge to real processes has still to be fully implemented in probiotic culture processes. Additional research is required to elucidate stress-response mechanisms and identify molecular targets and biomarkers, for example, using holistic “omics” methods that could be used to assess cell physiology and possibly health functionality during and after the process. Indeed cell systems cannot be reduced to a set of independent chemical reactions, meaning that modification of one physiological property may, positively or negatively, affect another one. Apart from getting high cell numbers, the effects of technology on functional properties of probiotic strains must be considered. Investigations in media formulation should also be pursued. Novel cultivation approaches should be developed with the aim to maximize viability and health functionality, and most importantly to enlarge the range of probiotic strains to sensitive probiotics exhibiting high functionality that cannot be produced with traditional technologies. Research for new technologies such as continuous cultures to produce cells with controlled physiology, and cell immobilization to increase cell density and productivity, enhance process stability, protect cells from environmental stresses, and induce stress tolerance should be pursued, also considering challenges for transferring these technologies to industrial setting. Encapsulation technologies are promising to sustain both functional properties and viability of probiotic cells when added to products and during transit in the upper digestive tract. Additionally, encapsulation matrices could be developed to target delivery probiotic cells to specific regions of the gut (e.g., small/large bowel), enhancing their efficacy while protecting cells from environmental stresses in earlier stages. Target delivery and activity of probiotics in specific locations of the gastrointestinal tract requires knowledge of the interaction between receptors and molecules on cell surfaces, the latter being possibly affected by process conditions. Current studies aim to identify precise mechanisms underlying health effects of probiotics, for example, microbial molecular patterns that interact with pattern-recognition receptors on eukaryotic cells. In the near future this knowledge could likely be exploited to develop and/or optimize technologies for improving such functional properties.

REFERENCES Adhikari, K., Mustapha, A., and Grun, I.U., 2003. Survival and metabolic activity of microencapsulated Bifidobacterium longum in stirred yogurt. J. Food Sci. 68: 275–280. Adhikari, K., Mustapha, A., Grun, I.U., and Fernando L., 2000. Viability of microencapsulated bifidobacteria in set yogurt during refrigerated storage. J. Dairy Sci. 83: 1946–1951.

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Ruiz, L., Sanchez, B., Ruas-Madiedo, P., de Los Reyes-Gavilan, C.G., and Margolles, A., 2007. Cell envelope changes in Bifidobacterium animalis ssp. lactis as a response to bile. FEMS Microbiol. Lett. 274: 316–322. Saarela, M., Rantala, M., Hallamaa, K., Nohynek, L., Virkajarvi, I., and Matto, J., 2004. Stationary-phase acid and heat treatments for improvement of the viability of probiotic lactobacilli and bifidobacteria. J. Appl. Microbiol. 96: 1205–1214. Salminen, S. and Ouwehand, A.C., 2002. Probiotics, applications in dairy products. In: H. Roginski (Ed.), Encyclopedia of Dairy Sciences, pp 2315–2322. Oxford: Elsevier. Sanchez, B., Champomier-Verges, M.C., Anglade, P., Baraige, F., de Los Reyes-Gavilan, C.G., Margolles, A., and Zagorec, M., 2005. Proteomic analysis of global changes in protein expression during bile salt exposure of Bifidobacterium longum NCIMB 8809. J. Bacteriol. 187: 5799–5808. Sanchez, B., Champomier-Verges, M.C., Collado Mdel, C., Anglade, P., Baraige, F., Sanz, Y., de los ReyesGavilan, C.G., Margolles, A., and Zagorec, M., 2007a. Low-pH adaptation and the acid tolerance response of Bifidobacterium longum biotype longum. Appl. Environ. Microbiol. 73: 6450–6459. Sanchez, B., Champomier-Verges, M.C., Stuer-Lauridsen, B., Ruas-Madiedo, P., Anglade, P., Baraige, F., de los Reyes-Gavilan, C.G., Johansen, E., Zagorec, M., and Margolles, A., 2007b. Adaptation and response of Bifidobacterium animalis subsp. lactis to bile: A proteomic and physiological approach. Appl. Environ. Microbiol. 73: 6757–6767. Sanchez, B., de los Reyes-Gavilan, C.G., and Margolles, A., 2006. The F1–F0-ATPase of Bifidobacterium animalis is involved in bile tolerance. Environ. Microbiol. 8: 1825–1833. Sanchez, B., Ruiz, L., de los Reyes-Gavilan, C.G., and Margolles, A., 2008. Proteomics of stress response in Bifidobacterium. Front Biosci. 13: 6905–6919. Sanders, J.W., Leenhouts, K.J., Haandrikman, A.J., Venema, G., and Kok, J., 1995. Stress response in Lactococcus lactis: Cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene. J. Bacteriol. 177: 5254–5260. Saxelin, M., 2008. Probiotic formulations and applications, the current probiotics market, and changes in the marketplace: A European perspective. Clinic. Infect. Dis. 46: S76–S79. Schachtsiek, M., Hammes, W.P., and Hertel, C., 2004. Characterization of Lactobacillus coryniformis DSM 20001T surface protein Cpf mediating coaggregation with and aggregation among pathogens. Appl. Environ. Microbiol. 70: 7078–7085. Schell, M.A., Karmirantzou, M., Snel, B., Vilanova, D., Berger, B., Pessi, G., Zwahlen, M.C., Desiere, F., Bork, P., Delley, M., Pridmore, R.D., and Arigoni, F., 2002. The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc. Natl. Acad. Sci. USA 99: 14422–14427. Schmidt, G. and Zink, R., 2000. Basic features of the stress response in three species of bifidobacteria: B. longum, B. adolescentis, and B. breve. Int. J. Food Microbiol. 55: 41–45. Serror, P., Dervyn, R., Ehrlich, S.D., and Maguin, E., 2003. csp-like genes of Lactobacillus delbrueckii ssp. bulgaricus and their response to cold shock. FEMS Microbiol. Lett. 226: 323–330. Shah, N.P., Lankaputhra, W.E.V., Britz, M.L., and Kyle, W.S.A., 1995. Survival of Lactobacillus acidophilus and Bifidobacterium bifidum in commercial yogurt during refrigerated storage. Int. Dairy J. 5: 515–521. Shah, N.P. and Ravula, R.R., 2000. Microencapsulation of probiotic bacteria and their survival in frozen fermented dairy desserts. Aust. J. Dairy Technol. 55: 139–144. Sheehan, V.M., Sleator, R.D., Fitzgerald, G.F., and Hill, C., 2006. Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118. Appl. Environ. Microbiol. 72: 2170–2177. Sheehan, V.M., Sleator, R.D., Hill, C., and Fitzgerald, G.F., 2007. Improving gastric transit, gastrointestinal persistence and therapeutic efficacy of the probiotic strain Bifidobacterium breve UCC2003. Microbiology 153: 3563–3571. Shimamura, S., Abe, F., Ishibashi, N., Miyakawa, H., Yaeshima, T., Araya, T., and Tomita, M., 1992. Relationship between oxygen sensitivity and oxygen metabolism of Bifidobacterium species. J. Dairy Sci. 75: 3296–3306. Sodini, I., Boquien, C.Y., Corrieu, G., and Lacroix, C., 1997. Microbial dynamics of co- and separately entrapped mixed cultures of mesophilic lactic acid bacteria during the continuous prefermentation of milk. Enz. Microb. Technol. 20: 381–388. Streit, F., Corrieu, G., and Beal, C., 2007. Acidification improves cryotolerance of Lactobacillus delbrueckii subsp. bulgaricus CFL1. J. Biotechnol. 128: 659–667.

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Streit, F., Delettre, J., Corrieu, G., and Beal, C., 2008. Acid adaptation of Lactobacillus delbrueckii subsp. bulgaricus induces physiological responses at membrane and cytosolic levels that improves cryotolerance. J. Appl. Microbiol. 105: 1071–1080. Sugimoto, S., Abdullah Al, M., and Sonomoto, K., 2008. Molecular chaperones in lactic acid bacteria: physiological consequences and biochemical properties. J. Biosci. Bioeng. 106: 324–336. Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., and Kailasapathy, K., 2000. Encapsulation of probiotic bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt. Int. J. Food Microbiol. 62: 47–55. Sun, W.R. and Griffiths, M.W., 2000. Survival of bifidobacteria in yogurt and simulated gastric juice following immobilization in gellan-xanthan beads. Int. J. Food Microbiol. 61: 17–25. Talwalkar, A. and Kailasapathy, K., 2003. Effect of microencapsulation on oxygen toxicity in probiotic bacteria. Aust. J. Dairy Technol. 58: 36–39. Talwalkar, A. and Kailasapathy, K., 2004. The role of oxygen in the viability of probiotic bacteria with reference to L. acidophilus and Bifidobacterium spp. Curr. Issues Intest. Microbiol. 5: 1–8. Teixeira, P., Castro, H., and Kirby, R., 1994. Inducible thermotolerance in Lactobacillus bulgaricus. Lett. App. Microbiol. 18: 218–221. Trauth, E., Lemaitre, J.P., Rojas, C., Divies, C., and Cachon, R.M., 2001. Resistance of immobilized lactic acid bacteria to the inhibitory effect of quaternary ammonium sanitizers. LWT Food Sci. Technol. 34: 239–243. van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S.D., and Maguin, E., 2002. Stress responses in lactic acid bacteria. Antonie Van Leeuwenhoek 82: 187–216. van der Heide, T., Stuart, M.C., and Poolman, B., 2001. On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine. EMBO J. 20: 7022–7032. Ventura, M., Canchaya, C., Zink, R., Fitzgerald, G.F., and van Sinderen, D., 2004. Characterization of the groEL and groES loci in Bifidobacterium breve UCC2003: Genetic, transcriptional, and phylogenetic analyses. Appl. Environ. Microbiol. 70: 6197–6209. Ventura, M., Kenny, J.G., Zhang, Z., Fitzgerald, G.F., and van Sinderen, D., 2005a. The clpB gene of Bifidobacterium breve UCC2003: Transcriptional analysis and first insights into stress induction. Microbiology 151: 2861–2872. Ventura, M., Zink, R., Fitzgerald, G.F., and van Sinderen, D., 2005b. Gene structure and transcriptional organization of the dnaK operon of Bifidobacterium breve UCC2003 and application of the operon in bifidobacterial tracing. Appl. Environ. Microbiol. 71: 487–500. Walker, D.C., Girgis, H.S., and Klaenhammer, T.R., 1999. The groESL chaperone operon of Lactobacillus johnsonii. Appl. Environ. Microbiol. 65: 3033–3041. Wouters, J.A., Jeynov, B., Rombouts, F.M., de Vos, W.M., Kuipers, O.P., and Abee, T., 1999. Analysis of the role of 7 kDa cold-shock proteins of Lactococcus lactis MG1363 in cryoprotection. Microbiology 145: 3185–3194.

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the Careers of 22 Tracking Grape and Wine Polymers Using Biotechnology and Systems Biology John P. Moore and Benoit Divol CONTENTS 22.1 Introduction .......................................................................................................................... 389 22.2 From Grape to Must: The Contribution of Grape Cell-Wall-Associated Polysaccharides and Proteins................................................................................................ 391 22.2.1 Genetic and Biochemical Changes Associated with the Cell Walls of Ripening Grape Berries ............................................................................. 391 22.2.2 Pathogenesis-Associated Changes in the Polymer Composition of Berries ............. 392 22.3 From Must to Wine: Improving Processing, Filtration, and Aroma during Winemaking .......393 22.3.1 Developing Polysaccharide-Degrading Yeast Strains for Improved Wine Processing ...................................................................................................... 393 22.3.2 Engineering Yeast Strains Which Release Aroma Compounds from Polymers ...... 393 22.4 Fermentation and Wine: Yeast Cell Wall Properties and Fermentation Phenotypes ........... 397 22.4.1 The Cell Wall of Wine Yeast and its Impact on Winemaking ................................. 397 22.4.2 Biotechnological Strategies Targeting Mannoproteins to Alter Wine Yeast Properties ............................................................................... 399 22.5 Future Trends: Tracking Wine Polymers Using Systems-Based Approaches ......................400 Acknowledgments..........................................................................................................................402 References ......................................................................................................................................402

22.1 INTRODUCTION The grapevine is the world’s most important agricultural fruit crop with 7,861,000 hectares of vineyards worldwide in 2008 and about 270 million hL of wine produced annually (OIV, 2009). Wine, an alcoholic beverage that dates back thousands of years, predating the Greek and Roman empires, is currently believed to have originated in the Middle East. Vitis vinifera, the cultivated grapevine, is a native species of the Caucasus, a mountainous region in Eurasia, and has become the primary fruit crop used for winemaking worldwide. Winemaking has traditionally been considered an art and wine itself conjures up romantic images of farmers laboring in trellised vineyards and winemakers working in rustic cellars. The art of winemaking goes hand-in-hand with the art of cooking and of fine cuisine. The reality could not be further from the truth. Winemaking can be considered as mankind’s first foray into the world of biotechnology. Indeed, wine itself is a product of a biotechnological process which involves the treatment of must (grape juice) with microorganisms, specifically with yeast 389 © 2010 Taylor and Francis Group, LLC

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(Saccharomyces cerevisiae) and in certain instances with bacteria such as Oenococcus oeni, with the primary aim of converting the grape sugars into alcohol and hence wine. The fermentation process modifies the chemical composition of wine in more subtle ways by the modification of preexisting grape compounds or the introduction of yeast or bacterial components which may enhance and/ or diminish the desirable characteristics of the final beverage. The overall fermentation process is in essence a biotechnological process involving the biologically driven conversion of biochemicals to achieve a desired product. Although the primary process is a fermentation to produce ethanol, the microorganisms present/or added are able to impart substantial complexity to wine through the introduction of components that are in low concentrations (nano- and femtogram levels) but which have significant human sensory impacts. Wine is both a chemically simple and complex beverage. It is simple because the main chemicals are water at around 70–85%, ethanol between 10% and 14%, organic acids at around 1–2% and tannins at around 0.5–1%. It is also complex because numerous components, such as minor phenolics, pyrazines, terpenes, anthocyanins, proteins and glycosides, occur in very low concentrations but play a major role from a sensory perspective. This simplicity and complexity provides wine with an enormous range of sensory characteristics which makes it difficult to pin down, thus imparting a number of different styles; these can be governed by the grape cultivar and the yeast and bacterial strain used, and cover a variety of consumer tastes. Moreover, all the technical operations performed by the winemaker, from harvest to bottling, have a significant impact on the final wine composition. Wine is evaluated by three primary senses, sight, smell, and taste. A wine should be translucent and hence should not show any turbidity or haziness, it should have a distinct aroma profile appropriate to the style, and a suitable taste again specific to the type of wine. The different biochemically derived components in the wine are able to interact with one another in synergy, or similarly could counteract one another if the wine is imbalanced, to cause the specific visual appearance and sensory (smell and taste) characteristics detected. Much research to date has focused on the chemical (relatively low-molecular–weight) components of wine as these can be more easily characterized using a number of standard chemical techniques. Many of these components have been identified, although much research still needs to be done, specifically on the phenolic compounds which undergo complex polymerization reactions during fermentation and aging, producing novel uncharacterized compounds, and also on the volatile compounds which occur at incredibly low concentrations to the degree that they are present below the detection threshold of modern instrumentation. Until recently, proteins and polysaccharides were a neglected group of wine components which have now become recognized as playing an integral role in imparting visual and sensory qualities to wine. Recent publications have confirmed that the nature and the amount of polysaccharide polymers evolves extensively throughout winemaking (Dols-Lafargue et al., 2007; Guadalupe and Ayestarán, 2007). The same phenomenon occurs for proteins because of their successive secretion and degradation by wine microorganisms. Both classes of macromolecules are capable of multiple interactions with other components in the must and wine as well as having the potential to trigger reception in the mouth and possibly impart health benefits to the imbiber. Nevertheless, to date, wine polysaccharides have mostly been studied because of their potential either to negatively (Gindreau et al., 2001; DolsLafargue et al., 2008) or positively (Charpentier et al., 2004; Carvalho et al., 2006) impact on the mouthfeel of wine. Research on wine proteins has been mainly focused on the enzymes of oenological interest secreted by microorganisms (Strauss et al., 2001; Mtshali, 2007), as well as mannoproteins and their indirect impact on flavor compound retention, mouthfeel (Chalier et al., 2007), filterability (Van Rensburg and Pretorius, 2000), and protein haze modulation. This chapter will discuss the chain of events, from the grape berry through maceration and fermentation to the final wine, which govern the complexity and contribution of polysaccharides and proteins in oenology. We will highlight the essential role that biotechnology and systems biology have begun to play in understanding these processes. Future approaches to trace and assess these wine polymers from the grape to the glass, using analytical, molecular, and spectroscopic methods, will be discussed.

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22.2

391

FROM GRAPE TO MUST: THE CONTRIBUTION OF GRAPE CELL-WALL-ASSOCIATED POLYSACCHARIDES AND PROTEINS

22.2.1 GENETIC AND BIOCHEMICAL CHANGES ASSOCIATED WITH THE CELL WALLS OF RIPENING GRAPE BERRIES The major contribution of polysaccharides and proteins to wine comes naturally from the grapes used (see Figure 22.1 for a diagrammatic summary). Grape berries can be subdivided into the seeds, pulp, and skin, with each region displaying a different chemical and macromolecular composition (Carmona et al., 2008). Ripening in V. vinifera, a nonclimacteric fruit, is generally described as showing double sigmoidal growth coinciding with distinct physiological and hormonal events. Berries develop from fertilized ovaries post-flowering (or anthesis) and are initially small, green, acidic, and firm (Carmona et al., 2008). At veraison, developmental and hormonal events trigger a switch from cell division to cell expansion; this is accompanied by a color change in red cultivars as anthocyanins accumulate as well as a parallel softening of the berry. Ripe berries are softer, sweeter (due to glucose and fructose accumulation) and display changes in the accumulation of apoplastic and/or cell-wall-associated proteins. The polysaccharide and cell wall composition of the grape berry during these events, as in many other fruits, is assumed to be important in defining the textural and biophysical changes that accompany ripening (Nunan et al., 1998; Goulao and Oliveira, 2008). However, it has only been relatively recently that the ripening-related cell wall changes in grape berries have been studied in more detail. Compositional analysis of berry cell walls indicates that the major cellulose and hemicellulose (e.g., the xyloglucan component) of the cell wall do not change to any appreciable degree (Nunan et al., 1998). In contrast, the pectin-associated components of the grape berry matrix undergo significant changes during ripening (Nunan et al., 1998; Vidal et al., 2001; Doco et al., 2003a); specifically, a marked reduction in the amount of arabinogalactan-II polysaccharides is evident (Nunan et al., 1998). More importantly, subtle changes in the pectin cross-linking and integrity within the wall matrix is implied by a shift in solubility of polymers in ripening berries towards being more water soluble

Arabinogalactan proteins

PECTINS (rhamnogalacturonan I & II, homogalacturonan and arabinans)

Structural proteins (Proline-rich proteins)

PR proteins (pathogenesis related) chitinases, thaumatin, PGIPs, oxidases) Phenolic polymers (Viniferins)

FIGURE 22.1 A diagrammatic summary of the role of grapevine (V. vinifera) in supplying “berry-derived” polysaccharides, proteins, and phenolic polymers to must and wine.

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(Nunan et al., 1998; Vicens et al., 2009). Arabinogalactan proteins and rhamnogalacturonan-II polymers have been found to be the main grape-derived polysaccharides in wine; however, rhamnogalacturonan-I and arabinans, also pectin components, have been detected (Doco et al., 2003b). The changes observed at the biochemical level have been borne out by the fact that increasing molecular biologicalbased evidence for cell-wall-mediated changes accompanying the growth phases in grape berries has been obtained. Transcriptomic (mainly microarray-based) approaches have uncovered numerous cell wall localized or functionally related genes that are upregulated at different stages of ripening (Pilati et al., 2007; Schlosser et al., 2008; Lücker et al., 2009). Genes include putative expansins, xyloglucanases, and pectin-modifying enzymes (Pilati et al., 2007; Schlosser et al., 2008; Lücker et al., 2009). Many of these genes are partially identified or show homology to other species but currently functional studies to confirm identity and localization are lacking. Few berry cell wall genes have been studied in depth; one such example is mRIP1 isolated from a complementary deoxyribonucleic acid (cDNA) library of the V. vinifera cultivar Pinotage (South Africa’s flagship red wine cultivar) (Burger et al., 2004). The full-length cDNA sequence has been obtained, followed by monitoring expression data and conducting messenger ribonucleic acid (mRNA) localization experiments (Burger et al., 2004). mRIP1 was found to code for a proline-rich protein that was expressed specifically in ripening berries post-veraison but functional assignment remains elusive (Burger et al., 2004). A structural analysis based on sequence data suggests that mRIP1 may function during berry ripening and may act in concert with other wall proteins and/or components to lock the wall in place and thereby check uncontrolled expansion following veraison (Burger et al., 2004). Osmotic processes significantly influence cell wall properties and this is evidenced by the fact that grapevine plants exposed to water or salinity stress show concomitant expression changes in cell-wall-related genes (Cramer et al., 2007).

22.2.2 PATHOGENESIS-ASSOCIATED CHANGES IN THE POLYMER COMPOSITION OF BERRIES Many berry-associated proteins appear to be expressed post-veraison and these include pathogenesis-related (PR) proteins such as chitinases, thaumatins, and enzyme-inhibitor proteins (Ferreira et al., 2002). Many of these PR proteins are very stable and they usually survive winemaking to become the major protein components in bottled wine (Ferreira et al., 2002). PR proteins, as their name suggests, are important in pathogenesis and as such they have attracted considerable interest to help elucidate plant–pathogen interaction mechanisms. PR proteins are expressed as defense proteins in response to or preparation for pathogen attack (Carstens et al., 2003a). Fungal pathogens such as Botrytis cinerea are able to induce modifications in the protein complements of infected berries, including upregulating defense proteins, thus modifying the final composition of the wine (Cillindre et al., 2008). Chitinases, for example, are antifungal and capable of degrading chitin, a unique polymer present in the cell walls of fungal pathogens (Carstens et al., 2003a). Molecular biological strategies to engineer fungal resistance into important crop plants have included overexpressing a chitinase gene from S. cerevisiae in a model tobacco plant background (Carstens et al., 2003b). Other types of antifungal genes include enzyme inhibitors such as VvPGIP1, which was shown to encode a polygalacturonase-inhibiting protein that displays antifungal activity against gray rot fungus (Botrytis cinerea) when expressed in tobacco (Joubert et al., 2006). Mechanisms of action include an ability to interact directly with fungal endo-polygalacturonases, inhibiting their action at the site of infection (Joubert et al., 2006). Although the exact role of VvPGIP1 in the postveraison berry is not clear, recent evidence suggests an indirect role in pathogen defense by regulating other wall-related events (Joubert et al., 2007). The overall objective of studying the genes and proteins involved in pathogenesis is to engineer grapevine plants able to withstand diseases and thereby limit crop loss (Vivier and Pretorius, 2000). The concept is to use these genes to genetically tailor grapevine plants with enhanced disease resistance for use by the wine industry (Vivier and Pretorius, 2002). However, many of these PR proteins are present in wine and contribute to undesirable phenomena such as haze formation resulting in

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“cloudy” wine (Ferreira et al., 2002). An interesting conundrum, still to be resolved, of these molecular engineering strategies, is whether it is possible to improve the disease resistance of commercial grapevine cultivars by overexpressing PR proteins, without compromising stability and the tendency to produce haze and other changes in the wine produced (Ferreira et al., 2004).

22.3

FROM MUST TO WINE: IMPROVING PROCESSING, FILTRATION, AND AROMA DURING WINEMAKING

22.3.1 DEVELOPING POLYSACCHARIDE-DEGRADING YEAST STRAINS FOR IMPROVED WINE PROCESSING Apart from the PR proteins, numerous other polysaccharides and proteoglycans are present in the fermenting must. The bulk of these originate from the grapes during maceration and they are predominantly located in the pulp tissue of the berries. These pectin polymers include homogalacturonan (a linear homopolymer of galacturonic acid), rhamnogalacturonan I, linear arabinans, and rhamnogalacturonan II. In contrast, nonpectin polymers such as xyloglucan and xylans do not appear to be present to any substantial degree in must and wine. The only exception is noble late harvest wines where Botrytis cinerea-derived glucans are present in substantial amounts in the juice extracted. The presence of these wall-derived polysaccharides and proteins is detrimental to wine processing as these viscous polymers tend to clog filters and impede processing steps. A common practice in winemaking is to add commercially available polysaccharide hydrolases (such as pectinases and glucanases) to the fermenting must to degrade the polysaccharides and hence reduce the viscosity. Table 22.1 summarizes the different families of enzymes whose addition to wine has been authorized by the International Organization for Vine and Wine (OIV) in order to enhance wine clarification and/or filterability. Most of the enzyme preparations (if not all) are blends of different enzymes, mainly originating from Aspergillus spp. The addition of these enzyme formulations is, however, an expensive exercise and therefore alternatives are sought which might improve filtration at less expense. One such alternative approach involves using a biotechnological strategy whereby genes encoding polysaccharide hydrolase enzymes are transferred into commercial wine yeast strains using genetic engineering methods and are tested for reducing viscosity. Examples of such successful strategies include expressing pectinase-, β-glucanase-, or hemicellulaseencoding genes in wine yeast (S. cerevisiae), resulting in improved processing (less viscous fermentation solutions) and improved aroma release (Table 22.1). The lack of specific pectin-degrading enzymes, in fermenting must, for rhamnogalacturonan II and arabinogalactan polymers during winemaking results in these polymers surviving and forming integral components of the final wine (Pellerin and O’Neill, 1998).

22.3.2

ENGINEERING YEAST STRAINS WHICH RELEASE AROMA COMPOUNDS FROM POLYMERS

Wine polysaccharides and proteoglycans have other both positive and negative properties (see Figure 22.2 for a diagrammatic summary of yeast impact on wine polysaccharide composition). Arabinogalactan proteins and colloidal polysaccharides can encapsulate volatile aroma compounds and thereby improve their retention in wine (Dufour and Bayonove, 2001). Similarly these polysaccharides can interact with other wine components such as proteins and phenolics producing colloidal aggregates which can result in haze (Escot et al., 2001). The interaction of these polymers with color pigments can also result in modified spectral properties and therefore modulate color properties and improve stability (Escot et al., 2001). Glycoside hydrolases also act on oligo- and polysaccharide conjugates such as those released from the cell vacuoles of grape berries during maceration. A common type of conjugate present in grape berry vacuoles consists of a volatile aroma compound, such as a monoterpene (the aglycone moiety), covalently attached to a saccharide moiety. The conjugation of a nonpolar volatile compound (e.g., linalool, a monoterpene) with a saccharide group substantially

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Enzyme Family β-Glucanases

Enzyme exo-β-1,3-Glucase, endo-1,31,4-glucanase, endo-β-1,4glucanase (also known as cellulase)

Authorized by OIV Resolution No. OENO 11/2004, 12/2004, 14/2004, 15/2004, 18/2004

Examples of Commercial Preparation Available for the Wine Industry Lallzyme MMX™ (Lallemand)

Enzyme Engineered in S. cerevisiae by Van Rensburg et al. (1994, 1996, 1997, 1998) and Louw et al. (2006)

Depectil Elevage (Martin Vialatte) Glucanex® 200 G (Lamothe-Abiet) Pectinases

Pectate lyase endo-Polygalacturonase

OENO 11/2004, 12/2004, 14/2004, 15/2004 OENO 11/2004, 12/2004, 14/2004, 15/2004

Van Rensburg et al. (1994, 2007) Lallzyme C™ (Lallemand) Rapidase® CB (DSM) Rapidase® Vinosuper (DSM) Rapidase® X-Press L (DSM) Depectil Clarification (Martin Vialatte) Ultrazym® Premium (Lamothe-Abiet)

Vilanova et al. (2000), Fernández-González et al. (2005), and Louw et al. (2006)

Biotechnology in Functional Foods and Nutraceuticals

TABLE 22.1 A Summary of the Different Families of Enzymes (Including Examples of Commercial Preparations)

OENO 11/2004, 12/2004, 14/2004, 15/2004

Pectin methyl esterase

OENO 11/2004, 12/2004, 14/2004, 15/2004

Rapidase® X-Press L (DSM) Depectil Clarification (Martin Vialatte) Rapidase® Vinosuper (DSM)

Christgau et al. (1996)

Rapidase® X-Press L (DSM) Pectinases + inactivated cells of specific S. cerevisiae strain Galactanase Rhamnosidase Rhamnogalacturonase

OENO 11/2004, 12/2004, 14/2004, 15/2004 OENO 9/2008 OENO 9/2008 n/a

Arabinase

OENO 9/2008

Arabinofuranosidase Xylanases

n/a n/a

Depectil EasyCLAR 32 FCE (Martin Vialatte) REDStyle™ (Lallemand) Lallzyme EX™ (Lallemand) Lallzyme BETA™ (Lallemand) Rapidase® Expression (DSM) Rapidase® X-Press L (DSM) Rapidase® Expression (DSM) Rapidase® X-Press L (DSM) n/a

n/a

Manzanares et al. (2003)

De Klerk (2008) Ganga et al. (1999), La Grange et al. (2001), Louw et al. (2006), and Van Rensburg et al. (2007)

Tracking the Careers of Grape and Wine Polymers

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Pectin lyase

Note: Biotechnology strategies (not authorized for commercial use) to engineer wine yeast with these enzyme genes are also indicated.

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Biotechnology in Functional Foods and Nutraceuticals Polysaccharide degrading enzymes (polygalacturonases, glucanases, aroma release)

Fermentation phenotypes (flocculation, velum, flor, haze, and lees)

Polysaccharides (glucans and mannans)

Cell wall (glucan, chitin, mannan, mannoprotein changes affect parietal absorption capacity)

Mannoproteins (enzymes, lectins, structural proteins, and aroma compound encapsulation)

FIGURE 22.2 A diagrammatic summary of the role of wine yeast (mainly S. cerevisiae) in supplying polysaccharides, mannoproteins, and polysaccharide-degrading enzymes to must and wine. Also indicated is the impact of cell wall changes on fermentation phenotypes (e.g., flocculation) and wine components (via parietal adsorption).

improves the water solubility of the aroma compound and thereby allows its storage in the cell vacuole. However, in this conjugated form these compounds are not able to contribute to the aroma potential of the final wine. Winemakers employ commercial hydrolase preparations, similarly to the pectinase preparations, to degrade these linkages in the fermenting must or wine and in so doing release the free aroma forms. The use of commercial preparations of β-glycosidase to enhance the release of flavoring substances has been approved by the OIV (resolutions OENO 16/2004, 17/2004). Again, commercial preparations are expensive and are usually mixed preparations thus lacking specificity of action. Unfortunately, only certain strains of S. cerevisiae seem to possess β-glucosidase and even those display low activity during alcoholic fermentation because of the harsh wine-related physicochemical conditions (Hernández et al., 2003). Biotechnology offers a feasible alternative by offering the opportunity to develop strains engineered with glycoside hydrolase genes which encode enzymes that target specific linkages, thus liberating aroma compounds during fermentation and simultaneously improving aroma. β-Glucosidases displaying activity at wine pH and temperature are found in several yeast species: Debaryomyces pseudopolymorphus (Cordero Otero et al., 2003; Arévalo Villena et al., 2006), Metschnikowia pulcherrima (González-Pombo et al., 2008), Brettanomyces spp. (Cordero Otero et al., 2003; Daenen et al., 2008), Debaryomyces hansenii (Yanai and Sato, 1999), and Candida molischiana (Genovès et al., 2003). Lactic acid bacteria can also potentially serve as a reservoir of enzymes of oenological interest; O. oeni is for instance known to possess β-glucosidase activity (Grimaldi et al., 2005). Most of these genes are, however, not yet sequenced and further studies are needed in order to test their potential for use in winemaking. Examples of the successful expression of β-glucosidase-encoding genes in wine strains of S. cerevisiae include expressing BGL1 and BGL2 genes from Saccharomycopsis fibuligera, resulting in improved aroma release during and after fermentation (Van Rensburg et al., 2005) and the BGLN gene from C. molischiana (Genovès et al., 2003). A larger quantity of flavoring monoterpenes have also been achieved by expressing the β-glucosidase-encoding genes of Aspergillus kawachii and © 2010 Taylor and Francis Group, LLC

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S. fibuligera and the arabinofuranosidase-encoding gene of Aspergillus awamori in a wine yeast strain (De Klerk, 2008), thereby coupling the two enzymatic reactions leading to the release of free monoterpenes.

22.4 22.4.1

FERMENTATION AND WINE: YEAST CELL WALL PROPERTIES AND FERMENTATION PHENOTYPES THE CELL WALL OF WINE YEAST AND ITS IMPACT ON WINEMAKING

Although the primary role of yeast in winemaking is to convert grape sugars (glucose and fructose) present in must into ethanol, and thereby produce wine, yeast’s contribution to winemaking is much more extensive. Yeast possesses a complex metabolism and is able to synthesize de novo compounds and to bioconvert (modifying existing compounds using enzymes) existing compounds present in the must (Lambrechts and Pretorius, 2000). Examples of de novo compounds produced by yeast include glycerol and sorbitol. Yeast is also able to bioconvert existing compounds, producing compounds such as diacetyl, butanedione and phenylethanol (Lambrechts and Pretorius, 2000). Many of these compounds are present in low concentrations in relation to grape components (e.g., tannins and acids); however, they are usually present at (or above) the sensory detection thresholds for humans and so are able to contribute significantly to wine taste and aroma. Apart from their metabolic versatility, yeasts are also able to impact significantly on fermentation processing, visual properties, and wine stability. Many of these other properties are not directly related to yeast metabolism but are rather derived from the polysaccharide-rich cell walls of wine yeast strains (see Figure 22.2 for a diagrammatic summary of yeast contribution to wine polysaccharide composition). The cell walls of wine yeast consist primarily of a layer of mannoproteins (a diverse group of glycoproteins where the glycan component is mainly mannose residues), glucan chains (β1-6 and β1-3 linked) and low amounts of chitin (a glucosamine-derived polymer). The bulk of the mannoproteins is found in the outer layers of the cell walls where they are implicated in processes related to cell recognition and adhesion. Certain yeast enzymes may be found at the cell exterior and may be mannosylated, therefore per definition are defined as mannoproteins (Klis et al., 2002). The surface properties of yeast cells appear to be governed by the composition of the cell wall mannoproteins and their degree of post-translational modification (Klis et al., 2002). The cell wall of wine yeast is a dynamic structure which undergoes modification and/or turnover as a function of physiological state, the phase of the cell cycle and the stage of fermentation (Klis et al., 2002). The cell wall changes that ensue can have direct impacts on the phenotype of yeast strain and can impart properties such as hydrophobicity or net positive (or negative) charge to the cell surface (Galichet et al., 2001, Klis et al., 2002). It has recently been shown that defects or changes in cell wall structure, as found in genetic deletion mutants, can result in the release of cell-wall-bound mannoproteins into the culture medium (Gonzales-Ramos and Gonzalez, 2006). Some of these mutants have been tested under winemaking conditions and have been shown to modify the mannoprotein and/or polysaccharide content of the final wine produced (Gonzales-Ramos and Gonzalez, 2006). The yeast cell wall also acts as an adsorbent surface during winemaking and is able to bind a variety of compounds and macromolecules present in the wine medium (Caridi, 2007) (see Table 22.2 for a summary of oenological methods and biotechnological strategies for fining and stabilizing wine). Much research has focused on the capacity of wine yeast strains to adsorb phenolic compounds, in particular the anthocyanin pigments, thus being able to modify the wine color or hue (Caridi, 2007). The nature and specificity of action between the wall composition and the compounds adsorbed (the ability to adsorb is referred to as the “parietal adsorption capacity” of the strain) have still to be investigated and are currently poorly understood (Caridi, 2007). A recent investigation has revealed that genetic mutants modified in the wall composition have different capacities to adsorb ethyl-phenol under oenological conditions (Pradelles et al., 2008). This capacity to adsorb different molecules from solution appears to be governed by the glucan, mannan, and

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TABLE 22.2 Examples of Different Fining-Related Treatments (Including Examples of Commercial Preparations and Yeast Strains) Authorized by OIV Resolution No.

Treatment

Examples of Commercial Preparations/Strains Available for the Wine Industry

Examples of Genetic Engineering Strategies in S. cerevisiae by

OENO 04/2001, OENO 26-2004, OENO 15-2005

Mannostab® (Laffort)

Gonzales-Ramos and Gonzalez (2006) and Gonzales-Ramos et al. (2008)

Yeast Lees

OENO 04/2001, OENO 26-2004 OENO 15-2005

Biolees® (Laffort), Opti-White® (Lallemand) Opti-Red® (Lallemand)

Gonzales-Ramos and Gonzalez (2006)

Induce yeast flocculation Fining agents (e.g., cellulose, bentonite, casein, polyvinylpolypyrrolidone (PVPP), diatomite, etc.)

N/A OENO 08/2002 OENO 10/2002

None Gelatine extra N°1®, Bentonite:Microalpha® (Laffort)

Govender et al. (2008) None

OENO 11/2003 OENO 13/2003 OENO 05-2004 OENO 01-2007 OENO 02-2007 Cold stabilization

OENO 02-2004

Mannostab® (Laffort)

Gonzales-Ramos and Gonzalez (2006) and Gonzales-Ramos et al. (2008)

Cell-wall-modified yeast (e.g., binding phenols)

N/A

Fermicru-XL® (DSM)

Pradelles et al. (2008)

Note: Examples of biotechnology strategies (not authorized for commercial use) to engineer wine yeast with relevant genes and/or to produce genetic mutant strains are also indicated.

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mannoprotein composition of the wall (Pradelles et al., 2008) and is probably also linked to the types of mannoproteins present at the surface of the cells. Since the addition of cell hulls to wine is already authorized, S. cerevisiae’s cell walls could be considered as a potential oenological tool to remove off-flavor compounds (produced by spoilage yeasts and lactic acid bacteria), such as ethylphenol, from wine. Moreover, recent research has focused on the cell wall components of flor-forming strains of S. cerevisiae which are commonly utilized in sherry production. The amount of mannoproteins released by these strains is high, as evidenced by the fact that mannose is the most abundant sugar in sherry (Villamiel et al., 2008). The high mannoprotein content present in sherry is because the yeast strains used are subjected to successive growth and autolysis cycles during sherry ageing (Villamiel et al., 2008). Genetic peculiarities, particularly in the ribosomal deoxyribonucleic acid (rDNA) genes of sherry strains, have been found, mostly consisting of single nucleotide polymorphisms, insertions, and deletions (van der Aa Kühle et al., 2001; Esteve-Zarzoso et al., 2004; Naumova et al., 2005; Divol et al., 2006). Even more interestingly, it has been shown that flor strains naturally express certain genes at a higher level than non-flor-forming strains. The functions of these highly expressed genes appear to be linked to stress adaptation (e.g., HSP12 [Zara et al., 2002] and SSU1 [Pérez-Ortin et al., 2002]), membrane protection (e.g., HYR1 [Avery and Avery, 2001]), and flocculation (e.g., FLO11 [Reynolds and Fink, 2001; Zara et al., 2005]). Very recently, Kovacs et al. (2008) also reported that the size of some cell wall proteins, namely Ccw7p and Hsp150p, is smaller than that of nonforming strains, leading to a reorganization of the cell wall probably as a consequence of stress adaptation. Flor formation during sherry ageing strongly impacts on wine composition and stability. Further studies on these strains could lead to a better understanding of autolysis and its impact on wine haze formation (and its prevention) and tartaric acid precipitation (and stabilization).

22.4.2 BIOTECHNOLOGICAL STRATEGIES TARGETING MANNOPROTEINS TO ALTER WINE YEAST PROPERTIES Mannoproteins are mainly released during the autolysis of yeast which usually occurs at the end of alcoholic fermentation. This natural process is nevertheless very slow and can be aided by the addition of hydrolytic enzymes. By breaking down the yeast wall polymer network, mannoproteins are released into the wine (Charpentier et al., 2004). Mannoproteins are usually considered beneficial, binding to various wine compounds such as polyphenols and aroma compounds, thereby increasing color stability, softening the tannins, and changing the global organoleptic profile. Moreover, mannoproteins enhance tartrate stabilization, protect against haze formation, and adsorb several molecules involved in oxidation reactions. A comprehensive list of the beneficial properties of mannoproteins has been reviewed by Dols-Lafargue and Lonvaud-Funel (2009). The use of mannoproteins in wine has been approved by the OIV (resolutions OENO 04/2001 and 15/2005) and commercial preparations are already available for the wine industry (Mannostab™, Laffort Oenologie). A class of mannoproteins encoded by the FLO gene family in S. cerevisiae is responsible for the flocculation ability of yeasts, thereby facilitating the precipitation of yeast cells at the end of alcoholic fermentation. Normally these modified surface phenotypes, such as flocculation, pseudohyphal formation, and invasive growth, are absent in industrial wine yeast strains (Carstens et al., 1998; Verstrepen and Klis, 2006). These responses in yeast have naturally evolved to facilitate survival under nutrient-limiting and/or stressful conditions by improving access to new nutrient sources and creating new microhabitats such as biofilms (Carstens et al., 1998). These processes are under genetic and environmental control, and numerous components of the signaling network, for example, in the case of pseudohyphal formation, have been elucidated (Lambrechts et al. 1996; Gagiano et al., 1999). Industrial wine yeast strains do not flocculate because the regulatory network is normally found to be genetically deactivated through mutation, although they possess the necessary flocculin “structural lectin-type mannoprotein”-encoding genes (FLO1, FLO5, FLO9, FLO10, FLO11) (Carstens et al., 1998).

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The most likely reason that industrial wine yeast strains lack the ability to flocculate is that efficient alcoholic fermentation of grape must requires the presence of widely dispersed cells having maximum access to the sugary medium and thus flocculation has been “selected against” in strains with good fermentative ability. Clearly, the ability to produce strains which are able to modify their wall composition (parietal adsorption capacity), release beneficial mannoproteins into the wine, and flocculate at the end of fermentation would be of great benefit to the wine industry. Certain novel strategies have recently been implemented with these goals in mind (see Table 22.2). Gonzales-Ramos et al. have produced a recombinant strain which overexpresses a mannoprotein under oenological conditions. The high quantity of this mannoprotein released from autolyzing cells at the end of fermentation was shown to protect the wine produced against undesirable protein haze (Gonzales-Ramos et al., 2008). Similarly, the ability to regulate the production of flocculins at the cell surface in order to facilitate flocculation in industrial strains has been attempted but until recently has been hindered by the observation that the associated genes (FLO1, FLO5, FLO9, FLO10, FLO11) are considered to be “un-clonable.” An elegant process to circumvent this problem has been carried out by Govender et al. where the authors engineered promoter replacement cassettes containing the late stationary phase promoters (Adh2p and Hsp30p) for each of the FLO1, FLO5, and FLO11 promoters in the industrial strains VIN13 and BM45. These engineered strains show “controlled flocculation” under oenological conditions, that is, they can perform alcoholic fermentation normally and thereafter undergo flocculation at stationary phase which facilitates their easy removal from the wine medium (Govender et al., 2008). This regulation of mannoprotein wall composition in industrial yeast strains holds great promise for the ability to “customize” or “tailormake” novel strains for numerous wall-associated properties without compromising on the oenological capacities of the parental/background strains used.

22.5 FUTURE TRENDS: TRACKING WINE POLYMERS USING SYSTEMS-BASED APPROACHES From the previous sections it is clear that the polysaccharide and protein composition of wine is governed by various components supplied by the grape berries as well as the yeast and bacterial species used for winemaking. In addition, the yeast and bacteria have the ability to modify certain of the preexisting components in the must during the various stages and types of fermentation the wine has undergone. Clearly, this indicates a multifactorial problem whereby every stage of winemaking from the choice of harvest time (i.e., berry physiological maturity and disease history), the addition of fermentation aids (e.g., enzyme preparations), the type of yeast strains used (e.g., highmannoprotein-release strains), and whether bacterial fermentations take place (e.g., malolactic fermentation) adds an additional (and substantial) layer of complexity to the final polymer composition of the wine. Each organism, whether grapevine (e.g., V. vinifera), yeast (e.g., S. cerevisiae), or bacteria (e.g., O. oeni) adds its own metabolic and polymeric signature to the wine produced. The traditional “highly empirical” approaches of the historical wine disciplines of viticulture and oenology involve studying each of the winemaking stages in isolation, focusing on specific processes and examining how these could be improved. Even the modern science of biotechnology has its roots in the “reductionist” approach found in the sciences of chemistry and molecular biology, where the focus has been on “one gene” coding for “one protein” possessing “one primary function.” These approaches have been highly successful and have been responsible for major advances in understanding and isolating the numerous genetic, protein/enzyme, metabolite, and environmental factors that are important in various wine-associated organisms. However, neither cataloguing all the components nor studying each group of related factors in isolation will ever allow us to understand how the entire vine and wine system works in a holistic and integrated manner. Recent advances in the biological sciences have begun to address these deficiencies by developing systems level approaches to studying entire organisms on a whole-cell and even whole-organism level. This approach is termed “systems biology” and the holistic methodologies are suffixed as

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“-omics” (see Kitano, 2002). The various “omics” approaches include genomics (functionally annotating an organism’s genome), transcriptomics (identifying and quantifying simultaneously expressed genes), proteomics (investigating the total protein complement and composition), metabolomics [the entire catalogue and quantification of an organism’s metabolites both released (exometabolome) and intracellular (endometabolome)], and fluxomics (investigating the network of metabolic reactions and the flux within each pathway). The ultimate goal of these approaches is summed up in the term “interactomics,” which is an attempt to integrate each of the previously described “omics” levels/approaches with one another, thus arriving at a predictive model which describes the particular organism under study. Each of the wine-associated organisms—grapevine, yeast, and bacteria—can be studied using a system’s biological approach and then the data from each stage of the winemaking process can be integrated to form a predictive model. Each of the input parameters can be modified which then allows predictive output results to be generated; this smart modeling approach is different from the “black box winery” modeling used to date because it takes into account the biological complexity of the organisms used, which will hopefully lead to the generation of more accurate winemaking models. Researchers in wine biotechnology have begun to make use of these newly emerging technologies and some results related to wine yeast studies have already been published, as reviewed by Divol and Bauer (2009). But more than this, the fundamental advances made in understanding the systems biology of these organisms could result in substantial new knowledge in the biological sciences of relevance to possibly unrelated disciplines and processes. The application of systems biology methods leads to the production of such large datasets that traditional univariate statistics is unable to handle efficiently the numbers of “interconnected” variables studied. To remedy this, “multivariate statistics” or “chemometrics” methods have been employed to handle the datasets generated from the various “omics” approaches. Chemometrics has been most extensively applied in the winemaking field to handle the multitude of variables generated from rapid spectral profiling tools used in on-line process monitoring of wine fermentations (Bauer et al., 2008). Near- and mid-infrared spectroscopic techniques have been used in winemaking for the rapid monitoring of fermentations for basic oenological parameters such as sugar content (total soluble solids), pH, and titratable acidity (Nieuwoudt et al., 2006; Swanepoel et al., 2007). More and more applications are being sought for such techniques because these spectroscopic tools are rapid, inexpensive, and provide quick data for real-time monitoring purposes. A recent example is the monitoring of glycerol levels in South African wines with the aim of developing of calibration models using chemometrics for use in the wine industry (Nieuwoudt et al., 2002, 2004). This combination of spectroscopic and chemometric tools has been applied to tracking polysaccharide polymer systems in both fruit and wine. Examples include monitoring uronic acid, neutral sugar, and methylesterification levels in pectic samples (Coimbra et al., 1998; Barros et al., 2002) and polymeric mannose levels in wine (Coimbra et al., 2005). These specific isolated studies indicate that it is possible to use spectroscopic tools coupled with multivariate statistics to track polymers in grapes during ripening, the release/modification of polymers by yeast and bacteria, through the winemaking process to the final bottled wine. Such an approach could be integrated with the systems biological approach to link the genetic and metabolic potentials of the different strains with the polymeric networks present at each stage of the winemaking process. These rapid spectroscopic tools could be used to screen new cultivars for different polymer compositions or novel yeast strains (generated from “traditional breeding” or “genetic engineering” projects) with the ability to modify/ release mannoproteins into the fermentation medium. This holistic approach to vine and wine science is exemplified in the philosophy and vision of the Institute for Wine Biotechnology and the Department of Viticulture and Oenology at Stellenbosch University, South Africa. This academic center is attempting to integrate the traditional disciplines of viticulture, oenology, wine chemistry, biology, and food science with the emerging disciplines of system biology and biological spectral profiling coupled through the use of (chemo)-metric and

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metabolomic techniques (see Moore et al., 2008). This ambitious approach, which most likely represents the future of the vine and wine sciences, is proposed in a multidisciplinary integrated system termed the “Wine Science Research Niche Area” (funded through the National Research Foundation of South Africa) (Moore et al., 2008). The aim, in the context of this review and the broader aims of the environment, would be not only to track but also to modify, through the use of biotechnological strategies, grape and wine polymers leading to (i) grapevines with improved abiotic/biotic resistance and berry quality; (ii) yeast and bacterial strains that improve aroma release, wine processing, visual appearance, and general wholesomeness of the wine; and (iii) better monitoring of wine fermentations and improved storage properties (e.g., reduced haze), while ensuring that all risks associated with the use of genetically modified wine organisms are thoroughly evaluated.

ACKNOWLEDGMENTS The authors would like to acknowledge gratefully the support of Stellenbosch University, Winetech, Technology and Human Resources for Industry Programme (THRIP, South African Government), National Research Foundation (South Africa), and the South African Table Grape Industry in financing their research programs in wine and grapevine biotechnology.

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Coimbra, M.A., Barros, A.S., Coelho, E., Gonçalves, F., Rocha, S.M., and Delgadillo, I., 2005. Quantification of polymeric mannose in wine extracts by FT-IR spectroscopy and OSC-PLS1 regression. Carbohydr. Polym. 61: 434–440. Cordero Otero, R.R., Ubeda Iranzo, J.F., Briones-Perez, A.I., Potgieter, N., Villena, M.A., Pretorius, I.S., and van Rensburg, P., 2003. Characterization of the β-glucosidase activity produced by enological strains of non-Saccharomyces yeasts. J. Food Sci. 68: 2564–2569. Cramer, R., Ergül, A., Grimplet, J., et al., 2007. Water and salinity stress in grapevines: Early and late changes in transcript and metabolite profiles Funct. Integr. Genomics 7: 111–134. Daenen, L., Saison, D., Sterckx, F., Delvaux, F.R., Verachtert, H., and Derdelinckx G., 2008. Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts. J. Appl. Microbiol. 104: 478–488. De Klerk, D., 2008. Co-expression of aroma liberating enzymes in a wine yeast strain. MSc dissertation, Stellenbosch University. Divol, B. and Bauer, F.F., 2010. Metabolic engineering of wine yeast and advances in yeast selection methods for improved wine quality and safety. In: A.G. Reynolds (Ed.), Managing Wine Quality: Viticulture and Wine Quality (Vol. 1), Cambridge: Woodhead (in press). Divol, B., Miot-Sertier, C., and Lonvaud-Funel, A., 2006. Genetic characterization of strains of Saccharomyces cerevisiae responsible for “refermentation” in Botrytis-affected wines. J Appl. Microbiol. 100: 516–526. Doco, T., Vuchot, P., Cheynier, V., and Moutounet, M., 2003a. Structural modification of wine arabinogalactans during aging on lees. Am. J. Enol. Viticult. 54: 150–157. Doco, T., Williams, P., Pauly, M., O’Neill, M.A. et al., 2003b. Polysaccharides from grape berry cell walls. Part II. Structural characterization of the xyloglucan polysaccharides. Carbohydr. Polym. 53: 253–261. Dols-Lafargue, M. and Lonvaud-Funel, A., 2009. Polysaccharide production by grapes, must, and wine microorganisms. In: H. König, G. Unden, and J. Fröhlich (Eds), Biology of Microorganisms on Grapes, in Must and in Wine, pp. 241–258. Heidelberg: Springer. Dols-Lafargue, M., Gindreau, E., Le Marrec, C., Chambat, G., Heyraud, A., and Lonvaud-Funel, A., 2007. Changes in red wine soluble polysaccharide composition induced by malolactic fermentation. J. Agric. Food Chem. 55: 9592–9599. Dols-Lafargue, M., Lee, H.Y., Le Marrec, C., Heyraud, A., Chambat, G., and Lonvaud-Funel, A., 2008. Characterization of gtf, a glucosyltransferase gene in the genomes of Pediococcus parvulus and Oenococcus oeni, two bacterial species commonly found in wine. Appl. Environ. Microbiol. 74: 4079–4090. Douglas, L.M., Li, L., Yang, Y., and Dranginis, A.M., 2007. Expression and characterization of the flocculin Flo11/Muc1, a Saccharomyces cerevisiae mannoprotein with homotypic properties of adhesion. Eukaryot. Cell 6: 2214–2221. Dufour, C. and Bayonove, C.L., 1999. Influence of wine structurally different polysaccharides on the volatility of aroma substances in a model system. J. Agric. Food Chem. 47: 671–677. Dupin, I.V.S., Stockdale, V.J., Williams, P.J., et al., 2000. Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: Evaluation of extraction methods and immunolocalization. J. Agric. Food Chem. 48: 1086–1095. Escot, S., Feuillat, M., Dulau, L., and Charpentier, C., 2001. Release of polysaccharides by yeasts and the influence of released polysaccharides on colour stability and wine astringency. Aust. J. Grape Wine Res. 7: 153–159. Esteve-Zarzoso, B., Fernandez-Espinar, M.T., and Querol, A., 2004. Authentication and identification of Saccharomyces cerevisiae “flor” yeast races involved in sherry ageing. Antonie van Leeuwenhoek 85: 151–158. Fernández-González, M., Úbeda, J.F., Cordero-Otero, R.R., Thanvanthri, V., and Briones, A.I., 2005. Engineering of an oenological Saccharomyces cerevisiae strain with pectinolytic activity and its effect on wine. Int. J. Food Microbiol. 102: 173–183. Ferreira, R.B., Monteiro, S.S., Piçarra-Pereira, M.A., and Teixeira. A.R., 2004. Engineering grapevine for increased resistance to fungal pathogens without compromising wine stability. Trends Biotechnol. 22: 168–173. Ferreira, R.B., Piçarra-Pereira, M.A., Monteiro, S.S., Loureiro, V.B., and Teixeira, A.R., 2002. The wine proteins. Trends Food Sci. Tech. 12: 230–239. Gagiano, M., van Dyk, D., Bauer, F.F., et al., 1999. Msn1p/Mss10p, Mss11p and Muc1p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae. Mol. Microbiolol. 31: 103–116.

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Galichet, A., Sockalingum, G.D., Belarbi, A., and Manfait, M., 2001. FTIR spectroscopic analysis of Saccharomyces cerevisiae cell walls: Study of an anomalous strain exhibiting a pink-coloured phenotype. FEMS Microbiol. Lett. 197: 179–186. Ganga, M.A., Piñaga, F., Vallés, S., Ramón, D., and Querol, A., 1999. Aroma improving in microvinification processes by the use of a recombinant wine yeast strain expressing the Aspergillus nidulans xlnA gene. Int. J. Food Microbiol. 47: 171–178. Genovès, S., Gil, J.V., Manzanares, P., Aleixandre, J.L., and Vallés, S., 2003. Candida molischiana_-Glucosidase Production by Saccharomyces cerevisiae and its application in winemaking. J. Food Sci. 68: 2096–2100. Gindreau, E., Walling, E., and Lonvaud-Funel, A., 2001. Direct polymerase chain reaction detection of ropy Pediococcus damnosus strains in wine. J. Appl. Microbiol. 90: 535–542. Gonzales-Ramos, D., Cobollero, E., and Gonzalez, R., 2008. A recombinant Saccharomyces cerevisiae strain overproducing mannoproteins stabilizes wine against protein haze. Appl. Environ. Microbiol. 74: 5533–5540. Gonzales-Ramos, D. and Gonzalez, R., 2006. Genetic determinants of the release of mannoproteins of enological interest by Saccharomyces cerevisiae. J. Agric. Food Chem. 54: 9411–9416. González-Pombo, P., Pérez, G., Carrau, F., Guisán, J.M., Batista-Viera F., and Brena, B.M., 2008 One-step purification and characterization of an intracellular beta-glucosidase from Metschnikowia pulcherrima. Biotechnol. Lett. 30: 1469–1475. Goulao, L.F. and Oliveira, C.M., 2008. Cell wall modifications during fruit ripening: When a fruit is not the fruit. Trends Food Sci. Tech. 19: 4–25. Govender, P., Domingo, J.L., Bester, M., Pretorius, I.S., and Bauer., F.F., 2008. Controlled expression of the dominant flocculation genes FLO1, FLO5 and FLO11 in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 74: 6041–6052. Grimaldi, A., Bartowsky, R., and Jiranek, V., 2005. A survey of glycosidase activities of commercial wine strains of Oenococcus oeni. Int. J. Food Microbiol. 105: 233–244. Guadalupe, Z. and Ayestaran, B., 2007. Polysaccharide profile and content during the vinification and aging of Tempranillo red wines. J. Agric. Food Chem. 55: 10720–10728. Hernández, L.F., Espinosa, J.C., Fernández-González, M., and Briones, A., 2003. Beta-glucosidase activity in a Saccharomyces cerevisiae wine strain. Int. J. Food Microbiol. 80: 171–176. Joubert, D.A., Kars, I., Wagemakers, L., et al., 2007. A polygalacturonase inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction. Mol. Plant Microbe Interact. 20: 392–402. Joubert, D.A., Slaughter, A.R., Kemp, G., et al., 2006. The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases. Transgenic Res. 15: 687–702. Kitano, H., 2002. Systems biology: A brief overview. Science 295: 1662–1664. Klis, F.M., Mol, P., Hellingwerf, K., and Brul, S., 2002. Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 26: 239–256. Kovacs, M., Stuparevic, I., Mrsa, V., and Maráz, A., 2008. Characterization of Ccw7p cell wall proteins and the encoding genes of Saccharomyces cerevisiae wine yeast strains: Relevance for flor formation. FEMS Yeast Res. 8: 1115–1126. La Grange, D.C., Pretorius, I.S., Claeyssens, M., and van Zyl, W.H., 2001. Degradation of xylan to d-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger beta-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes. Appl. Environ. Microbiol. 67: 5512–5519. Lambrechts, M.G., Bauer, F.F., Marmur, J., and Pretorius, I.S., 1996. Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast. Proc. Natl. Acad. Sci. USA 93: 8419–8424. Lambrechts, M.G. and Pretorius, I.S., 2000. Yeast and its importance to wine aroma. S. Afr. J. Enol. Vitic. 21: 97–129. Louw, C.T., Pretorius, I.S., and van Rensburg, P., 2006. The effect of polysaccharide-degrading wine yeast transformants on the efficiency of wine processing and wine flavour. J. Biotechnol. 125: 447–461. Lücker, J., Laszczak, M., Smith, D., et al., 2009. Generation of a predicted protein database from EST data and application to iTRAQ analyses in grape (Vitis vinifera cv. Cabernet Sauvignon) berries at ripening initiation BMC Genomics 10: 50. Manzanares, P., Orejas, M., Gil, J.V., De Graaff, L.H., Visser, J., and Ramón, D., 2003. Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus rhaA gene, encoding an alpha-l-rhamnosidase of enological interest. Appl. Environ. Microbiol. 69: 7558–7562.

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Moore, J.P., Divol, B.T., Young, P.R., et al., 2008. Wine biotechnology in South Africa: Towards a systems approach to wine science. Biotechnol. J. 3: 1355–1367. Mtshali, P.S., 2007. Screening and characterisation of wine-related enzymes produced by wine-associated lactic acid bacteria. MSc dissertation, Stellenbosch University. Naumova, E.S., Ivannika, Y.V., and Naumov, G.I., 2005. Genetic differentiation of the sherry yeasts Saccharomyces cerevisiae. Appl. Biochem. Microbiol. 41: 578–582. Nieuwoudt, H.H., Pretorius, I.S., Bauer, F.F., et al., 2006. Rapid screening of the fermentation profiles of wine yeasts by Fourier transform infrared spectroscopy. J. Microbiol. Methods 67: 248–256. Nieuwoudt, H.H., Prior, B.A., Pretorius, I.S., and Bauer, F.F., 2002. Glycerol in South African table wines: An assessment of its contribution to wine quality. S. Afr. J. Enol. Vitic. 23: 22–30. Nieuwoudt, H.H., Prior, B.A., Pretorius, I.S., et al., 2004. Principal component analysis applied to Fourier transform infrared spectroscopy for the design of glycerol calibration models in wine and for the detection and classification of outlier samples. J. Agric. Food Chem. 52: 3726–3735. Nunan, K.J., Sims, I.M., Bacic, A., et al., 1998. Changes in cell wall composition during ripening of grape berries. Plant Physiol. 118: 781–792. O.I.V. (International Organisation for Vine and Wine). 2009. State of Vitiviniculture World Report, March 2009. Available at http://news.reseau-concept.net/images/oiv_uk/Client/2009_note_conj_mars_EN_ compilee.pdf (accessed June 8, 2009) Pellerin, P. and O’Neill, M.A., 1998. The interaction of the pectic polysaccharide rhamnogalacturonan II with heavy metals and lanthanides in wines and fruit juices. Analusis 26: 32–36. Pérez-Ortin, J.E., Querol, A., Puig, S., and Barrio, E., 2002. Molecular characterization of chromosomal rearrangement involved in the adaptative evolution of yeast strains. Genome Res. 12: 1533–1539. Pilati, S., Perazzolli, M., Malossini, A., et al., 2007. Genome-wide transcriptional analysis of grapevine berry ripening reveals a set of genes similarly modulated during three seasons and the occurrence of an oxidative burst at veraison. BMC Genomics 8: 428. Pradelles, R., Alexandre, H., Ortiz-Julien, A., and Chassagne, D., 2008. Effects of yeast cell-wall characteristics on 4-ethylphenol sorption capacity in model wine. J. Agric. Food. Chem. 56: 11854–11861. Reynolds, T.B. and Fink, G.R., 2001. Baker’s yeast, a model for fungal biofilm formation. Science 219: 878–881. Schlosser, J., Olsson, N., Weis, N., et al., 2008. Cellular expansion and gene expression in the developing grape (Vitis vinifera L.). Protoplasma 232: 255–265. Strauss, M.L., Jolly, N.P., Lambrechts, M.G., and van Rensburg, P., 2001. Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts. J. Appl. Microbiol. 91: 182–190. Swanepoel, M., du Toit, M., and Nieuwoudt, H.H., 2007. Optimisation of the quantification of total soluble solids, pH and titratable acidity in South African grape must using Fourier transform mid-infrared spectroscopy. S. Afr. J. Enol. Vitic. 28: 140–149. Van der Aa Kühle, A., Jesperen, L., Glover, R.L., Diawara, B., and Jakobsen, M., 2001. Identification and characterization of Saccharomyces cerevisiae strains isolated from West African sorghum beer. Yeast 18: 1069–1079. Van Rensburg, P. and Pretorius, I.S., 2000. Enzymes in winemaking: Harnessing natural catalysts for efficient biotransformations—a review. S. Afr. J. Enol. Vitic. 21: 52–73. Van Rensburg, P., Stidwell, T., Lambrechts, M.G., et al., 2005. Development and assessment of a recombinant Saccharomyces cerevisiae wine yeast producing two aroma-enhancing β-glucosidase encoded by the Saccharomycopsis fibuligera BGL1 and BGL2 genes. Ann. Microbiol. 55: 33–42. Van Rensburg, P., Strauss, M.L., Lambrechts, M.G., Cordero Otero, R.R., and Pretorius, I.S., 2007. The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae. J. Appl. Microbiol. 103: 2248–2257. Van Rensburg, P., van Zyl, W.H., and Pretorius, I.S., 1994 Expression of the Butyrivibrio fibrisolvens endobeta-1,4-glucanase gene together with the Erwinia pectate lyase and polygalacturonase genes in Saccharomyces cerevisiae. Curr. Genet. 27: 17–22. Van Rensburg, P., van Zyl, W.H., and Pretorius, I.S., 1996 Co-expression of a phanerochaete chrysosporium cellobiohydrolase gene and a Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene in Saccharomyces cerevisiae. Curr. Genet. 30: 246–250. Van Rensburg, P., van Zyl W.H., and Pretorius, I.S., 1997. 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Verstrepen, K.J. and Klis, F.M., 2006. Flocculation, adhesion and biofilm formation in yeasts. Mol. Microbiol. 60: 5–15. Vicens, A., Fournand, D., Williams, P., et al., 2009. Changes in polysaccharide and protein composition of cell walls in grape berry skin (Cv. Shiraz) during ripening and over-ripening. J. Agric. Food Chem. 57: 2955–2960. Vidal, S., Williams, P., O’Neill, M.A., et al., 2001. Polysaccharides from grape berry cell walls. Part I. tissue distribution and structural characterization of the pectic polysaccharides. Carbohydr. Polym. 45: 315–323. Vilanova, M., Blanco, P., Cortés, S., Castro, M., Villa, T.G., and Sieiro, C., 2000. Use of PGU1 recombinant Saccharomyces cerevisiae strain in oenological fermentations. J. Appl. Microbiol. 89: 876–883. Villamiel., M., Polo, M.C., and Moreno-Arribas, M.V., 2008. Nitrogen compounds and polysaccharides changes during the biological ageing of sherry wines. LWT – Food Sci. Technol. 41: 1842–1846. Vivier, M.A. and Pretorius, I.S., 2000. Genetic improvement of grapevine: Tailoring grape varieties for the third millennium—a review. S. Afr. J. Enol. Vitic. 21: 5–26. Vivier, M.A. and Pretorius, I.S., 2002. Genetically tailored grapevines for the wine industry. Trends Biotechnol. 20: 472–478. Yanai, T. and Sato, N., 1999. Isolation and properties of β-glucosidase produced by Debaryomyces hansenii and its application in winemaking. Am. J. Enol. Viticult. 50: 231–235. Zara, S., Antonio Farris, G., Budroni, M., and Bakalinsky, A.T., 2002. HSP12 is essential for biofilm formation by a Sardinian wine strain of S. cerevisiae. Yeast 19: 269–276. Zara, S., Bakalinsky, A.T., Zara, G., Pirino, G., Demontis, M.A., and Budroni, A.T., 2005. FLO11-based model for air-liquid interfacial biofilm formation by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 71: 2934–2939.

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Impact of Supercritical 23 The Extraction and Fractionation Technology on the Functional Food and Nutraceutical Industry Andrés Moure, Beatriz Díaz-Reinoso, Herminia Domínguez, and Juan Carlos Parajó CONTENTS 23.1 Introduction ..........................................................................................................................408 23.2 Solubility of Nutraceuticals in SC-CO2 ................................................................................409 23.3 Extraction and Fractionation of Biomass Components ........................................................ 412 23.3.1 Fundamentals and Mechanism of SCFE .................................................................. 413 23.3.1.1 Kinetics ...................................................................................................... 413 23.3.1.2 Equipment .................................................................................................. 414 23.3.2 Major Variables Affecting SCFE ............................................................................. 415 23.3.2.1 Pressure and Temperature.......................................................................... 415 23.3.2.2 Modifier ..................................................................................................... 415 23.3.3 Extraction of Nutraceuticals from Microbial Sources .............................................. 416 23.3.3.1 Extraction of Carotenoids .......................................................................... 416 23.3.3.2 Extraction of α-Tocopherol ........................................................................ 421 23.3.3.3 Extraction of Lipids ................................................................................... 422 23.3.3.4 Extraction of Other Metabolites ................................................................ 424 23.3.4 Removal of Undesirable Compounds ....................................................................... 425 23.4 Inactivation of Microorganisms and Enzymes by Dense CO2 ............................................. 425 23.4.1 Fundamentals and Mechanism of Inactivation ......................................................... 427 23.4.1.1 Mechanism ................................................................................................. 427 23.4.2 Treatment Equipment and Operation........................................................................ 431 23.4.3 Inactivation and Disruption of Microbial Cells under Dense CO2 ........................... 432 23.4.3.1 Operational Conditions Affecting the Inactivation and Disruption of Microbial Cells under Dense CO2 ............................................................. 432 23.4.4 Morphological and Structural Alterations ................................................................ 435 23.4.5 Applications of Inactivation...................................................................................... 436 23.4.6 Applications of the Disruption and Fractionation of Biomass Components ............ 436 23.4.6.1 Recovery of Enzymes ................................................................................ 437 23.5 Perspectives of SC-CO2 in the Biotechnological Extraction of Nutraceuticals.................... 438 References ...................................................................................................................................... 439

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INTRODUCTION

Fluids reach a supercritical condition when they rise above their critical temperature and critical pressure (Figure 23.1a). Supercritical fluids (SCF) possess high compressibility, low surface tension, liquid-like density, gas-like viscosity, and diffusivity in between a liquids and gas condition (Figure 23.1b). The ability to manipulate the physical properties of a solvent by simply changing the pressure or temperature is unique to supercritical systems, enabling the control of extraction conditions, separation, and the reaction environment. The density of supercritical fluids is highly sensitive to pressure and temperature. Sensitivity to these variables is high, particularly near the critical point, and small changes in pressure lead to significant changes in density, dielectric constant, solubility, and partition coefficient. Due to these solvent properties, supercritical fluids show a greater ability to penetrate into pores and diffuse into solids than conventional organic solvents, thus improving the extraction yield of solutes from complex matrixes. The enhanced mass transfer rates of reactants to the active sites of enzymes dispersed in supercritical fluids favor reactions limited by the rates of diffusion, allowing higher substrate concentrations. Supercritical extraction of natural products is attractive for nutraceuticals of biotechnological origin. SCF can be optimal for enzyme-catalyzed reactions in nonaqueous solvents, due to the nonaggressive conditions and the feasibility of an accurate modulation of activity, selectivity, and stability by controlling pressure or temperature. Supercritical carbon dioxide extraction (SC-CO2E) is the most common method both for extracting natural compounds and for food processing. Due to its nonpolar nature, CO2 is a not suitable solvent for polar compounds. However, the addition of a polar entrainer (such as water, ethanol, or methanol) enables the extraction of polar compounds. In addition to the favorable properties of CO2 (nontoxic, nonflammable, inexpensive, relatively inert, environmentally and physiologically safe), its phase behavior and other thermophysical properties affecting process development are well known. CO2 supercritical conditions are achieved at temperatures above 31.1°C and pressures above 73.8 bar. The mild critical temperature prevents thermal degradation (or denaturation) of delicate biological materials and natural products, preserving heat labile components. The extract obtained using SC-CO2E is highly concentrated, as CO2 can be readily separated by depressurization, leaving no chemical residues in the product. Furthermore, the resulting CO2 gas stream can be recycled, resulting in an environmentally friendly process. The extracted solutes obtained with SCFs can be crystallized with an accurate control of crystal size by changing the process pressure and temperature

(b)

(a)

Pressure (bar)

0.1 72.8 Solid

Liquid

1

10 100 ρ (kg/m3)

Critical point

5.11 Triple point

SCF

G

Super critical fluid

1000

10,000

L

SCF

G

0.01

0.1

Gas

216.8 304.2 Temperature (K)

L

L

μ (mPa/s)

1

10

SCF G

0.0001 0.001

0.01 0.1 1 D (106 · m2/s)

10

FIGURE 23.1 (a) A typical pressure–temperature phase diagram of pure CO2. (b) The physical properties of SCF (density ρ, viscosity μ, and diffusivity D) in comparison to those of gases (G) and liquids (L).

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(Shariati and Peters, 2003). Additionally, CO2 considerably lowers the microbiological activity of the products and inactivates microbial cells. The main disadvantages of SCF include: low solubility of some biological molecules, lack of data about the physical properties of biological metabolites (which make the prediction of equilibrium difficult), comparatively high capital costs, and the necessity of expertise in high pressure technology. Supercritical CO2 (SC-CO2) is accepted for extraction, fractionation, purification, and recrystallization, and is commercially used for a wide range of applications involving biological materials such as the decaffeination of coffee, extraction of hop, separation of esters from fish oils, and synthesis. SCF technology is becoming relevant to the food, pharmaceutical, and nutraceutical industries, offering higher purity and quality, while keeping the bioactive properties and the “natural” character. This is of particular importance in a context defined by increasingly restrictive regulations concerning food additives produced by chemical synthesis and by growing consumer demand for foods and nutraceuticals from natural sources or from biotechnological origin. SCF processing—requiring low temperatures, and characterized by high mass transfer rates, reduced use of toxic solvents, and negligible solvent content of the final product—produces results which are particularly appropriate for the extraction and separation of high-value biological compounds with strict requirements for thermal stability and purity, which cannot be met by conventional extraction or distillation. SCF can also be used for cell disruption, which (in combination with extraction), produces larger numbers of bioactive fractions than those derived from conventional solvent extraction. Utilization of the same equipment for both disruption and extraction has been proposed to reduce processing time and labor and equipment costs (Khosravi-Darani and Vasheghani-Farahani, 2005). The combination of extraction with reaction or crystallization steps using SCF is of great interest in the bioprocessing industries. A number SC-CO2E applications in the field of nutraceutical products have been reported (Marentis et al., 2001). These include: the processing of fermentation broths containing vitamins and other bioactive compounds; the extraction of biomass for the recovery and purification of carotenoids; the extraction of valuable compounds from animal tissues, cells, and organs; enzymatic reactions (e.g., the conversion of lipids to methyl or ethyl esters); and the separation of enantiomers and fatty acid esters. Recent literature reviews have reported on the utilization of SC-CO2E at an analytical and processing scale for the recovery of natural bioactive compounds from plant materials (Díaz-Reinoso et al., 2006; Herrero et al., 2006; Reverchon and De Marco, 2006; Wang and Weller, 2006; Martinez, 2007), as a processing technique for pharmaceuticals (Cape et al., 2008; Daintree et al., 2008), and as an alternative to thermal technologies for microbial inactivation (Zhang et al., 2006; García-González et al., 2007). The increasing number of publications concerned with different applications of SCF (Sihvonen et al., 1999; García-González et al., 2007) and patents (Schütz, 2007) confirm the growing interest in the application fields of SCF during the past decade. Two general revisions of the applications of these fluids in biotechnological issues have been published (Williams et al., 2002; Khosravi-Darani and VasheghaniFarahani, 2005). The objective of this chapter is to give an overview of the SC-CO2E applications in the field of biotechnologically produced nutraceuticals. Only two relevant topics (in which supercritical fluids can be an alternative to conventional technologies) are considered: • The extraction and recovery of products obtained by biotechnological processes • Associated operations, such as microbial cell inactivation and disruption

23.2

SOLUBILITY OF NUTRACEUTICALS IN SC-CO2

The solubility of a solute in a given solvent is strongly influenced by heteromolecular interaction and, therefore, by the density of the solvent, which in turn depends on pressure and temperature.

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Increasing the pressure of SC-CO2 at a constant temperature increases both the density and solubility. The decreased intermolecular mean distance of the solvent molecules leads to an enhanced specific interaction between solute and solvent. However, solubility may be reduced at higher pressures because of repulsive solute–solvent interactions, which can become important (Williams et al., 2002). Solubility depends on two opposite trends: the density of the solvent, favored by the pressure (at constant temperature) and the vapor pressure of the solutes, favored by the temperature (at constant pressure). The opposite effects of temperature on density and volatility conform to a typical behavior, with a “crossover point” in isotherms. Below this critical pressure, an increase in temperature decreases the solvent power due to decreased fluid density; whereas above this pressure, an increase in temperature can improve the extraction efficiency (despite the relatively small decrease in fluid density), as the vapor pressure of the solute increases. Studies published in the past decade about the solubility of some nutraceuticals are listed in Table 23.1. The differences between sets of data in the literature can be attributed to differences in the purity of the sample and/or to limitations of experimental techniques (de la Fuente et al., 2006). Figure 23.2 summarizes the effects of pressure, temperature, and modifier on the solubility of some nutraceuticals that can be produced biotechnologically. The solubilities of carotenoids in SC-CO2 and in other SC fluids have been the most studied (Canela et al., 2002; Shi et al., 2007). SC-CO2 is suitable for extracting oils and lipophilic compounds owing to its nonpolar character. However, many natural products possessing biological activity and nutraceuticals are poorly soluble in SC-CO2, owing to their slightly polar character. Their solvent power can be increased by adding a small amount of another substance (called an entrainer, modifier, or cosolvent) with a volatility that falls intermediately between the solute and the supercritical fluid. Modifiers can increase the solvent density and interact chemically with the target compounds, and their optimal concentration should be established experimentally. The addition of some polar entrainers, such as methanol, requires increased temperatures to reach the supercritical state, and is not suitable for thermosensitive compounds (Hansen et al., 2001). To improve the solvent power of SC-CO2, ethanol, ethyl acetate, dichloromethane, chloroform, and

TABLE 23.1 Solubility of Some Nutraceuticals in SC-CO2 Compound

P (bar)

T (°C)

Solubility (×106)

Reference

Asthaxanthin

94.9–400 80–300 120–250 68.3–342.9 200–350 300 120–280 120–200 69–172 90–248 54.2–241 171–418 98.8–241.6 56–296 84.5–303

40–60 30–60 35–55 40–70 40–80 40–60 40–60 40–50 25–55 40–100 40–60 40–60 40–60 40–80 40–80

0.01–1.2 4.2–48.9 100–508 0.0013–2.2982 0.09–3.24 0.16–0.66 0.032–0.580 0.43–0.94 0–6350 90–8200 100–2000 0.28–1.5 100–1700 0–2330 20–1730

de la Fuente et al. (2006) Youn et al. (2007) Hansen et al. (2001) Hansen et al. (2001) Johannsen and Brunner (1997) Cocero et al. (2000) Sovova et al. (2001) Saldaña et al. (2006) Liang and Yeh (1991) Liong et al. (1992) Chen et al. (2000) de la Fuente et al. (2006) Chen et al. (2000) Fang et al. (2004) Skerget et al. (2003)

Capsaicin β-Carotene

DHA EPA Linoleic acid Lycopene α-Tocopherol

Source: Data from publications in 2000s, except for DHA and EPA.

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Impact of Supercritical Extraction and Fractionation Technology (b) 1.6

(a) 0.8 307.6 K

313.2 K

1.2

313 K

y × 106

y × 106

0.6

323 K

0.4

333 K

0.2

323.2 K

0.8

333.2 K

0.4

0 0

500

1000 P(bar)

1500

0

2000

(c) 1.6

100

200 P(bar)

300

(d) 1.6 313 K

313 K

1.2

1.2

323 K

0.8

y × 106

y × 106

411

333 K

0.4

323 K

0.8

333 K

0.4

0 50

0

100 150 200 250 300 350 400 450 P(bar)

100

150

200

250 300 P(bar)

350

400

450

(f) 3000

(e) 10,000

313 K

y × 106

y × 106

7500 5000 298.2 K 313.2 K 328.2 K

2500

333 K

125 P(bar)

0

200

(g) 120

373 K

1000

0 50

323 K

2000

50

150 200 P(bar)

90

300

313.1 K

30 y × 106

313 K 323 K

60 30

20 10

50

150

250

0 100

350

150

200

(j) 4000

(i) 4000

δ-tocopherol

308.2 K

3000

250

P(bar)

P(bar)

2000 1000

α-tocopherol 313 K

306.1 K 313.1 K 323.1 K 333.1 K

3000

318.2 K

y × 106

y × 106

250

(h) 40 305 K

y × 106

100

2000

333 K 353 K

1000

0 50

100

150 P(bar)

200

0 60

160 P(bar)

260

360

FIGURE 23.2 The effect of pressure and temperature on the solubility in SC-CO2 of some nutraceuticals that can be produced biotechnologically: (a) β-carotene, (b) β-carotene with ethanol, (c) astaxanthin, (d) lycopene, (e) EPA, (f) DHA, (g) Coenzyme Q10, (h) ascorbic acid, (i) vanillin, and ( j) δ-tocopherol, α-tocopherol. © 2010 Taylor and Francis Group, LLC

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natural entrainers (including vegetable oils and waxes) have been used. In order to preserve the major advantage of supercritical fluid extraction (SCFE), that it produces nontoxic extracts, the addition of toxic organic solvents should be avoided (Shi et al., 2007). Solvent modifiers may require downstream separation to obtain a pure product. Ethanol is the most suited entrainer for the SCFE of nutraceuticals, because of its low toxicity, large availability, and biorenewability. Mixtures of modifiers have also been used in SCFE. In order to keep the advantage of minimal solvent residues in the final product, it is desirable that the cosolvent is added at low concentrations, which in some cases provide the largest increase in solubility, as happens for β-carotene extraction by CO2 containing 1 wt% ethanol (Cygnarowicz et al., 1990). Static or dynamic methods can be used in the experimental assessment of phase equilibria at elevated pressures. In static methods, the components are placed within a closed volume and kept under defined conditions until equilibrium conditions are reached. Mass transfer can be enhanced by stirring or pumping. According to the type of vessel, static methods can be analytic or synthetic. In analytic methods, fluid mixtures are sampled from the equilibrium cell to measure solubility data. Sampling may affect the reliability of data, as the sampled volume must be small enough to avoid equilibrium perturbations. The synthetic method is very convenient for determining binary phase equilibria and phase envelopes in multicomponent systems. Solubility is determined in a variable volume cell in which the operating conditions can be controlled, and the vessel is equipped with sapphire windows to visually detect the cloud point. This operation is usually carried out by changing the pressure at constant temperature, and physical properties can be monitored. In dynamic methods, the condensed phase remains in equilibrium while the gaseous phase flows through, and the outlet streams are assumed to be in equilibrium under the selected operational conditions. This method is suitable for determining low solute concentrations in the gaseous phase. In complex mixtures, the solubility of the target compound may be affected by the presence of other solutes. The study of multicomponent systems is important because many potential applications of SCFE involve the removal of valuable compounds from a complex natural matrix. In the extraction of solid mixtures with a pure SCF, the solubilities of the individual solutes can be significantly greater than their respective binary solubilities. Experimental data suggest that the solubility in mixed-solid systems can be enhanced by strong solute–solute interactions, particularly when the solutes contain potential hydrogen-bonding sites (Lucien and Foster, 1996). Dobbs and Johnston (1987) proposed that the solubility of a solid in a ternary system will increase in respect of one of its binary systems proportionally to the solubility of the second solid.

23.3

EXTRACTION AND FRACTIONATION OF BIOMASS COMPONENTS

The industrial production of some fermentation metabolites may require a sequence of solvent extraction and crystallization stages. Supercritical fluids can be more efficient than traditional liquid solvents, since their selectivity can be adjusted by changing pressure and temperature. SCFE can be a suitable alternative, particularly for products with high sale value where the high operating and capital costs of SCFE would be acceptable (Cygnarowicz and Seider, 1990). SCFE allows the isolation of natural compounds directly from the fermentation broth or from the biomass, which can be ruptured by individual or combined mechanical, chemical, enzymatic, or SCFE methods. KhosraviDarani and Vasheghani-Faharani (2005) made the distinction between postfermentation extraction and in situ extraction of fermentation products, this latter method being particularly relevant when end-product inhibition occurs. SCFE fulfills the requirements for product recovery from a fermentation broth, mainly in terms of product concentration, broth viscosity, and biocatalyst properties. This technology has been successfully used to separate ethanol, acetone, and butanol, as well as for removing products from the cell environment, limiting their pernicious effects on cell metabolism. SCFE has been employed for ethanol removal from alcoholic beverages (Medina and Martinez, 1997), and for obtaining an aromatic fraction of distilled alcoholic drinks (Señoráns et al., 2003). SC or near critical CO2 has © 2010 Taylor and Francis Group, LLC

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been proposed for the in situ extraction of fermentation products, although other solvents are promising in biphasic systems (Berberich et al., 2000). Under controlled conditions, both microorganism killing and intracellular material extraction can be achieved in a subsequent stage. Many intra- and extracellular metabolites of commercial interest as nutraceuticals can be extracted from fermentation broths using SC-CO2. Countercurrent column operation is currently used for this purpose, without leaving solvent residues in the product. When SC-CO2 is added to a liquid medium, the overall viscosity decreases, facilitating the handling of the broth and enhancing mass transfer from the medium to the SC solvent (Khosravi-Darani and Vasheghani-Farahani, 2005). Biomass fractionation is based on the different solvation properties modulated by the temperature, pressure, and/or modifier (Castor and Hong, 2003). Nonpolar compounds can be extracted at the fermentation temperature with low energy requirements compared to distillation. Intracellular compounds can be extracted after cell rupture by different technologies, including SC-CO2. Solid–liquid extraction may be necessary if the cells were previously dried.

23.3.1

FUNDAMENTALS AND MECHANISM OF SCFE

Solid–liquid extraction is a multicomponent, nonsteady, heterogeneous, mass transfer operation. The sequential steps involved in the whole extraction process are: • • • •

Penetration of the solvent in the matrix Solubilization of the target compounds Transport of the solute(s) to the exterior of the solid matrix Transport of the solute(s) into the bulk solution

Further separation of the extract and spent solids can be performed in the same equipment or in a separate stage, whereas the solvent can easily be separated from the extract by changing the state and thermodynamic properties of the solvent. Separation with the aid of a third agent (adsorption or absorption) has also been considered. When the solute is located inside a solid matrix of complex cellular structure, or linked to cell walls, intraparticle solute diffusion is frequently the limiting step. In this case, particle size should be optimized to facilitate extraction (small particles are desirable) without limiting the performance of extractors (particles which are too fine may lead to compaction and channeling). In addition to particle size reduction, moisture conditioning should also be considered, since it affects yields, rates, and selectivity. Moisture complicates the solvent–solid contact, but water can act as a cosolvent for polar compounds and as a swelling agent of the solid matrix. Optimal grinding and drying should limit both energy requirements and thermal degradation of solutes. 23.3.1.1 Kinetics Typical time profiles of solute extraction in SC-CO2 extraction show that the process occurs rapidly at the first moments, and afterwards extraction continues at a slightly lower, constant rate. In the early stages, the solute at the surface is extracted, and the process takes place due to the solubilization of outer solutes and convection. The second zone corresponds to solute extraction from the inside of the cell matrix, controlled by diffusion. The slower kinetics is due to the depletion of the substrate. Reported mathematical models assume the plug flow of the supercritical fluid through the fixed bed of milled solids (vegetal or microbial biomass), with control by external mass transfer, internal mass transfer, or a combination of both (Sovová, 1994). Mathematical models fitting the SCFE curves include empirical and mass balance equations for the solute in solid and in fluid phases. Phenomenological models have been reviewed by Sovová (2005), whereas a revision of the theoretical basis underlying all kinetic SCFE models (based on desorption kinetics, mass transfer, or thermodynamic equations to describe the interactions at the interface) is available (Al-Jabari, 2002).

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When the desired compounds are present in a liquid stream, the key stages are the phase contact between solvent and solute-containing solutions and solubility. Liquid–liquid contact in SC-CO2 fractionation has been revised by Reverchon and De Marco (2006) and Gamse (2004). 23.3.1.2 Equipment Solid–liquid extraction to obtain a solute from a natural product is the most common application of SCFE. The basic SC-CO2E design consists of a gas compressor (a pump if the solvent is recirculated in liquid state), one extraction vessel containing a bed of particles, at least one separator (up to three separators are used frequently), heat exchangers, and measuring and controlling devices (see Figure 23.3). The pressurized solvent flows continuously through the extractor, and the pressure of the outlet stream is reduced in the separators, where the fluid expands to gaseous state, facilitating the precipitation of the solutes. Utilization of several separators in series, operating at different pressures and temperatures, enables the fractional separation of the extracted solutes, and facilitates their selective precipitation. Most systems operate in batch owing to difficulties in the management of solids at high pressures in continuous operation, although this latter method has also been employed (Voges et al., 2007). A continuous countercurrent operation can be simulated by a cascade of batch extractors (Koerner, 1985; Nguyen et al., 1991; Brunner, 2005). Sequential or fractional extraction and/or separation are frequently used to yield separate fractions with different physicochemical properties. Batch contact in liquid–liquid operations can be performed in stirred vessels with an adequate control of pressure and temperature, whereas continuous operation is performed in packed columns (usually with the liquid phase flowing downwards). In order to facilitate mass transfer between phases, an adequate selection of the packing material is desirable. In addition to pressure and temperature, which influence equilibrium solubility and mass transfer, other important design variables are flow rates, reflux ratio, and column height. Temperature gradients along the columns may facilitate operation and separation, enhancing the selectivity of the process. The equipment used for biotechnology-related applications (from analytical to commercial scale) has been revised by Williams et al. (2002). An overview of the different SCFE for solid and liquid

(a)

(b) Solvent + solubilized component(s)

Extraction column Separator

Enriching feed section

Separator Feed

Reflux

Solvent line

Extractor

Product Raffinate

Extract

Stripping section Gas fraction

Solvent

Raffinate

FIGURE 23.3 A basic set-up for SCF extraction of (a) solid materials, with stages of extraction and separation, and (b) liquid streams.

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samples has been published by Brunner (2005), whereas general dispositions have been presented in several revisions (Starmans and Nijhuis, 1996; Raventós et al., 2002; Reverchon and De Marco, 2006). Relevant data for estimation of SCFE operating costs have been reported by Meireles (2003). A recent and complete revision of the equipments used for the supercritical extraction of nutraceuticals has been published by Martínez and Vance (2008), who also considered aspects related to industrial implementation, auxiliary equipment, and cost analysis.

23.3.2

MAJOR VARIABLES AFFECTING SCFE

Both the extraction yield and the rate can be affected by sample preparation and operational variables, which should be optimized for each material. When the most influential variables are fixed at favorable levels, the effects of other variables may become negligible: for example, by using finely crushed materials, the modifier presence becomes nonimportant, and when a modifier is used the pressure and the amount of CO2 required can be reduced for a sufficiently prolonged time extraction. In general, the major operational variables affecting SCFE are pressure, temperature, solvent flow rate, solvent-to-feed ratio, cosolvent type, and cosolvent concentration. Due to the number of variables and to the interactions among them, experimental designs and response surface methodology have been proposed to establish optimal extraction conditions (Hu, 1999; Careri et al., 2001; Wang and Weller, 2006; Jaime et al., 2007; Choudhari and Singhal, 2008; Mendiola et al., 2008; Sajilata et al., 2008; Thana et al., 2008). Any previous conditioning of particle size and moisture content should also be considered in the optimization of each particular case. 23.3.2.1 Pressure and Temperature Pressure and temperature are influential on both equilibrium and kinetics, and control the density and solvating power of CO2. Their optimal values should be established for each system. An increased extraction pressure (at constant temperature) results in increased solvent density and solvating power (although this could result in decreased selectivity due to coextraction of other compounds), in a higher vapor pressure of the solute, and in a decrease of the diffusion coefficient (which reduces the penetration capacity of the SC solvent). An increased temperature influences the properties of the solid matrix, decreases the solvent power by decreasing its density, and may affect the thermal stability of the solutes; however, it increases solubility by increasing the vapor pressure of the solute. Mild temperatures (below 40–60°C) are desired to extract biological materials. 23.3.2.2 Modifier The major effects caused by the presence of a modifier are altered density, polarity, and viscosity of the SC solvent, enhanced solubility of the solutes in the extracting mixture, breakage of solute– matrix polar interactions, and the swelling of the solid matrix (leading to increased surface area). This latter effect can be relevant if the inner structure of the matrix interferes with the release of some metabolites due to interactions with the protein environment. The optimal modifier concentration should be established experimentally to maximize the extraction of the target compound, since too high entrainer concentrations could decrease both the yield (due to the lower solvent density) and the selectivity (resulting in coextraction of other components). In the presence of a modifier, higher solute amounts can be obtained under mild operational conditions (pressure, temperature, and flow rate). The cosolvent can be introduced into the process mixed with the SC-CO2 (i.e., by flowing the solvent through a bed of glass wool wetted with the cosolvent, according to Larson (1986), or mixed uniformly with the dry biomass before being placed in the extractor (Gouveia et al., 2007). Increased CO2 flow rates can shorten the residence time and increase the solvent–solute interactions, thus favoring its dissolution. Excessively high solvent flow rates limit the contact time between solute and solvent (which could limit the extraction yield), and may also cause sample

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compaction. External mass transfer resistances are reduced by increasing the CO2 flow rate up to a given threshold, this effect being more noticeable at the early stages (when extraction of the solute attached to the surface occurs). However, increasing CO2 flow rate may not significantly enhance extraction rates or yields if the solute is easily extractable or if another variable controls the extraction (e.g., when internal diffusion is the controlling step, as usually happens in the later extraction stages). A negligible effect on increased flow rates has also been reported when CO2 is not evenly distributed throughout the extractor (Machmudah et al., 2006).

23.3.3 EXTRACTION OF NUTRACEUTICALS FROM MICROBIAL SOURCES The extraction of nutraceuticals from the biomass by organic solvents (methanol, acetone, hexane, etc.) requires further purification by suitable techniques (e.g., chromatography or fractional crystallization). Some of these solvents are potentially toxic, and their use for food and pharmaceutical applications is limited by legal regulations. Treating suitable raw materials by SCFE under suitable experimental conditions enables the recovery of valuable compounds (including lipids, proteins, nucleotides, and saccharides), or the removal of undesired components. The extraction of a number of microbial metabolites to be used in the chemical, pharmaceutical, or food industries has been revised (Williams et al., 2002; Khosravi-Darani and Vasheghani-Farahani, 2005). SCFE of compounds produced by algae has recently been considered (Mendes, 2008). Table 23.2 summarizes the extraction yields of some microbial metabolites in experiments at laboratory or pilot scale. Control extractions (used as a reference) were carried out with conventional organic solvents such as acetone (Lim et al., 2002; Mendes et al., 2003; Macías-Sánchezet al., 2005; Nobre et al., 2006), methanol (Macías-Sánchez et al., 2008), hexane (Mendes et al., 2006), dimethylformamide (DMF) (Montero et al., 2005; Macías-Sánchez et al., 2008), ethanol (Mendes et al., 2003), and a mixture of chloroform, methanol, and water (Bligh and Dyer method) (Mendes et al., 2003, 2006). 23.3.3.1 Extraction of Carotenoids Carotenes are usually added to foods to impart colors ranging from yellow to red, and can be commercially produced by microalgae. β-carotene is a highly conjugated polyprenoid hydrocarbon with provitamin A, antioxidant, and anticancer activities. Cis and trans isomers of β-carotene have different biological values, since the cis isomer is more easily absorbed in the human body. Astaxanthin, a red ketocarotenoid used as a feed supplement for poultry and marine fish, is a precursor of vitamin A associated with cell reproduction and embryo development. This compound has higher antioxidant potency than β-carotene, lutein, lycopene, cantaxanthin, and vitamin E (Miki, 1991). Astaxanthin may be produced by chemical synthesis or obtained from natural sources. The products obtained by this latter method exhibit greater stability and activity than the synthetic ones (Higuera-Ciapara et al., 2006). Lycopene is a red-colored carotenoid with anticarcinogen and antioxidant activities used as a feed supplement and nutraceutical. Commercial lycopene is extracted from natural sources (i.e., tomatoes), although the production of a mixture of carotenoids in plants and algae could be a feasible alternative. The pigment profile of various microorganisms varies, and may show a different distribution along the extraction period (Montero et al., 2005). These aspects have to be considered when designing the extraction process. Microalgae are interesting natural sources of carotenoids, because of their fast growth and easy manipulation. SCFE of carotenoids has been proposed for Chlorella vulgaris (Mendes et al., 1995; Gouveia et al., 2007; Zhengyun et al., 2007), Dunaliella salina (Macías-Sánchez et al., 2008), Haematococcus pluvialis (Nobre et al., 2006; Krichnavaruk et al., 2008), Nannochloropsis sp. (Andrich et al., 2005; Macías-Sánchez et al., 2008), Schizochytrium sp. (Zinnai et al., 2006), Spirulina sp. (Andrich et al., 2006; Mendiola et al., 2007, 2008; Valderrama et al., 2008), and Synechococcus sp. (Macías-Sánchez et al., 2008). Spirulina platensis contains β-carotene, inositol, niacin, γ-linolenic acid (GLA), eicosapentaenoic acid (EPA), and low levels of tocopherol © 2010 Taylor and Francis Group, LLC

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TABLE 23.2 Examples of Products Obtained Biotechnologically and Extracted with SC-CO2 Compounds

Sources

Arachidonic acid Astaxanthin

Morteriella sp. H. pluvialis

550, 70

Astaxanthin

H. pluvialis

300, 60, E (10%)

Astaxanthin

H. pluvialis

500, 80

Astaxanthin

H. pluvialis

300, 60, E (9.4%)

Astaxanthin

500, 40

Carotenoids

Phaffia rhodozyma Chlorella vulgaris Chlorella vulgaris D. salina

Carotenoids

D. salina

400, 60, E (5%)

Carotenoids

N. gaditana

500, 60, E (5%)

Carotenoids

N. gaditana

400, 60

Carotenoids

180, 30

Chlorophyll

Spirulina maxima Synechococcus sp. Synechococcus sp. Synechococcus sp. Spirulina platensis D. salina

Chlorophyll

N. gaditana

500, 60, E (5%)

Chlorophyll

Synechococcus sp. Synechococcus sp. Liagora boergesenii Nanochloropsis sp.

300, 60, E (5%)

Carotenoids Carotenoids

Carotenoids Carotenoids β-Carotene β-Carotene

Chlorophyll EPA EPA

Conditions (bar, °C) —

350, 55 300, 40, E (5%) 443, 27.5

300, 50 300, 50, E (5%) 300, 50 300, 48 400, 60, E (5%)

500, 60 34.5, 55 550, 55

Yield SCFE (CSE) Purity SCFE (CSE) 28.7% — 2.67% (3.43% DMC) 12.3% 0.16% (1.8% A) — 0.228% (2.75% A) — 1.7% — 0.2% (0.3% A) — 5% (3% H to 4% A) — 0.299% (0.426% A) — 6.58% 7.199% 0.9629% (1.41% M, 2.77% DMF) — 0.2893% (0.22% M, 0.69% DMF) — 0.0343% — 2.27a — 0.151% (0.135% M) — 0.186% (0.14% M, 0.33% DMF) — 75%b 80%c 0.071% 7.78% 0.07%, (0.25% M, 0.31% DMF) — 0.0369% (2.64% M, 4.15% DMF) — 0.2218% (0.041% M, 0.96% DMF) — 0.071% (0.409% M) — 21.5% 6.78%d — 33% (31.1% H)

Reference Totani and Oba (1988) Machmudah et al. (2006) Nobre et al. (2006) Thanaet al. (2008) Valderrama et al. (2008) Lim et al. (2002) Mendes et al. (1995) Gouveia et al. (2007) Jaimeet al. (2007) Macías-Sánchez et al. (2008) Macías-Sánchez et al. (2008) Macías-Sánchezet al. (2005) Youn et al. (2007) Macías-Sánchez et al. (2007) Macías-Sánchez et al. (2008) Montero et al. (2005) Wang et al. (2007) Macías-Sánchez et al. (2008) Macías-Sánchez et al. (2008) Macías-Sánchez et al. (2008) Macías-Sánchez et al. (2007) Chen and Chou (2002) Andrich et al. (2005)

continued

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TABLE 23.2 (continued) Examples of Products Obtained Biotechnologically and Extracted with SC-CO2 Compounds GLA GLA GLA Lutein Lycopene Tocopherol

Sources Arthorspira maxima Spirulina platensis Spirulina platensis Chlorella pyrenoidosa Blakeslea trispora Spirulina platensis

Conditions (bar, °C) 350, 60, E (10%) 400, 40 400, 40, E (0.44 mL/g biomass) 50, 250, E (5 0%) 350, 45, A (1 mL/g biomass) 220, 83

Yield SCFE (CSE) Purity SCFE (CSE) 0.44% (0.68% E, 0.63% A) — — 0.29%d 0.51% (0.49% B&D) — 87%e — 0.003817% — 0.62% 3.161%

Reference Mendes et al. (2003) Hu (1999) Sajilata et al. (2008) Zhengyun et al. (2007) Choudhari and Singhal (2008) Mendiolaet al. (2008)

Note: E, ethanol; A, acetone; AC, acetyl chloride; M, methanol; B&D, Bligh and Dyer; DMF, dimethyl formamide; H, hexane. Expressed in mg. b Of DMF extraction. c Of total carotenoid content. d Of crude lipid. e Of initial amount of lutein. a

(Canela et al., 2002). Synechococcus strains contain β-carotene, zeaxanthin, chlorophyll a, cryptoxanthin, myxoxanthophyll, and equinenone (Montero et al., 2005). Chlorella vulgaris is a producer of astaxanthin, cantaxanthin and, in minor amounts, β-carotene and lutein. Nannochloropsis gaditana is an important source of pigments (chlorophyll a, β-carotene, violaxanthin, and vaucheriaxanthin). Commercially grown H. pluvialis accumulates the highest levels of astaxanthin as the main carotenoid (between 1.5 and 3.0 wt%), followed by lutein, β-carotene, and canthaxanthin (Nobre et al., 2006). Although the concentration of astaxanthin in the red yeast Phaffi a rhodozyma (500–2000 μg carotenoids/g dry yeast, 45–95% of which correspond to astaxanthin) is lower than that in H. pluvialis, the growth rate and productivity are higher. SC-CO2 extraction of astaxanthin from Phaffi a rhodozyma was proposed (Lim et al., 2002). Spirulina is composed of proteic pigments (phycobiliproteins), suitable to be used in foods, feeds, and cosmetics. Numerous studies on SCFE of lycopene from tomato products and byproducts have been followed by a recent study on the SCFE extraction of lycopene from Blakeslea trispora (Choudhari and Singhal, 2008). Lutein is used as a food colorant and additive, to prevent age-related macular degeneration and atherosclerosis, and to stimulate the immune response. SC-CO2 extraction of lutein from Chlorella pyrenoidosa leads to yields comparable to those obtained from marigold (Zhengyun et al., 2007). SC-CO2 extraction of carotenoids is an alternative to solvent extraction because it provides a rapid extraction at low temperatures and enables a simple purification. In the presence of more polar pigments (such as xanthophylls or chlorophyll a), SC-CO2 can be more selective for carotenoids than conventional solvents (i.e., hexane), resulting in more limpid and fluid extracts. Another advantage of SCFE lies in its selectivity towards the cis–trans isomers of β-carotene, since their conventional separation is costly. Based on their different physicochemical properties, the 9-cis isomer is highly soluble in pressurized CO2, all-trans is practically insoluble in oil, and both carotenoids (in the natural mixture) present higher solubilities than the synthetic trans isomer. SC-CO2 of the microalga D. salina allowed the extraction of products with higher cis:trans ratio than conventional solvents

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(Mendes et al., 2003). The use of an adsorbent (silica gel) in the second extractor (operating at 20.0 MPa and 313.1 K) increased the cis:trans ratio (Mendes et al., 2003; Nobre et al., 2000). Selective extraction of cis–trans isomers from Dunaliella bardawil was achieved by SC-CO2 extraction from a carotenoid concentrate, based on their favorable dissolution rate (Gamlieli-Bonshtein et al., 2002). Although the antioxidant activity of the pure 9-cis isomer is higher than all-trans (Jiménez and Pick, 1993; Bohm et al., 2002), a synergistic behavior was observed. The maximum antioxidant activity was obtained at high concentrations of the two major β-carotene isomers (cis and trans) (Jaime et al., 2007). 23.3.3.1.1 Effect of the SC-CO2 Extraction Conditions 23.3.3.1.1.1 Mechanical and Chemical Pretreatment The degree of crushing strongly influenced the extraction yield of carotenoids from cells by SC-CO2. An increased number of disrupted cells enhances the accessibility of the solvent, limiting the mass transfer resistance. Some microalgae lack a proper cell wall (i.e., D. salina), but others have a thick wall with pigments located in the chloroplast (i.e., N. gaditana), near to the cell wall (i.e., Synechococcus sp.), or in the cytoplasm membrane (i.e., Phaffia rhodozyma). A previous mechanical treatment of microalgae enhanced the SC-CO2 extraction yield of carotenoids for completely crushed freeze-dried Chlorella vulgaris (Gouveia et al., 2007; Mendes et al., 1995, 2003), H. pluvialis (Nobre et al., 2006), and improved selectivity for isomer separation from D. bardawil (Gamlieli-Bonshtein et al., 2002). Zhengyun et al. (2007) reported on the utilization of superfine, pulverized Chlorella pyrenoidosa for lutein extraction. The lower recovery rate of some compounds (canthaxanthin) in comparison to other compounds suggests that different carotenoids are differently bound to the algal structure (Nobre et al., 2006). As astaxanthin is intracellular and surrounded by thick, impermeable cell walls, crushing (by cutting mills), and manual grinding (with dry ice) increased the total astaxanthin extract yield from H. pluvialis (Valderrama et al., 2008). A similar behavior has been reported for Phaffia rhodozyma disrupted by ball milling and further spray dried before SC-CO2 extraction (Lim et al., 2002). Although disruption of Synechococcus cells by sonication in 0.9% ammonium formate did not enhance the carotenoid extraction yield, this treatment solubilized phycobilins in the aqueous medium. Similarly, detergent treatments causing protein denaturation did not result in improved carotenoid recovery, but the extraction of chlorophylls and phycobilins was enhanced (Montero et al., 2005). 23.3.3.1.1.2 Effects of Pressure and Temperature Table 23.2 summarizes the operational conditions leading to maximal extraction yields in the SC-CO2 extraction of carotenoids. Optimal pressures lie in the range of 30–50 MPa operating at 50°C. At a given temperature, a higher pressure results in higher CO2 density and higher solvent power. Increased carotenoid extraction yields at increased pressures were reported in experiments with Spirulina pacifica (in the range 15–35 MPa) (Careri et al., 2001), and with Chlorella pyrenoidosa (in the range 20–30 MPa) (Zhengyun et al., 2007). The yields of extractable material and astaxanthin yield from H. pluvialis increased with temperature and pressure in the ranges 313–353 K and 20–55 MPa, respectively (Machmudah et al., 2006). Both variables were the most influential on the extraction of astaxanthin from H. pluvialis (Thana et al., 2008) and on the extraction of β-carotene from D. salina (Jaime et al., 2007). However, the utilization of high extraction pressure at a commercial scale is not recommended due to safety considerations and high operational costs. Decreased extraction yields obtained at high pressures can be attributed to the opposing effects of increased density (and solvating power) and decreased diffusivity (and solvent–solute interactions in the solid matrix). Operating at a given pressure, higher temperatures increased the extraction yields of carotenoids and astaxanthin, owing to the increased vapor pressure of solutes. This behavior has been reported for experiments with Spirulina pacifica (Careri et al., 2001) and H. pluvialis (Machmudah et al., 2006). Increased temperatures (up to 343 K) contributed to the decomposition of cell walls, enhancing the availability of astaxanthin and increasing both the extraction yield and product purity.

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However, no improvements were observed in the temperature range 343–353 K (Machmudah et al., 2006). Temperature should be kept below the values which lead to the thermal degradation of solutes. Thermal degradation of carotenes was evident as the temperature increased from 30°C to 70°C (Canela et al., 2002), whereas the highest lutein yield was obtained at 50°C (Zhengyun et al., 2007). The amount of astaxanthin extracted from H. pluvialis increased with temperatures up to 70°C, but a further increase to 80°C did not have a significant effect, and visual observation revealed thermal deterioration (Krichnavaruk et al., 2008). Decreased extraction yields observed at increased temperatures was due to the decreased SC-CO2 density (Mendes et al., 1995; Macías-Sánchez et al., 2005, 2008). The solubility of solutes increases by decreasing the temperature near the critical pressure; but at high pressures, solubility increases with temperature. This crossover effect is due to the opposite contributions of solvent density reduction and vapor pressure increase. The crossover range for β-carotene was found between 15 and 17.5 MPa (Mendes et al., 1999) and near 25 MPa for Coenzyme Q10 (Matias et al., 2004). End-temperature crossovers were apparent at approximately 12 and 16 MPa for astaxanthin and lycopene, respectively (de la Fuente et al., 2006). Higher severity (defined by pressure, temperature, and contact time) leads to higher extraction yields, but selectivity and extract purity could be maximal under moderate severity conditions. Higher selectivity in the separation of carotenoids from chlorophylls was obtained as the operation pressure decreased from 50 to 20 MPa; however, in all cases, selectivity was higher than that obtained by conventional solvent extraction (Macías-Sánchez et al., 2005). The operation of SC-CO2 flow rate is usually selected where this variable does not exert a limiting effect. The flow rate showed a limiting effect on the initial extraction stages of astaxanthin and carotenoids from Phaffia rhodozyma (Lim et al., 2002) and in the separation of astaxanthin from H. pluvialis biomass (Krichnavaruk et al., 2008). The amounts of the total extractable material and astaxanthin increased slightly by increasing the CO2 flow rate from 2 to 4 mL/min, operating at 50 MPa and 323 K, but the astaxanthin content of the extract remained constant (Machmudah et al., 2006). The effects of the CO2 flow rate depend on the solute under consideration: for example, the recovery of lycopene operating at flow rates of 4 and 8 kg/h was not significantly different, but operation at 4 kg/h was more favorable than at 8 kg/h for the recovery of β-carotene (Baysal et al., 2000). Different effects of flow rates on the extraction yield at different temperatures were reported for lycopene and β-carotene (Sabio et al., 2003). 23.3.3.1.1.3 Effect of Entrainer The enhanced solubility of carotenoids in SC-CO2–ethanol mixtures is due to the polar character of the modifier, which makes the formation of hydrogen bonds easier. Although different solvents (including methanol, ethanol, and acetone) have been proposed (e.g., to extract lycopene from Blakeslea trispora) (Choudhari and Singhal, 2008), ethanol has been used most frequently. The yield and selectivity of carotenoid extraction was significantly increased when ethanol was used as a cosolvent in proportions up to 15 wt% for processing the biomass of Synechococcus sp. (Montero et al., 2005), Chlorella vulgaris (Gouveia et al., 2007), and Spirulina platensis (Mendiola et al., 2005). Ethanol (in the range 1–15 vol%) has been reported to improve the extraction of astaxanthin from Phaffia rhodozyma (Lim et al., 2002) and H. pluvialis (Machmudah et al., 2006; Nobre et al., 2006; Krichnavaruk et al., 2008; Valderrama et al., 2008). A similar method (using 5–15 wt% ethanol) has been employed for extracting zeaxanthin, β-cryptoxanthin, β-carotene (Careri et al., 2001), canthaxanthin, and lutein (Nobre et al., 2006). The optimal modifier concentration depends on the types of solute and solid considered, as well as on the operational conditions (temperature and pressure) (Lim et al., 2002). The extraction of astaxanthin from H. pluvialis was enhanced by more than twice using 1.67 vol% ethanol (Machmudah et al., 2006). However, higher ethanol concentration (50%) was required for extracting 87.0% of the lutein present in Chlorella pyrenoidosa (Zhengyun et al., 2007). In the recovery of astaxanthin, the extraction yield increased with the ethanol concentration up to 5 vol%, but decreased at higher concentrations (up to 7.5 vol%). Indirect separation by increasing the concentration of unextracted substances from

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the residue has been observed for phycocyanine, as a result of removing the CO2-soluble fraction from Spirulina maxima, using 10 wt% ethanol (Valderrama et al., 2008). Vegetable oils have been proposed as modifiers to enhance the yields of carotenoids from microalgae. Some protective effects of vegetable oils against the degradation of Chlorella vulgaris carotenoids have been reported (Mendes et al., 1995), whereas carotenoids without any oil protection (for instance, in acetone) were rapidly degraded (Gouveia et al., 2007). Conversely, pigments can contribute to soybean oil stability due to their antioxidant effect. Soybean oil and olive oil were proposed as cosolvents (concentration, 10%) for enhancing the astaxanthin extraction from H. pluvialis (Krichnavaruk et al., 2008). These protective effects could relate to the higher solubility of carotenoids in the mixture of soybean oil–SC-CO2 than in pure SC-CO2, as well as to the improved mass transfer through the swollen solid matrix imbibed in the solvent mixture. Although soybean oil was less efficient than ethanol as a cosolvent for astaxanthin extraction, olive oil showed comparable effects to this latter solvent. On the other hand, utilization of olive oil avoids the need for subsequent product separation. 23.3.3.1.1.4 Two-Stage Extraction Two-step, pressure gradient operation was proposed to extract astaxanthin from Phaffia rhodozyma. The first extract was obtained at 30 MPa, and the second at 50 MPa (operating with the same sample). Higher lipid extraction yields were achieved in the first stage, and products which were more concentrated in astaxanthin were produced in the second extraction step, regardless of the extraction temperature (40°C or 60°C). In comparison with acetone extraction, this operational mode allowed for higher astaxanthin concentrations in the second fractions (4–10 times higher), whereas the carotenoid concentration increased by 3.6–13 times depending on temperature (Lim et al., 2002). Following the method known as two-step pressure gradient operation (or fractional extraction), Arthorspira maxima was subjected to pure SC-CO2 (25.0 MPa, 323 K), and to a mixture of CO2 and ethanol (10 mol%), and the residue of the latter extraction was extracted at a higher pressure and temperature (Hu, 1999), to enable the separate extraction of different valuable fractions. This operational mode is suitable for fractionating compounds on the basis of their different solubility in the SC solvent. A short period of static extraction before dynamic extraction facilitates the penetration of the fluid in the matrix more than the dynamic mode. The desorption of pigments from Spirulina pacifica (Careri et al., 2001) and from Synechococcus sp. during extraction with ethanol-modified SC-CO2 was also enhanced as a result of the matrix environment alteration (Montero et al., 2005). The processing of crude fractions obtained by conventional solvent extraction (with solvents such as hexane, acetone, or ethanol, or with vegetable oils) and further concentration and purification have been carried out using a combination of different techniques (including molecular distillation, column chromatography, steam distillation, treatment with a SC fluid, liquid–liquid extraction, and saponification). Acetone extracts of mechanically crushed Phaffia rhodozyma cells were processed by adding a silver nitrate solution to the filtrate, and extracted with SC-CO2 using ethanol as a cosolvent (Mishima et al., 1996). The direct fractionation of conventional solvent extracts is sometimes not feasible, and a sequence of steps is desired. The blue–green microalgae Spirulina maxima are a source of the blue pigment phycocyanin, which is not soluble in CO2 but is soluble in water. However, the water extract is a mixture of polar and nonpolar substances which are difficult to fractionate. Selective extraction of fatty acids and carotenoids of Spirulina maxima with CO2 was achieved at 150–200 bar and temperatures below 50°C, to yield extracts rich in essential fatty acids, GLA, palmitic acid, palmitoleic acid, linoleic acid, and stearic acid (Canela et al., 2002). 23.3.3.2 Extraction of α-Tocopherol Natural vitamin E is formed by tocopherols and tocotrienols, the first group being assimilated and used to correct vitamin E deficiencies. α-Tocopherol is a lipid-soluble antioxidant which provides protection against cardiovascular diseases, by preventing lipoprotein oxidation and platelet

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aggregation (Landvik, 2004; Clarke et al., 2008). Major sources of α-tocopherol include vegetable oils (olive, sunflower, and safflower oils), nuts, whole grains, and green leafy vegetables. Applications of SC-CO2 for tocopherol analysis include the characterization of lipid components from seeds, marine and vegetable oils, and the analysis of antioxidant samples and deodorizer distillates, vegetable oils, and pharmaceutical preparations. A process based on SCFE at the pilot scale has been optimized to obtain fractions highly enriched in α-tocopherol from Spirulina platensis, which contains 0.011–0.014 mg tocopherol/g dried microalga (Mendiola et al., 2008). Temperature and pressure were the most influential variables on the concentration of vitamin E in extracts. The maximum extraction yield, calculated from an experimental design, corresponded to operation at 362 bar and 55°C, and enabled an enrichment factor of 12, with a tocopherol content of 29.4 mg tocopherol/g extract (Mendiola et al., 2007). 23.3.3.3 Extraction of Lipids Organic solvents (such as hexane, chloroform, acetone, and methanol) are commonly used for oil extraction from lipid-bearing vegetal and microbial biomass. SC-CO2 is an alternative solvent for industrial processes, with advantages derived from its safety, mild operating conditions, no environmental impact, high product quality, and solvent-free extracts. The extraction of lipids from biological samples has been revised by Khosravi-Darani and Vasheghani-Faharani (2005). Table 23.2 presents comparative data on the extraction yields of representative products obtained biotechnologically, using SC-CO2, conventional solvents, or the Bligh and Dyer routine method for lipid extraction (which is presented for comparative purposes). In some cases, cell breakage (by sonication, homogenization, freezing, or grinding) may be necessary to allow a better penetration of the solvent into the cell and to increase the lipid yield (Robles Medina et al., 1998). 23.3.3.3.1 Polyunsaturated Fatty Acids The n-3 polyunsaturated fatty acids (PUFA) group consists mainly of α-linolenic acid (ALA), EPA, docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA). The first is found in vegetable oils, and the last three from marine sources. The n-6 PUFA group is made up of linoleic acid, GLA, dihomo-γ-linolenic acid, and arachidonic acid (AA). The therapeutic value of PUFAs in relation to the prevention of cardiovascular and coronary heart diseases, blood platelet aggregation, cancer, type 2 diabetes, inflammatory bowel disorders, asthma, arthritis, kidney and skin disorders, depression, and schizophrenia has been confirmed. When the diet does not contain the daily requirement of bioactive fatty acids, supplementation is recommended. Tissues (from brain, adrenals, or reproductive organs) of mammals (pork, beef, bovine, and ox) and fish (cod, menhaden, herring, tuna, anchovy, and sardine) are used for PUFAs production. n-3 PUFAs used in functional foods, nutraceuticals, and pharmaceuticals are principally obtained from fish. PUFAs from fish oils can be affected by the presence of cholesterol or may have unpleasant odor, and they may be contaminated by heavy metals. On the other hand, they may present a complex fatty acid profile (with up to 50 different fatty acids), which are difficult to purify. In contrast, microalgae are a renewable source of bioactive lipids having a high proportion of PUFAs, with a simpler fatty acid profile than fish oil. The PUFA content of algae depends on the species and on operational variables (including composition of the medium, aeration rate, light intensity, duration of the photoperiod, temperature, and culture age). Representative PUFA-producing organisms are microalgae (Chlorella minutissima, Isochrysis galbana, Phaeodactylum tricornutum, Porphyridium cruentum, and Monodus subterraneus) and fungi (including Mortierella alpine and Pythium irregulare). Although microalgae have several advantages over fish oils, their production cost is high. The recovery, concentration, and purification of PUFAs from microalgae have been extensively revised (Robles Medina et al., 1998). The cultivated microalgae biomass is recovered from the culture broth by filtration or centrifugation, and processed to obtain highly pure PUFAs in a fast and efficient way to avoid product degradation. PUFAs are easily attacked by oxygen, in a process accelerated by light, heat, oxygen,

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moisture, and the presence of catalysts such as metal ions. Any exposure of PUFAs to these agents must be minimized during processing and storage. Solvent extraction is usually employed to recover PUFAs from the microalgal biomass, and the recovery of highly pure PUFAs is a multistep process due to the complexity of the mixtures. The furthest developed separation techniques include the formation of urea inclusion compounds, low-temperature fractional crystallization, salt solubility, gas and liquid chromatography, thin-layer chromatography, and liquid–liquid fractionation. However, not all these methods are suitable for implementation at an industrial scale. A thorough revision of the extraction of fatty acids was published by Robles Medina et al. (1998). The extraction and separation of PUFAs by SCF looks quite promising, since the extraction with CO2 is carried out at moderate temperatures (thus limiting auto-oxidation, decomposition, and/or polymerization), and the inert CO2 atmosphere excludes oxygen. The use of SC-CO2 to extract lipids (particularly, arachidonic acid) from crushed Mortierella sp. cells has been claimed (Totani and Oba, 1988). Koerts et al. (1995) proposed the utilization of SC-CO2 to extract fatty acids and fatty acid alkyl esters, whereas sugars and salts remained unextracted. 23.3.3.3.2 γ-Linolenic Acid GLA has an important role in human metabolism, particularly as a precursor of one kind of prostaglandin. It has been used in several medical applications, such as the treatment of schizophrenia, multiple sclerosis, dermatitis, premenstrual syndrome, atopic eczema, diabetes, and rheumatoid arthritis (Fan and Chapkin, 1998; Kapoor and Huang, 2006). Lipids extracted from the cyanobacteria Arthrospira (Spirulina) maxima contain large amounts of GLA. Several Spirulina species have been processed by SC-CO2E. The effects of pressure on extraction are different for oleic acid than for linoleic and linolenic acids, and the GLA yields increased with pressure up to 35 MPa (Hu, 1999). Using ethanol-modified CO2 at constant temperature, the GLA yield increased with pressure, whereas operating at a constant pressure, the yield increased slightly with temperature (Mendes et al., 2006; Sajilata et al., 2008). A quantitative recovery of GLA from Spirulina platensis was attained at 400 bar, using ethanol as entrainer (Sajilata et al., 2008). Although SC-CO2E of lipids from Arthrospira maxima resulted in a lower GLA yield and decreased GLA content in comparison with acetone, ethanol, or mixtures of conventional solvents (chloroform, methanol, and water), the use of ethanol-modified CO2 increased the yield of lipids and GLA, as well as the GLA selectivity compared to the values obtained with acetone. The modification of extractant polarity resulted in different distributions of lipids: with pure CO2, 70% of the extracted GLA was in the neutral lipid fraction, whereas with ethanol-modified CO2, 73% was in the glycolipid fraction (Mendes et al., 2006). In the presence of a modifier, selectivity increased with temperature (in the range 50–60°C) and pressure (in the range 250–350 bar) (Mendes et al., 2006). Temperatures of 50°C or lower were desirable in order to avoid excessive coextraction of triglycerides. The pressure enabling a maximal yield of fatty acids and carotenoids at minimum extraction time was 150 bar. The fatty acids extracted from Spirulina maxima showed a constant profile during the entire extraction process, GLA being the most abundant fatty acid (Canela et al., 2002). 23.3.3.3.3 Eicosapentaenoic Acid EPA shows beneficial effects, including the prevention of arthritis and cardiovascular disease. A possible source of this fatty acid is filamentous fungi and microalgae. The recovery of lipids from Saprolegnia parasitica increased with pressure, and when ethanol (10 wt%) was used as a modifier, the recovery increased 2-fold compared to operating with pure CO2 (Cygnarowicz-Provost et al., 1992). Certain species such as Chlorella minutissima, Nannochlorosis sp., and Monodus subterraneus are high in EPA and low in other PUFAs, a fact facilitating downstream purification (Wen and Chen, 2003). No particular differences were found in the fatty acid profiles of SCFE products of Nannochloropsis compared to those obtained using n-hexane. Moreover, a slight increase in EPA and DPA (with a corresponding decrease in C16:0, C16:1, C18:0, and C18:1) was

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reported when going from the mildest to the severest SCFE conditions. Under all conditions, SC-CO2E was faster than extraction with hexane, although both led to comparable yields and fatty acid composition of extracts (Andrich et al., 2005). 23.3.3.4 Extraction of Other Metabolites Wood et al. (2000) claimed the utilization of SC-CO2 or other SC fluids to extract meat flavor, produced by the reaction between the free amino groups of amino acids and/or peptides with sugars. This process is based on the solid state fermentation of a mixture containing sources of proteins and carbohydrates of vegetal origin, inoculated with microbial species traditionally used in the preparation of fermented cooked meat products. The meat flavor is extracted from the fermented mixture. The antioxidant properties of SC-CO2 extracts from the microbial biomass have recently been pointed out. The antioxidant activity of Spirulina platensis extracts (containing flavonoids, β-carotene, vitamin A, and α-tocopherol) was characterized in relation to synthetic antioxidants and α-tocopherol (Wang and Weller, 2006). The radical scavenging activity of Chlorella pyrenoidosa extracts was significantly increased by ethanol, suggesting that polar compounds were responsible for the antioxidant activity. Pressure also showed a positive effect, but prolonged extraction periods increased the coextraction of nonactive components (Hu, 1999). Compounds with antioxidant activity (such as glutathione, thioredoxin, and rubredoxin) are naturally present in some microorganisms or could be produced by genetic manipulation (Hugenholtz and Smid, 2002). 23.3.3.4.1 Vanillin Vanilla is one the most common flavoring compounds used worldwide, with applications in foods, pharmaceuticals, and cosmetics; it is used in some indigenous cultures for its therapeutic properties. The limited supply and high cost of the natural source (vanilla pods) has fostered both chemical and biotechnological processes (Priefert et al., 2001). Vanilla contains more than 200 components, but the aroma and flavor of vanilla extract is attributed mainly to the presence of vanillin (4-hydroxy-3methoxybenzaldehyde), although the antioxidant properties (in emulsion and as a radical scavenger) were higher for 4-hydroxy-3-methoxybenzyl alcohol and 4-hydroxybenzyl alcohol (Shyamala et al., 2007). SC-CO2 extraction has been applied successfully to the extraction of vanillin (Nguyen et al., 1991). However, the ratio of the major constituents of vanilla flavor (vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid) differed from that of products obtained by alcoholic extraction (Sinha et al., 2008). 23.3.3.4.2 Prebiotics SC-CO2 has recently been applied to the separation of oligosaccharide mixtures produced by chemical methods (Montañés et al., 2008), but this technology could also be relevant for those produced biotechnologically (Ahmed, 2001). The solubility of carbohydrates in SC-CO2 increases in the presence of a suitable cosolvent. The successful and selective extraction of tagatose or lactulose from complex commercial solid carbohydrates (containing tagatose–galactose and lactulose–lactose mixtures) has been possible under selected extraction conditions (temperature, pressure, and type and amount of cosolvent). A two-step process has been proposed for the purification of mixtures with a high monosaccharide content: in an initial step, the extraction of monosaccharides could be achieved before lactulose separation (Montañés et al., 2008). Other prebiotics (such as fructans, homopolysaccharides produced by bacteria and fungi) could be target products in the future (Hugenholtz and Smid, 2002). 23.3.3.4.3 Flavonoids and Other Phenylpropanoid-Derived Natural Products A large number of secondary plant metabolites can be produced by genetically modified microorganisms. In particular, Saccharomyces cerevisiae has an enormous potential for producing several of the most important secondary metabolites in nature (Huang et al., 2008). Flavonoids represent

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a large and diverse group of natural plant products, with long-term beneficial effects on human health. However, the productivity of flavonoids and stilbenoids is limited by their low concentrations and by the limited growth rates of plants. Exploitation of the structural genes encoding enzymes involved in phenylpropanoid metabolism (including flavonoid and stilbenoid), has allowed the production of flavanones, naringenin, pinocembrin, or resveratrol glycosides. Among isoprenoids, a large and structurally diverse group with biological roles in plants, monoterpenols (geraniol and linalool), sesquiterpenes, and cytochrome P450 could be produced. The biosynthesis of vitamin C (l-ascorbic acid, a water-soluble potent antioxidant) from d-glucose by metabolically engineered yeasts may have a substantial impact on its biotechnological production (Branduardi et al., 2007). In future, the importance of this application field is likely to increase, due to the improved knowledge of biosynthetic pathways and genetic engineering.

23.3.4

REMOVAL OF UNDESIRABLE COMPOUNDS

SC-CO2 may remove undesirable compounds from the biomass or from a conventional solvent extract. The removal of volatile off-flavor compounds from the ruptured cell suspension after the CO2 release and deodorization of particular proteins by hydrostatic pressurization has been proposed by Lin (1991). Spirulina biomass (rich in protein, GLA, β-carotene, and polysaccharides) presents a particular stench smell which is a major obstacle to the acceptance of the product. SC-CO2 extraction has been proposed to separate and purify the active component of Spirulina and to remove its stench smell. The quality of Spirulina was preserved, since the reductions in lipids and total amino acids were 2.32% and 3.12%, and the content of the essential amino acids did not decrease significantly (Hu, 1999). SC-CO2E was used to remove bitterness (caused by α-acids) from powdered brewing yeast (operating at 25–45°C and 1300–2500 psi) (Choi et al., 2001) or from a concentrated and pulverized yeast extract (Yasuma et al., 1989). SCFE has also been proposed to purify crude extracts obtained with conventional solvents. The beneficial effects of reducing odor and enhancing the coloring properties and light resistance of extracts from dried yeast Phaffia sp. have been claimed (Nobuhisa and Toru, 1991).

23.4 INACTIVATION OF MICROORGANISMS AND ENZYMES BY DENSE CO2 The inactivation of the microbial biomass is relevant for the manufacture of sterilized and microbiologically safe nutraceutical products, as well in the cases where the biomass produces part of the product. Both the intracellular components described in the previous section and fractions of the cell walls (i.e., yeast glucan) are potential nutraceutical supplements. Inactivation can be either simultaneous or subsequent to extraction. Lactic acid bacteria (LAB) are of particular interest, as they are considered as “cell factories” for the production of important nutraceuticals. Several examples have been reported of the production of certain nutraceuticals, newly induced through the overexpression and/or disruption of relevant metabolic genes (Hugenholtz and Smid, 2002). Thermal sterilization is the most common conventional process for microorganism inactivation in the food and pharmaceutical industries. However, at the high temperatures required, degradation of thermosensitive compounds (vitamins and proteins) and the production of toxic metabolites can occur, affecting the nutritive value and organoleptic properties of the product. A variety of alternative sterilization techniques (based on physical and chemical methods) have been developed for extending shelf life while retaining the original flavor and nutritional properties. Conventional, nonthermal treatments include dehydration, cold preservation, and fermentation. Nonthermal treatments have gained importance as potentially valuable technologies to replace (or at least to complement) the traditional processes for microbial inactivation, avoiding drawbacks such as the denaturation of nutrients, the production of side reactions leading to toxic products, and the alteration of properties. Novel nonthermal processes include high hydrostatic pressure, pulsed high electric fields, intense light pulses, irradiation, oscillating magnetic fields, ohmic heating by superheated

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steam, ultrafiltration, SC-CO2, antimicrobials, or combinations of these methods. The implementation at an industrial scale of nonthermal technologies is limited by high capital and operating costs, low effectiveness in actual operation, and a lack of specific regulations on product safety (GarcíaGonzález et al., 2007). High-pressure treatments are attractive for microorganism inactivation in foodstuffs, since they are environmentally friendly and nontoxic chemicals are used. High-hydrostatic pressure treatment (HPT) is the most studied process, but a major limitation to its wider industrial use is the control of the extremely high pressure employed. Pressurized CO2 has become a more economical alternative to HPT, since it allows operation under milder temperatures and pressures, and can be controlled and managed more easily. However, it has not yet been implemented in the food industry. Whereas HPT operates at 2000–7000 bar, food preservation by SC-CO2 can be achieved at pressures lower than 200 bar. As an example, comparable results with Escherichia coli could be obtained after 1 min by HPT (operating at 275 MPa and 35°C) and SC-CO2 (operating at 4 MPa and 35°C) (Watanabe et al., 2007). CO2 plays an important physiological role in microorganisms, stimulating or inhibiting growth and reproduction, and it is known for its bacteriostatic action and inhibitory effects. Microbial inactivation by dense CO2 has been known about since the 1950s, when it was presented as a very simple, rapid, and inexpensive method consisting of the sudden release of gas pressure (Fraser, 1951). A systematic study of this effect was addressed by Kamihira et al. (1987), and there is a growing scientific and economic interest in its practical application. The antimicrobial action of compressed CO2 comes from its physical and chemical effects. The higher efficiency of compressed CO2 under supercritical rather than subcritical conditions has been ascribed to both microscopic and macroscopic effects. These are related to density, as the major viability drop occurs as the phase change occurs (Isenschmid et al., 1995). SC-CO2 is more effective than both gaseous and liquid CO2 for inactivating microbial cells, since the mass transport properties (viscosity and diffusivity) of SC-CO2 are closer to those of gases, and its low surface tension facilitates penetration into microporous materials (Ishikawa et al., 1995; García-González et al., 2007). SC-CO2 is bactericidal, whereas vapor CO2 causes a bacteriostatic effect (Oule et al., 2006). Under appropriate conditions, the survival ratio of bacteria, molds, and yeast can be dramatically reduced by pressurized CO2, as summarized in several comprehensive compilations dealing with vegetative microbial forms (Spilimbergo et al., 2003; Zhang et al., 2006; García-González et al., 2007). Since the dense CO2 technology used in different studies is not standardized, the lethal effects depend on the equipment and the operational conditions, and comparisons should be interpreted with care (Gunes et al., 2006). Microorganisms differ in their resistance to the inhibitory effect of CO2, and different conditions can be required for inactivation. These differences could be related to factors such as membrane composition, CO2 permeability, type of microorganism, phase of growth, and type of medium (Isenschmid et al., 1995; Debas-Louka et al., 1999; Wu et al., 2007). A knowledge of the cellular biology and physiology of some microorganisms facilitates the understanding of inactivation mechanisms by high-pressure CO2. The most common microorganisms employed in literature studies are Escherichia coli (Kamihira et al., 1987; Isenschmid et al., 1995; Ballestra et al., 1996; Dillow et al., 1999; Erkmen, 2001; Kincal et al., 2005; Gunes et al., 2006; Kim et al., 2007a) and LAB. These latter are acid-tolerant fermentative microorganisms, viable over a wide pH range in the presence of organic acids and high CO2 concentration (Hong et al., 1999; Hong and Pyun, 1999). High-pressure CO2 is suitable for inactivating microorganisms at room temperature, with the exception of bacteria with spores. Bacteria species produce spores in the stationary phase of their life cycle under adverse conditions. Spores are the most resistant forms of bacteria and survive physical and chemical treatments, requiring a higher temperature and longer contact in comparison to other microorganisms. The two-layer membrane of spores is less fluid than that of the vegetative forms. Spores are considered dehydrated forms, since the bound and free water content are about 65% and 74% of the respective values found in the vegetative forms (Ishihara et al., 1999). It has been suggested that the cells can transform into spores resistant to the effect of CO2 under certain © 2010 Taylor and Francis Group, LLC

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conditions (Spilimbergo et al., 2003; Wu et al., 2007). Literature addressing spore inactivation by SC-CO2 is scarce, and the data are contradictory (Spilimbergo et al., 2003). The individual effect of CO2 pressure in a single batch was not sufficient to promote spore inactivation, which required the coupled action of CO2 with a higher temperature, longer treatment time, and/or the addition of ethanol or acetic acid (Spilimbergo et al., 2002). A different contact system such as the microbubble method (Ishikawa et al., 1997) can be suitable for spore inactivation. In some cases, relatively high temperatures (above 60°C) are efficient, but are not practical for industrial applications. Nonsignificant inactivation of Bacillus subtilis was achieved at a higher temperature, longer treatment time, or higher operating pressure even in the presence of peptone (an efficient antiaggregant, which avoids the formation of spore-clusters complicating CO2 penetration inside their structure) (Spilimbergo et al., 2002). The use of cyclical pressure variations at physiological temperature appears promising, probably because the sudden change in pressure induce germination yet does not allow the spores to restore their latent status before the next cycle. Most of them can be activated cycle after cycle, becoming sensitive to CO2 penetration inside their structure (Spilimbergo et al., 2002). Bacillus subtilis and Pseudomonas aeruginosa spores could be partially inactivated by a fast CO2 cyclic treatment (30 cycles/h, 80 bar, 30 min, 35°C). The coupled effect of CO2 together with pulsed electric field (PEF) showed interesting results against spores of Bacillus cereus at 40°C, a particularly mild temperature (Spilimbergo et al., 2003).

23.4.1

FUNDAMENTALS AND MECHANISM OF INACTIVATION

Most authors support the idea that the major effect causing microbial inactivation is the ability of SC-CO2 to diffuse readily into the cell, altering the cytoplasmic pH. Under pressure, it is possible that CO2 molecules can penetrate into cells at a much faster rate than other fluids, which do not produce acidification in solution, and can dramatically increase its permeability. Further diffusion of CO2 through the membrane, favored by pressure and cell injury, would lead to a decrease of the internal pH, exceeding the buffer capacity of the cytoplasm. The key factor is considered to be the concerted effects of intracellular acidification (due to CO2 penetration) and damage to cell membranes (with subsequent leakage of intracellular substances), leading to a disturbance of biological processes within the cells by inhibiting certain enzymes and metabolic reactions. It has also been suggested that inactivation could occur when the CO2 pressure within the cell is released and bicarbonate converts to carbonate, which is able to precipitate intracellular calcium, magnesium, and similar ions from the cell and cell membrane, causing lethal changes to the biological system. 23.4.1.1 Mechanism Various hypotheses have been proposed to explain the microbicidal activity of CO2, but there is no definite evidence about the precise inactivation mechanism. Spilimbergo et al. (2005) discussed which mechanism could be considered as primary or complementary, and proposed a series of disturbances of microbial metabolism during cell inactivation. For vegetative forms, the rate limiting step of inactivation could be governed by CO2 dissolution inside the cell (since its solubility in water is relatively low, conversely to lipids). Zhang et al.(2006) summarized all the mechanisms proposed and classified them in two groups: those based on cell bursting at the depressurization stage, and those based on physiological deactivation (including decrease of intracellular pH, enzyme deactivation, extraction of cell content, and bursting). Related information on this topic has been reported by Spilimbergo et al. (2005) and García-González et al. (2007). According to the above ideas, the major stages with incidence on inactivation are • Solubilization of pressurized CO2 and acidification of the suspended medium • Chemical modification of the lipid double-layer of the membrane, increasing its permeability, and penetration of CO2 into the cell membrane • Irreversible acidification of cytoplasm

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• Inactivation of key enzymes of the microbial metabolism • Interference with the intracellular electrolyte balance • Removal of vital constituents from cells and cell membranes 23.4.1.1.1 Acidification The water solubility of CO2 depends on pressure and temperature. CO2 dissolves in aqueous solutions to form carbonic acid, helping decrease the pH of the medium. This can inhibit cell metabolism and attenuate the microorganism resistance to inactivation, but can not account for the antimicrobial action of CO2, which shows a greater inhibitory effect than other acids (hydrochloric acid, phosphoric acid, etc.) that hardly penetrate the cells. The pH of the cell suspension during the treatment is not directly related to the lethal effect, since viable cells can resist low environmental pH (particularly, LAB, due to their ability for pH regulation). Although microorganisms can live in low pH extracellular environments, the internal (cytoplasmatic) pH has a greater effect on cellular activity. Its regulation is fundamental for cellular processes (such as ATP synthesis, RNA and protein synthesis, DNA replication, and cell growth), and plays an important role in the transport of sugars, amino acids, and peptides, as well as in proton translocation. When the difference between the intra- and extracellular pH approaches to zero, the cell is unable to restore a favorable cytoplasmatic environment, resulting in viability loss (Spilimbergo et al., 2005). In a study of Bacillus subtilis, Spilimbergo et al. (2005) considered the effect of SC-CO2 (80 bar, 303 K) on the intra- and extracellular pH. The intracellular pH depletion (down to 3) inside the cell caused by the action of CO2 correlated with the inactivation ratio, as confirmed by cytometry analysis. The extracellular pH, strongly influenced by the amount of CO2 dissolved, was acidified below 3. The value of the extracellular pH was very close to that of the intracellular pH, suggesting that under these conditions the microbial cells were unable to maintain cytoplasmatic pH homeostasis (Spilimbergo et al., 2002). The inactivation in aqueous solution under pressurized CO2 was a little more effective in the presence of NaHCO3, although this alkaline buffer kept the pH above 7. The reason is that CO2 can penetrate cells more quickly in an aqueous solution of NaHCO3 (where HCO3− and CO32− already exist, and further dissociation is not necessary). This behavior confirms that the diffusion rate of CO2 into cells is the determining step in the antimicrobial effect of pressurized CO2 (Wu et al., 2007). The mechanical impact of high pressure is lethal to cells. For example, sterilization by ultrahigh HPT at 100–300 MPa can unfold and inactivate proteins and induce a phase transition of the cytoplasmatic membrane to the gel phase, with a leakage of sodium and calcium ions and increased permeability. Pressures greater than 300 MPa induce an irreversible denaturation of proteins. However, the pressure used for microbial inactivation by SC-CO2 is much lower and the mechanism should be different. Pressurization with N2 was used in several studies as a control to distinguish the specific interaction between SC-CO2 and the cell. Microbial inactivation was not achieved at the same temperature and pressure. Little reduction of viable cells is caused by the poor solubility of N2 gas in aqueous solution (Wu et al., 2007; Nakamura et al., 1994). Nitrogen at the same pressure, time, and temperature used in SC-CO2 processing (137 atm, 35°C, 2 h) neither reduced the microbial counts of Listeria and Salmonella sp. nor affected pH (Wei et al., 1991). Environmental acidification created by a acidic buffer (7 MPa, 30°C) had no lethal effect on cells exposed to high N2 pressure (Hong and Pyun, 1999, 2001), whereas pressurization with a mixture of N2 and O2 (80/20%) at 8.5 MPa was not suitable for reducing the viable cells of Escherichia coli, Saccharomyces cerevisiae, and Enterococcus faecalis (Debas-Louka et al., 1999). Similarly, when nitrogen was used instead of CO2 with initial pH 3.3 at the same temperature and pressure (near 7.8 MPa), more than 80% of Escherichia coli cells survived (Wu et al., 2007). Nitrogen experiments did not result in a sterilizing effect on this microorganism (Dillow et al., 1999). The lethal effects caused by treatments with CO2 and N2O (relatively soluble in water), were not achieved with N2 or Ar (40 atm, 40°C, 4 h), suggesting that cell death may be highly correlated with gas absorption by the cells.

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23.4.1.1.2 Modification of Cell Membrane and Extraction of Cell Wall Lipids The cell membrane consists of a double layer of phospholipids, the inner one having a lipophilic character. The unique properties of the lipo- and hydrophilicity of CO2 could facilitate dissolution in the phospholipids, and thus membrane penetration. Accumulation of CO2 in the lipid phase may cause structural and functional disorders in the cell membrane. SC-CO2 treatment can alter the total cellular lipid content; for example, the quantity of fatty acids from Listeria monocytogenes gradually decreased with the treatment time, in a nonselective process (Kim et al., 2008). This step, highly dependent on temperature, increases the fluidity and hence the permeability of the membrane. Alteration of the optimal surface charge density and membrane function is known as “anesthesia” (Isenschmid et al., 1995). Electronic microscopy has confirmed the hypothesis that the mechanism does not involve cell rupture caused by increased internal pressure: both cell walls and external cell shape remain unchanged, but leakage of cytoplasm occurs (Hong and Pyun, 1999). On the other hand, it was suggested that the damage of the initial cellular envelope is not lethal for cells, since it starts after about 2 min of treatment (when the rate of cell survival is still 100%), and increases with longer treatment times (Bertoloni et al., 2006). Explosive decompression and cell rupture was initially proposed as a cause of microbial inactivation. It was suggested that after a pressurization stage causing penetration of CO2 gas into the cells, a sudden release of the pressure (“flash decompression”) caused a rapid gas expansion within the cells. As a consequence, most of them could be mechanically ruptured due to the resulting unbalanced pressure difference. Influence of SC-CO2 on the activity of key enzymes for cell metabolism: Under pressure, CO2 passes through the membrane and lowers the internal pH by exceeding the buffer capacity of the cytoplasm. This internal acidification could cause the selective precipitation of enzymes with an acidic isoelectric point. The inactivation of the enzymes involved in metabolic processes leads to the collapse of the cell. It has been suggested that both the molecular CO2 and the bicarbonate formed can interfere with key enzymatic and biochemical pathways. Loss of enzymatic activity in SC-CO2-treated cells was observed in relation to either the release of cellular enzymes into the cell suspension or the hydrolysis and inactivation of enzymes (Ballestra et al., 1996; Hong and Pyun 2001; Bertoloni et al., 2006). Another possible cause of cell inactivation by CO2 is the bicarbonate conversion to carbonate when the CO2 pressure within the cell is released, and intracellular precipitation of calcium and magnesium ions occurs (Lin et al., 1993). Certain types of calcium- and magnesium-sensitive proteins may be parts of the essential proteins involved in intracellular regulation. Using Escherichia coli cells, chromosomal DNA became complexed with coagulated cytoplasmic proteins caused by acidification of the cytoplasm, and could not be recovered, suggesting that acidification could be the major inactivation mechanism (Watanabe et al., 2007). Different effects of SC-CO2 on the intracellular enzymes of some microorganisms have been reported. Treatment at 70 bar and 30°C reduced the viability of Lactobacillus plantarum cells by several log cycles and inactivated some enzymes (cystine arylamidase, α-galactosidase, α- and β-glucosidase, and N-acetyl-β-glucosaminidase), whereas ATPase of the cell membrane was not affected (Hong and Pyun 2001). The different behavior of enzymes was also reported for alkaline phosphatase (10–57%), acid phosphatase (70%), and ATPase (5%) from Escherichia coli (Bertoloni et al., 2006). A decreased activity of alkaline phosphatase, leucine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, β-glucuronidase and α-glucosidase in Escherichia coli and Escherichia coli O157:H7 was found (Kim et al., 2007a). Other enzymes (lipase, leucine arylamidase, β-galactosidase, phosphatase (acid and alkaline) or naphthol AS-BI-phosphohydrolase) were not affected. The most pronounced effect caused on the treatment of Listeria monocytogenes cells was probably due to the direct inactivation of the enzymes, including esterase, acid phosphatase, and β-glucosidase. Either increased or no reduction of activity was found for alkaline phosphatase, esterase lipase, and naphtol-AS-BI-phosphogydrolase (Kim et al., 2008). The initial increase in

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enzymatic activity might result from a release into the suspension through the damaged cell walls (Hong and Pyun, 2001; Bertoloni et al., 2006; Kim et al., 2008). 23.4.1.1.3 Extraction of Intracellular Substances CO2 concentrations within the cells above a given threshold results in leakage of some vital constituents, such as phospholipids, nucleic acid, proteins, amino acids, and lipids, due to the increased permeability of the cell wall (Lin et al., 1992; Kim et al., 2007a). Leakage of UV-absorbing (260– 280 nm) substances from the cytoplasm of Escherichia coli increases with SC-CO2 treatment time (Kim et al., 2007a). Cell membrane injury would not be the primary cause of cell death, since severe membrane damage of yeast cells required a much longer duration under elevated pressured CO2. Wu et al. (2007) observed that both Escherichia coli cell leakage and cell inactivation occurred during the earlier treatment phase (20 min), suggesting that inactivation could be due to the injury of the cell membrane. The level of protein leakage was very low during the initial hours, suggesting that the injury to the membrane was not serious. Increased values reached with prolonged treatment times could be the result of membrane disruption and rupture (Wu et al., 2007). 23.4.1.1.4 Kinetics Inactivation of microbial cells in two phases was observed (Lin et al., 1994; Ballestra et al., 1996; Hong et al., 1997; Erkmen, 2001, 2003; Spilimbergo and Mantoan, 2005). The rate of inactivation progressed slowly in the initial (“lag”) phase, whereas in the second (“death”) phase it increased sharply with first-order kinetics. The duration of the first stage and the inactivation rate of the second stage were sensitive to pressure. The dissolution of CO2 into the aqueous solution and then its diffusion into cells controlled the duration and inactivation rate of the early stage, which was a limiting step. Once the CO2 concentration inside cells reached a critical level, inactivation proceeded at a high rate (Wu et al., 2007). Each strain presented a characteristic curve defining its resistance (Debas-Louka et al., 1999). Microbial death caused by pressurized CO2 has been assessed by firstorder kinetics, for example, in the case of Salmonella typhimurium (Erkmen and Karaman, 2001), Listeria monocytogenes (Erkmen, 2000a), Saccharomyces cerevisiae (Kumagai et al., 1997; DebasLouka et al., 1999; Shimoda et al., 2001; Spilimbergo and Mantoan, 2005), Escherichia coli (Ballestra et al., 1996; Debas-Louka et al., 1999, 2005; Kim et al., 2007a), and Enterococcus faecalis (Debas-Louka et al., 1999). Most studies showed a slow stage followed by a fast one, but a fast initial rate followed by a slow deactivation stage has also been observed. It was suggested that when the pressure increases, the kinetic behavior of the system changed from “slow to fast” to “fast to slow.” This pattern has been ascribed to differing efficiencies of the contact between CO2 and microorganisms. The kinetic curves also showed a “fast to slow” behavior in experiments employing: (i) cells deposited on filter paper and covered only by a thin liquid (Debas-Louka et al., 1999), (ii) microbubbles to increase contact area between CO2 and liquid media, and contact between CO2 and the microorganism (Ishikawa et al., 1997), and (iii) high pressure, which facilitated gas transfer through the media and increased the contact between CO2 and cells (Zhang et al., 2006; Hong and Pyun, 1999). However, at a lower pressure (1.2–10 MPa), the behavior observed was “slow to fast” kinetics. The modified Gompertz model for the microbial inactivation curve includes three stages: the slower first stage (“lag” phase or “shoulder”), the second stage (exponential inactivation), and the stationary phase (i.e., a “tail” or “resistant” phase) (Erkmen, 2001, 2003; Kim et al., 2007a, 2007b). Assessment by a stochastic model was proposed by Spilimbergo and Mantoan (2005). Explanations for the lag phase include: the presence of clumps of microorganisms in the suspension (all cells in the clump should be inactivated before the colony-forming ability of the clump is lost); the ability to resynthesize a vital component (death would happen only when the rate of destruction exceeds the rate of resynthesis); and the cumulative feature of microorganism inactivation (i.e., multiple targets must be hit before inactivation can be reached). A need has been stated for further research in

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relation to mechanistic studies and mathematical modeling of the inactivation processes with SC-CO2 (Zhang et al., 2006).

23.4.2

TREATMENT EQUIPMENT AND OPERATION

Most inactivation studies have been performed using batch or multibatch equipment, in laboratory or pilot reactors (Spilimbergo and Mantoan, 2005). A typical batch apparatus at the laboratory scale consists of a CO2 pump, a high-pressure vessel, and a discharge system to release pressure. The high-pressure vessel is usually constructed of stainless steel with a volume of 10–5000 mL, which can be stirred (e.g., with a magnetic device) (Kamihira et al., 1987; Lin et al., 1992, 1993, 1994; Isenschmid et al., 1995; Ballestra et al., 1996; Debas-Louka et al., 1999; Hong and Pyun, 1999; Erkmen, 2001, 2007; Erkmen and Karaman, 2001; Spilimbergo et al., 2002; Wu et al., 2007; Kim et al., 2007a). A filter may be placed before the outlet valve to prevent any loss of media and contamination of lines via back-diffusion during pressurization/depressurization steps. Temperature and pressure should be monitored during operation, and automated operation is preferred. The operations using this apparatus include the following steps: (i) introducing the cell suspension into the high-pressure cell; (ii) tightly closing the vessel and switching the temperature control until the target value is reached; (iii) pressurization by pumping CO2 at the target pressure; (iv) keeping the system under the selected conditions for the desired time; and (v) depressurizing the cell and removing the treated material. After each run, the system must be cleaned (e.g., with sterile distilled water and 70% ethanol). Continuous processes have been designed and constructed on a laboratory or industrial scale (Shimoda et al., 2001, 1998). Liquid CO2 and a saline solution are simultaneously pumped through a CO2 dissolving vessel. Liquid CO2 is brought to a supercritical state, and dispersed into the aqueous saline solution from a 10 μm mesh filter placed in the bottom of the vessel. Microorganisms are introduced into the stream of saline solution, and the suspension circulated through the residence coil (4–10 MPa, 30–38°C), and sent to a pressure control valve (Erkmen, 2000b). It has been suggested that the inactivation mechanism is different in batch and continuous operation (Shimoda et al., 1998). In continuous operation, microbial inactivation is influenced by CO2 concentration, which is determined by the CO2 flow rate. Complete inactivation is caused by cell bursting induced by the explosive expansion of the CO2 contained in cells. Continuous technology has been used successfully to inactivate Escherichia coli, Saccharomyces cerevisiae, and Torulopsis versatilis. It has been suggested that in batch treatments, characterized by a gentle CO2 expansion, cell death is mainly due to the anesthesia effect and inactivation of enzymes, rather than to cell bursting. However, in a continuous treatment, explosive expansion of CO2 may cause cell bursting (Shimoda et al., 2001, 1998). The profile of the survival curve is different: whereas in batch operation the survival ratio decreases markedly for densities about 0.8 g CO2/ cm3 or higher, in the continuous method the microorganisms are inactivated at densities of 0.16– 0.9 g CO2/cm3 (Shimoda et al., 1998). Significant differences in inhibitory activity can be achieved by changing the concentration of dissolved CO2 in the cell suspension. Advanced designs include devices to increase mass transfer (i.e., microbubbling of pressurized CO2 to facilitate the CO2 dissolution in the sample and to enable an effective inactivation of microorganisms) (Ishikawa et al., 1995, 2000; Shimoda et al., 1998). Lactobacillus brevis, Saccharomyces cerevisiae, Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus polymyxa, and Bacillus coagulans were more efficiently sterilized by the microbubble method than by conventional treatment (Ishikawa et al., 2000). Other modifications have been revised by Spilimbergo et al. (2003). Equipment design for continuous processing is an important subject of patenting. Inventions related to sterilization by this method have been revised recently (Zhang et al., 2006; GarcíaGonzález et al., 2007).

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Cell disruption involves a sudden release of pressure and expansion of CO2 within the microbial cells, causing the breakage of microorganisms. As the extracted biomaterials of nonvolatile character are retained in the vessel, the solid and liquid phases can be separated by centrifugation. Protein and enzymes can be recovered from the supernatant (Lin, 1991). Supercritical and near-critical fluids can be used to fractionate the microbial biomass in two stages: in a first step, the biomass is exposed to the dense fluid to cause cell disruption, enabling the release of structural biomass constituents, whereas in the second one, the disrupted biomass can be subjected to one or more supercritical or near-critical fluid extraction stages.

23.4.3

INACTIVATION AND DISRUPTION OF MICROBIAL CELLS UNDER DENSE CO2

23.4.3.1 Operational Conditions Affecting the Inactivation and Disruption of Microbial Cells under Dense CO2 The inactivation effectiveness of high-pressure CO2 treatments of cell suspensions is affected by several factors, including the type of medium, cell moisture, pressure, temperature, entrainers and treatment time, number of cells, and cell cultivation history (Erkmen, 1997, 2000a, 2000b, 2000c, 2000d, 2001; Wu et al., 2007; Kim et al., 2008). Among the above parameters, increased temperature, pressure, and moisture increase the effectiveness of treatments by increasing the CO2 diffusivity. The effect of different variables has been summarized in recent revisions (Spilimbergo et al., 2003; Zhang et al., 2006; García-González et al., 2007). 23.4.3.1.1 Pressure Microbial inactivation under high CO2 pressure is governed by penetration into cells, and it is improved by the enhanced transfer rate (Lin et al., 1994). Pressure is a key variable since it controls both the CO2 solubilization rate into the aqueous solution and the diffusion into cells through the cellular membrane (Spilimbergo and Bertucco, 1994). The pressure usually employed is under 20 MPa. Effective inactivation has been observed at pressures under 10 MPa, and critical conditions were also effective for defi ned cases. In studies dealing with Escherichia coli inactivation, higher pressures allowed shorter exposure times to achieve the same effects (Wu et al., 2007). A clear dependence between the inactivation ratio and pressure increase was observed for Saccharomyces cerevisiae (Isenschmid et al., 1995; Erkmen, 2003; Spilimbergo and Mantoan, 2005; Shimoda et al., 2001), Kluyveromyces fragilis (Isenschmid et al., 1995), Candida utilis (Isenschmid et al., 1995), Lactobacillus sp. (Hong et al., 1997; Hong and Pyun, 2001), Listeria monocytogenes (Erkmen, 2001; Kim et al., 2008), and Salmonella typhimurium (Erkmen and Karaman, 2001). The unsteady period needed to reach the target pressure exerted an important effect on the survival of Salmonella typhimurium (Erkmen and Karaman, 2001). The behavior during inactivation was strain dependent: Escherichia coli had the least pressure resistance, whereas only a slight effect on Enterococcus faecalis cells was reported (Debas-Louka et al., 1999). Similarly, no effect of temperature and pressure on Escherichia coli was observed at 25–45°C and 7–48.3 MPa, due to the high sensitivity of these cells to dense CO2 (Zhang et al., 2006), for which optimal conditions corresponded to 7.8 MPa, a relatively low pressure (Wu et al., 2007). Trichoderma cells were scarcely resistant to SC-CO2, as relatively low severity conditions (100 bar, 40°C, 15 min) were sufficient for cell disruption (Egyházi et al., 2004). Inactivation rates at room temperature appear insensitive to pressures below certain values (e.g., Escherichia coli, Saccharomyces cerevisiae, and Enterococcus faecalis under 3.5 MPa). An increase of pressure from 100 to 300 bar at 55–60°C do not cause significant effects on the water solubility of CO2, but operating and investment costs increase significantly (Spilimbergo et al., 2005). No effects of pressure or exposure time were observed on the pH drop: the end-values of the treated sample were 1.5 times lower than those of the control sample (pH 6.8), but they remained constant when different experimental conditions were applied (Debas-Louka et al.,

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1999). Temperature- and pressure-dependent inactivation rates have been reported for Listeria monocytogenes (Lin et al., 1994), Escherichia coli (Erkmen, 2001), and Bacillus subtilis (Spilimbergo et al., 2003). Conflicting results on the effect of the decompression rate and pressure cycling have been reported. A compilation of data from research studies was published by Zhang et al. (2006). Early studies assumed that the cells were mechanically ruptured by the fast expansion of CO2 during flash decompression, and it was assumed that a fast depressurization rate would favor cell bursting and/ or enhance mass transfer across cell membranes. More recent studies have more exhaustively controlled the decompression conditions, and similar rates of destruction have been observed for different decompression under subcritical conditions (Debas-Louka et al., 1999). The differences in the rate of inactivation between flash release of pressure and slow release of pressure were not distinct enough (0.5 logs of decrease). Other authors did not find an improved inactivation with the repetitious release of pressure (Shimoda et al., 2001). The levels of deactivation and protein release were nearly the same for fast (4.8 MPa/min) and slow (0.033 MPa/min) decompression. It was proposed that much of the deactivation should occur during pressurization, since a more prolonged operation and smaller number of repetitions was more lethal than a shorter operation repeated more frequently. Lower decompression rates to atmospheric pressure resulted in neither a decreased reduction of remaining cells nor a smaller release of total cellular proteins (Enomoto et al., 1997). When the process is used for disruption, the efficiency of the method depends on the characteristics of the cell walls. Disruption rates are sensitive to variables such as temperature, pressure, and cosolvents. Cell breakage is strengthened by higher pressures. The treatment time influences the efficiency of the process in a strain-dependent way: whereas Trichoderma cells could be disrupted by treatments at 40°C and 100 bar for 15 min (Egyházi et al., 2004), harsher conditions are needed for Saccharomyces cerevisiae (2 h of incubation time were required to disrupt 80% of cells operating at 35°C and 210 bar) (Lin, 1991). The repeated release of pressure during a disruption process either at supercritical or subcritical temperatures has been suggested as a strategy to improve the disruption rates of cells. A static apparatus was used to apply a repeated release of pressure by recharging CO2 into the vessel at the experimental pressure immediately after it was released. Cell breakage occurred as a result of gas expansion within the cells when the vessel pressure was suddenly released (Lin et al., 1992). Various enzymes (alcohol dehydrogenase, invertase, glucose-6phosphate dehydrogenase, and fumarase) in the ruptured cell suspension were active, confirming that the process preserved the functional properties of proteins. Operating at 35°C, the repeated release of pressure enabled a reduction in the exposure time from 7 to 3.5 h or 2 h (when the pressure was released/repressurized once or twice, respectively) (Lin et al., 1992). 23.4.3.1.2 Temperature This variable influences different aspects related to inactivation by SC-CO2. Higher temperatures favor the diffusivity of CO2 in aqueous solution and cells, increase the fluidity of the cellular membrane to accelerate the gas penetration inside the cytoplasm (Isenschmid et al., 1995; Spilimbergo et al., 2003), and favor the solute solubility in CO2, but decrease CO2 solubility in the liquid phase. Too high temperatures lower the extraction power, and could promote food degradation. As a general trend, up to certain limits, a more prolonged exposure to CO2 allows for better sterilization. Alternatively, the exposure time can be decreased by increasing the temperature. Temperature increase was effective to inactivate Escherichia coli cells and shortened the duration of the first stage of inactivation, for example, from 10 min at 25°C, to more than 5 min at 35°C and to less than 5 min at 45°C (Wu et al., 2007). Temperature was found to be more influential than pressure, suggesting that the inactivation may have been due to a combination of physical and chemical effects. The susceptibility of generic Escherichia coli to inactivation by SC-CO2 is more dependent on temperature than that of Escherichia coli O157:H7 (Kim et al., 2007a), although both strains are less temperature dependent than in the case of conventional thermal sterilization (Shuler and Kargi, 2002). The relevance of this variable has been confirmed in studies with Lactobacillus sp. (Hong

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et al., 1997, 1999), Saccharomyces cerevisiae (Shimoda et al., 2001), and Listeria monocytogenes (Lin et al., 1994; Kim et al., 2008). Moisture is essential to the antimicrobial action of pressurized CO2, since the gas must dissolve in an aqueous solution to form carbonic acid, which dissociates into bicarbonate and hydrogen ions, lowering the extracellular pH. Another effect was in relation to the swelling of cell walls that became more permeable to CO2. A threshold water content was required for inactivation, since dry cells (water content less than 10%) were not inactivated (Kamihira et al., 1987). Similar inactivation rates and pH drop were observed for Escherichia coli and Saccharomyces cerevisiae for water contents in the range 37–75%, but for samples dried at 30°C for 7 h showed a remarkable decrease in the rate of destruction (Debas-Louka et al., 1999). The sterilization rate of Saccharomyces cerevisiae correlated well with the CO2 uptake of cells, whereas the increase in the sterilization rate was only slight when free water existed (Kumagai et al., 1997). The role of water for the efficiency of the inactivation and the inactivation rates was confirmed for Listeria monocytogenes (Lin et al., 1994; Kim et al., 2008), Escherichia coli (Hong and Pyun, 1999), and Micrococcus luteus (Cinquemani et al., 2007). 23.4.3.1.3 Entrainer The sterilization efficiency and rates obtained with pressurized CO2 can be improved by the addition of a specific entrainer. Ethanol enhances the solvent power and facilitates the penetration of CO2 into the cells by altering the cell walls and membranes, causing denaturalization and precipitation of protein in cells and the inhibition of certain enzymes (Wu et al., 2007). The addition of ethanol allows a reduction in severity due to the synergistic effects of ethanol and CO2, although the increase of CO2 solubility induced by 10% ethanol is only about 15%. This effect is dependent on the microbial strain and on the ethanol content. Increased ethanol concentration (5–20%) facilitates complete inactivation of Escherichia coli, Lactobacillus brevis, Saccharomyces cerevisiae, and Agelastica coerulea, but it is not efficient with Bacillus subtilis, probably because the cells transform into spores (Wu et al., 2007). Other additives have been used as inactivation aids: for example, 30 ppm of sulfur dioxide improved the inactivation of wet cells of Saccharomyces cerevisiae (Lin et al., 1992). The importance of CO2/product mass ratio depends on the case considered: it can be important, as in Escherichia coli (Gunes et al., 2006); or of minor importance, as in continuous flow systems (Shimoda et al., 1998). Agitation facilitates the dissolution of CO2 into the aqueous solution and its contact with cells (Lin, 1991), enhancing the inactivation effectiveness. Limited agitation could be unfavorable for the process (Lin et al., 1992, 1994; Hong et al., 1997; Dillow et al., 1999). Other factors influencing the bactericidal effectiveness of compressed CO2 include the environmental conditions of cell growth, cell age, and media composition. The fat or oil content of the growth media of Listeria monocytogenes increased cell resistance to CO2 treatment, probably due to some protection of the cell wall structure and/or to membrane porosity modification. Similarly, bacteria were more difficult to inactivate when suspended in the medium with fat or oil (Lin et al., 1994): oleic acid, a model lipophilic substance, protected against inactivation. However, the addition of a surfactant (sucrose monolaurate, 0.001% w/v) significantly enhanced the antimicrobial efficiency of SC-CO2, although this effect was not concentration dependent, possibly because of effects derived from the toxicity of the surfactant itself (Kim et al., 2008). The use of a selective medium (with increased NaCl concentration with respect to the complete medium) enhanced the lethality of the CO2 treatment (7.8 MPa, 35°C) on Escherichia coli (Wu et al., 2007). The baroprotective effect of high solute concentration on the viability of Listeria monocytogenes cells (6.05 MPa) was decreased by 22% and 24% in brain heart infusion agar and physiological saline solution, respectively (Erkmen, 2001). Higher count reductions after CO2 treatment of Lactobacillus plantarum cells (7 MPa, 30°C, 10 min) were determined for a selective medium containing 3% NaCl rather than for a complex medium (Hong et al., 1999; Hong and Pyun, 2001). Comparative data concerning the effects of media composition on the inactivation efficiency of several microorganisms have been compiled by Zhang et al. (2006). The efficacy of microbial inactivation can be reduced in some protein-rich systems and

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in buffered media. The slow inactivation in buffer solution was attributed to decreased pH changes caused by the dissolution of CO2 in the cell suspension. The inactivation efficacy of Escherichia coli in the presence of phosphate buffer solution diminished with respect to experiments with phosphate solutions regardless of temperature, making longer treatment times necessary for inactivation. After SC-CO2 treatment, the pH of the phosphate solution of Escherichia coli decreased rapidly from 5.8 to below 4.0, in comparison with a pH drop from 7.0 to 5.3 when using phosphate buffer solution. In parallel, the duration of the lag phase was 15 min longer at 35°C and 5 min longer at 45°C in the phosphate buffer than in the phosphate solution, respectively (Kim et al., 2007a). Listeria monocytogenes cells suspended in phosphate buffer required longer times to inactivation than those suspended in phosphate solution, regardless of reaction temperature (Kim et al., 2008). Cell lytic enzymes can be used as CO2 entrainers to aid in cell disruption. The operation with repeated pressure release can be used in combination with lytic enzymes to improve the efficiency of cell disruption without protein denaturation. Cell age has been suggested as an influential factor in the inactivation efficiency (Lin et al., 1994). Young cells are more susceptible to CO2 treatment than mature ones since, in the stationary phase of growth, bacteria synthesize new proteins able to protect cells against a variety of adverse conditions including high temperature, oxidative stress, high salt concentrations, and high pressure (Kashket, 1987; Mackey et al., 1995). PEF showed a synergistic effect with high-pressure CO2 in Escherichia coli, Staphylococcus aureus, and Bacillus cereus suspended in glycerol solution (PEF up to 25 kV/cm). Cell viability decreased with increasing electrical field strength and number of pulses. Complete inactivation of bacterial species and decreased count of spores by at least three orders of magnitude was observed when PEF was followed by a batch treatment with SC-CO2 (200 bar at temperature not higher than 40°C). In this case, inactivation was enhanced by an increase of contact time between CO2 and the sample (Spilimbergo et al., 2003).

23.4.4 MORPHOLOGICAL AND STRUCTURAL ALTERATIONS High-pressure CO2 can damage the structure and functions of the cell. In addition to physicochemical changes such as acidification, loss of salt tolerance, leakage of intracellular materials, and inactivation of enzymes, morphological changes in the cells were also confirmed by microscopic observations. Some features found in the treated cells included deformations and irregularities in cell surface, enlarged periplasmic space between the wall and the cytoplasmic membrane, fractures in membranes, bursting, and the empty aspect of the cytoplasm. Modifications of the structural features and extraction of cellular materials were observed during the lag phase, whereas dissolution and extraction of cellular materials by SC-CO2 occur during the death phase at a higher inactivation rate (Wu et al., 2007). Damage to the balance of the biological system can cause cell death without rupture or shrinking of the cell wall: in this case, cells lose viability but do not break down and they maintain their cell shape and integrity (anesthesia effect). The reason for the viability loss is probably more related to a microscopic inactivation mechanism than to a solvent effect (Isenschmid et al., 1995). This viability loss of cells with apparently intact membranes has been reported for Leuconostoc dextranicum (Lin et al., 1993) and Lactobacillus plantarum cells (Hong and Pyun, 1999). Cell walls remained unchanged, and no external deformation was observed after the treatment (Dillow et al., 1999; Hong and Pyun, 1999). Alternatively, exposure of Lactobacillus plantarum cells to pressurized CO2 has been reported to result in loss of cell membrane integrity, which was correlated with the decrease of cell viability (Nakamura et al., 1994). Scanning electron microscopy (SEM) observations showed deformation of cell walls of some Escherichia coli cells treated with SC-CO2 (5 MPa, 35°C) (Ballestra et al., 1996), as well as changes in Saccharomyces cerevisiae cell surfaces with bursting of cells and possible leakage of cytoplasmatic material (treatment conditions, 6 MPa and 35°C) (Shimoda et al., 1998).

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A modification of cell surfaces has been confirmed by different techniques: in SEM images, Escherichia coli O157:H7 cells treated at 100 bar and 35°C appeared with alterations in the cell surfaces, whereas transmission electron microscopy (TEM) images showed uneven distribution, aggregation of cytoplasmic substances and isolation of the cell membrane from the cytoplasm (Hong and Pyun, 1999; Kim et al., 2007a). However, SEM showed that about 25% of cells had intact plasma (in comparison with less than 2.5% viability). SEM observation of Escherichia coli cells treated at 7.8 MPa and 35°C showed that the cell wall was intact, and that the mean diameters hardly changed with the increase of treatment time, but TEM images showed great differences between untreated and treated cells: these latter cells presented a reduction in their cytoplasm density, injury of membrane, leakage of intracellular substances, and vacant space between the wall and cytoplasm (Wu et al., 2007). Although the cell membrane may have been damaged enough to release cellular materials, the changes could not be visually shown by SEM or TEM. Neither SEM nor TEM images revealed significant ultrastructural differences between the treated and untreated Listeria monocytogenes cells, probably due to the thicker peptidoglycan layer in the cell wall structure of this Gram-positive bacteria in comparison with Gram-negative ones (Kim et al., 2008).

23.4.5

APPLICATIONS OF INACTIVATION

Most inactivation studies with dense CO2 were performed on microorganisms (including Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Brochothrix thermosphacta, Escherichia coli, Enterococcus faecalis, and Yersinia enferocolitica) growing on the surface or inside solid food samples or in liquid products. Data on this topic have been recently revised by GarcíaGonzález et al. (2007). In these systems, the presence of carbohydrates, fats, and other organic compounds should be considered since they can affect the inactivation rate (Erkmen, 1997, 2000a, 2000b, 2000c, 2000d, 2001). Particular attention has been paid to LAB, which generally proliferate at a late stage of the natural mixed fermentation of vegetables. The overall acceptability of treated, fermented vegetable foods was better than those of the untreated ones, without significant effects on color, flavor, or texture (Hong et al., 1999). Pressurized CO2 treatment is also an attractive option for the sterilization of thermally and hydrolytically sensitive materials used in pharmaceutical formulations, biomedical applications, and medicine devices (including porous materials, implants, and liquid products) (Spilimbergo et al., 2003). Some potential advantages of this technology are (i) CO2 may sterilize at mild temperatures through a nonreactive process and (ii) the ability to inactivate a wide variety of microorganisms in the absence of organic solvents or irradiation (Dillow et al., 1999; Zhang et al., 2006). SC-CO2 has been used to disinfect fabrics in hospitals without affecting the physical properties of the textile materials (Cinquemani et al., 2007).

23.4.6

APPLICATIONS OF THE DISRUPTION AND FRACTIONATION OF BIOMASS COMPONENTS

Microbial cells are an important source of commercial products of biotechnological origin (biochemicals, antibiotics, and enzymes). An increasing demand for biotechnological products in industry and medicine has fostered considerable efforts to develop efficient technologies for the recovery of intracellular enzymes and organelles from microbial cells. Disruption techniques commercially available at a large scale include mechanical and chemical methods. Conventional organic solvents and enzymatic lysis are widely used, but can cause degradation and denaturation of proteins. In general, mechanical methods are nonspecific, but their efficiency is higher and their applications broader than those of alternative methods. Disruption of microbial cells by pressurized CO2 at either subcritical or supercritical temperatures has been applied to a variety of cells. Under optimum conditions, over 80% of cells can easily be ruptured in 1 h. Selective extraction conditions can be established, allowing the fractionation of

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the biomass in order to obtain one or more compounds. Cell disruption by dense CO2 is relatively simple and inexpensive. As the operation is carried out at moderate temperatures, enzymes are not inactivated, and the functional properties of proteins are preserved. SC-CO2 is an attractive technology for cell disruption and product recovery. It is not as effective as other disruption techniques for cells with robust and rigid cell walls (Egyházi et al., 2004), but the results are particularly encouraging for the extraction of sensitive materials. Khosravi-Darani and Vasheghani-Faharani (2005) have revised some examples of fractionation of different types of cells by this method. Disruption of the cells of some microorganisms (e.g., Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae) is well known, but data in the literature are scarce for other microorganisms. Significant differences in their susceptibility to disruption were observed for different strains. The following order of resistance was reported: Kluyveromyces fragilis > Saccharomyces cerevisiae > 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.

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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

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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

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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 (<1 μm) colloidal systems (Brigger et al., 2002). Nanonization possesses many advantages, such as increasing compound solubility, reducing medicinal doses, and improving the absorbency of herbal medicines compared to the respective crude drug preparations (Brigger et al., 2002). The nanotechnology of drug formulation not only enhances the absorption of poorly water-soluble drugs but also improves the therapeutic effectiveness of drugs in pharmaceutical research. Nanoparticle formulation is a novel drug delivery systems which possesses various advantages, including increasing drug solubility, enhancing dissolution rate, improving bioavailability, and decreasing the dosage required for the same effect, compared with crude or micronized medicines (Liversidge and Cundy, 1995; Müller and Peters, 1998; Liversidge et al., 2003). There are several reports relating to the nanoparticle formulation of traditional Chinese medicine. Liu et al. (2008) reported a comparison of the efficacy of the medicinal plant material Salvia miltiorrhiza prepared using nanotechnology and the traditional grinding method. Stronger antioxidant bioactivities were observed for the extracts prepared using nanotechnology in all tested assays. These results suggest a greater liberation of active components in the Danshen samples prepared using this novel technique. A further investigation could be carried out to see whether nanonization processes improve the absorption of bioactive compounds in the gastrointestinal system, by monitoring the constituents with the in vitro Caco-2 cell lines model

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(Liu et al., 2008). Another case is reported by Yen et al. (2008), who utilized the nanosuspension method to develop ethanolic extracts of the Cuscuta chinensis loaded PF68 nanoparticles system. An oral dose of C. chinensis nanoparticles 5 times lower than the equivalent ethanolic extract of C. chinensis exhibited similar hepatoprotective and antioxidant effects. In addition, results demonstrated that the hepatoprotective and antioxidant effects of 50 mg/kg C. chinensis nanoparticles were more effective than the ethanolic extract at a dose of 125 mg/kg. Curcuminoids are one of the best recognized antioxidants isolated from the rhizome of Curcuma longa Linn. Curcuminoids are obtained as a yellowish pigment and consist primarily of three phenolic compounds: curcumin, demethoxycurcumin, and bisdemethoxycurcumin (Sreejayan and Rao, 1994; Ahsan et al., 1999; Khopde et al., 1999). Tiyaboonchai et al. (2007) reported that curcuminoid loaded solid lipid nanoparticles were successfully prepared by microemulsion technique at a moderate temperature. The process parameters, such as the amount of lipid and emulsifier, were crucial factors for the resulting mean particle size and the efficacy of drug incorporation. At optimal conditions, the mean particle size of curcuminoid loaded solid lipid nanoparticles was 447 nm and the incorporation efficacy of curcuminoids was 70% (w/w). Furthermore, this study demonstrates that the stability of curcuminoids in a cream containing curcuminoids incorporated into solid lipid nanoparticles was significantly better than free curcuminoids in a cream formulation.

24.4 24.4.1

IMPROVEMENTS IN THE BIOACTIVITY OF FUNCTIONAL FOODS AND NUTRACEUTICALS HEPATOPROTECTIVE

Hepatitis is an inflammatory liver disease induced by various causes, such as virus infection, alcohol, autoimmune response, drugs, or xenobiotic injury. Although many unsolved problems still remain concerning the related mechanisms, it is generally accepted that activated T cells are involved in both acute and chronic hepatitis (Napoli et al., 1996; Chisari, 1997; Lapierre et al., 2007). Cytokines secreted by lymphocytes dominantly modulate the immune reaction in the liver, ultimately resulting in liver dysfunction, hepatic cirrhosis, and even liver failure. Accordingly, several types of drugs have been developed to treat hepatitis, such as interferon and purine analogs for viral hepatitis, and prednisone and azathioprine for autoimmune chronic hepatitis (Wu et al., 2008). In addition, silymarin and glycyrrhizin have frequently been used as adjunct liver-protective drugs (Ye and Lu, 2004). An ethanolic extract from C. chinensis has been proved to have hepatoprotective and antioxidant activities that helps prevent hepatotoxicity induced by acetaminophen in rats (Yen et al., 2007). The hepatoprotective effects of the C. chinensis ethanolic extract and C. chinensis nanoparticles were determined and compared by assessing levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), and also by examining the histopathological sections of the liver, all of which are associated with hepatic integrity (Yen et al., 2008). The results showed that the hepatoprotectivity of C. chinensis nanoparticles were more effective than traditional ethanolic extracts. Thus, the author suggested that the nanoparticle system could be adapted and further applied to overcome other poorly water-soluble herbal medicine instances and, moreover, to decrease treatment dosages.

24.4.2

ANTIOXIDANT

Many reactive oxygen species (ROS), including the hydroxyl radical, hydrogen peroxide, and the peroxide radical, are known to cause oxidative damage to living systems. ROS also play a significant role in human diseases such as cancer, atherosclerosis, hypertension, and arthritis (Halliwell and Gutteridge, 1984; Frenkel, 1992). Given this negative impact, there is increasing interest in discovering natural antioxidants derived from traditional Chinese medicines as it is believed that they protect the human body from the attack of free radicals and retard the progress of many

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chronic diseases (Pryor, 1991). Furthermore, these natural antioxidants are likely to be more desirable than chemically produced analogs because some of the latter are reportedly carcinogenic (Imaida et al., 1983). Antioxidants are substances which counteract free radicals and prevent the damage caused by them. Antioxidants can greatly reduce damage due to oxidants by scavenging them before they react with biologic targets, preventing chain reactions, or inhibiting the activation of oxygen to highly reactive species (Azzi et al., 2004). Except for anaerobes, oxygen is vital for all living systems. However, the paradox of aerobic life is that oxidative damage occurs at the key biological sites, threatening their structure and function. Oxygenic threat is met by an array of antioxidants that have evolved in parallel with our oxygenic atmosphere (Benzie, 2003). Our bodies implement various antioxidants, some of which are dietary-derived antioxidants to help restrain potential free radical damage. It has been estimated that more than two-thirds of human cancers, which are caused by mutations in multiple genes, could be prevented by a changed lifestyle including dietary modification. In the modern-day environment, people are exposed to a variety of toxins, some of which are potential oxidants. Taking the increased environmental pressures of oxidant damage combined with our unbalanced contemporary food supply into consideration, the value of antioxidant supplementation becomes more apparent. These agents should possess characteristics such as good bioavailability, stability, and selectivity to the damaged or transformed cells. Formulating antioxidants is particularly interesting because of their relative lack of toxicity, preventive and therapeutic roles in diverse diseases, and encouraging evidence from epidemiology. The human diet has evolved over years, but the study of this evolution reveals that the modern day intake of antioxidants is far less than in ancient times. Organized agriculture which was instigated some thousands of years ago has deprived us of a previously antioxidant-rich diet (Benzie, 2003). The human antioxidant defense system is incomplete without dietary antioxidants. For instance, humans lack the ability to synthesize ascorbic acid endogenously, but have an absolute requirement for it (Nishikimi and Yagi, 1991). These ascorbic acid requirements must be met from dietary sources. Other antioxidants such as vitamin E, CoQ10, carotenoids, and polyphenols are obtained from external sources and play important roles in maintaining human health. Liu et al. (2008) reported that S. miltiorrhiza nanoparticles displayed stronger antioxidant bioactivities in three in vitro biological assays, including DPPH free radical scavenging, ferrous ion chelation, and reduction power. Yen et al. (2008) also reported that C. chinensis nanoparticles enhanced antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and diminished lipid peroxidation by lowering malondialdehyde (MDA) levels. Their findings also demonstrated that C. chinensis nanoparticles showed stronger antioxidant effects than C. chinensis ethanolic extract.

24.5

NANOTECHNOLOGY FUNCTIONAL FOODS AND DRUG DELIVERY SYSTEMS

Nanoparticles can enter the body and its vital organs, including the brain, much more easily than larger particles. The incorporation of bioactive compounds, such as vitamins, probiotics, bioactive peptides, or antioxidants into food systems provides a simple way to develop novel functional foods that may have physiological advantages or reduce the probability of diseases. As a vital macronutrient in food, proteins possess unique functional properties including their ability to form gels and emulsions, which make them an ideal material for the encapsulation of bioactive compounds. The development of delivery systems for small molecules, proteins, and DNA has been impacted to an enormous degree over the past decade by nanotechnology, and has led to the development of entirely new and somewhat unpredicted fields. It is expected that novel drug delivery systems can make a significant contribution to global pharmaceutical sales. This is illustrated by the fact that approximately 13% of the current global pharmaceutical market is accounted for by sales of products incorporating a drug delivery system (Roco and Bainbridge, 2002). Several methods have been described

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in the literature for the preparation of lipid nanoparticles, such as high-pressure homogenization, emulsification-solvent evaporation method, and solvent-injection method (Barichello et al., 1999; Cavalli et al., 2001; Jenning et al., 2002; Schubert and Müller-Goymann, 2003; Garcia-Fuentes et al., 2004; Langguth et al., 2005; Liu et al., 2005). In one of these cases, surfactants were added to the formulation of lipid nanoparticles to stabilize the emulsion formed during particle preparation. The type of surface active substances or stabilizers involved in the fabrication procedure obviously has an important effect on the physical and pharmaceutical properties, such as particle size and zeta potential values which are important for physicochemical stability as well as the biopharmaceutical properties of the preparation. However, many of these stabilizers have a low biocompatibility (Youan et al., 2003). The multidisciplinary fields of nanotechnology promise to deliver a technological breakthrough and are moving very fast from concept to reality. The flexibility to modify nanotechnology to meet the standards of pathologic conditions either for therapeutic applications or as a diagnostic tool is an important characteristic of this technique. Nanoemulsions and nanoparticles have been developed using a range of materials. Polymer structures such as block-co-polymer micelles, polyelectrolyte capsules, colloidosomes and polymersomes, or gelled macromolecules have already been applied in drug delivery systems (Förster and Konrad, 2003). The release of drugs from these structures may be triggered by exposure to appropriate environmental conditions, such as adjusting the pH or salt concentration, or by the application of a stress such as ultrasound. In addition, by appropriate control of the surface properties of the polymers, interaction with specific proteins and cells in the body may be modulated, thus providing a suitable means for targeting distribution in the human body. It should be noted that most examples given in this chapter derive from the pharmaceutical industry. Cases of nanotechnology applications in the food industry for the target delivery of bioactive constituents are not so common and the use of pharmaceutical products made with non-food-grade additives is usually not acceptable in food requirements. However, there are parallels that can be drawn between the delivery of drugs for the treatment of disease and that of bioactive food components in functional food applications for improving health and reducing the risk of disease. Research into food-grade encapsulants is needed in order to enable the delivery of desirable bioactive components through the food supply system. Hence, many of the encapsulation systems being developed for the delivery of bioactive components through food have capitalized on the self-assembly behavior of natural ingredients such as proteins, carbohydrates, lipids, or mixtures of these, or of ingredients functionalized by physical or enzymatic means for encapsulation.

24.6 NANOTECHNOLOGY FOR FOOD AND GLOBAL CLIMATE ISSUES Global warming is the increase in the average measured temperature of the Earth’s near-surface air and oceans since the mid-twentieth century, and its projected continuation. Increasing global temperatures are expected to raise sea levels, increase the intensity of extreme weather events, and significantly change the amount and pattern of precipitation, probably leading to an expanse of tropical areas and an increase of desertification (Climate Change, 2007). Desertification is the degradation of land in arid, semi-arid, and dry subhumid areas resulting primarily from human activities and influenced by climatic variations. Current desertification is taking place much faster worldwide than historically and usually arises from the demands of increased populations who settle on the land in order to grow crops and graze animals. A major impact of desertification is the loss of biodiversity and productive capacity, for example, by the transition from land dominated by shrublands to non-native grasslands. In the semi-arid regions of southern California, many coastal sage scrub and chaparral ecosystems have been replaced by non-native, invasive grasses due to the shortening of fi re return intervals. This can create a monoculture of annual grass that cannot support the wide range of animals found in the original ecosystem (Wilson, 2001).

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Desertification is widespread in many areas of the People’s Republic of China. The populations of rural areas have increased since 1949 for political reasons as more people have settled there. While there has been an increase in livestock, the land available for grazing has decreased. The import of European cattle such as Friesian and Simmental, which require higher food intake, has made things worse (Brown, 2006). Human overpopulation is leading to the destruction of tropical wet forests and tropical dry forests, due to the increasing practice of slash-and-burn and other methods of subsistence farming necessitated by famines in less developed countries. A sequel to this deforestation is typically large-scale erosion, loss of soil nutrients, and sometimes total desertification. Most of the important plant materials used in traditional Chinese medicine are cultivated in regions such as the Inner Mongolian grasslands which are attracting much environmental concern; the large-scale harvest of raw plant materials is inducing serious environmental problems such as dust storms. Hence, the application of nanotechnology to commonly used traditional Chinese medicine plant materials may not only improve their bioactivity but should also reduce the amount of the nanopharmaceuticals required and thereby decrease the environmental degradation associated with the harvesting of the raw products. As reported by Yen et al. (2008) the nanoparticle counterpart is 2–3 times more effective than the crude extract. That is, the required raw products for nanoparticle formulation may only be one-third of the medicinal plant materials harvested today. This should decrease the area of cultivation significantly and therefore slow down the desertification process.

24.7

SUMMARY

Nanotechnology has already been widely applied in various food science fields. The advantages of nanotechnology applications have been discussed above. Generally, the quantity of raw materials can be reduced and the bioactivity of the food is improved. However, it must be noted that nobody can promise that all nanotechnology foods will be safe and more bioactive. Each new product prepared by nanotechnology must be examined for toxicity before marketing. In addition, nanotechnology foods should also be evaluated for their effect on the human body just as GM foods have been.

ACKNOWLEDGMENTS The authors would like to thank the National Center for High-Performance Computing, Taiwan, People’s Republic of China, for computer time and facilities relating to reference compilation.

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Part IV Quality Assurance and Safety: Design and Implementation

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the Nutritional 25 Enhancing Quality of Fruit Juices Advanced Technologies for Juice Extraction and Pasteurization Robert D. Hancock and Derek Stewart CONTENTS 25.1 Introduction ..........................................................................................................................465 25.2 Optimization of Fruit Extraction for Enhancing the Nutritional Content of Juice Products ...................................................................................................................466 25.3 Novel Technologies for the Pasteurization and Preservation of Fruit Juices: Impact on Nutritional Quality ..............................................................................................469 25.3.1 Pulsed Electric Fields ............................................................................................... 470 25.3.2 High Hydrostatic Pressures ...................................................................................... 472 25.3.3 Dense Phase CO2 ...................................................................................................... 475 25.3.4 Ultrasound ................................................................................................................ 476 25.3.5 Ultraviolet Light........................................................................................................ 477 25.3.6 Ionizing Radiation .................................................................................................... 478 25.4 Conclusions ........................................................................................................................... 478 References ...................................................................................................................................... 479

25.1 INTRODUCTION For many years epidemiological studies have highlighted a strong negative correlation between fruit and vegetable intake and incidence of degenerative diseases such as numerous forms of cancer (Block et al., 1992), cardiovascular diseases (Ness and Powles, 1997), and other diseases including age-related macular degeneration (Goldberg et al., 1988), cataract (Mares-Perlman et al., 1995), bronchitis, asthma, peptic ulcers, gallstones, liver cirrhosis, kidney stones, and arthritis (La Vecchia et al., 1998). One suggestion for the protective effects of diets high in fruits and vegetables is their high content of antioxidant phytochemicals, which are proposed to protect vital biomolecules such as DNA, proteins, and lipids from oxidation caused as a result of the production of free radicals under both normal metabolism and conditions of stress. Over time and with insufficient repair, the damage caused by molecular oxidation is proposed to result in disease (Diplock et al., 1998). As a result of the epidemiological evidence, many governments have introduced campaigns to educate citizens with regard to the importance of fruit and vegetable consumption (e.g., the United Kingdom 5-a-day campaign), and a recent World Health Organization expert consultation suggested the consumption of at least 400 g of fruits and vegetables per day to minimize the risk of chronic disease (WHO/FAO, 2003). Fruit juices can make an important contribution to fruit and vegetable consumption, with fruit juice comprising approximately 18% of the total intake of the adult UK 465 © 2010 Taylor and Francis Group, LLC

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population (Henderson et al., 2002). Furthermore, several intervention studies have shown improved antioxidant status following fruit juice consumption (Netzel et al., 2002; Bub et al., 2003; García-Alonso et al., 2006; McGhie et al., 2007). However, in recent years there has been increased skepticism regarding the antioxidant hypothesis, with several intervention trials suggesting that antioxidant supplementation may have adverse effects (Selman et al., 2006; Ristow et al., 2009) and a recent systematic review suggesting that antioxidant supplementation may increase all-cause mortality (Bjelakovic et al., 2007). As a result of these findings, researchers are now beginning to focus on mechanisms of protection unrelated to the in vitro antioxidant capacity of phytochemicals (Hancock et al., 2007; Stevenson and Hurst, 2007). Whatever the merits of the antioxidant hypothesis, consumer opinion is firmly in favor of antioxidant consumption and many juice manufacturers have exploited these beliefs in their marketing campaigns. Furthermore, there is a strong consumer demand for premium and “natural” products with minimum processing and no additives. As a result, a key to improving competitiveness for juice manufacturers will be to provide high-quality, fresh-tasting juices that maintain a nutrient profile as close as possible to that of fresh fruits. In the current chapter we discuss modern manufacturing technologies for maximizing the nutritional properties of fruit juices. In particular, the impacts of nonthermal extraction and pasteurization technologies on the antioxidant capacity and content of antioxidant compounds in fruit juices are highlighted. Furthermore, the benefits of such technologies in maintaining the nutritional quality of fruit juices over shelf life are discussed.

25.2 OPTIMIZATION OF FRUIT EXTRACTION FOR ENHANCING THE NUTRITIONAL CONTENT OF JUICE PRODUCTS Historically, fruit juice manufacturers have focused on maximizing juice yield achieved through physical and (bio)chemical treatments designed to weaken plant cell walls and loosen membranes and through process engineering solutions designed to optimize press design and juice recovery. More recently, it has become clear that fruit pretreatments and extraction methods can have significant impacts on the nutritional quality of the product. The present section focuses on recent work aimed at maximizing the extraction of wall-associated polyphenols by optimization of enzymatic maceration treatments. Hydrolytic enzymes such as pectinases, cellulases, and amylases have been used in the juice processing industry for many years to improve juice yields, improve rheological properties, remove suspended matter in the production of clear juices, and improve cloud stability in cloudy juices (Kashyap et al., 2001). Pectinases are by far the most widely used, accounting for 25% of global sales of food enzymes (Jayani et al., 2005) with an estimated annual market value of $150 million in 2005 (Kashyap et al., 2001). Acidic pectinases used in fruit juice production are mainly produced by fungi and yeasts in either submerged or solid state culture with Aspergillus niger, the most commonly used species for commercial production (Favela-Torres et al., 2006). Commercially available pectinases are not single purified proteins but mixtures of enzymatic activities including polygalacturonase, pectin lyase, pectin methylesterase, endoglucanase, β-glucanase, xylanase, mannanase, α-arabinosidase, β-arabinosidase, β-galactosidase, and β-glucosidase activities (Table 25.1). Specific enzyme preparations are tailored for and find specific applications. In recent years it has become clear that careful selection of the appropriate pectinase preparation is required not only to maximize juice yield and obtain desirable physical properties but also to maximize both initial yield and subsequent stability of health promoting phytochemicals. Several parameters must be considered for the optimization of enzymatic maceration treatments to maximize the recovery of nutritionally active components into fruit juices. Firstly, the physical association of nutritionally active compounds with insoluble fruit fractions can have a negative impact on recoveries. For example, polyphenolic antioxidants are frequently associated with macromolecular components found in the cell wall and are subsequently lost in fruit pomace. Secondly,

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TABLE 25.1 Relative Glycosidase Activities of Commercial Pectinase Preparations Glycosidase Activity Product

PG

PL

PME

EG

β-Glu

Xyl

Man

α-Ara

β-Ara

β-Gal

β-Glc

E CE B CCM B 8X Roh PS P S XXL P BE 3L P BEX P SP-L P 3XL

100 100 100 100 100 100 100 100 100 100

0 0 NA NA NA 890 0 NA NA NA

0 14 16 0 22 0 17 17 9 17

1300 4 5 10 6 38 8 8 6 5

4700 100 NA NA NA 98 180 NA NA NA

2300 48 25 135 2 39 180 72 3 7

160 8 5 14 89 0 11 10 55 5

50 5 7 1 2 0 25 26 2 27

0 0.2 NA NA NA 0 2 NA NA NA

5 2 2 4 5 0 23 12 5 11

5 0.4 0 2 0 0 3 0 0 0

Sources: All data were obtained from Buchert et al. (2005), Koponen et al. (2008), and Puupponen-Pimiä et al. (2008) Pectinase preparations were E CE, Econase CE; B CCM, Biopectinase CCM; B 8X, Biopectinase Super 8X; Roh, Rohapect; P S, Pectinex Smash; P S XXL, Pectinex Smash XXL; P BE 3L, Pectinex BE 3-L; P BEX, Pectinex BE XXL; P SP-L, Pectinex Ultra SP-L; and P 3XL, Pectinex 3 XL. Enzyme activities were PG, polygacturonase; PL, pectin lyase; PME, pectin methylesterase; EG, endoglucanase; β-Glu, β-glucanase; Xyl, xylanase; Man, mannanase; α-Ara, α-arabinosidase; β-Ara, β-arabinosidase; β-Gal, β-galactosidase; and β-Glc, β-glucosidase. NA, data not available.

conditions of enzymatic maceration are an important consideration with temperature, time, and aeration affecting the recovery of chemically unstable compounds, including many of the antioxidant components. Finally, the specific enzyme activities present in the pectinase preparation selected can have important consequences for both the release of nutritionally important compounds and their subsequent stability. Pectinase preparations are commonly used in the preparation of berry juices to reduce the viscosity and increase the processibility and juice yields of these high pectin containing fruits (Hilz et al., 2005). Juice yield improvements are typically in the region of 20% (Buchert et al., 2005) and viscosity reduction of expressed juice is up to 80% (Semenova et al., 2006). Enzymatic treatments have been observed to improve total polyphenol (Landbo et al., 2007) and anthocyanin (Koponen et al., 2008) recoveries, and to produce juices with improved antioxidant properties as measured by in vitro radical scavenging capacity (Puupponen-Pimiä et al., 2008) and protection of human low-density lipoprotein (LDL) from oxidation (Landbo and Meyer, 2004). The positive impact of pectinase treatment on polyphenol recovery is linked to the improved solubilization of cell walls preventing them from acting as an adherent to which polyphenols can bind, thus partitioning them into the fruit pomace. This mechanism is supported by the observed linear correlation between cell wall degradation and phenolic release in pectinase-treated blackcurrants (Bagger-Jørgensen and Meyer, 2004). Furthermore, berry anthocyanins, which frequently form a large proportion of the total polyphenol pool and impart the vibrant colors desired by consumers, are mainly located in the vacuoles of lysis-resistant epidermal cells, which become significantly more extractable following enzymatic treatments (Ros Barcelo et al., 1994). At least two studies have systematically examined the impact of varying enzymatic maceration parameters (temperature, time, and enzyme/substrate ratio) on the yield and nutritional attributes of berry juices. When preparing juice from elderberry, maceration time, maceration temperature, and enzyme dose were all positively correlated with juice yields within the range of 7–50 min, 30–62°C, and 0–0.36% enzyme/substrate ratio, respectively (Landbo et al., 2007). However, maceration time had no impact on recovery of anthocyanins or total phenols and only maceration temperature was

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correlated with recovery of phenols. Following optimization, ideal maceration conditions were determined as 63°C for 30 min at an enzyme/substrate ratio of 0.12%. The pectinase preparation used for maceration also had an impact, with Pectinex BE Color consistently providing the highest juice yields and phenolic and anthocyanin contents. Similar work undertaken in blackcurrants demonstrated that maceration time (0–60 min), temperature (30–50°C), and enzyme/substrate ratio (0–0.1%) were all positively correlated with phenol recoveries. Of the four enzyme preparations tested, Macer8 [FJ] and Pectinex Ultra SP-L were the most effective in improving both juice yield and phenol concentration; however, the juice produced following Pectinex maceration demonstrated greater inhibition of LDL oxidation in vitro (Bagger-Jørgensen and Meyer, 2004). These data emphasize the need to optimize fruit maceration parameters both in terms of physical parameters (temperature, time, and enzyme/substrate ratios) and regarding the specific pectinase preparation used. Side activities found in many commercial pectinases are capable of hydrolyzing not only cell wall components but also sugar–phenol conjugates generating their aglycone moieties. A number of studies have observed correlations between side activities and the release and subsequent stability of berry juice phenolic components. The importance of judicial selection of the pectinase preparation was highlighted by a comparison of the effects of pectinase treatment on anthocyanin recoveries in bilberry and blackcurrant. Bilberry anthocyanins are highly heterogeneous consisting of glucosides, galactosides, and arabinosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin (Riihinen et al., 2008), whereas blackcurrant anthocyanins are dominated by four main species, glucosides and rutinosides of delphinidin and cyanidin (McDougall et al., 2005). Treatment of bilberry mash with low doses (1 nkat polygalacturonase activity/g) of Econase CE, Biopectinase CCM, Pectinex Smash XXL, or Pectinex BE 3-L enhanced the recovery of anthocyanin glucosides, arabinosides, and galactosides compared with untreated mash. In addition, increased enzyme dosage up to 100 nkat/g further improved the recovery of glucosides and arabinosides (with the exception of Pectinex BE 3-L), while high doses of all enzymes with the exception of Pectinex Smash XXL reduced the recovery of galactosides (Koponen et al., 2008). Table 25.1 shows the catalytic activities associated with the various enzyme preparations used and suggests that the reduced recovery of galactosides is associated with the β-galactosidase side activity present in all preparations, with the exception of Smash XXL. The nutritional significance of these results relates to the instability of the aglycones, which are subsequently rapidly broken down (Sadilova et al., 2006). On the contrary, the stability of glucosides following Econase CE treatment is more problematic given that the enzyme preparation contains equivalent glucosidase and galactosidase activities. One explanation may arise from the results obtained following the digestion of blackcurrant mash, where no aglycone anthocyanidins were detected and recoveries of both glucosides and rutinosides were enhanced (Buchert et al., 2005; Koponen et al., 2008). These data imply that in blackcurrant, enzymatic digestion improved the release of anthocyanins from the cell wall and rutinosidase and rhamnosidase activities were absent from the pectinase preparations used. The observation that glucosides were spared despite the fact that some preparations contained glucosidase activity suggests that anthocyanidin glucosides are poor substrates for such enzymes or alternatively that blackcurrant extracts contained a glucosidase inhibitor. The latter suggestion was supported by the observation that maceration of blackcurrant with Econase CE, which contains equivalent amounts of galactosidase and glucosidase activity, resulted in a slight reduction of glucosides significantly lower than that observed when the same enzyme was used to macerate bilberries. Further support for the hypothesis that side activities may have negative impacts on anthocyanin stability was obtained following the preparation of juices from strawberry and raspberry where extended incubation periods (2–6 h) resulted in losses of anthocyanins compared with nonenzyme macerated controls. On the contrary, enzymatic maceration resulted in improved or neutral recoveries of quercetins and ellagic acids compared with nonenzymatic maceration (Versari et al., 1997). One issue affecting a number of juices, and in particular berry fruit juices, is the production of a haze consisting of polyphenol–protein complexes on storage (Siebert, 1999). Numerous fining

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agents are used to remove either haze-active proteins (e.g., silica gel) or haze-active polyphenols (e.g., polyvinylpolypyrrolidone); however, the use of such fining agents can result in significant losses in polyphenol and antioxidant content. An alternative to the use of finings is the degradation of proteins by the use of proteases, thus maintaining nutritionally significant polyphenols in solution. In one study, the impact of protease-assisted clarification of blackcurrant juice was compared with conventional clarification using gelatin and silica sol (Landbo et al., 2006). Impacts on haze formation and retention of nutritionally significant polyphenols were examined. Following tests of five different acid fungal protease preparations on immediate turbidity reduction and rate of turbidity increase following storage, Enzeco Fungal Acid Protease was demonstrated to be at least as effective as conventional gelatin–silica treatment in preventing haze formation. However, unlike conventional clarification treatments that removed approximately 30% of the anthocyanins present in juices, protease treatment only decreased juice anthocyanin content by 12%. As a hard fruit, enzymatic maceration is necessary to improve apple juice yields from below 50% in the absence of enzymes up to 80% in their presence (van der Sluis et al., 2002). Unlike berry fruit, apples contain high endogenous levels of polyphenol oxidase (PPO) and peroxidase, resulting in enzymatic losses of polyphenols through oxidation. This results not only in a reduction in nutritional value but also in reduced organoleptic desirability caused by browning reactions, which can become extensive during extended enzymatic maceration. The impact of PPO was highlighted by the observation that during a 2-hour enzymatic maceration, substantial losses were observed for the PPO substrates phloridzin, chlorogenic acid, and catechin while the nonsubstrates cyanidin galactoside and quercetin glycosides were not affected (van der Sluis et al., 2002). Losses of polyphenolic compounds can be reduced using nonoxidative enzymatic maceration in which ascorbic acid is added, resulting in particularly strong preservation of chlorogenic acid, procyanidins, and epicatechins (Mihalev et al., 2004). Several authors have examined the impact of using pectinases in conjunction with other hydrolytic enzymes to improve the yields and recovery of nutritionally active biomolecules in apple processing. Frank Will et al. (2000) developed a two-stage process for the production of a premium, cloudy juice following pectinase treatment followed by pomace liquefaction with different pectinases and cellulases for the production of extraction juice. Extraction juices contained between 1.5 and 2.5 times the polyphenol content of premium juices and in addition contained certain polyphenolic substances, including procyanidins and quercetin derivatives, that were absent in the premium juices. Extraction juices also contained approximately 10–20-fold higher colloids (mainly consisting of cell wallderived oligo- and polysaccharides) than premium juices, acting as a potential source of dietary fiber. Although sensory data were not presented, it would appear that extraction juice could have significant nutritional advantages over premium juices and although pomace liquefaction is currently banned in the European Union it has been used in some North and South American countries. Enzyme-assisted juice extraction is now widespread in modern juice manufacture, with enzymes often chosen for their yield maximizing potential and impact on juice physical and organoleptic properties. However, it is now becoming clear that the choice of enzymes can also have a significant impact on the nutritional qualities of fruit juices, and manufacturers need to be aware that the choice of enzyme and specific maceration conditions can have both positive and negative impacts.

25.3 NOVEL TECHNOLOGIES FOR THE PASTEURIZATION AND PRESERVATION OF FRUIT JUICES: IMPACT ON NUTRITIONAL QUALITY In 1998 the U.S. Food and Drug Administration (FDA) introduced new labeling requirements for fruit juices that had not been treated to achieve a 5 log reduction in pathogen load (Freidman and Shalala, 1998). Taken together with the recognition that traditional thermal pasteurization can have significant negative impacts on the nutritional quality of fruit juices, this ruling has spurred research

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into nonthermal methods for the reduction of microbial pathogens while maintaining high nutritional quality. The most promising technologies are discussed.

25.3.1

PULSED ELECTRIC FIELDS

Although pulsed electric field (PEF) technology was first introduced in the 1960s (Wouters et al., 2001a), it was not until the development of a continuous treatment chamber in the late 1980s that the use of PEF in food processing became a realistic opportunity (Dunn and Pearlman, 1987). PEF technology involves the application of short pulses (<10 μs) of high voltage (20–80 kV cm−1) to fluid foods placed between two electrodes. Pulses can be generated at rates up to 2000 per second and foods are typically subject to a series of pulses where the total time of application is normally less than 1 s (Min et al., 2003a). At present there are a number of commercially available PEFprocessed juices and juice blends available in the United States; however, European Union novel food regulations will require the demonstration of substantial equivalence before such products can go on sale in Europe (Min et al., 2007). One particular area of concern is the transfer of metal ions from PEF electrodes into the medium being processed, although under typical processing conditions, such transfer remains below legislation values for fruit juices producing lower contamination than that outlined for water under the European Union Drinking Water Directive (Roodenburg et al., 2005). Ultrastructural and biophysical data suggest that microbial inactivation occurs as a result of the formation of membrane pores induced by structural fatigue caused by electric field-induced compression and tension. Induction of such pores allows the flow of ions and water, creating further membrane stress that finally leads to cellular swelling or shrinkage and the disruption of cytoskeletal structures (Chang and Reese, 1990). Additional factors affecting eukaryotic microorganisms include organellar disruption and loss of ribosomes (Harrison et al., 1997). Microbial inactivation is dependent on process parameters that can be controlled externally in addition to the intrinsic characteristics of both the microorganism and the product being treated (Table 25.2). For the majority of process parameters, there is a positive correlation with increasing microcidal activity, primarily as a result of increasing energy entering the system. The limiting factors are therefore the time and cost implications of increasing the parameters and the negative impacts of increased energy input on nutritional components, as discussed below. Although limited data are currently available, it appears that square wave pulses in which maximal applied voltage is rapidly achieved and then

TABLE 25.2 Factors Affecting the Efficacy of Pulsed Electric Field Treatments in Microbial Sanitization Process Parameters

Product Characteristics

Electric field strength (+) Pulse width (+) Pulse number (+) Total pulse duration (+)

Composition

Pulse shape Treatment temperature (+)

pH Water activity (+) Density (+) Viscosity (−)

Heterogeneity (−) Conductivity (−) Ionic strength (−)

Microbial Characteristics Cell size (+) Cell morphology Cellular complexity (+) Cell type (vegetative/ spore) Growth phase

Note: Further details are found in the text. +, microcidal effect increases as parameter increases; −, microcidal effect decreases as parameter increases.

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removed are more microcidal than pulses in which voltage decays exponentially (Zhang et al., 1994; Pothakamury et al., 1996). The ionic strength and conductivity of the processed food correlate negatively with the microcidal action of PEF, where low medium electrical conductivity exacerbates the differences between medium and microbial cytoplasm, thereby increasing ionic flow across microbial membranes (Pothakamury et al., 1996). Heterogeneity can lead to ineffective microcidal activities as a result of the formation of microenvironments in which PEF treatment may be less effective and increased viscosity will favor the formation of a heterogeneous fluid. On the contrary, increased water activity is associated with improved microcidal activity of PEF (Min et al., 2002) while higher densities limit temperature changes allowing greater energy input without adversely affecting nutritional qualities. Although pH has been shown to affect microcidal activity using a range of microbes (Vega-Mercado et al., 1996; Liu et al., 1997; Wouters et al., 2001b), the impact of pH was species dependent. Although different microbial spoilage organisms show very different responses to PEF treatments, a few generalizations can be drawn. Cellular size, morphology, and complexity all affect the microcidal activity of PEF, with smaller, simpler cells being more resistant than larger, more complex eukaryotic cells. For example, bacterial and fungal spores show greater resistance than vegetative bacterial cells, which in turn show greater resistance than yeasts (Grahl and Markl, 1996), while larger cells of the same species show greater susceptibility than smaller cells (Wouters et al., 2001b). In addition, actively dividing cells are more susceptible than those in the stationary phase of growth (Pothakamury et al., 1996; Wouters et al., 1999). Numerous studies have analyzed the impact of PEF treatments in reducing the microbial load in fruit juices, and these have been previously summarized (Wouters et al., 2001a; Min et al., 2007). Reductions in microbial counts greater than 5 log have been observed in apple, orange, cranberry, grape, and tomato juices. Studies have demonstrated a greater than 5 log reduction against specific microorganisms directly inoculated into the product, including Saccharomyces cerevisiae, Escherichia coli, and various Leuconostoc species. At the commercial scale, PEF treatments were observed to reduce both total aerobic microorganisms and yeasts and moulds by at least 6 log to <10 CFU mL−1 in both orange and tomato juice showing microbial shelf lifes of 196 and 112 days, respectively, at 4°C (Min et al., 2003a, 2003b). In addition to pore induction in microbial membranes, PEF can induce pore formation in the cells of plant material being processed for juice manufacture and several groups have exploited this capacity for the improvement of processing yields and efficiency. In apple juice production, the impact of PEF as an alternative to enzymatic mash maceration prior to pressing and juice collection has been examined by several laboratories. Perhaps due to the wide variation in experimental design, the results have been mixed, with some investigators demonstrating significant yield benefits following mash PEF treatment (Bazhal and Vorobiev, 2000; Lebovka et al., 2004; Schilling et al., 2007) while other researchers working at the pilot scale (220 kg apples) failed to demonstrate enhanced yields following mash treatments with either PEF or pectinases compared to untreated mash (Schilling et al., 2008). Despite the lack of consistent data regarding yields, PEF treatment may also provide benefits in terms of nutritional quality, where juices produced following PEF treatment of mash demonstrated enhanced antioxidant capacity and phenol content over juices produced by enzymatic maceration (Schilling et al., 2008). Such improvements were only observed under oxidative maceration treatments, suggesting that the higher phenol content of PEF-treated juices was a result of the reduced incubation time between mashing and pressing (Schilling et al., 2007). As a nonthermal microcidal treatment, PEF processing has been demonstrated to protect key antioxidant nutrients in a range of juice products when compared against standard thermal pasteurization treatments. Following PEF treatment, strawberry, orange, and tomato juices retained close to 100% of the vitamin C content of untreated control juices. On the contrary, juices sterilized by heat treatment (90–95°C, 30–90 s) lost between 5% and 20% of initial ascorbic acid immediately following treatment (Yeom et al., 2000; Min et al., 2003a, 2003b; Odriozola-Serrano et al., 2008a). Furthermore, juices sterilized by PEF retained more ascorbic acid during subsequent storage than

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either heat-treated or untreated juices such that orange juice retained 25 mg ascorbate per 100 mL [equivalent to the vitamin C recommended daily allowance (RDA) in a single serving] for between 14 and 16 days longer than heat-treated juice when stored at 4°C (Yeom et al., 2000; Min et al., 2003a). In the production of strawberry juice, PEF treatment also demonstrated benefits over conventional thermal treatments with regard to the maintenance of levels of phenolic compounds. PEF treatment resulted in a marginal, nonsignificant decline in total phenolic content compared to untreated juices, which was equivalent to that observed in juices that had been thermally processed at 90°C for 30 s. Increasing the thermal treatment period to 60 s resulted in a significant decline in the total phenol content from 47.3 mg 100 g−1 to 43.6 mg 100 g−1. Both heat and PEF treatment resulted in a slower degradation of phenolic compounds than observed in untreated juice on storage, with PEF treatment being the most effective (Odriozola-Serrano et al., 2008a). The reasons for the slower degradation kinetics of both ascorbic acid and anthocyanins are unclear, although mechanisms may include the inactivation of oxidative or hydrolytic enzymes as has been demonstrated for lipoxygenase and β-glucosidase (Aguiló-Aguayo et al., 2009) and lower initial production of oxidizing compounds in PEF than in heat-treated juices. In addition to hydrophilic antioxidants, extensive research has been undertaken regarding the impact of PEF treatment on lipophilic antioxidants, with particular emphasis on carotenoids. Heat treatments have been found to have negative impacts on total carotenoid content in orange juice, with thermal treatment reducing total carotenoids and vitamin A by approximately 12.5% and 15.6%, respectively (Cortés et al., 2006a, 2006b). On the contrary, heat treatment only marginally reduced the lycopene content of tomato juice (<2%) and enhanced the concentration of both carotenoids (9%) and vitamin A (8%) in a 20% carrot–orange juice mix (Min et al., 2003b; Torregrosa et al., 2005). PEF treatments proved to be beneficial in comparison, resulting in only a 6.7% and 7.5% loss in carotenoids and vitamin A, respectively, from orange juice, a marginally improved retention of lycopene in tomato juice when compared with heat treatment, and significant further increases in carotenoid and vitamin A yields in carrot–orange juice over those observed for heat treatments. As observed for ascorbic acid, PEF treatments resulted in a greater retention of carotenoids and vitamin A on storage (Min et al., 2003a, 2003b; Cortés et al., 2006a). Analyses of changes in individual carotenoids following a range of PEF treatments (variable field strengths and treatment times) in orange and carrot–orange juice revealed varying effects on individual carotenoids. For example, treatment of orange juice at 35 or 40 kV cm−1 resulted in improved recoveries of 13-cis-violaxanthin and 7,8,7′,8′tetrahydrolycopene while the concentrations of β-cryptoxanthin, phytoene, and β-carotene decreased (Cortés et al., 2006b). One mechanism proposed was PEF-induced cis/trans isomerization. Similar results were observed in carrot–orange juice mixtures; however, under appropriate conditions the concentrations of all carotenoids were increased, perhaps suggesting improved extraction and recovery prior to high performance liquid chromatography (HPLC) analysis as a result of PEF-induced macromolecular disruption (Torregrosa et al., 2005). This discussion highlights the requirement to define optimal PEF treatments for maximal nutritional benefit. A number of studies have addressed these issues, and it has been observed that while ascorbic acid concentration is best retained following low-intensity treatments (low field strengths, fewer, lower frequency and narrower pulses), higher phenolic and carotenoid retention is favored following intermediate- to high-intensity treatments (Odriozola-Serrano et al., 2007, 2008b, 2009; Oms-Oliu et al., 2009).

25.3.2

HIGH HYDROSTATIC PRESSURES

High hydrostatic pressures (HHPs) represent an alternative method for the inactivation of microbial activity through the induction of membrane damage under reduced heating regimes, providing better retention of many antioxidant compounds (Knorr, 2003). It was originally developed as an alternative to pasteurization and sterilization for the reduction or removal of microbial activity (Guerrero-Beltran et al., 2005) and the inactivation of enzyme activities associated with loss of quality (Rastogi et al., 2007). For example, HHPs have been used to inactivate pectin methylesterase

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in orange juice, thereby preventing the hydrolysis of pectin and maintaining the desirable cloud (Polydera et al., 2004). The fact that high-pressure-treated, extended shelf life products are available in Europe, the United States, and Japan is testament to the utility and cost effectiveness of the process (Butz et al., 2004; San Martin et al., 2002). HHPs have been shown to be highly effective in reducing microbial activity in a range of fruit purees and juices. For example, the treatment of carrot and apple juices at 250 MPa (~25 atm) at 35°C for 15 min was sufficient to reduce the initial bacterial loads of 4.5–5.5 log CFU mL −1 to below the limit of detection (<1 CFU mL −1) over subsequent storage for 30 days at 25°C (Dede et al., 2007). High-pressure treatments have also been demonstrated to be specifically effective against pathogenic bacteria, with E. coli O157 inactivated in apple and orange juices following treatment at 250 MPa for 20 min at 4°C or 25°C (Noma et al., 2004) and in apple, orange, apricot, and sour cherry juices following treatment at 250 MPa for 5 min at 40°C (Bayindirli et al., 2006). Bayindirli et al. (2006) also showed that other pathogenic microorganisms, including Staphylococcus aureus and Salmonella enteritidis, were inactivated under similar conditions; however, Listeria monocytogenes was more resistant to high pressures, requiring 300 and 400 MPa for 20 min for complete inactivation in peach and orange juice, respectively (Dogan and Erkmen, 2004). Given that commercial operation tends to be in excess of 400 MPa (Buzrul et al., 2008), the majority of vegetative cells are likely to be inactivated; however, spores are resistant to pressures up to 1200 MPa (Cheftel, 1995), requiring specific techniques such as high-pressure sterilization at temperatures in excess of 100°C for inactivation. Such techniques are known to negatively affect the functional properties of foods (Oey et al., 2008). The low operating temperatures usually applied under HHP processing result in protection of antioxidant vitamins and total antioxidant capacity in the material processed. Ascorbic acid shows little degradation under typical high-pressure processing conditions in citrus juices (SánchezMoreno et al., 2005). Similarly, ascorbic acid in noncitrus juices and nectars, including juices of carrot and tomato (Dede et al., 2007) and purees of guava (Yen and Lin, 1996) and strawberry (Sancho et al., 1999), was stable under high-pressure treatment. High-pressure treatment compared well with thermally processed juices, maintaining higher or equivalent levels of ascorbic acid in tomato, carrot, and orange juices (Bull et al., 2004; Dede et al., 2007; Sánchez-Moreno et al., 2005) and strawberry and guava purees (Sancho et al., 1999; Yen and Lin, 1996). Literature regarding the postprocessing stability of ascorbic acid in fruit juices presents mixed results, with some researchers reporting improved stability in pressure-treated juices compared with untreated or thermally treated juices (Dede et al., 2007; Yen and Lin, 1996) and others reporting few differences (Sancho et al., 1999; Bull et al., 2004). In one report, pressure-treated orange juice rapidly lost ascorbic acid on storage; however, no comparisons with other treatments were made (Nienaber and Shellhamer, 2001). Differences in the rates of ascorbic acid loss on storage could be attributed to the impact of microbial activity, packaging materials (García et al., 2001), or differences in gaseous headspace (Polydera et al., 2003). Plant foods represent the most important contributor to dietary folate in the adult population; therefore, the impact of processing on stability and availability of folate is an important area of study (Scott et al., 2000). While there are a number of studies examining the kinetics of folate degradation in model systems, fewer reports are available concerning its stability under pressure in foods and juices. However, several forms of folic acid did show reasonable stability in orange juice following pressure treatment at 600 MPa for 5 min at 25°C: 5-methyl-tetrahydrofolate (5-methylTHF) showed greater stability than 5-formyl-THF, which in turn was more stable than THF. Under these conditions all forms retained at least 80% of the content found in untreated juices; however, at a higher temperature of 80°C stability was reduced, and although more than 80% of the 5-methylTHF was retained less than 80% and 70% THF and 5-formyl-THF were retained, respectively (Butz et al., 2004). Similar results were obtained by Indrawati et al. (2004), who observed a strong protective effect of ascorbic acid. In this study 5-methyl-THF was relatively baro- and thermostable in both orange juice and kiwi puree containing relatively high levels of ascorbic acid; however,

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stability in carrot juice containing low levels of ascorbic acid was greatly reduced and the stability could be improved by its addition. High-pressure treatments had the additional benefit of increasing the bioavailability of folate in orange juice with an increased free 5-methyl-THF concentration following pressure treatments of 150 and 200 MPa (Indrawati et al., 2004). In addition to analysis of the impact of high-pressure treatment on water-soluble vitamins, a number of studies have examined its impact on total antioxidant capacity. This parameter has normally been measured as the capacity of juices to scavenge either the 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS+•) or the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) and is generally reported as trolox equivalent antioxidant capacity (TEAC). Different authors have used reaction media of different polarity, affecting the antioxidant contribution from the lipophilic phase and making direct comparison between laboratories difficult; however, a number of generalizations can be made. Where direct comparisons have been undertaken, high pressure-treated tomato or carrot juice retained more antioxidant capacity than juices given a thermal treatment, resulting in equivalent microbial reduction (Dede et al., 2007). On the contrary, in a detailed kinetic analysis of the evolution of antioxidant capacity in thermally treated orange or carrot juice in the presence or absence of HHPs, an increase in antioxidant capacity following thermal treatment at atmospheric pressure that was substantially inhibited by high pressures was observed (Indrawati et al., 2004). However, the authors suggested that the increased antioxidant capacity observed under atmospheric pressure heating was a result of the generation of Maillard reaction products that had adverse effects on appearance and could potentially result in the formation of mutagenic compounds. For this reason, high-pressure processing (300–600 MPa) at mildly elevated temperatures (40–50°C) was recommended for the maintenance of antioxidant capacity and juice quality. Other authors failed to observe an improvement in the antioxidant capacity of thermally pasteurized orange juice (80°C, 60 s) and found that high-pressure-treated juice (600 MPa, 40°C, 4 min) had a higher initial antioxidant capacity that was also more stable to storage (Polydera et al., 2005). In one study, high-pressure treatment of orange juice significantly improved the recovery of the major flavonones naringenin and hesperetin, with treatment at 350 MPa for 2.5 min at 30°C improving recovery by 13% and 34%, respectively, against untreated juice (Sánchez-Moreno et al., 2003). Despite the improvement in flavonone recovery, total antioxidant capacity was unchanged from that of untreated juice, probably because the two flavonones combined accounted for less than 10% of the total antioxidant capacity of orange juice (Miller and Rice-Evans, 1997). Regarding lipophilic antioxidants, several studies have demonstrated that high-pressure treatment improves the recovery of carotenoids in orange juice (de Ancos et al., 2002; SánchezMoreno et al., 2005) and persimmon puree (de Ancos et al., 2000), probably as a result of enhanced carotenoid release from denatured proteins and membranes. Enhancements of carotenoid recovery of up to 54% and 19% were observed in orange juice and persimmon puree, respectively. Recoveries of carotenoids were not linearly correlated with pressure, with low (50 MPa) and high (350 MPa) pressures resulting in greater carotenoid recovery from orange juice than intermediate pressures (de Ancos et al., 2002). These data may explain the lack of impact of high-pressure treatment on carotenoid recovery from orange juice treated at 600 MPa for 1 min (Bull et al., 2004). In general, high-pressure treatment appears to be an effective way of controlling microbial activity in fruit juices without the loss of potentially health-beneficial antioxidants that are frequently unstable under thermal pasteurization and sterilization procedures. In addition, thermal treatments may actually improve the recovery and bioavailability of specific antioxidant compounds such as folates and carotenoids, which are found in close association with other macromolecules. Despite the useful data obtained to date, there are still several gaps in our knowledge regarding the impact of high-pressure treatments on a number of antioxidant compounds such as tocopherols and polyphenols. Future research should be geared toward filling in these important gaps in our knowledge.

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25.3.3 DENSE PHASE CO2 The capacity of dense phase CO2 to cause microbial inactivation was first observed in the 1950s (Fraser, 1951), although it was not until the late 1980s and early 1990s that researchers began to recognize its potential as a nonthermal, antimicrobial treatment for fruit juices. Continuous systems were introduced in the late 1990s, improving throughput and antimicrobial efficiency (Shimoda et al., 2001; Kincal et al., 2005). Two commercial systems were produced by Praxair and Air Liquide in the early 2000s (Damar and Balaban, 2006) and at least one system was installed by a U.S.-based juice manufacturer (Anonymous, 2003), although it is currently unclear whether commercial systems are still available or whether any dense phase CO2-treated juices are marketed. Dense phase CO2 sterilization is undertaken at pressures significantly lower than those used for HHP treatment, being typically in the region of 5–30 MPa (Spilimbergo and Bertucco, 2003). Several mechanisms have been proposed for microbial inactivation by dense phase CO2, including physical disruption of cells following pressure release, modification of cell membrane properties and permeability, reduction of cytoplasmic pH, inactivation of enzymes through the formation of arginine–bicarbonate complexes, and precipitation of intracellular Ca2+ and Mg2+ (Damar and Balaban, 2006). It appears that specific properties of CO2 are responsible for microcidal action as other gases (nitrogen and argon) were ineffective when used under identical conditions; however, high-pressure nitrous oxide (N2O) has also been demonstrated to exhibit antimicrobial properties (Enomoto et al., 1997; Spilimbergo et al., 2007). The microcidal activity of dense phase CO2 is comparable with pasteurization and performs favorably in comparison with other nonthermal technologies. In general, continuous systems show greater levels of inactivation than batch systems: for example, a 60 min treatment at 30 MPa was required to produce a 6 log reduction of E. coli in apple juice in a batch system (Liao et al., 2007), whereas 12 min at 20 MPa was sufficient to induce a similar reduction in a continuous system (Kincal et al., 2005). In addition to microcidal activity against bacteria, dense phase CO2 has been demonstrated to be effective against fungi (Spilimbergo et al., 2007) and even highly resistant spores, where a greater than 6 log inactivation of Saccharomyces cerevisiae and Alicyclobacillus acidoterretis spores was observed within 10 min of dense phase CO2 treatment of orange juice (Sims and Estigarribia, 2002). It has been proposed that the high inactivation capacity of dense phase CO2 against spores may be a result of its ability to cause germination (Furukawa et al., 2004). The few studies that have examined the impact of dense phase CO2 treatment on the nutritional quality of juices have shown that antioxidant nutrients are generally well maintained and are not degraded as a result of treatment. In addition, the juices demonstrated improved storage stability. Likely mechanisms of protection include the inactivation of oxidative enzymes (Park et al., 2002) and the displacement of dissolved oxygen (Del Pozo-Insfran et al., 2006a). In muscadine grape juices treated with either dense phase CO2 (34.5 MPa, 6.25 min, 8–16% CO2) or heat pasteurization (75°C, 15 s), the latter treatment caused an immediate loss of approximately 20% of anthocyanins, soluble phenolics, and antioxidant capacity and 10% of ascorbic acid compared with untreated juice while there were no significant differences between untreated juice and dense phase CO2treated juice (Del Pozo-Insfran et al., 2006a, 2006b). Dense phase CO2 reduced the yeast and mould counts by at least 5 log and microbiological stability was identical between CO2 and heattreated juice for at least 5 weeks (Del Pozo-Insfran et al., 2006a). Not only did CO2-treated juices display superior nutritional quality to thermally treated juices immediately following processing, but the rate of loss of anthocyanins, soluble phenolics, antioxidant capacity, and ascorbic acid was reduced on storage in CO2-treated juices. One potential problem identified was the acidification of juice caused by the dissolution of CO2; however, neither pH nor titratable acidity changed following CO2 treatment and, furthermore, CO2-treated juices retained better organoleptic qualities than thermally treated juices. Similar experiments undertaken with apple juice using either dense phase CO2 or N2O (batch process, 10 MPa, 10 min) leading to complete inactivation of spoilage microorganisms had no impact on the levels of ascorbic acid or total polyphenols, although there were

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perceptible changes in juice quality and marked differences in a number of volatile aroma compounds (Gasperi et al., 2009).

25.3.4

ULTRASOUND

Ultrasound has found a wide range of applications in the food industry, including nondestructive inspection, characterization of physicochemical properties (texture and viscosity), surface cleaning of foods and food processing equipment, enzyme inactivation, and ultrasound-assisted extraction, crystallization, emulsification, filtration, drying, and freezing (Knorr et al., 2004). Like the PEF and HPP treatments, ultrasound treatments have been investigated for their capacity to inactivate microorganisms, which occurs by disruption to the cellular membrane caused by cavitation in which microbubbles are induced in the liquid. Subsequent collapse generates exceptionally high local temperatures (5500°C) and pressures (50 MPa). Despite these high local changes, through careful process control, bulk temperature elevation can be kept to a minimum, resulting in improved product quality and nutritional characteristics. To date, experiments to determine the impacts of ultrasound on microbial inactivation have shown mixed results, with many studies suggesting that ultrasound in isolation will be insufficient to meet the 5 log reduction required by the U.S. FDA. Thus Cheng et al. (2007) observed only marginal reductions of total microbial load from 4.05 to 3.94 log CFU mL −1 in untreated and sonicated (35 kHz, 30 min) guava juice, respectively. Similarly, yeast and mould survival was only marginally reduced from 3.22 to 3.06 log CFU mL −1. As samples were treated in a commercially available sonic cleaning bath and no efforts were made to record energy input, it could be argued that treatments were insufficiently aggressive to have significant antimicrobial impact. However, detailed experiments analyzing a number of process parameters in both batch and continuous processing in orange juice also failed to confirm the utility of ultrasound for commercial pasteurization (Valero et al., 2007). In an initial series of experiments, juices were batch treated for 15 min at frequencies of either 500 or 23 kHz and power ratings between 120 and 600 W. All treatments caused a rise in temperature from 18°C to between 35°C and 88°C. Under these conditions, the maximum log reduction was 1.7 log CFU mL−1 when using 23 kHz frequency at 600 W; however, this treatment also caused the maximum rise in temperature, which was shown to have a considerable impact on microorganisms in the absence of sonication. Treatment at high frequency using 240 W caused a 1.08 log decrease in total aerobic plate count while raising the temperature to only 51°C, a temperature shown to have a negligible impact on microorganisms alone. Clearly, the impact of sonication was well below the 5 log reduction required by the U.S. FDA, and treatments were almost completely ineffective when considering moulds and yeasts. Suspended solids also had a negative impact on microcidal activity, where juice containing pulp added at a ratio of 1% or 10% prevented any microbial inactivation in a continuous system where ultrasound was applied to circulating juice for up to 180 min. Similarly, a variety of ultrasound treatments (up to 700 W, 60 min) were shown to be insufficiently effective at inactivating Alicyclobacillus acidoterrestris, a major spoilage organism in apple juice (Yuan et al., 2009). On the contrary, one study found sonication to be capable of reducing E. coli by up to 5 log cycles in apple cider, a traditional, nonalcoholic, minimally processed apple juice (Ugarte-Romero et al., 2006). In these experiments, apple cider was treated at a range of temperatures (40–60°C) with 20 kHz ultrasound and an acoustic energy density of 0.46 W mL −1. In the absence of sonication, E. coli inactivation at 40°C and 45°C was negligible and only a 0.67 log reduction was observed after 20 min at 50°C. Effective microbial inactivation was not achieved until temperatures were raised to 60°C, where a 5 log reduction was observed within 4 min. In the presence of sonication, E. coli inactivation kinetics were significantly enhanced at lower temperatures so that a 5 log reduction was achieved following around 15 min treatment at 40°C, 45°C, or 50°C or in just longer than 10 min at 55°C. At 60°C, sonication had little impact on inactivation kinetics and cell inactivation was only increased by 0.1 log cycles over temperature treatment alone.

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An alternative to ultrasound-induced cavitation is hydrodynamic cavitation induced by subjecting liquid foods to extreme flow conditions. In one experiment in which cavitation was induced by subjecting juices to mixing in a high-speed rotor, a 5-log reduction of Lactobacillus plantarum, Lactobacillus sakei, and Zygosaccharomyces bailii in apple juice was achieved along with a concomitant rise in temperature up to 77° (Milly et al., 2007). Taken together, the available data suggest that while ultrasound may be insufficient alone to achieve the microbial reduction required, combination with other treatments may offer an opportunity to reduce the harshness of microbial inactivation treatments, thereby improving the nutritional quality of juices. Although several studies have analyzed the impact of sonication on juice quality, direct comparisons with alternative sterilization technologies (e.g., thermal treatments) are limited as are studies that combine analysis of juice quality with impact on microbial inactivation. In one study, treatment of guava juice in an ultrasonic cleaning bath at 35 kHz resulted in improved ascorbic acid content. Moreover, it was suggested that sonication caused the elimination of dissolved oxygen, resulting in lower oxidative degradation (Cheng et al., 2007). However, the same study showed that PPO activity, a key enzyme implicated in reduced nutritional and sensory quality as a result of enzymatic browning, was increased. Furthermore, the treatment conditions used were only marginally effective for microbial activation. In recent years, a group based at University College Dublin has been particularly active in examining the impact of ultrasound on the nutritional quality of juices. Ascorbic acid degradation was observed to be dependent on treatment time and acoustic energy density when treated at a constant frequency of 20 kHz in both strawberry (Tiwari et al., 2009a) and orange juice (Tiwari et al., 2009b), although maximal losses were below 15% and 5%, respectively. Sonicated orange juice samples had slower ascorbic acid degradation kinetics on storage, although the same effect was not observed in strawberry juices. Anthocyanins were found to be more resistant to ultrasound-induced degradation than ascorbic acid in both strawberry and blackcurrant juice, exhibiting a maximum loss of 5% (Tiwari et al., 2009a, 2009c). Both increased acoustic energy density and treatment time resulted in greater degradation; however, at low treatment times and energy densities an enhancement of anthocyanin content was observed in strawberry juice, probably as a result of anthocyanin release from suspended pulp particles. At present, only limited data are available to allow assessment of the potential for ultrasound in juice processing. Although ultrasound appears to cause low losses of important nutritional components, further comparative studies are required. However, the mixed results regarding microbial inactivation, the potential energy inputs required, and the difficulty in designing high-throughput continuous systems suitable for commercial-scale processing suggest that it is likely to lag behind other alternatives to thermal pasteurization.

25.3.5

ULTRAVIOLET LIGHT

Ultraviolet (UV) light is currently commercially used as an alternative to thermal pasteurization in the production of a number of fruit juices, including apple cider produced in Northeast America (Hanes et al., 2002) and a number of juices produced in South Africa (Keyser et al., 2008). One of the most appealing aspects concerning the use of UV is its potential microcidal activity at low temperatures; however, the use of UV in the processing of fruit juices has not received the same attention as some other potential processes. Some literature is available regarding its germicidal activity in fruit juices, and a small number of papers dealing with its impact on quality and nutritional parameters will be outlined here. An excellent recent review provides further details regarding theory and practical considerations for the UV treatment of juices (Koutchma, 2009). One problem associated with UV inactivation of microbes in fruit juices is related to the high absorbance coefficients of the products, resulting in 90% absorbance as light penetrates into the first 10–100 μm of juice. This problem has been overcome by reactor design in which juices are exposed to light either under a narrow laminar flow or under conditions of high turbulence, where the juice is rapidly mixed resulting in all parts being exposed to the UV-producing surface. Laminar flow

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systems were shown to effectively reduce the loads of E. coli, Listeria innocua, and the protozoan parasite Cryptosporidium parvum by at least 5 log cycles in apple cider; however, microcidal activity against Saccharomyces cerevisiae was not as effective, causing only a 1.34 log reduction under equivalent process conditions (Hanes et al., 2002; Guerrero-Beltrán and Barbosa-Cánovas, 2005). The inefficiency of UV treatment in reducing yeast and mould numbers has also been reported elsewhere (Choi and Nielsen, 2005) and it has been suggested that hot filling (63°C) of apple cider bottles is more effective at reducing their numbers (Tandon et al., 2003). Similarly, UV was less efficient in reducing yeasts and mould (~2 log reduction) than total aerobes (~3 log reduction) in orange juice (Tran and Farid, 2004). The impact of juice type and UV dose was examined in a turbulent system by Keyser et al. (2008), who did not observe consistent differences between the susceptibility of total aerobes and yeasts and moulds but did observe a strong reduction in microcidal activity in turbid (orange and tropical mix) or concentrated (strawberry and mango nectar) compared with less turbid juices (apple, guava, and pineapple). Nutritional components with absorption maxima close to the UV output maximum (~254 nm) of current commercial systems are those most likely to suffer detrimental effects from UV treatment. These include ascorbic acid with maximal absorbances at 243 and 265 nm in acid and neutral media, respectively (Hancock et al., 2008), a number of carotenoids that have absorption maxima in the range 240–280 nm (Du et al., 1998), and several classes of phenolic compounds such as flavonoids, which include the important color-providing anthocyanins with absorption maxima in the range 250–275 nm (Harborne, 1989). Such predictions were confirmed following UV treatment of orange juice, where losses of β-carotene and riboflavin were close to 50% while ascorbic acid losses were in the region of 20% (Koutchma, 2009).

25.3.6 IONIZING RADIATION A number of authors have investigated the impact of ionizing radiation on the microbiological safety and nutritional quality of fruit juices. While irradiation was shown to be highly effective in reducing microbial contamination in a range of juices (Buchanan et al., 1998; Wang et al., 2006; Song et al., 2007), it has been demonstrated to have negative impacts on ascorbic acid content (Fan et al., 2002; Song et al., 2007), anthocyanin content (Alighourchi et al., 2008), and organoleptic quality (Wang et al., 2006; Song et al., 2007). In addition, ionizing radiation has been shown to promote the formation of potentially toxic aldehydes (formaldehyde, acetaldehyde, and malondialdehyde) in apple juice (Fan and Thayer, 2002). For these reasons, irradiation of fruit juices is unlikely to provide any nutritional benefits over standard pasteurization technologies.

25.4

CONCLUSIONS

As consumers continue to seek products with improved nutritional value and functionality, juice producers have the opportunity of improving product marketability through the use of novel technologies that maintain levels of key phytonutrients. Indeed, many juices are currently marketed for their proposed nutritional benefits, including many brands that claim high antioxidant capacity (e.g., POM Wonderful pomegranate juice and Ribena blackcurrant juice). Laboratory and pilot-scale research have demonstrated the advantages of nonthermal extraction and pasteurization technologies in maximizing the nutritional value and fresh qualities of fruit juices; however, with the exception of enzyme-aided extraction it remains unclear which, if any, of the technologies can provide sufficient advantage at an economically viable cost. As food labeling and nutritional claims rules become stricter (e.g., Official Journal of the European Union, http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2006:404:0009:00 25:EN:PDF), it will become necessary to provide further evidence in support of ascribed functional properties and simple statements such as “high in antioxidants” are unlikely to be sufficient. Indeed, there is a growing consensus within the scientific community that antioxidant capacity per se is not

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relevant to the health benefits of fruit and vegetable consumption and, in fact, pharmacological interactions with individual compounds that happen to have a high antioxidant capacity are more likely mediating such benefits (Stevenson and Lowe, 2009). In future it may therefore be necessary to conduct more detailed studies in which the impact of processing on individual compounds and their chemical transformations are examined.

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McGhie, T.K., Walton, M.C., Barnett, L.E., Vather, R., Martin, H., Au, J., Alspach, P.A., Booth, C.L., and Kruger, M.C., 2007. Boysenberry and blackcurrant drinks increased the plasma antioxidant capacity in an elderly population but had little effect on other markers of oxidative stress. J. Sci. Food Agric. 87: 2519–2527. Mihalev, K., Schieber, A., Mollov, P., and Carle, R., 2004. Effect of mash maceration on the polyphenolic content and visual quality attributes of cloudy apple juice. J. Agric. Food Chem. 52: 7306–7310. Miller, N.J. and Rice-Evans, C.A., 1997. The relative contributions of ascorbic acid and phenolic antioxidants to the total antioxidant activity of orange and apple fruit juices and blackcurrant drink. Food Chem. 60: 331–337. Milly, P.J., Toledo, R.T., Harrison, M.A., and Armstead, D., 2007. Inactivation of food spoilage microorganisms by hydrodynamic cavitation to achieve pasteurization and sterilization of fluid foods. J. Food Sci. 72: M414–M422. Min, S., Evrendilek, G.A., and Zhang, Q.H., 2007. Pulsed electric fields: Processing system, microbial and enzyme inhibition, and shelf life extension of foods. IEEE Trans. Plasma Sci. 35: 59–73. Min, S., Jin, Z.T., Yeom, H.W., Min, S.K., and Zhang, Q.H., 2003a. Commercial-scale pulsed electric field processing of orange juice. J. Food Sci. 68: 1265–1271. Min, S., Jin, Z.T., and Zhang, Q.H., 2003b. Commercial scale pulsed electric field processing of tomato juice. J. Agric. Food Chem. 51: 3338–3344. Min., S., Reina, L., and Zhang, Q.H., 2002. Water activity and the inactivation of Enterobacter cloacae inoculated in chocolate liquor and a model system by pulsed electric field treatment. J. Food Process. Preserv. 26: 323–337. Ness, A.R. and Powles, J.W., 1997. Fruit and vegetables, and cardiovascular disease: A review. Int. J. Epidemiol. 26: 1–13. Netzel, M., Strass, G., Kaul, C., Bitsch, I., Dietrich, H., and Bitsch, R., 2002. In vivo antioxidative capacity of a composite berry juice. Food Res. Int. 35: 213–216. Nienaber, U. and Shellhammer, T.H., 2001. High-pressure processing of orange juice: Combination treatments and a shelf life study. J. Food Sci. 66: 332–336. Noma, S., Tomita C., Shimoda M., and Hayakawa, I., 2004. Response of Escherichia coli O157:H7 in apple and orange juices by hydrostatic pressure treatment with rapid decompression. Food Microbiol. 21: 469–473. Odriozola-Serrano, I., Aguiló-Aguayo, I., Soliva-Fortuny, R., Gimeno-Añó, V., and Martín-Belloso, O., 2007. Lycopene, vitamin C, and antioxidant capacity of tomato juice as affected by high-intensity pulsed electric fields critical parameters. J. Agric. Food Chem. 55: 9036–9042. Odriozola-Serrano, I., Soliva-Fortuny, R., Gimeno-Añó, V., and Martín-Belloso, O., 2008b. Kinetic study of anthocyanins, vitamin C, and antioxidant capacity in strawberry juices treated by high-intensity pulsed electric fields. J. Agric. Food Chem. 56: 8387–8393. Odriozola-Serrano, I., Soliva-Fortuny, R., and Martín-Belloso, O., 2008a. Phenolic acids, flavonoids, vitamin C and antioxidant capacity of strawberry juices processed by high-intensity pulsed electric fields or heat treatments. Eur. Food Res. Technol. 228: 239–248. Odriozola-Serrano, I., Soliva-Fortuny, R., and Martín-Belloso, O., 2009. Impact of high-intensity pulsed electric fields variables in vitamin C, anthocyanins and antioxidant capacity of strawberry juice. LWT—Food Sci. Technol. 42: 93–100. Oey, I., Van der Plancken, I., Van Loey, A., and Hendrickx, M., 2008. Does high pressure processing influence nutritional aspects of plant based food systems? Trends Food Sci. Technol. 19: 300–308. Oms-Oliu, G., Odriozola-Serrano, I., Soliva-Fortuny, R., and Martín-Belloso, O., 2009. Effects of high-intensity pulsed electric field processing conditions on lycopene, vitamin C and antioxidant capacity of watermelon juice. Food Chem. 115: 1312–1319. Park, S.-J., Lee, J.-I., and Park, J., 2002. Effects of combined process of high pressure CO2 and high hydrostatic pressure on the quality of carrot juice. J. Food Sci. 67: 1827–1833. Polydera, A.C., Galanou, E., Stoforos, N.G., and Taoukis, P.S., 2004. Inactivation kinetics of pectin methylesterase of greek Navel orange juice as a function of high hydrostatic pressure and temperature process conditions. J. Food Eng. 62: 291–298. Polydera, A.C., Stoforos, N.G., and Taoukis, P.S., 2003. Comparative shelf life study and vitamin C loss kinetics in pasteurised and high pressure processed reconstituted orange juice. J. Food Eng. 60: 21–29. Polydera, A.C., Stoforos, N.G., and Taoukis, P.S., 2005. Effect of high hydrostatic pressure treatment on post processing antioxidant activity of fresh Navel orange juice. Food Chem. 91: 495–503. Pothakamury, U.R., Vega, H., Zhang, Q., Barbosa-Cánovas, G.V., and Swanson, B.G., 1996. Effect of growth stage and processing temperature on the inactivation of E. coli by pulsed electric fields. J. Food Protect. 59: 1167–1171.

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26 Probiotics Health Benefits, Efficacy, and Safety Nagendra P. Shah CONTENTS 26.1 Introduction .......................................................................................................................... 485 26.1.1 Commercially Important Probiotics ......................................................................... 485 26.2 Probiotic Bacteria ................................................................................................................. 487 26.3 Health Benefits of Probiotic Bacteria ................................................................................... 488 26.3.1 Effectiveness Against Diarrhea ................................................................................ 489 26.3.2 Improvement in Lactose Metabolism ....................................................................... 489 26.3.3 Antimutagenic Properties ......................................................................................... 490 26.3.4 Anticarcinogenic Properties ..................................................................................... 490 26.3.5 Reduction in Serum Cholesterol ............................................................................... 491 26.3.6 Inflammatory Bowel Disease ................................................................................... 491 26.3.7 Immune System Stimulation .................................................................................... 491 26.4 Efficacy ................................................................................................................................. 491 26.5 Safety of Probiotic Foods ..................................................................................................... 492 26.6 Conclusions ........................................................................................................................... 494 References ...................................................................................................................................... 494

26.1

INTRODUCTION

The word “probiotic” originates from the Greek meaning “for life.” Probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (Shah, 2007). Several health benefits derived from the consumption of foods containing Lactobacillus acidophilus, Bifidobacterium, and Lactobacillus casei are now well documented. These benefits include the control of gastrointestinal infections, improvement in lactose metabolism, antimicrobial and antimutagenic properties, reduction in serum cholesterol, immune system stimulation, and improvement in the infections of bowel disease.

26.1.1

COMMERCIALLY IMPORTANT PROBIOTICS

Probiotic cultures have been exploited extensively by the dairy industry as a tool for the development of novel functional products. It is estimated that there are approximately 70 probioticcontaining products marketed in the world (Shah, 2004), and this list is continuously expanding. Probiotics have traditionally been incorporated into yoghurt, edible spreads, and cheese (Ong et al., 2006) or cheese-based dips (Tharmaraj and Shah, 2004). Probiotic organisms are also available commercially in milk, sour milk, fruit juices, ice cream, single shots, and oat-based products. Lunebest, Olifus, Bogarde, and Progurt are some examples of commercial fermented dairy products 485 © 2010 Taylor and Francis Group, LLC

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containing probiotics available on the international market with a steady increase in the market shares. Probiotic products are very popular in Japan where there are more than 53 different types of probiotic-containing products on the market. Commercial cultures used in these applications mainly include strains of Lactobacillus spp. and Bifidobacterium spp.; some of these are listed in Tables 26.1 and 26.2. The probiotic strains are mainly used as adjunct cultures due to their poor growth in milk, which extends the fermentation time (Shah, 2004). Lactobacilli are ubiquitous in nature, found in carbohydrate-rich environments. They are Gram-positive, non-spore-forming microorganisms, catalase negative with noted exceptions, appearing as rods or coccobacilli. They are fermentative, microaerophilic, and chemoorganotrophic. Considering the DNA base composition of the genome, they usually have a guanine and cytosine (GC) content of less than 54 mol%. The genus Lactobacillus belongs to the phylum Firmicutes, class Bacilli, order Lactobacillales, family Lactobacillaceae, and its closest relatives are the genera Paralactobacillus and Pediococcus. This is the most numerous genus, comprised of 106 described species. L. acidophilus, L. salivarius, L. casei, L. plantarum, L. fermentum, L. reuteri, and L. brevis are the most common Lactobacillus species isolated from the human intestine (Mitsuoka, 1992). The functional properties and safety of particular strains of L. casei, L. rhamnosus, L. acidophilus, and L. johnsonii have been extensively studied and well documented. Bifidobacteria were first isolated and visualized by Tissier in 1900 from the faces of breast-fed neonates. These rod-shaped, non-gas-producing and anaerobic organisms were named Bacillus bifidus due to their bifurcated morphology. They are generally characterized as Gram-positive, nonspore-forming, nonmotile, and catalase-negative anaerobes with a special metabolic pathway which

TABLE 26.1 Lactobacilli Used as Probiotic Cultures Species L. acidophilus L. acidophilus L. acidophilus Johnsonii L. acidophilus L. acidophilus L. bulgaricus L. lactis L. casei Immunitas L. plantarum L. rhamnosus L. rhamnosus L. rhamnosus L. reuteri L. rhamnosus L. plantarum L. reuteri L. casei L. paracasei L. fermentum L. helveticus

Strains LA-1/LA-5 (Chr. Hansen) NCFM (Rhodia) La1 (Nestle) DDS-1 (Nebraska Cultures) SBT-2062 (Snow Brand Milk Products) Lb12 L1A (Essum AB) (Danone) 299v, Lp01 GG (Valio) GR-1 (Urex Biotech) LB21 (Essum AB) SD2112/MM2 (Biogaia) 271 (Probi AB) (Probi AB) SD2112 (also known as MM2) Shirota (Yakult) CRL 431 (Chr. Hansen) RC-14 (Urex Biotech) B02

Source: Adapted from Krishnakumar, V. and Gordon, I.R., 2001. Dairy Ind. Int. 66: 38–40; Holm, F., 2003. The World of Ingredients 2: 52–55; Playne, M.J. et al., 2003. Aust. J. Dairy Technol. 58: 242–204; Shah, N.P. 2004. Agro Food Ind. Hi Tech 15: 13–16.

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TABLE 26.2 Bifidobacterium Cultures Used as Probiotic Cultures Species Bifidobacterium adolescentis Bifidobacterium longum Bifidobacterium longum Bifidobacterium breve Bacillus bifidus Bifidobacterium lactis Bifidobacterium essensis Bifidobacterium lactis Bifidobacterium infantis Bifidobacterium infantis Bifidobacterium infantis Bifidobacterium infantis Bifidobacterium laterosporus Bifidobacterium lactis Bifidobacterium longum Bifidobacterium lactis

Strains ATCC 15703, 94-BIM BB536 (Morinaga Milk Industry) SBT-2928 (Snow Brand Milk Products) Yakult Bb-11 (Reclassified as Bifidobacterium animalis) Bb-12 (Chr. Hansen) Danone (Bioactivia) Bb-02 Shirota Immunitass 744 01 CRL 431 LaftiTM, B94 (DSM) UCC 35624 (UCCork) DR10/HOWARU Danisco

Source: Adapted from Krishnakumar, V. and Gordon, I.R., 2001. Dairy Ind. Int. 66: 38–40; Holm, F., 2003. The World of Ingredients 2: 52–55; Playne, M.J. et al., 2003. Aust. J. Dairy Technol. 58: 242–204; Shah, N.P., 2004. Agro Food Ind. Hi Tech 15: 13–16.

allows them to produce acetic acid in addition to lactic acid in the molar ratio of 3:2. Due to their fastidious nature these bacteria are often difficult to isolate and grow in the laboratory. The taxonomy of bifidobacteria has changed continuously since they were first isolated. They were initially assigned to the genera Bacillus, Bacteroides, Nocardia, Lactobacillus, and Corynebacterium, before being recognized as separate genera in 1974. Due to their high (>50 mol%) G + C content, bifidobacteria are phylogenetically assigned to the actinomycete division of the Gram-positive bacteria. This family consists of five genera: Bifidobacterium, Propionibacterium, Microbacterium, Corynebacterium, and Brevibacterium. New fermented products containing L. acidophilus, Bifidobacterium spp., L. casei Shirota, L. rhamnosus GG, and L. reuteri have been developed in Europe. However, L. acidophilus and Bifidobacterium spp. are most commonly used as probiotics. It is estimated that over 70 products containing L. acidophilus and Bifidobacterium spp., including yoghurt, buttermilk, frozen desserts, and milk powder are produced worldwide. Probiotic organisms are also available as powders, capsules, and tablets.

26.2 PROBIOTIC BACTERIA A number of genera of bacteria and yeast are used as probiotics, including Lactobacillus, Leuconostoc, Pediococcus, Bifidobacterium, and Enterococcus, but the main species believed to have probiotic characteristics are L. acidophilus, Bifidobacterium spp., and L. casei. Members of the genera Lactobacillus and Bifidobacterium have a long and safe history in the manufacture of dairy products and are also found as part of the gastrointestinal microflora. At present, 56 species of the genus Lactobacillus have been recognized (Table 26.1). L. acidophilus is the most commonly suggested organism for dietary use. Growth of L. acidophilus occurs at temperatures as high as 45°C, however, the optimum growth temperature is between 35–40°C. The

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organisms grow in slightly acidic media at pH of 6.4–4.5, but growth ceases when a pH of 4.0–3.6 is reached. The acid tolerance of the organisms varies from 0.3% to 1.9% titratable acidity, with an optimum pH of 5.5–6.0 (Curry and Crow, 2003; Shah, 2002). L. acidophilus tends to grow slowly in milk, leading to the risk of overgrowth of undesirable microorganisms. Ironically, most strains of L. acidophilus do not survive well in fermented milk due to the low pH, and it is difficult to maintain large numbers in the product. The poor growth is partly related to the low concentration of small peptides and free amino acids in milk, which would be insufficient to support bacterial growth. Bifidobacterium is a normal inhabitant of the human gastrointestinal tract. Recent in vivo scientific studies using animals or human volunteers have shown that consumption of live Bifidobacterium has an effect on the gut microflora. Selected strains survive stomach and intestinal transit and reach the colon in abundant numbers. Newborns are colonized with Bifidobacterium within days after birth and the population appears to be relatively stable until advanced age, when a decline in their numbers occurs. However, diet, antibiotics, and stress are reported to influence the population of Bifidobacterium in the intestine. There are presently 32 species in the genus Bifidobacterium (Table 26.2), 14 of which are isolated from human sources (i.e., dental caries, faces, and vagina), 12 from animal intestinal tracts or rumen, and 3 from honeybees. Bifidobacterium species found in humans are Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum are found in human infants; Bifidobacterium adolescentis and Bifidobacterium longum are found in human adults (Shah and Lankaputhra, 2002). The optimum pH for the growth of Bifidobacterium is 6.0–7.0, with virtually no growth at pH 4.5–5.0 and below or at pH 8.0–8.5 and above. Optimum growth occurs at a temperature of 37–41ºC; the minimum and maximum growth temperatures are 25–28ºC and 43–45ºC, respectively. The main probiotic organisms that are currently used worldwide belong to the genera Lactobacillus and Bifi dobacterium. A limited number of investigations have also been carried out into the potential properties of genera including Pediococcus, Leuconostocs, and Propionibacterium and also of Enterococcus faecium. Ent. faecium is more pH stable than L. acidophilus and produces bacteriocins against some enteropathogens. These properties make this organism attractive as a probiotic. From published reviews, the strains with the most published clinical data are L. rhamnosus GG (ATCC 53103), L. casei Shirota, L. johnsonii La1, L. acidophilus NCFB 1478, Bifidobacterium animalis Bb-12, L. reuteri, and Saccharomyces cerevisiae Boulardii (Shah, 2004, 2006b).

26.3

HEALTH BENEFITS OF PROBIOTIC BACTERIA

A number of health benefits are claimed for products containing probiotic organisms, including antimicrobial activity against gastrointestinal infections, improvement in lactose metabolism, antimutagenic properties, anticarcinogenic properties, reduction in serum cholesterol, antidiarrheal properties, immune system stimulation, improvement in inflammatory bowel disease, and suppression of Helicobacter pylori infection (Kurmann and Rasic, 1991; Shah, 2000a, 2006b, 2004). Some of the health benefits are well established, while other benefits have shown promising results in animal models. However, additional studies are required in humans to substantiate these claims. Health benefits imparted by probiotic bacteria are strain specific, and not species or genus specific. It is important to note that no strain will provide all the proposed benefits, not even strains of the same species, and not all strains of the same species will be effective against particular health conditions. The strains L. rhamnosus GG (Valio), S. cerevisiae Boulardii (Biocodex), L. casei Shirota (Yakult), and Bifidobacterium animalis Bb-12 (Chr. Hansen) have the strongest human health efficacy data with respect to management of lactose malabsorption, rotaviral diarrhea, antibiotic-associated diarrhea, and Clostridium difficile diarrhea (Playne et al., 2003; Shah, 2006a,

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2006b). There is sufficient evidence to support the view that oral administration of Lactobacillus and Bifidobacterium is able to restore the normal balance of microbial populations in the intestine (Shah, 2006b).

26.3.1

EFFECTIVENESS AGAINST DIARRHEA

A major problem associated with antibiotic treatment is appearance of diarrhea, often caused by Cl. difficile. This organism is found in small numbers in the healthy intestine, but disruption of indigenous microflora due to antibiotic treatment leads to an increase in their number and in toxin production, which causes the development of diarrhea. Treatment with metonidazole or vancomycin is usually effective but recurrences are common. Probiotics have proved to be useful as a prophylactic regimen with antibiotic-associated diarrhea as well as for treatment after the onset of antibioticinduced diarrhea. A daily dose of Lactobacillus GG has been found to be effective in the termination of diarrhea. Studies with a yeast preparation containing S. cerevisiae Boulardii has also been effective in treatment of Cl. difficile-related colitis (Shah, 2004, 2006b). Rotavirus is one of the most common causes of acute diarrhea in children worldwide. During the diarrheal stage of infection, the permeability of gut epithelial cells is increased to intact proteins. Probiotics are claimed to shorten the duration of rotavirus diarrhea in children (Saavedra et al., 1994). The strongest evidence of a beneficial effect of defined strains of probiotics has been established using L. rhamnosus GG and Bifidobacterium animalis Bb-12 for the prevention and treatment of diarrhea and acute diarrhea in children mainly caused by rotaviruses. Selected probiotic strains are also effective against antibiotic-associated diarrhea. Certain probiotic strains can inhibit the growth and adhesion of a range of enteropathogens. Studies have indicated beneficial effects against pathogens such as Salmonella typhimurium and Salmonella enteriditis. Bifidobacterium longum SBT-2828 has shown inhibition of enterotoxigenic Escherichia coli. A pediatric beverage containing a mix of Bifidobacterium animalis, L. acidophilus, and L. reuteri has been found useful in the prevention of rotavirus diarrhea (Guandalini et al., 2000). There is also strong evidence that probiotic strains can prevent traveler’s diarrhea (Hilton et al., 1997), which is caused by bacteria, particularly enterotoxigenic E. coli. Several studies have been carried out to assess the effects of probiotic preparations as prophylaxis for traveler’s diarrhea; however, the results are conflicting. In one study, Danish tourists on a 2-week trip to Egypt were given a mixture of live freeze-dried preparation of L. acidophilus, Bifidobacterium animalis, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus at a daily dose of 109 cfu. The administration of this probiotic preparation reduced the frequency of diarrhea. A similar study conducted with Finnish tourists using a lyophilized preparation of Lactobacillus GG also showed a reduction in the occurrence of traveler’s diarrhea. On the other hand, Katelaris et al. (1995) and de dios PozoOlano et al. (1978) found no effect in people suffering from traveler’s diarrhea when L. fermentum KLD and L. acidophilus, and Lactobacillus bulgaricus were given in separate studies. Yoghurt containing Bifidobacterium longum was found to be effective in reducing the course of erythromycin-induced diarrhea. Fecal counts of Lactobacillus GG indicated that the organisms colonized the intestine despite erythromycin treatment. Probiotic preparations containing 4 × 109 cfu of Bifidobacterium animalis Bb-12 and L. acidophilus La-5 showed similar results when volunteers received ampicillin along with the probiotic preparation. Several studies have shown a reduction in diarrhea in subjects taking S. cerevisiae Boulardii during the period of antibiotic treatment (Shah, 2004, 2006a).

26.3.2

IMPROVEMENT IN LACTOSE METABOLISM

Relief of the symptoms of lactose malabsorption is probably the most widely accepted health benefit of probiotic organisms. Lactose malabsorption is a condition in which lactose, the principal carbohydrate of milk, is not completely hydrolyzed into its component monosaccharides, glucose,

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and galactose. Since lactose is cleaved into its constituent monosaccharides by the enzyme β-d-galactosidase, lactose malabsorption results from a deficiency of this enzyme. Lactose malabsorbers often complain of “gastric distress” after consuming fresh, unfermented milk or milk products due to the formation of hydrogen gas by microbial action on undigested lactose in the gut (Onwulata et al., 1989; Savaiano et al., 1984; Shah, 1993; Shah et al., 1992). The traditional cultures used in making yoghurt (i.e., L. delbrueckii ssp. bulgaricus, and Str. thermophilus) contain substantial quantities of β-d-galactosidase (Shah, 2000b), and so both yoghurt and probiotic yoghurt are tolerated well by lactose malabsorbers (McDonough et al., 1987). However, reduced levels of lactose in the fermented products due to partial hydrolysis of lactose during fermentation are only partly responsible for this greater tolerance for yoghurt. The slower gastric emptying of both semi-solid and pasteurized yoghurt results in better absorption of lactose (in the latter, enzyme activity and bacteria are destroyed due to heat treatment; Shah et al., 1992).

26.3.3 ANTIMUTAGENIC PROPERTIES An antimutagenic effect of fermented milks has been detected against a range of mutagens and promutagens in various test systems based on microbial and mammalian cells. Probiotic organisms are reported to bind mutagens to the cell surface (Orrhage et al., 1994). Probiotic bacteria are reported to reduce fecal enzymatic activity, including β-glucuronidase, azoreductase, and nitroreductase, which are involved in activation of mutagens (Goldin and Gorbach, 1984). Lankaputhra and Shah (1998) studied the antimutagenic activity of organic acids produced by probiotic bacteria against several mutagens and promutagens. The TA-100 mutant of Sal. typhimurium (His−) strain was used as a mutagenicity indicator organism. The mutagenicity test was carried out using the Ames Salmonella test. In their study, butyric acid showed a broad-spectrum antimutagenic activity against all mutagens or promutagens studied and live bacterial cells showed higher antimutagenicity than killed cells. Inhibition of mutagens and promutagens by probiotic bacteria was permanent for live cells and temporary for killed cells. The results emphasized the importance of consuming live probiotic bacteria and of maintaining their viability in the intestine to provide efficient inhibition of mutagens.

26.3.4

ANTICARCINOGENIC PROPERTIES

Bacteria and metabolic products such as genotoxic compounds (nitrosamine, heterocyclic amines, phenolic compounds, and ammonia) are responsible for colorectal cancer. The consumption of cooked red meat, especially barbequed meat, and a low consumption of fiber are reported to play a major role in causing colorectal cancer. The colonic flora is also reported to cause carcinogenesis mediated by microbial enzymes such as β-glucuronidase, azoreductase, and nitroreductase, which convert procarcinogens into carcinogens. Certain strains of L. acidophilus and Bifidobacterium spp. are reported to decrease the levels of enzymes such as β-glucuronidase, azoreductase, and nitroreductase responsible for activation of procarcinogens and consequently decrease the risk of tumor development (Yoon et al., 2000). Short-chain fatty acids produced by L. acidophilus, Bifidobacterium, L. plantarum, and L. rhamnosus are reported to inhibit the generation of carcinogenic products by reducing enzyme activities (Cenci et al., 2002). The anticarcinogenic effect of probiotic bacteria is reported to be due to the removal of sources of procarcinogens (or the enzymes that lead to their formation), an improvement in the balance of intestinal microflora, normalized intestinal permeability (leading to prevention or delaying of toxin absorption), strengthening of intestinal barrier mechanisms, and activation of nonspecific cellular factors (such as macrophages and natural killer cells) via regulation of γ-interferon production. Orally administered Bifidobacterium is also reported to play a role in increasing production of IgA antibodies and functions of Peyer’s patch cells (Singh et al., 1997).

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26.3.5

491

REDUCTION IN SERUM CHOLESTEROL

The level of serum cholesterol is a major factor for coronary heart disease, and elevated levels of serum cholesterol, particularly LDL-cholesterol, have been linked to an increased risk (Liong and Shah, 2005, 2006a). There is a high correlation between dietary saturated fat or cholesterol intake and serum cholesterol level. The feeding of fermented milk containing very large numbers of probiotic bacteria (~109 bacteria/g) to hypercholesterolemic human subjects has resulted in lowering cholesterol from 3.0 to 1.5 g/L. Probiotic bacteria are reported to deconjugate bile salts: deconjugated bile acid does not absorb lipids as readily as its conjugated counterpart, leading to a reduction in the cholesterol level. L. acidophilus is also reported to take up cholesterol during growth and this makes it unavailable for absorption into the blood stream (Mann and Spoerry, 1974; Klaver and Meer, 1993).

26.3.6

INFLAMMATORY BOWEL DISEASE

Inflammatory bowel disease (ulcerative colitis and Crohn’s disease) is related to the intestinal microflora. Symptoms of inflammatory bowel disease include a disturbance in bowel habits and mucosal inflammation. In the intestine of people with inflammatory bowel disease the numbers of Lactobacillus and Bifidobacterium are lower and those of coccoids and anaerobes are higher. Probiotics do not cure the disease, but once patients are in remission through treatment with corticosteroids, some probiotics can prolong the remission period, thus reducing the incidence of relapse and the use of corticosteroids. This improves the quality of life of patients.

26.3.7

IMMUNE SYSTEM STIMULATION

The intestine is the body’s largest immune organ and the intestinal microflora and metabolic activity of the intestine is equivalent to that of the liver. Probiotics may directly, by changing the composition or activity of the intestinal microflora, or indirectly influence the body’s immune function (Marteau et al., 1997). Probiotic cultures produce γ-interferon by T-cells and stimulate cytokines as represented by TNF-α (tumor necrosis factor) and IL-6 and IL-10 (interleukines 6 or 10). Immunomodulation by L. acidophilus and Bifidobacterium has been observed, in particular IgA levels and nonspecific immunity. Ingestion of probiotic yoghurt has been reported to stimulate cytokine production in blood cells and enhance the activities of macrophages. A more comprehensive review of the health benefits of probiotic bacteria and probiotic products can be found in the reviews of Shah (2000b, 2004, 2006a, 2006b, 2007), Ouwehand et al. (2002, 2003), Chandan and Shah (2006), and Vasiljevic and Shah (2007).

26.4 EFFICACY Health effects including the role of probiotic bacteria in reducing the symptoms of lactose malabsorption and the shortening of rotavirus diarrhea are very well documented and generally accepted. There is evidence for the therapeutic use of probiotics in infectious diarrhea in children, and recurrent Cl. difficile infections. Their role in immune modulation is also well documented. However, more work must be carried out in humans in order to substantiate health claims. Promising results are available for other health benefits of selected probiotic strains, including treatment of irritable bowel disease and inflammatory bowel syndrome. Further work is needed on other health benefits, including the effect of probiotics on reducing serum cholesterol. It must be remembered that these benefits are strain-specific. A number of studies have been carried out to evaluate these health benefits. Naidu et al. (1999) reviewed and recorded 143 human clinical trials using probiotics between 1961 and 1998, comprising 7500 individuals. Probiotics did not cause any illness.

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Pedone et al. (1999) studied the effect of L. casei on the prevention of diarrhea in children. A group of 127 children received yoghurt containing L. casei; yoghurt without any probiotics was used as a control. The severity of the diarrhea was reduced. Guandalini et al. (2000) administered L. rhamnosus GG through rehydration solution to 127 children at 11 day care centers in 10 different European countries. The administration of the probiotic reduced the duration of diarrhea. In a study conducted by Saavedra et al. (2004), 180 infants aged 7 ± 2.9 months were given infant formula supplemented with Bifidobacterium lactis and Str. thermophilus for 210 ± 127 days. The formula resulted in a lower frequency of colic or irritability and a lower frequency of antibiotic use. The organisms were found to be well tolerated. A comprehensive report on the clinical studies on the health benefits of probiotics and their efficacy can be found in Mogensen et al. (2002).

26.5 SAFETY OF PROBIOTIC FOODS The most common microorganisms used in fermented products belong to the genera Lactococcus, Leuconostoc, Pediococcus, and Lactobacillus. Based on safety records, microorganisms can be placed in three groups: safe strains (Lactococcus, Leuconostoc, Pediococcus, Lactobacillus, Oenococcus, Str. thermophilus, Bifidobacterium, Carnobacterium, E. saccharolyticus, Ent. faecium), doubtful strains (Enterococcus, L. rhamnosus, L. catenaforme, Vagococcus, Bifidobacterium dentium), and risky strains Peptostreptoccus, Streptoccus) (Mogensen, 2003). Lactic acid bacteria (LAB) are consumed in large quantities. It is estimated that per capita consumption of fermented milk in Europe is 22 kg; this amounts to approximately 8.5 billion kg/year, a total of 8.5 × 1020 LAB (assuming 108 CFU/g), and 3400 tones of LAB cells (assuming each cell weighs 4 × 10−12 g). The ingestion of large numbers of bacteria requires an assurance of safety. One of the criteria used for the selection of probiotics is their adhering property, so their ability to survive in gastric conditions and adhere to gastric epithelium may pose a risk of translocation. The organisms translocate the epithelial cells in the gastrointestinal tract, entering the lymph or blood vessels and causing systemic infections. However, translocation is usually seen in compromised individuals only. Translocation has been observed in sterile-born mice; however, lactobacilli did not cause any harm and the organisms cleared in 2–3 weeks (Mogensen, 2003). Other experiments have shown that L. delbrueckii ssp. bulgaricus, L. rhamnosus, and Bifidobacterium lactis did not translocate. Aguirre and Collins (1993) and Gasser (1994) have reviewed clinical cases involving LAB and bifidobacteria between 1938 and 1993; the results are summarized in Table 26.3. An analysis of cases of infections revealed that out of 155 infections involving LAB or bifidobacteria, 95 cases involved Lactobacillus spp., 33 of Leuconostoc spp., 18 of Pediococcus spp., and 9 cases involved Bifidobacterium spp. (Table 26.3) (Gasser, 1994). Endocarditis was the most frequent infection in which Lactobacillus species have been involved; strains of the species L. rhamnosus/casei have most often been isolated. Only about 180 cases of septicemia in humans involving LAB have been reported. In only one of these cases was the identified LAB identical to a commercially available dairy strain. Ent. faecium and Ent. faecalis are more frequently involved in clinical infection. In most cases of infection, people were reported to be infected by their own flora; however, in a few cases the consumption of probiotics was a potential source. About 30 cases of fungemia have been reported in patients treated with Saccharomyces boulardii (Gasser, 1994) and two cases of infection have been with food-borne L. rhamnosus (MacKay et al., 1999). Saxelin et al. (1996) studied the prevalence of bacteremia caused by the Lactobacillus species in Southern Finland and compared the characteristics of the blood culture isolates and probiotic dairy strains. Lactobacillus was identified in eight of 3317 blood culture isolates; however, there was no isolate from the dairy strain.

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TABLE 26.3 Clinical Cases in which LAB or Bifidobacteria have been Isolated Clinical Outcome Endocarditis Bacteremia Other infection Total

Lactobacillus

L. acidophilus

L. casei

L. plantarum

L. rhamnosus

Bifidobacterium

Leuconostoc

Pediococcus

Total

7 8 19 34

3 3 2 8

12 — — 12

11 2 1 14

19 5 3 27

— 9 — 9

2 23 8 33

— 11 7 18

54 61 40 155

Source: Adapted from Mogensen, G. et al., 2002. Bulletin of International Dairy Federation, Brussels, Belgium, 377, pp. 4–9.

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In a study by Kalliomaki et al. (2001), L. rhamnosus GG was given to 132 women who were at high risk of their babies developing atopic dermatitis. There was no report of adverse effects in the mothers, indicating that the probiotic was safe. Reports by Salminen et al. (2002) suggest that L. rhamnosus GG has been widely used in Finland since the late 1980s and despite the long-term use of this probiotic organism, there have been only a few cases of bacteremia (0.05 cases per 100,000 cases). Lara-Villoslada et al. (2007) carried out a safety assessment on two probiotic strains including Lactobacillus coryniformis CECT5711 and Lactobacillus gasseri CECT5714 using 20 Balb/C mice, which were orally treated with L. coryniformis CECT5711 or L. gasseri CECT5714 for 30 days, and reported that these organisms were not present in liver or spleen, suggesting that there was no treatment-associated bacterial translocation. Zhou et al. (2000) administered L. acidophilus. Bifidobacterium lactis, and L. rhamnosus to 78 mice at three levels including 5 × 107, 109, 1010 cfu/day and found that the organisms were safe and no adverse effects were observed. Liong and Shah (2006a, 2006b) administered L. casei and Bifidobacterium infantis to 24 rats and no probiotics were detected in the spleen, liver, and kidney, suggesting that the organisms were not translocated to these organs. Cannon et al. (2005) reviewed 241 reported cases of Lactobacillus infection and Husni et al. (1997) reviewed 45 cases of Lactobacillus infection and the organisms causing infections were characterized. Four out of 241 cases were associated with dairy consumption. The organisms were associated with 129 cases of bacteremia and 73 cases of endocarditis. L. casei and L. rhamnosus were common isolates among Lactobacillus species. The patients had severe underlying conditions such as cancer, diabetes, and transplantation, which may have predisposed them to infections in general. In relation to a consumption of about 20 million tons of fermented milk annually, the above numbers are negligible (Mogensen, 2003). There is no foundation for safety concerns in relation to probiotic dairy products on the market today. Probiotics are generally considered safe. As evidenced by epidemiologic studies, bacteremia, or sepsis from lactobacilli is extremely rare. Several probiotics have had a long history of safe use and no health concerns have been observed. A long history of safe use is still the most credible safety test.

26.6 CONCLUSIONS Probiotic products containing L. acidophilus, Bifidobacterium spp., and L. casei are becoming increasingly popular. Other probiotic organisms including Ent. faecium, S. cerevisiae Boulardii, and Propionibacterium have the potential to be used in probiotic products. Several health benefits have been claimed for probiotic bacteria. While some health benefits including the management of lactose malabsorption, rotavirus diarrhea, antibiotic-associated diarrhea, and Cl. difficile diarrhea have been well studied and accepted, other health benefits will require further investigation. Although a number of infections have resulted from the consumption of probiotic organisms, they are generally considered safe.

REFERENCES Aguirre, M. and Collins, M.D., 1993. Lactic acid bacteria and human clinical infection. J. Appl. Bacteriol. 75: 95–107. Cannon, J.P., Lee, T.A., Bolanos, J.T., and Danziger, L.H., 2005. Pathogenic relevance of Lactobacillus: A retrospective review of over 200 cases. Eur. J. Clin. Microbiol. Infect. Dis. 24: 31–40. Cenci, G., Rossi, J., Throtta, F., and Caldini, G., 2002. Lactic acid bacteria isolated from dairy products inhibit genotoxic effect of 4-nitroquinoline-1-oxide in SOS-chromotest. Syst. Appl. Microbiol. 25: 483–490. Chandan, R.C. and Shah, N.P., 2006. Functional foods and disease prevention. In: R.C. Chandan (Ed.), Manufacturing Yogurt and Fermented Milks, pp. 311–325. Iowa: Blackwell Publishing Professional. Curry, B. and Crow, V., 2003. Lactobacillus spp.: General characteristics. Encyclopedia of Dairy Sciences, Vol. 3, pp. 1479–1484. London, UK: Academic Press.

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de dios Pozo-Olano, J., Warram, J.H., Gomez, R.G., and Cavazos, M.G., 1978. Effect of lactobacilli preparation on traveller’s diarrhoea. A randomized, double blind clinical trial. Gastroenterology 74: 829–830. Gasser, F., 1994. Safety of lactic acid bacteria and their occurrence in human clinical infections. Bull. Inst. Pasteur 92: 45–67. Goldin, B.R. and Gorbach, S.L., 1984. The effect of milk and Lactobacillus feeding on human intestinal bacterial enzyme activity. Am. J. Clin. Nutr. 39: 756–761. Guandalini, S., Pensabene, L., Zikri, M.A., et al., 2000. Lactobacillus GG administered in oral rehydration solution to children with acute diarrhoea: A multicenter European trail. J. Pediatr. Gastroenterol. Nutr. 30: 460. Hilton, E., Kolakowski, P., Singer, C., and Smith, M., 1997. Efficacy of Lactobacillus GG as a diarrhoeal preventive in Travellers. J. Travel Med. 4: 41–43. Holm, F., 2003. Gut health and diet: The benefits of probiotic and prebiotics on human health. The World of Ingredients 2: 52–55. Husni, R.N., Gordon, S.M., Washington, J.A., and Longworth, D.L., 1997. Lactobacillus bacteremia and endocarditis: Review of 45 cases. Clin. Infect. Dis. 25: 1048–1055. Kalliomaki, M., Salminen, S., Arvilommi, H., Kero, P., Koskinen, P., and Isolauri, E., 2001. Probiotics in primary prevention of atopic disease: A randomized placebo-controlled trial. Lencet 357: 1076–1079. Katelaris, P.H., Salam, I., and Farthing, M.J., 1995. Lactobacilli to prevent traveller’s diarrhoea? New Engl. J. Med. 333: 1360–1361. Klaver, F.A.M. and Meer, R.V.D., 1993. The assumed assimilation of cholesterol by lactobacilli and Bifidobacterium bifidum is due to their bile salt deconjugating activity. Appl. Environ. Microbiol. 59: 1120–1124. Krishnakumar, V. and Gordon, I.R., 2001. Probiotics: Challenges and opportunities. Dairy Ind. Int. 66: 38–40. Kurmann, J.A. and Rasic, J.L., 1991. The health potential of products containing bifidobacteria. In: R.K. Robinson (Ed.), Therapeutic Properties of Fermented Milks, pp. 117–157. London: Elsevier Applied Science Publishers. Lankaputhra, W.E.V. and Shah, N.P., 1998. Antimutagenic properties of probiotic bacteria and of organic acids. Mutat. Res. 39: 169–182. Lara-Villoslada, F., Sierra, S., Martin, R., Delgado, S., Rodriguez, J.M., Olivares, M., and Xaus, J., 2007. Safety assessment of two probiotic strains, Lactobacillus coryniformis CECT5711 and Lactobacillus gasseri CECT5714. J. Appl. Microbiol. 103: 175–184. Liong, M.T. and Shah, N.P., 2005. Acid and bile tolerance and cholesterol removal ability of Lactobacilli strains. J. Dairy Sci. 88: 55–66. Liong, M.T. and Shah, N.P., 2006a. Sorbitol, maltodextrin, inulin, and Bifidobacterium infantis modify serum lipid profiles, intestinal microbial population and organic acids concentrations in rats. Int. J. Probiotics and Prebiotics 1 (2): published on line. Liong, M.T. and Shah, N.P., 2006b. Effects of Lactobacillus casei synbiotic on serum lipoprotein, intestinal microflora, and organic acids in rats. J. Dairy Sci. 89: 1390–1399. MacKay, A., Taylor, M., Kibbler, C., and Hamilton Miller, J., 1999. Lactobacillus endocarditis caused by a probiotic microorganism. Clin. Microbiol. Infect. 5: 290–292. Mann, G.V. and Spoerry, A., 1974. Studies of a surfactant and cholesterolemia in the Massai. Am. J. Clin. Nutr. 27: 464–469. Marteau, P., Vaerman, J.P., Bord, J.P., et al., 1997. Effects of intrajejunal perfusion and chronic ingestion of Lactobacillus johnsonii strain LA1 on serum concentrations and jejunal secretions of immunoglobulins and serum proteins in healthy humans. Gastroenterol. Clin. Biol. 21: 293–298. McDonough, F.E., Wong, N.P., Hitchins, A., and Bodwell, C.E., 1987. Alleviation of lactose malabsorption from sweet acidophilus milk. Am. J. Clin. Nutr. 42: 345–346. Mitsuoka, T., 1992. The human gastrointestinal tract. Lactic Acid Bacteria 1: 69–114. Mogensen, G., 2003. Safety aspects of fermented products. Bulletin of International Dairy Federation, Brussels, Belgium, pp. 144–158. Mogensen, G., Salminen, S., O’Brien, J., et al., 2002. Food microorganisms-health benefits, safety evaluation and strains with documented history of use in foods. Bulletin of International Dairy Federation, Brussels, Belgium, 377, pp. 4–9. Naidu, A.S., Bidlack, W.R., and Clemens, R.A., 1999. Probiotic spectra of lactic acid bacteria (LAB). Crit. Rev. Food Sci. Nutr. 39: 13–126. Ong, L., Henriksson, H., and Shah, N.P., 2006. Development of probiotic Cheddar cheese containing Lactobacillus acidophilus, Lb. casei, Lb. paracasei and Bifidobacterium spp. and their influence on proteolytic patterns and production of organic acid. Int. Dairy J. 16: 446–456. Orrhage, K., Sillerström, E., Gustafsson, J.Ä., Nord, C.E., and Rafter, J., 1994. Binding of mutagenic heterocyclic amines by intestinal and lactic acid bacteria. Mutat. Res. 311: 239–248.

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Ouwehand, A.C., Bianchi Salvadori, B., Fonden, R., Mogensen, G., Salminen, S., and Sellars, R., 2003. Health effects of probiotics and culture-containing dairy products in humans. Bulletin of the International Dairy Federation, Brussels, Belgium, 380: 4–19. Onwulata, C.I., Ramkishan Rao, D., and Vankineni, P., 1989. Relative efficiency of yoghurt, sweet acidophilus milk, hydrolysed-lactose milk, and a commercial lactase tablet in alleviating lactose maldigestion. Am. J. Clin. Nutr. 49: 1233–1237. Ouwehand, A.C., Salminen, S., and Isolauri, E., 2002. Probiotics: An overview of beneficial effects. Antón. Van Leeuw. 82: 279–289. Pedone, C.A., Bernabeu, A.U., Postaire, E.R., Bouley, C.F., and Reinert, P., 1999. The effect of supplementation with milk fermented by Lactobacillus casei (strain DN-114001) on acute diarrhoea in children attending day care centres. Int. J. Clin. Pract. 53 (3): 179–184. Playne, M.J., Bennet, L.E., and Smithers, G.W., 2003. Functional dairy foods and ingredients. Aust. J. Dairy Technol. 58: 242–264. Saavedra, J.M., Abi-Hanna, A., Moore, N., and Yolken, R.H., 2004. Long term consumption of infant formulas containing live probiotic bacteria: Tolerance and safety. Am. J. Clin. Nutr. 79: 261–267. Saavedra, J.M., Bauman, N.A., Oung, I., Perman, J.A., and Yolken, R.H., 1994. Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus. Lancet 344: 1046–1049. Salminen, M.K., et al., 2002. Lactobacillus bacteremia during a rapid increase in probiotic use of Lactobacillus rhamnosus GG in Finland. Clin. Infect. Dis. 35: 1155–1160. Savaiano, D.A., ElAnouar, A.A., Smith, D.J., and Levitt, M.D., 1984. Lactose malabsorption from yoghurt, pasteurized yoghurt, sweet acidophilus milk, and cultured milk in lactase-deficient individuals. Am. J. Clin. Nutr. 40: 1219. Saxelin, M., Chuang, N.H., Chassy, B., et al., 1996. Lactobacilli and bacteriemia in Southern Finland, 1989– 1992. Clin. Infect. Dis. 22: 564–566. Shah, N.P., 1993. Effectiveness of dairy products in alleviation of lactose intolerance. Food Aust. 45: 268–271. Shah, N.P., 2000a. Probiotic bacteria: Selective enumeration and survival in dairy foods. J. Dairy Sci. 83: 894–907. Shah, N.P., 2000b. Some beneficial effects of probiotic bacteria. Bioscience Microflora 19: 99–106. Shah, N.P., 2002. Bifidobacterium spp.: Applications in fermented milks. In: H. Roginski, J. Fuquay, and P. Fox (Eds), Encyclopedia of Dairy Science, pp. 147–151. London: Academic Press. Shah, N.P., 2004. Probiotics and prebiotics. Agro Food Ind. Hi Tech 15: 13–16. Shah, N.P., 2006a. Health benefits of yogurt and fermented milks. In: R.C. Chandan (Ed.), Manufacturing Yogurt and Fermented Milks, pp. 327–340. Iowa: Blackwell Publishing Professional. Shah, N.P., 2006b. Microorganisms and health attributes (probiotics). In R.C. Chandan (Ed.), Manufacturing Yogurt and Fermented Milks, pp. 341–354. Iowa: Blackwell Publishing Professional. Shah, N.P., 2007. Functional cultures and health benefits. Int. Dairy J. 17 (11): 1262–1277. Shah, N.P., Fedorak, R.N., and Jelen, P., 1992. Food consistency effects of quarq in lactose absorption by lactose intolerant individuals. Int. Dairy J. 2: 257–269. Shah, N.P. and Lankaputhra, W.E.V., 2002. Bifidobacterium spp.: Morphology and physiology. In: H. Roginski, J. Fuquay, and P. Fox (Eds), Encyclopedia of Dairy Science, pp. 141–146. London: Academic Press. Singh, J., Rivenson, A., Tomita, M., Shimamura, S., Ishibashi, N., and Reddy, B.S., 1997. Bifidobacterium longum and Lactobacillus acidophilus producing intestinal bacteria inhibits colon cancer and modulates the intermediate biomarkers of colon carcinogenesis. Carcinogenesis 18: 833–841. Tharmaraj, N. and Shah, N.P., 2004. Survival of Lactobacillus acidophilus, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Bifidobacterium animalis and Propionibacterium in cheese-based dips and the suitability of dips as effective carriers of probiotic bacteria. Int. Dairy J. 14: 1055–1066. Vasiljevic, T. and Shah, N.P., 2007. Fermented milk health benefits beyond probiotic effect. In: Y.H. Hui (Ed.), Handbook of Food Products Manufacturing, vol. 2, pp. 99–115. Hoboken: Wiley. Yoon, H., Benamouzig, R., Little, J., Francois-Collange, M., and Tome, D., 2000. Systematic review of epidemiological studies on meat, dairy products, and egg consumption and risk of colorectal adenomas. Eur. J. Cancer Prev. 9: 151–164. Zhou, J.S., Shu, Q., and Rutherford, K.J., Prasad, J., Birtles, M.J., Gopal, P.K., and Gill, H.S., 2000. Safety assessment of potential probiotic lactic acid bacteria strains Lactobacillus rhamnosus HN001, Lb acidophilus HN017, and Bifidobacterium lactis HN019 in BALB/c mice. Int. J. Food Microbiol. 56 (1): 87–96.

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of High Pressure 27 Use Technology to Inactivate Bacterial Spores in Foods Noriyuki Igura, Seiji Noma, and Mitsuya Shimoda CONTENTS 27.1 Introduction .......................................................................................................................... 497 27.2 Bacterial Spores in Foods ..................................................................................................... 498 27.3 Several Techniques for Spore Inactivation ........................................................................... 499 27.4 Spore Inactivation by High-Pressure Treatment................................................................... 499 27.5 Food Quality after High-Pressure Treatment .......................................................................500 27.6 Conclusion ............................................................................................................................ 502 Acknowledgments.......................................................................................................................... 502 References ...................................................................................................................................... 503

27.1 INTRODUCTION The human race has used fire directly and/or indirectly for the processing of foods for several hundred thousand years. We have also discovered that heated foods can be preserved for a longer time than nonheated foods. Since the invention of electricity, we have cooked and processed foods with heat generated not only by fire but also by electricity. A variety of methods have been employed to preserve foods by heat, depending on the technology available at the time. One of the oldest methods is by drying. Dried foods, such as jerked meats and hard chunk, were taken along on early expeditions. Smoked meats were used for long-time storage. Even now, these preservation methods of foods are still used around the world, giving a unique flavor and texture to the foods; of course this also means that the foods lose their original flavor and texture. In the early modern period, alternative preservation methods which maintained the original flavor and texture of the foods were developed. Denis Papin, a French physicist and inventor in the late seventeenth century, invented a steam digester, and recorded its food preservation capability. The Royal Society reported that his steam digester could preserve fruits without loss of flavor and texture. About a hundred years after Papin’s invention, Nicolas Apert, a French inventor and confectioner, developed an airtight food preservation method. He filled a glass bottle with food and sealed it tightly with a cork, then boiled it for a certain length of time. This method is the precursor of “canning.” Nowadays, food preservation by heating is achieved by the inactivation of microorganisms and enzymes. As the canning method kills almost all microorganisms, canned foods have become a common method of storing foods at a normal temperature. Canning is better than drying of foods; however, it is well known that foods exposed to excessive heat by this canning method also lose their original flavor and texture. Today’s consumers desire not only safe and good foods but also “delicious” foods. Because of this demand, nonthermal inactivation methods have been researched and developed. One nonthermal inactivation method is the high-pressure treatment. 497 © 2010 Taylor and Francis Group, LLC

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Percy Williams Bridgman, an American physicist, researched the physics of high pressures. He won the 1946 Nobel Prize in Physics for his work. Bridgman (1914) reported that the coagulation of egg albumin can occur through high-pressure treatment just as it does through heat treatment. On the other hand, Bert Hite reported an important study for food scientists and industries, the extension of raw milk shelf life by high-pressure treatment (Hite, 1899). Since then there have been numerous research projects on the application of high-pressure treatments to foods. High pressure is a process commonly used for particular food processing, such as fruit jam, ham, rice, and open-shell oysters (Horie et al., 1991; Kimura et al., 1994; Yamazaki and Sasagawa, 2000; Butz and Tauscher, 2002; Torres and Velazquez, 2005). Although these processes are successful in maintaining the original flavor of the foods, there are some problems in microbial sterility assurance because of the existence of “spores.” Some bacteria show two types of morphology; one is a vegetative form and the other is a dormant form or bacterial spore. In the vegetative form, almost all bacteria are easy to kill by heat treatment below 100°C, whereas some of the spores may survive heat treatment. The spores show resistance not only to heat but also to some chemicals and to high pressure. In order to apply high-pressure treatment to certain foods or pharmaceutical products, we need to seek effective methods to inactivate spores. This chapter will fi rst give a brief background on bacterial spores and spore inactivation methods and then will introduce some examples of the inactivation of spores by high-pressure treatment and the effects of this treatment on foods.

27.2 BACTERIAL SPORES IN FOODS Food spoilage and food poisoning are mainly caused by microorganisms. Almost all of these microorganisms can be inactivated by heat treatment at 60–80°C; the number of viable pathogens in foods is reduced by this heat treatment (pasteurization). On the other hand, some microorganisms can survive after pasteurization. Typical microorganisms which survive after pasteurization include spore-forming bacteria. When these spore-forming bacteria are in a fertile environment, they tend to multiply prolifically. Under these conditions, the bacteria are known as being in a vegetative form and are sensitive to heat. When the spore-forming bacteria are in an unfavorable environment, for example in a starvation state, they halt their multiplication metabolism, and produce a particle or endospore which contains DNA and enzymes. This endospore, also known as a bacterial spore or simply spore, shows a high resistance to heat, chemical agents, and pressure. When the spores are under a fertile environment again, the spores germinate and initiate their metabolism for multiplication. Typical spore-forming bacteria in foods are of the Bacillus and Clostridium genera. Bacteria belong to the Bacillus genus are aerobic rod-shaped bacteria used in food processing. For instance, Bacillus subtilis is used in Japan for fermented food such as natto, and the genome of Bacillus thuringiensis is incorporated into crops (corn and cotton) to obtain resistance to nematode pests (Marroquin et al., 2000). On the other hand, some Bacillus species cause health concerns. Bacillus coagulans is known as a flat sour causing bacteria, Bacillus cereus as a food-borne illness, and Bacillus anthracis as anthrax (Kalogridou-Vassiliadou, 1992; Hanna and Ireland, 1999; Brackett, 2001; Schoder et al., 2007). The genus Clostridium, anaerobic rod-shaped bacteria, includes Clostridium thermocellum, Clostridium acetobutylicum, Clostridium perfringens, and Clostridium botulinum. C. thermocellum and C. acetobutylicum are utilized for producing alternative fuels (Lamed and Zeikus, 1980; Bahl et al., 1982; Yu et al., 1985), whereas C. perfringens and C. botulinum are pathogens which cause food poisoning (Regan et al., 1995; Kreyden et al., 2000; Barth et al., 2004). Because these spore-forming bacteria are common in the environment, particularly in soil, processed foods which contain plants or raw materials previously in contact with soil are highly likely to be contaminated with bacterial spores. These contaminated foods are difficult to sterilize safely by heating or pasteurization.

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27.3 SEVERAL TECHNIQUES FOR SPORE INACTIVATION There are several techniques for the reduction or inactivation of the bacterial spores; however, these spores cannot be completely inactivated by boiling at 100°C because bacterial spores are highly resistant to heat. In order to inactivate bacterial spores completely, higher temperatures such as heating in an autoclave (121°C under saturated steam at ca. 2 atm for more than 15 min), and dry heat (180°C for 30 min or 160°C for more than 60 min) is necessary. Other inactivating methods for bacterial spores include gaseous sterilization using ethylene oxide and formalin, and γ-ray sterilization. Filtering is another method for reducing bacterial spores. Intermittent sterilization is a specialized procedure for spore inactivation. This sterilization method is achieved by repeating hot and cool cycles several times. During cooling the bacterial spores germinate and turn into their vegetative form which is sensitive to heat. The treatment using autoclave “canning” is widely used for the sterilization of bacterial spores in foods. The other treatments are usually used for the sterilization of instruments and containers, and are not suitable for food sterilization. During the canning process, foods are loaded into a can, bottle, or heat-resistant pouch, and then heated at 120–130°C for 20–60 min in a large-scale autoclave. After canning, foods remain preserved for several years at normal temperature. However, there are problems associated with this method, including the degradation of nutrients, the generation of off-flavor, and excessive denaturing of food protein, causing less organoleptic quality. In this respect, novel techniques which could sterilize bacterial spores under lower temperature would be preferred.

27.4 SPORE INACTIVATION BY HIGH-PRESSURE TREATMENT Research into the inactivation of microorganisms by high-pressure treatment has been actively carried out since the late twentieth century. It is well known that high-pressure treatment at 600 MPa can inactivate almost all vegetative microorganisms under normal temperature. Some bacterial spores may be resistant to high pressure, and can survive even after high-pressure treatment at 1500 MPa at normal temperature. Past research on bacterial spores unfortunately indicated that it was difficult to inactivate them completely by high-pressure treatment alone. Consequently, researchers have used the application of a hurdle technology (Leistner, 1994), that is, high pressure must be combined with heating or/and another factors. The combined effect of high pressure and heating is well reviewed by several researchers (Rastogi et al., 2007; Wilson et al., 2008; Zhang and Mittal, 2008). Wilson et al. (2008) summarized a myriad of research on the effects of pressure and temperature on the inactivation of Clostridium and Bacillus spores. An investigation into the optimum conditions for the combined treatment of high pressure and heating using a response surface methodology is currently being carried out (Gao et al., 2006; Gao and Ju, 2007; Ju et al., 2008). When we inactivate bacterial spores, we have to consider the germination phenomenon. Bacterial spores initiate germination by recognizing a germinant, such as l-alanine for B. subtilis, at germination receptors. After germinants bind to the receptors, spores release dipicolinic acid and cations, followed by the hydrolysis of the spore’s peptideglycan cortex by cortex lytic enzymes and germ cell wall expansion. Through these reactions, spores germinate and vegetative growth is restored (Moir et al., 2002; Setlow, 2003). Interestingly, it has been revealed that high pressure also initiates germination (Clouston and Wills, 1969). Relatively moderate-pressure treatment (100 MPa) induces spore germination by activating the germinant receptor. In contrast to germination by moderate-pressure treatment, relatively high-pressure treatment (500–600 MPa) induces germination by short cuts in some physiological germination pathways (Wuytack et al., 1998, 2000; Paidhungat et al., 2002). Black et al. (2007) reviewed the pressure-induced germination of spores at various conditions. During this germination, spores lose their resistance to heat, chemicals, and pressure. Thus it is thought that pressure-induced germination can reduce the pressure and temperature necessary to inactivate bacterial spores. It is logical that reciprocal pressure treatment showed a higher inactivation ratio of bacterial spores than single-pressure treatment (Hayakawa et al., 1994; Furukawa et al.,

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2000). Pressure-induced germination depends on the treatment temperature and pressure. There may be an optimum temperature for pressure-induced germination at the lower pressure of 40–65 MPa, whereas at a higher pressure (>100 MPa), pressure-induced germination requires an increase in temperature and pressure (Murrel and Wills, 1977; Furukawa and Hayakawa, 2001). In combination with low-pressure treatment (100 MPa) and relatively high temperature (80–100°C), pressure-induced germination is effective to inactivate bacterial spores (Furukawa et al., 2001; Islam et al., 2006). In addition to heating, other treatments, such as ultrasonic energy, pulsed electric fields, irradiation, high-pressure carbon dioxide, and antimicrobial reagents are combined with high-pressure treatment, and these combinations are successful in reducing applied pressure and treatment time to inactivate microorganisms (Rastogi et al., 2007; Black et al., 2008; Gao and Ju, 2008; Zhang and Mittal, 2008).

27.5 FOOD QUALITY AFTER HIGH-PRESSURE TREATMENT It is said that high-pressure treatment can maintain food quality without the loss of nutrients and flavor (Cheftel, 1992). We have investigated the quality of three kinds of foods: curry, stew, and soy soup (Japanese traditional soup) after a combinational treatment with high pressure and heating. We prepared two types of samples; one is a sample inoculated with bacterial spores (B. coagulans, Bacillus megaterium, Bacillus licheniformis, and Geobacillus stearothermophilus, each initial spore concentration being about 105 CFU/mL) for the sporicidal test, and the other is a noninoculated sample for the sensory test. Each sample was poured into a plastic bag coated with aluminum. After the plastic bag was airtight, heat sealed, and subjected to high-pressure treatment with heating (600 MPa, 95°C for 2 min holding time) or canning using a laboratory autoclave (121°C for 15 min), survival spores were counted in the inoculated sample. A sensory test was performed on the noninoculated sample. Table 27.1 shows the survival spores suspended in cooked foods after canning or high-pressure treatment. As shown in the table, there are few survival spores after both treatments. This result indicates that high-pressure treatment combined with heating can inactivate bacterial spores by more than a 4-log order, which is comparable with canning. TABLE 27.1 Survival Spores after Canning and High-Pressure Treatment (HPT) in Several Cooked Foods after 0 and 7 Days at 25°C 0 Day Sample Curry

Stew

Soy soup

7 Days

Spores

Intact

Canning

B. coagulans B. licheniformis B. megaterium G. stearothermophilus B. coagulans B. licheniformis B. megaterium G. stearothermophilus B. coagulans B. licheniformis B. megaterium G. stearothermophilus

1.1 × 105 1.3 × 105 1.2 × 105 1.5 × 105 1.4 × 105 1.2 × 105 8.4 × 104 1.4 × 105 8.6 × 104 1.2 × 105 1.1 × 105 1.6 × 105

3.3 × 100 3.3 × 100 n.d. n.d.

n.d. n.d. n.d. n.d.

3.3 × 100 n.d.

3.3 × 100 n.d. n.d. n.d.

n.d., not detected.

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3.3 × 100 n.d. 6.7 × 100 n.d. n.d. n.d.

HPT

6.7 × 100 3.3 × 100 n.d.

Intact 3.4 × 104 9.8 × 104 4.8 × 104 6.9 × 103 1.1 × 105 9.8 × 104 1.4 × 105 1.1 × 105 2.4 × 104 1.3 × 105 3.9 × 104 1.3 × 105

Canning

HPT

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

n.d.

3.3 × 100 n.d.

6.7 × 100 n.d.

3.3 × 100 n.d. n.d. n.d. n.d. 6.7 × 100 n.d. 2.7 × 101 n.d.

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Curry

Stew

Soy soup

Intact

Canning

HPT

FIGURE 27.1 The appearance of cooked foods after canning and high-pressure treatment (HPT).

Figure 27.1 shows the appearance of the treated foods. Although the curry samples show no difference between each treatment, stew and soy soup samples differ in appearance after canning as distinct from intact and high-pressure treatment. Nonenzymatic browning occurs in the canning samples of stew and soy soup. Figure 27.2 shows the results of the sensory evaluation. Here the sensory evaluations were carried out compared to intact samples. The scores are as follows: like Appearance

Curry

Canning HPT

Flavor Texture Taste Appearance-

Stew

Flavor Texture Taste Appearance-

Soy soup

Flavor Texture Taste –2.0 –1.5 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0

FIGURE 27.2

A sensory evaluation of cooked foods after canning or high-pressure treatment (HPT).

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+2, relatively like +1, no difference 0, relatively dislike −1, and dislike −2, compared to untreated samples. The appearance scores were low in stew and soy soup after canning, whereas the scores for the appearance of other samples were nearly zero, which means they were almost the same as intact samples. The scores for flavor, texture, and taste of curry and stew samples after canning were between −0.5 and −1.0, and those of soy soup after canning showed worse values. On the other hand, the scores after high-pressure treatment were between −0.5 and 0.5. These results suggest that high-pressure treatment with heating exhibits an efficiency equivalent to that of canning in the inactivation of bacterial spores suspended in cooked foods. Furthermore, treatment can maintain a food quality (appearance, flavor, texture, and taste) comparable to non inoculated cooked foods. Pressure has a unique characteristic of adiabatic compressive heating. The temperature of water increases about 3–4°C per 100 MPa when it is pressurized. In our experiments, we consider that the temperature of the foods under pressurization reached about 120°C. The exposure time to this high temperature in high-pressure treatment, however, is short because the temperature rapidly decreases to its initial temperature along with decompression. On the other hand, the heating time of canning is long because it takes more than 1 h to heat up and cool down, and the subjected materials are exposed to a high temperature for the duration of the process. As a result, the quality of foods was better maintained after high-pressure treatment with heating. While there is a large body of research on the inactivation of bacterial spores under high pressure and heating conditions (Ananta et al., 2001; Ahn et al., 2007; Wilson et al., 2008), there are few studies about the quality change in foods under these conditions (Leadley et al., 2008). More research should be conducted to study the effect of high-pressure treatment with heating on food quality. Pressure-induced germination is also effective in the inactivation of bacterial spores. Islam et al. (2006) reported that more than 105 orders of bacterial spores can be inactivated in low-acid foods at 100 MPa, 75–95°C for 3–12 h, the condition under which pressure-induced germination can occur. They also reported that potatoes and carrots showed good qualities after this treatment compared to canning or comparable to conventional boil cooking (Islam et al., 2003, 2007). It may be possible to supply fresh foods contaminated with only the vegetative cells of microorganisms by treating them with high pressure at a normal temperature. If the foods are contaminated with bacterial spores, it may be more difficult to preserve them. However, we can maintain a high quality of cooked foods by treating them with high pressure and heating. Even so, the optimization of high pressure with heat treatment must be determined to attain high sporicidal conditions in order to maintain food qualities.

27.6 CONCLUSION The application of high-pressure treatment for spore inactivation of foods was described in this chapter. As mentioned above, although bacterial spores are difficult to inactivate by high-pressure treatment alone, combination with other treatments such as heating can inactivate the bacterial spores. In order to sterilize foods, however, there are numerous problems that need to be solved. These concerns include food quality loss by heating, sterilization loss by food matrices (sugars, pH, and ions), costs of facilities, and so on. In order to solve these problems, further research on the mechanisms of bacterial spore inactivation at various conditions and the development of new facilities is warranted.

ACKNOWLEDGMENTS The authors gratefully acknowledge to Emeritus Professor, Hayakawa Isao at Kyushu University for his sincere supervision, and thank the Ministry of Economy, Trade and Industry, Japan for financial support.

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REFERENCES Ahn, J., Balasubramaniam, V.M., and Yousef, A.E., 2007. Inactivation kinetics of selected aerobic and anaerobic bacterial spores by pressure-assisted thermal processing. Int. J. Food Microbiol. 113: 321–329. Ananta, E., Heinz, V., Schlüter, O., and Knorr, D., 2001. Kinetic studies on high-pressure inactivation of Bacillus stearothermophilus spores suspended in food matrices. Innov. Food Sci. Emerg. Technol. 2: 261–272. Bahl H., Andersch, W., and Gottschalk, G., 1982. Continuous production of acetone and butanol by Clostridium acetobutylicum in a 2-stage phosphate limited chemostat. Euro. J. App. Microbiol. Biotechnol. 15(4): 201–205. Barth H., Aktories, K., Popoff, M.R., and Stiles, B.G., 2004. Binary bacterial toxins: Biochemistry, biology, and applications of common Clostridium and Bacillus proteins. Microbiol. Mol. Biol. Rev. 68(3): 373–402. Black, E.P., Setlow, P., Hocking, A.D., et al., 2007. Response of spores to high-pressure processing. Compre. Rev. Food Sci. Food Safety 6: 103–119. Black, E.P., Linton, M., McCall, R.D., et al., 2008. The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk. J. Appl. Microbiol. 105: 78–87. Brackett, R.E., 2001. Fruits, vegetables, and grains. In: M. Doyle, L.R. Beuchat, and T.J. Montville (Eds), Food Microbiology, Fundamentals and Frontiers, pp. 127–138. Washington, DC: ASM Press. Bridgman, P.W., 1914. The coagulation of egg albumen by pressure. J. Biol. Chem. 19(4): 511–512. Butz, P. and Tauscher, B., 2002. Emerging technologies: Chemical aspects. Food Res. Int. 35(2–3): 279–284. Cheftel, J.C., 1992. Effects of high hydrostatic pressure on food constituents: An overview. In: C. Balny, R. Hayashi, K. Heremans, and P. Masson (Eds), High Pressure and Biotechnology, pp. 195–209. Montrouge: Colloques INSERM. Clouston, J.G. and Wills, P.A., 1969. Initiation of germination and inactivation of Bacillus pumilus spores by hydrostatic pressure. J. Bacteriol. 97(2): 684–690. Furukawa, S., Nakahara, A., and Hayakawa, I., 2000. Effect of reciprocal pressurization on germination and killing of bacterial spores. Int. J. Food Sci. Technol. 35(5): 529–532. Furukawa, S., and Hayakawa, I., 2001. Effect of temperature on the inactivation of Bacillus stearothermophilus IFO 12550 spores by low hydrostatic pressure treatment. Biocontrol Sci. 6(1): 33–36. Furukawa, S., Matsuoka, A., Nakamichi, Y., et al., 2001. Inactivation of spores of three Bacillus strains by low hydrostatic pressure (LHP) treatment J. Fac. Agric. Kyushu Univ. 45(2): 525–530. Gao, Y.L., Ju, X.R., and Jiang, H.H., 2006. Studies on inactivation of Bacillus subtilis spores by high hydrostatic pressure and heat using design of experiments. J. Food Eng. 77: 672–679. Gao, Y.L. and Ju, X.R., 2007. Statistical prediction of effects of food composition on reduction of Bacillus subtilis As 1.1731 spores suspended in food matrices treated with high pressure. J. Food Eng. 82: 68–76. Gao, Y.L. and Ju, X.R., 2008. Exploiting the combined effects of high pressure and moderate heat with nisin on inactivation of Clostridium botulinum spores. J. Microbiol. Method 72: 20–28. Hanna, P.C. and Ireland, J.A.W., 1999. Understanding Bacillus anthracis pathogenesis. Trends Microbiol. 7(5): 180–182. Hayakawa, I., Kanno, T., Yoshiyama, K., and Fujio, Y., 1994. Oscillatory compared with continuous highpressure sterilization on Bacillus stearothermophilus spores. J. Food Sci. 59(1): 164–167. Hite, B.H., 1899. The effects of pressure in the preservation of milk. Morgantown. Bull WV Univ. Agric. Exp. Sta. Morgantown 58: 15–35. Horie, Y., Kimura, K., and Hori, K., 1991. Development of a new fruit processing method by high hydrostaticpressure. J. Jpn. Soc. Biosci. Biotechnol. Agrochem. 65(10): 1469–1474. Islam, M.S., Igura, N., Shimoda, M., and Hayakawa, I., 2003. Effects of low hydrostatic pressure and moderate heat on texture, pectic substances and color of carrot. Eur. Food Res. Technol. 217(1): 34–38. Islam, M.S., Inoue, A., Igura, N., Shimoda, M., and Hayakawa, I., 2006. Inactivation of Bacillus spores by the combination of moderate heat and low hydrostatic pressure in ketchup and potage. Int. J. Food Microbiol. 107(2): 124–130. Islam, M.S., Igura, N., Shimoda, M., and Hayakawa, I., 2007. The synergistic effect of moderate heat and pressure on the physical properties and pectic substances of potato tissue. Int. J. Food Sci. Technol. 42(4): 434–440. Ju, X.R., Gao, Y.L., Yao, M.L., and Qian, Y., 2008. Response of Bacillus cereus spores to high hydrostatic pressure and moderate heat. LWT—Food Sci. Technol. 41: 2104–2112. Kalogridou-Vassiliadou, D., 1992. Biochemical activities of Bacillus species isolated from flat sour evaporated milk. J. Dairy Sci. 75: 2681–2686. Kimura, K., Ida, M., Yoshida, Y., et al., 1994. Comparison of keeping quality between pressure-processed jam and heat-processed jam: Changes in flavor components, hue, and nutrients during storage. 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Kreyden, O.P., Geiges, M.L., Boni, R., and Burg, G., 2000. Botulinum toxin: From poison to medicine. A historical review. Hautarzt 51(10): 733–737. Lamed, R. and Zeikus, J.G., 1980. Ethanol production by thermophilic bacteria: Relationship between fermentation product yields of and catabolic enzyme activities in Clostridium thermocellum and Thermoanaerobium brockii. J. Bacteriol. 144(2): 569–578. Leadley, C., Tucker, G., and Fryer, P., 2008. A comparative study of high pressure sterilization and conventional thermal sterilisation: Quality effects in green beans. Innov. Food Sci. Emerg. Technol. 9: 70–79. Leistner, L., 1994. Further developments in the utilization of hurdle technology for food preservation. J. Food. Eng. 22: 421–432. Marroquin, L.D., Elyassnia, D., Griffitts, J.S., Feitelson, J.S., and Aroian, R.V., 2000. Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. Genetics 155: 1693–1699. Moir, A., Corfe, B.M., and Behravan, J., 2002. Spore germination. Cell. Mol. Life Sci. 59: 403–409. Murrel, W.G. and Wills, P.A., 1977. Initiation of Bacillus spore germination by hydrostatic pressure: Effect of temperature. J. Bacterial. 129(3): 1272–1280. Paidhungat, M., Setlow, B., Daniels, W.B., et al., 2002. Mechanisms of induction of germination of Bacillus subtilis spores by high pressure. Appl. Environ. Microbiol. 68(6): 3172–3175. Rastogi, N.K., Raghavarao, K.S.M.S., Balasubramaniam, V.M., Niranjan, K., and Knorr, D., 2007. Opportunities and challenges in high pressure processing of foods. Crit. Rev. Food Sci. Nutr. 47: 69–112. Regan, C.M., Syed, Q., and Tunstall, P.J., 1995. A hospital outbreak of Clostridium perfringens food poisoning: Implications for food hygiene review in hospitals. J. Hosp. Infect. 29(1): 69–73. Setlow, P., 2003. Spore germination. Curr. Opin. Microbiol. 6: 550–556. Schoder, D., Zangerl, R., Manafi, M., et al., 2007. Bacillus cereus: A food-borne pathogen of different risk potential for the dairy industry. Wiener Tierarztliche Monatsschrift 94(1–2): 25–33. Torres, J.A. and Velazquez, G., 2005. Commercial opportunities and research challenges in the high pressure processing of foods. J. Food Eng. 67: 95–112. Wilson, D.R., Dabrowski, L., Stringer, S., Moezelaar, R., and Brocklehurst, T.F., 2008. High pressure in combination with elevated temperature as a method for the sterilization of food. Trends Food Sci. Technol. 19: 289–299. Wuytack, E.Y., Boven, S., and Michiels, C.W., 1998. Comparative study of pressure-induced germination of Bacillus subtilis spores at low and high pressures. Appl. Environ. Microbiol. 64(9): 3220–3224. Wuytack, E.Y., Soons, J., Poschet, F., and Michiels, C.W., 2000. Comparative study of pressure- and nutrientinduced germination of Bacillus subtilis spores. Appl. Environ. Microbiol. 66(1): 257–261. Yamazaki, A. and Sasagawa, A. 2000. Development of rice food products processed by high pressure treatment J. Jpn. Soc. Biosci. Biotechnol. Agrochem. 74(5): 619–623. Yu, E.K.C., Chan, M.K.H., and Saddler, J.N., 1985. Butanol production from cellulosic substrates by sequential co-culture of Clostridium thermocellum and Clostridium acetobutylicum. Biotechnol. Lett. 7(7): 509–514. Zhang, H. and Mittal, G.S., 2008. Effects of high-pressure processing (HPP) on bacterial spores: An overview. Food Rev. Int. 24: 330–351.

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Part V Legal, Social, and Regulatory Aspects of Food Biotechnology

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of Biotechnology 28 Regulations Generally Recognized as Safe (GRAS) and Health Claims Ryan R. Simon, Earle R. Nestmann, Kathy Musa-Veloso, and Ian C. Munro CONTENTS 28.1 Introduction ..........................................................................................................................507 28.2 Regulation of Biotechnology-Derived Food Products .......................................................... 508 28.3 Role of GRAS in the Regulation of Food Products of Biotechnology ................................. 510 28.3.1 Chemistry and Manufacturing ................................................................................. 510 28.3.2 Intended Uses............................................................................................................ 511 28.3.3 Safety ........................................................................................................................ 511 28.3.3.1 Substantial Equivalence ............................................................................. 512 28.3.3.2 History of Safe Use .................................................................................... 513 28.3.3.3 Safety of Novel Proteins ............................................................................ 514 28.3.4 General Recognition of Safety ................................................................................. 517 28.4 Foods Derived from Biotechnology and Health Claims in the United States ...................... 518 28.4.1 Permissible Food Claims in the United States ......................................................... 518 28.4.2 Nutrient Content Claims ........................................................................................... 521 28.4.2.1 Definition and Scope.................................................................................. 521 28.4.2.2 Applicable Regulations, Scientific Requirements, and Guidance Documents ................................................................................................. 523 28.4.3 Structure/Function Claims ....................................................................................... 523 28.4.3.1 Definition and Scope.................................................................................. 523 28.4.3.2 Applicable Regulations, Scientific Requirements, and Guidance Documents ................................................................................................. 523 28.4.4 Health Claims ........................................................................................................... 524 28.4.4.1 Definition and Scope.................................................................................. 524 28.4.4.2 Applicable Regulations, Scientific Requirements, and Guidance Documents ................................................................................................. 524 28.4.5 Claims as They Relate to Crops Genetically Modified for Output Traits ................ 527 References ...................................................................................................................................... 527

28.1 INTRODUCTION Products of biotechnology intended for use as functional foods or nutraceuticals are subject to regulations that have evolved in various jurisdictions around the world. The focus of this chapter is regulation, in the United States, of these products of biotechnology that are intended for use as foods or additives or ingredients in foods. For safety considerations, regulation is guided by provisions 507 © 2010 Taylor and Francis Group, LLC

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in the Federal Food Drug and Cosmetic Act (FFDCA) concerning “generally recognized as safe, under the conditions of intended use,” or GRAS for short. Much has been written on the legal definition and applications of the GRAS concept (Kessler et al., 1992; Vetter, 1996), and this chapter is not intended to address such legal issues. Rather, the focus of this chapter deals, in two separate parts, (1) with the scientific basis for achieving GRAS status for specified uses of biotechnology-derived food products and (2) with the standards of evidence to obtain regulatory approval of claims related to nutrient composition/health benefits. As the name GRAS implies, this aspect of regulation concerns safety of the public who would be consumers of the product under the intended conditions of use, whether or not health benefits are claimed for such uses. Health claims are not subject to the well-established GRAS safety standard, but are considered separately.

28.2 REGULATION OF BIOTECHNOLOGY-DERIVED FOOD PRODUCTS Regulation of biotechnology has been evolving gradually from a principal focus on the process of gene or recombinant deoxyribonucleic acid (rDNA) technology to the inherent safety and intended use of the product itself. Historically, in the United States for instance, initial concerns about the potential risks from developments in rDNA in the early 1970s were expressed (Berg et al., 1974) and a cautions approach to the uses of these new research methods was adopted (Berg et al., 1975), following the famous Asilomar Conference at that time. Regulatory developments over the ensuing years have been summarized in previous publications (OSTP, 1986, 1992; Franklin et al., 1987; FDA, 1992, 2001; Nestmann et al., 2002; McHughen and Smyth, 2008). Techniques of genetic modification used in the development of novel plant cultivars, like all plant breeding techniques, have the potential to impart changes to the plant that can affect the legal status of foods derived from such crops. Sections 409 and 402 of the FFDCA pertaining to additive, foods, and adulteration of food give the U.S. Food and Drug Administration (FDA) widespread jurisdiction over the safety of foods and food ingredients, including those derived from genetically modified (GM) plants. By strict definition, any substance that is not an inherent constituent of food or whose level in food has been increased by human intervention is considered “added” within the meaning of section 402(a)(1) of the FFDCA (FDA, 1992). Therefore, nonpesticidal substances added to foods using genetic modifications could be viewed as food additives, unless the substance and its use are generally recognized as safe; foods containing added substances imparting pesticidal or herbicidal resistance traits to a GM crop are not subject to FDA oversight, and are under the purview of the U.S. Environmental Protection Agency (EPA). Although it is clear that the FDA has the legal jurisdiction to regulate “added” substances in GM foods and the corresponding foods derived from GM sources as food additives, in practice, interaction between developers of a GM crop and the FDA is usually limited to informal consultation with the agency via a voluntary premarket biotechnology notification (PBN) process (FDA, 2001). This process has been described in detail previously (Nestmann et al., 2002), and ensures that all market entry decisions made by the industry are made consistently and in full compliance with the standards of the FFDCA. Dialogue between the FDA and the notifier continues until all safety concerns of the agency are addressed, resulting in a letter issued by the agency stating that it has no further questions. Only rarely has the FDA conducted premarket evaluations of GM foods as food additives (i.e., FlavrSavr Tomato). The vast majority of GM crops currently introduced to the U.S. marketplace are those modified for various agronomic traits, and typically involve the addition of substances that impart insect or disease resistance to the plant or those substances conferring herbicidal resistance to the plant (i.e., Round up Ready crops). Since compositional changes resulting from these types of genetic modifications are almost exclusively limited to the “added” pesticidal substance(s), the safety of GM foods derived from these plants is typically based on a substantial equivalence approach. Using the substantial equivalence approach, compositional testing of the GM plant is used to support the fact that any introduced GM traits do not alter the levels of constituents that are of nutritional or toxicological (naturally occurring toxins) concern relative to those occurring in the non-GM comparator

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cultivars, and therefore by nature are considered GRAS; foods produced from these plants are permitted for introduction to the U.S. marketplace without the requirement for premarket review or GRAS evaluation, provided no concerns have been raised following consultation with the EPA, United States Department of Agriculture (USDA), or FDA under the biotechnology notification process. In contrast to GM crops modified for agronomic purposes, genetic modifications producing “nutritionally enhanced” foods, by definition, would result in the production of a plant cultivar that, from a compositional perspective, is no longer substantially similar to its traditional non-GM counterpart. Although examples of such nutritionally enhanced crops are numerous in the literature, most of these crops are developed to meet the needs of developing or third-world nations (e.g., golden rice); therefore, the introduction of foods derived from such crops has not been widespread in the United States. In fact, and unfortunately so, the introduction of nutritionally enhanced crops worldwide has been a slow process that has encountered significant regulatory and political roadblocks, and golden rice has yet to be introduced in any country, many of which are in desperate need of such a food source (i.e., third-world or developing nations). Currently, the use of GM technology to produce nutritionally enhanced foods intended for the U.S. marketplace is limited to GM soybean and canola cultivars that have been altered to produce novel fatty acid profiles, from which canola and soybean oils with the desired nutritional properties (e.g., low saturated fat, high oleic acid, low linolenic acid, and low linoleic acid contents) are produced. Progress in the development of nutritionally enhanced products of biotechnology is moving rapidly, and it is easy to envision examples where genetic modifications may be used to introduce novel proteins with unique organoleptic properties (and no prior history of human exposure), naturally occurring bioactive substances, or naturally occurring non-nutritive sweeteners (Stevia glycosides), to agricultural commodities; such uses of genetic modification would clearly constitute food additive uses. The legal introduction of food products derived from plants containing such genetic modifications without premarket approval as a food additive would require a self-affirmed GRAS determination specific to the intended uses of such products in food. Thus, it is the use of the expressed novel protein, and the resulting secondary effects on plant composition, both intended and unintended, that trigger the need for premarket evaluation via a food additive petition or formal GRAS evaluation. A general rule that can be followed when determining whether changes in crop composition as a result of genetic modification warrant premarket approval or formal GRAS evaluation is “when the substance present in the food is one that is already present at generally comparable or greater levels in currently consumed foods, there is unlikely to be a safety concern sufficient to call into question the presumed GRAS status of such naturally occurring substances and thus warrant formal premarket review and approval by FDA” (FDA, 1992, p. 22,990). As defined in 21 CFR 170.3, the basis supporting the GRAS status of a substance can be determined through one of two means, either by scientific procedures or based on evidence of safety through history of use in food prior to January 1, 1958 (FDA, 2008a). Since cases of successful GRAS determinations for foods established using a history of use in food prior to January 1, 1958, are difficult to support, the majority of GRAS determinations typically follow the scientific procedures approach. Producers or manufacturers who intend to conduct a self-affirmed GRAS determination for the use of a food ingredient derived from a GM food source under a specified set of conditions of use in the U.S. marketplace should be aware that two important standards must be met to qualify forexemption from premarket review as a food additive: first, the information that is used to support the GRAS use of a substance under a set of proposed uses requires the same quantity and quality of scientific evidence as is required of food additive petitions; second, this information must be generally available, and ordinarily is obtained from studies published in the peer-reviewed literature. The GRAS route was intended to be a dynamic process, and the classic “check-box” guidelines to food safety that are typically applied by regulatory authorities worldwide do not exist, and each GRAS use is evaluated on a case-by-case basis by experienced qualified scientific experts. However, in order to clarify the criteria and types of information that are expected for exempting a food ingredient from premarket evaluation as a food additive, the FDA has published a proposed ruling on

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substances Generally Recognized as Safe (62 FR 18937). Since the considerations that are required to establish the safety of a food ingredient under a set of given conditions of use are handled on a case-by-case basis, it is not possible to comprehensively detail all of the requirements of a typical GRAS determination; however, most GRAS evaluations will contain the following types of information: data pertaining to the chemistry and manufacturing of the ingredient; estimates of exposure under the intended conditions of use; data supporting safety; and a description of the basis for general recognition of these data. A general overview of these data requirements is discussed below.

28.3 ROLE OF GRAS IN THE REGULATION OF FOOD PRODUCTS OF BIOTECHNOLOGY 28.3.1

CHEMISTRY AND MANUFACTURING

Most major agricultural commodities (e.g., canola, soybean, and wheat) are not consumed as whole foods and are processed to produce flour, protein concentrates, or oils, which in turn are used as food ingredients for a variety of different food applications. One of the initial steps in a scientific procedures GRAS determination involves the characterization of data and information pertaining to the chemistry and manufacturing of the ingredient. Additionally, since the type of data supporting a scientific procedures GRAS determination must be of the same quality and quantity of scientific data required of a food additive petition, the manufacturing process used in the production of the GM food is expected to be conducted in compliance with current Good Manufacturing Practice (cGMP), using suitable food grade raw materials and processing aids. All processing aids used in the manufacture of the ingredient must be used in accordance with appropriate federal regulations. It is not uncommon for manufacturers to unknowingly use common food grade processing aids under a use that is not regulated. Appropriate specifications need to be established. Specifications should adequately characterize the principal constituents of the material, provide limits for any inherent naturally occurring toxins, and, where applicable, include specifications limiting microbial contamination. Lead is ubiquitous in the environment, and the potential presence of lead in food is always a particular concern of the FDA, particularly for foods produced from natural sources and foods where the proposed uses result in significant exposures in the U.S. population. Although the use of GM technology is unlikely to result in changes in the plant that affect the naturally occurring lead content, producers of GM foods should be aware that the FDA expects all food ingredients sold in the United States to meet strict limits for lead contamination. Although specific regulations for maximum permissible lead levels in food ingredients do not exist for most foods, nevertheless lead levels should always be kept to a minimum, and the suitability of a lead limit will be determined by the intended uses of the ingredient. Since a GRAS determination is specific to the intended uses of the food/food ingredient, there may be instances where information characterizing the novel physicochemical properties should be included. For example, many traditional vegetable oils are unsuitable for frying or spraying applications because of their high content of polyunsaturated fatty acids, which are unstable at elevated temperatures and in highly oxidative environments. Vegetable oils produced from GM oil seed, containing high thermal and oxidative stability, should include a measure of this physicochemical property in the specification (e.g., Active Oxygen Method and Peroxide Value). Assurance that the manufacturing conditions produce a consistent product meeting the product specifications is required. Certificates of analyses from a minimum of three to five nonconsecutive lots of the food ingredient, meeting the product specifications, are required to show that the manufacturing process is sufficiently controlled so that the specifications are truly representative of the product. The composition of a food derived from a GM plant will not only be influenced by the processing/manufacturing method but, more importantly, will also be influenced by the inherent variability in the composition of the whole food, which is highly influenced by environmental and other agronomic factors. Thus, establishing the agronomic stability of the modified trait during the

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development process is important. Compositional data obtained from several different generations in different growing locations (representative of the anticipated growing locations for the crop) should be produced, so that suitable/reliable product specifications can be developed. This can present a difficulty to the producer of the GM plant, since manufacturing conditions for food commodities typically involve large quantities of product, which may not be available during product development. Both bulk stability of the GM food ingredient under suitable storage conditions, and stability data of the food ingredient under the various intended conditions of use of the food ingredient are required. In some instances, these data can be obtained from the literature if stability studies from comparable materials are determined to be applicable to the stability of the GM substance under various food uses.

28.3.2

INTENDED USES

Exposure assessments form a critical component of any safety assessment. The introduction of nutritionally enhanced foods derived from GM plants to the marketplace can have significant beneficial or adverse nutritional impacts on frequent consumers of such foods who have historically consumed foods derived from the non-GM counterparts. Furthermore, the safety of a newly introduced protein, or a genetically induced secondary metabolite, can only be determined if quantitative estimates of exposure are known. Multiple factors need to be considered when conducting exposure estimates, and the types of end-products for which a GM food is intended for use need to be thoroughly characterized. Furthermore, consistent with federal regulations, a typical GRAS determination requires that the proposed food uses and corresponding food types be grouped according to Title 21, section 170.3 of the Code of Federal Regulations. Estimates of exposure can become complex when nutritionally enhanced food contains numerous compositional changes, and is intended to replace a widely consumed food in the diet. Food uses such as deep-frying oils can also present challenges to exposure estimation. Once the intended food use and use levels have been determined, exposure estimates are then produced using consumption estimates obtained from various food survey databases in conjunction with statistical modeling software. Intake estimates can also present challenges to seed producers of GM foods who are usually not end-users of the product, and therefore may not have an appreciation for the various types of intended uses for which foods produced from their crops/seeds may be used. Although the development of intake estimates for nutritionally enhanced foods produced from GM crops can be very labor intensive, careful consideration of the intended food uses during a GRAS determination can provide marketing opportunities by increasing the number of food uses and, therefore, the number of potential companies interested in purchasing their product.

28.3.3 SAFETY The safety of foods derived through biotechnology must be evaluated against existing standards of food safety that are applied to traditional foods. The World Health Organization’s food safety standard is reasonable certainty that no harm will result from consumption of a food, and in the United States, “bioengineered food should be just as safe as conventional or traditional food” (FDA, 1992) is used as a guiding principle for GRAS determinations. Thus, the focus is on the product, not the process by which it was derived, recognizing that rDNA technology inherently does not confer a unique or novel potential for hazard. Historically, various food crops were bred and selected because they could be safely eaten, recognizing that many, if not all, food plants contain toxins such as solanine (potato), tomatine (tomato), cucurbiticin (cucumber), psoralens (celery), and hydrogen cyanide (cassava). Plant breeders have long recognized the potential for toxic substances to be present in foods, and varieties have been selected on the basis of acceptably low levels along with other agronomic traits. One notable example of a cultivar that was not recognized as unsafe until after

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it was introduced to the market was the Lenape potato, which contained high levels of solanine (Akeley et al., 1968; Zitnak and Johnston, 1970). The levels of toxins found in many conventional vegetables may be only 2–5 times lower than amounts that can cause toxicity, but this level of risk is considered acceptable in view of the fact that these foods provide nutritional value to the human diet. As a result, it is recognized that human food supply is not without risk, and a theoretical concept of “zero risk” is unattainable, due to endogenous toxic factors. Consequently, inherent risk at an acceptable level is a basic tenet of food safety and is consistent with the standard of “reasonable certainty of no harm.” For perspective, the methods used in plant breeding have long depended on the laborious practice of cross breeding, to enhance desirable traits and to minimize those that are not. More recently, methodology has expanded to include mutagenesis, cell fusion, and embryo rescue, but these can also lead to rather large or multiple changes in genetic material in comparison to rDNA technology, which is considered to be more directed and precise. Hence, modern plant breeding with rDNA aims to add or extract specific genes in order to enhance specific traits without causing significant unintended changes in existing traits or composition that are not beneficial. It goes without saying that any gross, phenotypic differences in appearance of the new crop would raise serious doubts about consumer acceptance, which would negate any benefits obtained from the agronomic or nutritional enhancements introduced to the food. Part of any safety assessment of a biotech food is a consideration of the donor genetic construct that has been transferred in terms of its size, its intended and known insertion sites in the recipient genome, and details about affiliated promotor sequences and marker genes. Of equal importance is information about the donor organism to ensure that there is little or no potential for inadvertent transfer of genetic material encoding for pathogenicity, allergenicity, or toxins. It is the expression product that is subject to the most scrutiny, because it represents novel protein produced in a new cellular environment in the recipient organism (i.e., crop). Protein safety will be discussed in detail in a subsequent section. 28.3.3.1 Substantial Equivalence The intent of the biotechnology process is to produce a food product that is “substantially equivalent” to the traditional food for all its characteristics except for the intended effect of the new trait. Therefore, the safety evaluation of foods derived from GM sources is typically initiated with a comparative or substantial equivalence approach, where characteristics of the food derived from the GM plant (or the plant itself) are compared to those of corresponding foods derived from the non-GM near-isogenic counterpart. The use of substantial equivalence in the evaluation of GRAS substances is recognized by the FDA, and in the Proposed Rule regarding Substances Generally Regarded as Safe, the agency has stated it “believes that in certain instances the concept of substantial equivalence may have applicability to the technical element of a GRAS determination” (FDA, 1997). The agency further states that “a critical factor that must be considered when applying the concept of substantial equivalence is any difference in composition or characteristic properties between the substances being compared” (FDA, 1997). A typical substantial equivalence comparison requires the generation of extensive bioanalytical data and involves actual measurements of concentrations of inherent constituents of the food/plant. All agricultural foods contain a vast number of compositional constituents, and it is impossible to comprehensively analyze every constituent present in the food component of a plant. Due to the obvious logistic and financial limitations that are inherent to analytical sampling, some level of selectivity in the analytical endpoints will be required. In fact, this limitation has been raised as a caveat to safety evaluations that are based almost exclusively on substantial equivalence (Millstone et al., 1999). The generation of an analytical sampling plan should consider the effect of the genetic modification on various metabolic pathways and should focus on the specific food components of a given crop or food type that are believed to have beneficial (nutrients and bioactive factors) and potential adverse (endogenous allergens, toxins, and antinutritional factors) effects on human

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health. The specific nutrients and bioactive components should be selected with the aim of efficiently characterizing the food in a manner that is reasonable given the financial costs of analytical testing, yet comprehensive enough to ensure safety. Although the nutrient compositions of most agricultural commodities are well established, the discovery of plant constituents with previously unrecognized bioactive properties continues. Therefore, the analytical plan should take into account the state of current thinking in the nutritional and toxicological sciences and should also consider the existence of adequate validated analytical methods and the availability of comparator data from the traditional counterpart. Chemical analysis can be a side-by-side comparison of the GM plant with its near-isogenic control; however, given the natural variability in the levels of many constituents, a composition database is a more comprehensive resource for this purpose. Several have been developed and are accessible through the internet. Examples include the International Network of Food Data Systems (INFOODS), established in 1984 (FAO, 2003), the USDA National Nutrient Database for Standard Reference (latest update: USDA, 2008), and the International Life Sciences Institute (ILSI) Crop Composition Database (ILSI, 2004). In some instances the comparison of a novel food to its conventional counterpart using the substantial equivalence approach may form the basis for evaluating the safety of a novel food (OECD, 1993; FDA, 1997). This approach recognizes that traditional toxicology testing of whole food or macromolecular materials is not practical and may be prone to artifactual results due to possible nutritional imbalance (Munro et al., 1996). Substantial equivalence does not infer that the safety of a food produced from a GM plant is based entirely on the compositional comparison of a food with food derived from its traditional counterpart; however, in conjunction with an understanding of the metabolic implications of the genetic transformation to the host plant, the identification of compositional similarities and differences between the novel plant/food and its traditional counterpart provides a starting point by which the safety of a food derived from a novel plant can be determined. In instances where the nutritional impact of significant changes in the nutritional profile or level of naturally occurring toxin cannot be determined with respect to the intended conditions of use of the food, or in instances where the safety of a newly introduced protein cannot be determined, the requirement for human and animal studies may be warranted; however, the specific requirements in this regard (i.e., what constitutes the requirement for animal or human safety studies) cannot be universally defined and are determined on a case-by-case basis. Guidance and criteria for evaluation have been suggested for microbial enzymes used in food processing using a decision tree approach (Pariza and Johnson, 2001), and recently for protein products of agricultural biotechnology using a tiered, weight-of-evidence approach (Delaney et al., 2008). The FDA has also issued a statement of policy regarding Foods Derived from New Plant Varieties*; therefore the safety assessment of foods derived from GM plants should be conducted in line with the above guidance documents, and in a manner that is consistent with the proposed ruling on GRAS substances (FDA, 1997). 28.3.3.2 History of Safe Use The rationale for using traditional foods as comparators in assessing the acceptability of novel biotech foods for human consumption is that these foods have been consumed by human populations for many years, usually generations, and as such they have a “history of safe use” (HOSU) (Constable et al., 2007). For foods that are in common use, in one country or another, it is logical to assume that through time these foods, often through processes of trial and error, develop a HOSU. It becomes common knowledge and practice to avoid certain foods unless they are cooked or otherwise prepared, and to benefit from other foods in season, for example, when they ripen sufficiently. People have learned to exploit native foods that are generally available and to adopt others that can * The term “variety” was defined in the proposed ruling as a general term to describe subgroups (whether varieties or cultivars) of plants within a species developed from desirable traits and therefore includes plants derived through traditional breeding practices, and those developed using biotechnology.

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be produced under local conditions. Through all this experience, food traditions become established and can be said to have a HOSU. This is not to say that traditional food is safe in an absolute sense, because due to sensitivities, metabolic conditions, and allergies, many consumers can experience adverse reactions to certain foods, even death; thus, it may be more appropriate to use the terminology “history of apparent safe use.” The standard for a biotech food, using the rationale of “HOSU” as part of the weight of evidence to support its safety, is that novel food use is just as safe as the traditional counterpart. 28.3.3.3 Safety of Novel Proteins Regardless of the fact that the novel trait in a biotech food may have a HOSU in the traditional food used as its source, and that the biotech food used as its recipient may be substantially equivalent to a traditional food that does not have this trait, a safety assessment is not complete until the newly introduced and expressed protein responsible for this trait undergoes satisfactory evaluation. Proteins by their nature would not be considered to be hazardous, since they represent a major class of food and are required for biochemical and structural aspects of all living organisms. Nonetheless, certain proteins are toxins (Aktories and Just, 2000) or allergens (Goodman et al., 2005) and have also been associated with pathogenicity (Falkow, 1997). Thus, the potential for a novel protein in a GM plant to exhibit such undesirable effects is subject to comprehensive evaluation in the course of safety determinations, including GRAS. One such methodical approach has been proposed by the ILSI International Food Biotechnology Committee Task Force on Protein Safety (Delaney et al., 2008), using a “scientifically based two-tiered, weight-of-evidence strategy to assess the safety of novel proteins used in the context of agricultural biotechnology.” Tier I is intended to examine the potential for hazard, and Tier II is proposed to characterize any hazard that may be identified in the first tier. The various aspects suggested by Delaney et al. (2008) for consideration in Tier I relate to the characterization of potential hazard and include the following: (1) mode of action as it relates to the biological function and intended application of the protein; (2) assessment of HOSU, as described above; (3) bioinformatics involving amino acid sequence analysis with comparisons to known proteins with similar functions as well as known toxins, antinutrients, and allergens; (4) chemicophysical properties of the protein, such as digestibility and stability, to assist in predicting a possible hazard potential; and (5) expression level and dietary intake, to provide some indication of potential exposure. In certain cases, and such examples will be discussed in a subsequent section, Tier I analysis will reveal no hazard potential and will permit a conclusion to be drawn with confidence that the protein would be reasonably expected to cause no harm. If not, further study may be necessary to assess whether the protein does pose a hazard. Types of studies involved in the characterization of hazard at the Tier II level include acute toxicity testing and/or other tests for allergenicity, mode of action, or repeated dose toxicity. This structured scheme for assessing the safety of novel proteins provides a scientifically derived comprehensive basis for the determination of GRAS status for a GM food product. Various aspects of this evaluation framework are discussed in more detail in the sections that follow. This suggested approach, based on weight of evidence, is an alternative to decision tree approaches that provide yes/no answers that lead to specific next steps (Pariza, 1992; Pariza and Johnson, 2001). The following sections provide a brief introduction to each of the Tier I parameters. 28.3.3.3.1 Tier I: Mode of Action and Specificity Before a genetic alteration is performed to produce a novel organism, an intended use was envisioned for the purpose of fulfilling some function or imparting some characteristic that otherwise is not possible in the recipient organism. Examples are diverse, from enhanced nutritional properties of a functional food to insect or herbicide resistance of crop plants. These are not random experiments, but concerted efforts to duplicate known functions by known proteins encoded by known genes, and to introduce these traits into other organisms. Thus, at the outset, much should be known about the protein and its role within the organism that would serve as its source. If the protein is an enzyme,

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for example, detailed knowledge about its activity would provide the opportunity of contemplating how this activity could be exploited in an alternate genetic environment within an organism that lacks this function. Once the genetic alteration has been performed, continuing study would confirm that the new protein fulfils the intended function via the same mode of action and with the same specificity as in the donor organism. Any genetic construct that does not express the desired trait will not be subject to further development. Hence, any characterization of the mode of action and specificity of a protein in its native environment, including relevance for humans, would serve as the basis for its evaluation in the novel organism. The protein would be expected to have the same activity and mode of action, but this would need study, characterization, and confirmation to support the safety of the modified organism. 28.3.3.3.2 Tier I: HOSU The concept of HOSU has already been discussed above in connection with the evaluation criterion known as substantial equivalence, but as one of five guiding components in Tier I identification of hazard potential, further consideration is warranted. In the context of the regulatory approach to the determination of GRAS, the FDA has adopted the HOSU concept by stating that further hazard characterization of a protein is not needed if it is the same or similar to proteins that exist in the food supply and thereby can be considered as GRAS (57 FR 22984). Other agencies also recognize the usefulness of HOSU for guiding evaluation and testing strategies (Codex, 2003; EFSA, 2006). The key to reliance on this concept is the degree to which it can be demonstrated that the protein in question is the same or sufficiently similar to a protein or proteins with a HOSU. Case-by-case consideration of available information, such as source organism and function of the protein, as well as consumption level, will be necessary to evaluate HOSU. The absence of a demonstrated HOSU, of course, does not mean that a novel protein is not safe. Rather, its safety would have to be determined through other means, possibly other aspects of Tier I or Tier II. 28.3.3.3.3 Tier I: Bioinformatics The general goals of bioinformatics are to show that a protein’s amino acid sequence is not similar to sequences that are associated with adverse effects (e.g., toxicity, allergenicity, or pathogenicity) and that it does share homology with proteins with similar functions or that are known to be in the common diet. Searchable databases contain millions of known sequences, and more are added on a regular basis. Computer-assisted analysis has become widely used with tools such as FASTA* (Pearson and Lipman, 1988) and the basic local alignment search tool (BLAST) (Altschul et al., 1990), providing an assessment and ranking of similarity to known sequences as well as a propensity for tertiary structures. Confidence in the safety of a novel protein is reinforced when its sequence is shown to have no similarity to the various groups of proteins with established toxicity and is shown to be similar to proteins that have the same or similar functions or modes of action. Such analysis may also provide evidence for relationships to foods that have well-established HOSUs for intended uses in food or feed. 28.3.3.3.4 Tier I: Stability Proteins in the diet that are readily digested or broken down during food processing are not likely to demonstrate unwanted, residual enzymatic activity or other properties, including allergenicity. Thus, one characteristic of proteins examined in the Tier I battery for characterization of hazard potential is protein stability. This could possibly be predicted by sequence comparisons with other proteins that are or are not stable, but typically an in vitro digestibility study is performed to see whether or not the protein can survive digestion using simulated gastric fluid. Similarly, protein function is a useful indicator of stability, following exposure to simulated temperature and/or pH changes that would normally be encountered during processing. * FASTA denotes FAST-All and refers to the bioinformatic software used to analyze DNA and protein sequence alignments.

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28.3.3.3.5 Tier I: Expression Level and Dietary Intake The necessity for, and various considerations required during dietary intake estimations of all substantial compositional changes, including the introduction of novel proteins, as a result of introducing food products of genetic engineering to the food supply are discussed above; however, with respect to the introduction of novel proteins to GM foods, the dietary intake estimations in conjunction with history of use and protein stability can, in some instances, form the basis for safety, without the requirement of additional toxicological testing. Novel GM proteins are unlikely to be introduced to a plant for nutritional purposes. In the vast majority of cases, the introduced protein is usually an enzyme, which mediates secondary effects on metabolic pathways resulting in compositional changes or the introduction of insecticidal/pesticidal resistance traits to the plant. Therefore, quantitatively the introduced protein is usually present in the food portion of the plant in minute quantities. When the introduced protein is from a nonallergenic plant or organism with a history of safe consumption in the diet, trivial dietary exposures are often sufficient to support the safety of the introduced protein. When the estimated consumption of a GM protein is greater than that occurring from historical consumption via its traditional food, or when the protein is from a potentially allergenic donor organism, Tier II hazard characterization is required; however, an exception to this are highly refined oils prepared from GM plants. For oils produced from GM plants, the introduced protein is not expected to be present in the oil; therefore, provided the oil is highly refined and produced with standard oil refining techniques using cGMP, allergenicity and toxicity concerns resulting from the introduction of novel proteins to the plant are not warranted. However, the introduction of food oils to the food supply without a comprehensive safety evaluation of the GM protein presents a due-diligence safety concern, in that it can be logistically difficult to ensure that by-products of the oil refining process (lecithin, protein concentrations) are not introduced to the food supply. For example, Starlink corn containing a genetically inserted Bt protein was permitted for commercial sale in the United States, with a stipulation that food products containing the protein did not enter the food supply due to potential allergenicity concerns over the protein. Various food products containing the Bt protein were inadvertently used in the production of food—Taco Bell shells being a notable and particularly well-publicized example—resulting in a public relations nightmare for the producer of the crop, and the GM industry in general. 28.3.3.3.6 Tier II: Hazard Characterization When Tier I evaluation is insufficient to establish the safety of a plant-incorporated protein, the safety assessment requires hazard characterization of the protein. As proposed by Delaney et al. (2008), the types and numbers of studies required to establish safety under Tier II evaluation are determined on a case-by-case basis, but typically involve acute toxicity, subchronic toxicity studies, and hypothesis-based testing strategies. When a GM protein does not have a HOSU in the diet and/or is determined to be homologous to a known toxin as determined via bioinformatic analyses, the first step in the Tier II evaluation process would be to conduct an acute toxicity study. Because of the difficulty and cost in preparing sufficient quantities of protein required of toxicity studies, the use of mice is generally the preferred species of choice for most Tier II testing studies. Limit doses of 1000 mg/kg, or 100- to 1000-fold above the estimated exposures, and administration of the compound via gavage administration are recommended. Although the use of parenteral exposures is not typically considered a suitable administration route for the safety evaluation of food ingredients, there may be instances where parenteral exposures are appropriate. For example, for a GM protein shown to be homologous to a known toxin, for which high-quality data characterizing its toxicity are limited to intraperitoneal studies, the use of the intraperitoneal route for acute toxicity testing may be justified. Consistent with the fact that proteins are nutrients, acute toxicity studies conducted with GM proteins have not revealed any evidence of toxicity to date. As concluded by Delaney et al. (2008), for plant-introduced proteins without a history of use in the food supply, which are not stable to food processing conditions and/or gastrointestinal digestion, not

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homologous to known toxins, and shown to be nontoxic following acute toxicity testing, additional safety information is unlikely to be provided by conducting repeat-dose toxicity studies. If safety concerns of prolonged exposure to a GM protein are raised during the Tier I safety evaluation (e.g., the protein is determined to be homologous to a known antinutritional compound), then further Tier II evaluation using repeat-dose toxicity studies may be required. Since most GM foods are introduced to the diet as macronutrients, administering the food in the diet may not provide a sufficient margin of safety for exposure to the GM protein to support safety. Therefore, the use of purified transgenic protein is typically necessitated. Given the high cost of producing enough protein for use in a repeat-dose rodent toxicity study, the need to conduct repeat-dose toxicity studies often represents the end of the GM cultivar, and further research and development is typically abandoned at this point. Finally, the use of various hypothesis-based testing strategies, including in vitro mechanistic studies, has been proposed as an additional means of characterizing the hazard of a GM protein for use in food. However, the lack of validated in vitro models that can be extrapolated to in vivo outcomes in animals precludes the use of these types of studies as providing pivotal information supporting safety, and these types of data are usually presented as corroborating evidence in conjunction with acute toxicity testing.

28.3.4

GENERAL RECOGNITION OF SAFETY

As mentioned previously, there are two important aspects to the requirements of the GRAS standard. The first is that the types of scientific data that are used to support a scientific procedures GRAS determination must be of the same quality and quantity as that required to support a food additive petition, and the second is that the pivotal data used to determine the safety of the ingredient must be generally available. The general availability of such data is typically defined as scientific studies published in quality peer-reviewed journals. Furthermore, to meet the definition of general recognition, any pivotal studies used to support a self-affirmed GRAS determination must be generally available in the public domain for a sufficient period of time so that such information can become disseminated within the scientific community. Although not specifically defined by U.S. law or in guidance documentation, the minimum period of time for which published studies are required to be present in the public domain, and therefore qualify as generally available, is 6 months. The general recognition standard also implies that all data, both negative and positive, are considered during a GRAS determination. In instances where some data related to the safety of a novel food ingredient indicating concern are present in the literature, the overall weight of evidence must scientifically refute such data. Finally, general recognition also requires that the totality of evidence used to support a GRAS determination is recognized by scientific experts within the community who are qualified by scientific training and experience to evaluate the safety of a given food ingredient under its proposed uses in food; this is established by convening a panel of such experts who are then charged with evaluating and rendering an opinion on the GRAS status of a food ingredient for a given set of food uses. Following a unanimous consensus within the Expert Panel that the ingredient would be considered GRAS under the proposed uses in food, the company can then legally market the food in the United States without premarket approval under section 409 of the FFDCA. Since the GRAS process is one that does not require preview by regulatory authorities before entry into the U.S. marketplace, and a GRAS self-affirmation is essentially conducted “behind closed doors” within the company, the FDA has established a voluntary notification procedure to provide some level of transparency for the safety evaluation of foods entering the U.S. marketplace. During this premarket notification procedure, the FDA reviews the types of data that are used to support general recognition, and renders an opinion on the qualification of such data under the legal requirements set forth in the GRAS rule. Following a review of the scientific data, one of three outcomes are possible: the agency issues a letter stating that the information provided to the agency

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does not form the basis for GRAS; the agency issues a letter stating that it does not disagree with the company’s GRAS determination; finally, a third option of withdrawing the notification is permitted, and typically occurs following failure to adequately address questions raised by the agency during the review process. It should be noted that a positive letter issued by the FDA is not an official endorsement by the agency regarding the GRAS status of the ingredient. Although the GRAS notification process is optional, a successful GRAS notification has obvious marketing advantages for the producer or distributor of a novel food ingredient, and in fact the GRAS notification procedure has become a standard by which most large food manufacturers expect food ingredients they purchase for sale to the U.S. public to meet. In addition to foods produced using GM microorganisms, novel foods produced from GM crops have also been notified to the FDA under the GRAS provisions. Stearidonic acid soybean oil, and a soybean oil with reduced palmitic and linolenic acid, and increased oelic acid are two examples. Since the fatty acid profiles of these soybean oils are no longer substantially equivalent to soybean oils produced from their traditional counterparts, the HOSU status of soybean oil does not apply to these oils and their use in various food applications, therefore requires GRAS self-affi rmation before introduction to the U.S. marketplace. As other nutritionally enhanced GM foods are developed, the occurence of these food ingredients on the FDA notification list is expected to become commonplace in the near future.

28.4

FOODS DERIVED FROM BIOTECHNOLOGY AND HEALTH CLAIMS IN THE UNITED STATES

The genetic modification of a crop results in the intentional introduction of input/output traits. Input traits protect the crop from disease or damage, either by increasing resistance to, for example, a virus or an insect or by providing tolerance to herbicides or pesticides (ILSI, 2003). Output traits affect the nutritional composition (i.e., protein or amino acids; lipids or fatty acids; carbohydrates; vitamins, minerals, and antioxidants; enzymes or enzyme activity; or antinutrients) of the crop and are intended to improve the technical properties of the crop or the nutritional status of the end consumer (ILSI, 2007). Crops that are genetically modified for output traits are being developed either for incorporation into animal feed to improve the growth/performance of the animal or for incorporation into the human diet to improve the overall nutritional status. Examples of crops genetically modified for output traits and intended for improving the nutritional status of humans are summarized in Table 28.1. The substantiation of human health claims in the United States requires data from human intervention studies, with animal studies considered to be supporting evidence only. Thus, it is necessary to consider how human intervention studies should be designed in order to assess the efficacy of a crop genetically modified for an output trait. The process and requirements for the substantiation of health claims in the United States apply to foods (including those obtained from GM sources) and dietary supplements/nutraceuticals. In the following sections, the types of claims permissible in the United States will be reviewed, as will the scientific requirements for each type of claim. The implications of these scientific requirements, with respect to the efficacy evaluation (in humans) of crops genetically modified for output traits, are reviewed.

28.4.1

PERMISSIBLE FOOD CLAIMS IN THE UNITED STATES

The part of the FDA that regulates the use of claims is The Office of Nutritional Products, Labeling, and Dietary Supplements of the Center for Food Safety and Applied Nutrition (CFSAN). While the focus of the current review is on claims that can be made on functional foods, it should be noted that claims on dietary supplements/nutraceuticals are also regulated by the same body. Three broad categories of food claims are permissible in the United States: (i) nutrient content claims, (ii) structure/function claims, and (iii) health claims.

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TABLE 28.1 Examples of Crops GM for an Output Trait Reference Cited in Sun (2008) Katsube et al., 1999. Plant Physiol. 120: 1063–1073 Zhang et al., 2003. Transgenic. Res. 12: 243–250 Molvig et al., 1997. Proc. Natl. Acad. Sci. USA 94: 8393–8398 Liu, Q.Q., 2002. Genetically engineering rice for increased lysine. PhD dissertation, Yangzhou University and the Chinese University of Hong Kong Chakraborty et al., 2000. Proc. Natl. Acad. Sci. USA 97: 3724–3729 Lai and Messing, 2002. Plant J. 30: 295–402 Zhu and Galili, 2003. Plant Cell 15: 845–853 Ye et al., 2000. Science 287: 303–305 Paine et al., 2005. Nat. Biotech. 23: 482–487 Visser et al., 1991. Mol. Gen. Genet. 225: 289–296 Voelker et al., 1992. Science 257: 72–74 Hood et al., 1997. Mol. Breeding 3: 291–306 Boothe et al., 1997. Drug Develop. Res. 42: 172–181 Woodard et al., 2003. Biotech. Appl. Biochem. 38: 123–130

Crop

Output Trait

Rice

Increased methionine content

Cassava

Increased protein content

Lupine

Increased sulfur amino acid content

Rice

Increased lysine content

Potato

Increased essential amino acid content

Corn

Increased methionine content

Arabidopsis

Increased lysine content

Rice

Potato

Synthesis of β-carotene (as a source of vitamin A) (“Golden Rice 1”) Increased synthesis of β-carotene (as a source of vitamin A) (“Golden Rice 2”) Expression of amylopectin instead of amylose

Canola Maize

Increased lauric acid content Introduced presence of egg white avidin protein

Rapeseed oil

Introduced presence of hirudin (anticoagulant)

Maize

Introduced presence of bovine trypsin

Rice

Cited in EFSA GMO Panel Working Group (2008) Avraham et al., 2005. Plant Biotechnol. Alfalfa Increased cysteine and methionine content J. 3: 71–79 ILSI, 2004 Canola Increased vitamin E content ILSI, 2004 Canola Increased lauric acid content ILSI, 2004 Canola Increased γ-linolenic acid content ILSI, 2004 Canola Increased omega-3 fatty acid content ILSI, 2004 Canola Increased β-carotene content Husken et al., 2005. Theor. Appl. Genet. Canola Increased resveratrol glucoside content 111: 1553–1562 ILSI, 2004 Cassava Decreased cyanogenic glycoside content Siritunga and Sayre, 2004. Plant Mol. Cassava Decreased cyanogenic glycoside (linamarin) content Biol. 56: 661–669 ILSI, 2004 Coffee Decreased caffeine content Decreased lignin content and increased lignin Chen et al., 2004. Funct. Plant Biol. 31: Fescue grass digestibility 235–245 continued

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TABLE 28.1 (continued) Examples of Crops GM for an Output Trait Reference

Crop

Output Trait

Wu et al., 2005. Nat. Biotechnol. 23: 1013–1017 ILSI, 2004 ILSI, 2004

Indian mustard

ILSI, 2004 Drakakaki et al., 2005. Plant Mol. Biol. 59: 869–880 O’Quinn et al., 2000. J. Anim. Sci. 78: 2144–2149 ILSI, 2004 ILSI, 2004 ILSI, 2004 ILSI, 2004 ILSI, 2004 ILSI, 2004 ILSI, 2004 ILSI, 2004 ILSI, 2004 ILSI, 2004 ILSI, 2004 ILSI, 2004 Karunanandaa et al., 2005. Metabolic Eng. 7: 384–400 ILSI, 2004 ILSI, 2004 ILSI, 2004 Davuluri et al., 2005. Nat. Biotechnol. 23: 890–895 Health Canada (2008)

Maize Maize

Increased very long-chain polyunsaturated fatty acid content (including AA, EPA, and DHA) Decreased fumonism Increased production of protein with a favorable amino acid profile Increased vitamin C content Increased bioavailable iron content

Maize

Increased lysine content

Potato Potato Potato Potato Rice Rice Rice Soybean Soybean Soybean Soybean Soybean Soybean

Increased starch content Increased inulin content Increased sulfur-rich protein content Decreased solanine content Increased β-carotene content Increased iron content Decreased production of allergenic proteins Increased sulfur amino acid content Increased oleic acid content Increased oleic acid content Decreased content of immunodominant allergen Increased isoflavone content Increased tocochromanols (including tocotrienol)

Sweet potato Tomato Tomato Tomato

Increased protein content Increased provitamin A and lycopene content Increased flavonoid content Increased provitamin A, lycopene, and flavonoid content

Crop

Output Trait

Company

Maize Maize

Monsanto Canada Pioneer Hi-Bred International First Venture Technologies Corporation Pioneer Hi-Bred International Dr. van Vuuren, University of British Columbia Monsanto Canada Inc. Martek

Soybean Corn Yeast Soybean Yeast

Enhanced stearate content Increased digestibility and energy availability Produces alcohol with low ethylcarbamate content Low linolenic acid content Produces wine with low malic acid content

Maize Sunflower

Archer Daniels Midland Company

Sunflower

Martek Biosciences Corporation

Sunflower

Increased lysine content High oleic acid-containing sunflower oil and oil extracted from the unicellular alga Crypthecodinium cohnii is used to make DHA-rich DHASCO® oil Moderate oleic acid sunflower oil has longer shelf life and is more stable during cooking High oleic acid-containing sunflower oil is combined with oil extracted from the unicellular alga Crypthecodinium cohnii to make DHASCO, and with oil extracted from the unicellular fungus Mortierella alpina to make ARASCO® (ARA-rich oil) continued

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TABLE 28.1 (continued) Examples of Crops GM for an Output Trait Company

Crop

Output Trait

Canola Council of Canada Agriculture and Agri-Food Canada

Canola Soybean

DuPont Canada Inc. Pioneer Hi-Bred International Calgene FDA (2009a) Syngenta Seeds, Inc. Agritope DuPont Agritope Calgene DNA Plant Technology Zeneca

Soybean Canola Canola

Reduced erucic acid and aliphatic glucosinolate content Low linolenic acid content (but higher than corn or olive oil) High oleic acid content High oleic acid and low linolenic acid content High laurate and myristate content

Corn Cantaloupe Soybean Tomato Canola Tomato Tomato

α-Amylase expression Delayed fruit ripening due to reduced ethylene synthesis High oleic acid soybean oil Delayed fruit ripening due to reduced ethylene synthesis High laurate canola oil Delayed ripening due to reduced ethylene synthesis Delayed softening due to reduced pectin degradation

Some examples contain uses relevant to industrial applications, domestic animal feed, or have been developed for research purposes only. FDA (2009a): http://www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?rpt=bioListing Health Canada (2008): http://www.hc-sc.gc.ca/fn-an/gmf-agm/appro/index-eng.php EFSA GMO Panel Working Group (2008). Sun (2008). FDAMA: http://www.fda.gov/RegulatoryInformation/Legislation/FederalFoodDrugandCosmeticActFDCAct/ SignificantAmendmentstotheFDCAct/FDAMA/default.htm

28.4.2

NUTRIENT CONTENT CLAIMS

28.4.2.1 Definition and Scope Nutrient content claims describe the level of a nutrient or dietary substance in food by using terms such as “free,” “low,” “high,” “rich in,” “excellent source of,” “good source of,” “contains,” “provides,” and so on; alternatively, nutrient content claims may compare the level of a nutrient/dietary substance in food to that of another similar food by using relative terms such as “reduced,” “less,” “more,” and so on. (FDA, 2008b). Other acceptable types of nutrient content claims include statements that describe the percentage of a nutrient in relation to a reference daily intake (RDI) [21 CFR 101.13(q)(3)(i)], and for those nutrients/dietary substances that are not associated with RDIs, the percentage level of the nutrient/dietary substance in the food may be declared, so long as the actual amount of the nutrient/dietary substance per serving is also declared [21 CFR 101.13(q)(3) (ii)(A)] (FDA, 2008a): for example, “contains 40% omega-3 fatty acids or 20 mg per 50 g serving.” Comparative percentage claims are also permitted, provided the actual amounts of the nutrient/ dietary substance per serving in both foods are also stated (e.g., “brand A potato chips have X amount of salt per serving, which is 25% less than brand B potato chips, which have XX amount of salt per serving”) [21 CFR 101.13(q)(3)(ii)(B)] (FDA, 2008a). Nutrient content claims (e.g., “Product X is an excellent source of omega-3 fatty acids”) are generally expressed per Reference Amount Customarily Consumed per eating occasion (i.e., per serving) (21 CFR 101.12) (FDA, 2008a). Nutrient content claims may only be made for nutrients or dietary substances that have established daily values (Table 28.2); however, for nutrients/dietary substances that do not have daily values, such as carotenoids, amino acids, polyphenols, and so on, the amount of nutrient per serving may be declared quantitatively (e.g., contains X g of sulfur amino acids per 100 g serving). When a nutrient content claim is made on a food, and the food contains

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TABLE 28.2 Nutrients for which Nutrient Content Claims are Permissible Nutrient Total fat Saturated fat Cholesterol Sodium Potassium Total carbohydrate Dietary fiber Protein Vitamin A Vitamin C Calcium Iron Vitamin D Vitamin E Vitamin K Thiamin Riboflavin Niacin Vitamin B6 Folate Biotin Pantothenic acid Phosphorus Iodine Magnesium Zinc Selenium Copper Manganese Chromium Molybdenum Chloride Vitamin B12

Unit of Measure g g mg mg mg g g g IU mg mg mg IU IU μg mg mg mg mg μg μg mg mg μg mg mg μg mg mg μg μg mg μg

Daily Value 65 20 300 2400 3500 300 25 50 5000 60 1000 18 400 30 80 1.5 1.7 20 2 400 300 10 1000 150 400 15 70 2 2 120 75 3400 6

Source: FDA, 2008a. U.S. Code of Federal Regulations (CFR). Title 21—Food and Drugs (Food and Drug Administration). Washington, DC: U.S. Government Printing Office (GPO). Available at http://www.access.gpo. gov/nara/cfr/cfr-table-search.html#page1 (See Table at the end of the reference list for CFR sections.); FDA, 2008b. Guidance for industry: Substantiation for dietary supplement claims made under Section 403(r) (6) of the Federal Food, Drug, and Cosmetic Act. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN). Available at http://www.fda.gov/Food/ GuidanceComplianceRegulatoryInformation/GuidanceDocuments/ DietarySupplements/ucm073200.htm

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TABLE 28.3 Nutrient Levels for which a Product with a Nutrient Content Claim would Require a Disclosure Statementa Nutrient Total fat Saturated fat Cholesterol Sodium

Foods Other than Main Dishes and Meal-Type Products 13.0 g 4.0 g 60.0 mg 480 mg

Main Dish Productsb 19.5 g 6.0 g 90.0 mg 720 mg

Meal-Type Productsc 26.0 8.0 120 mg 960 mg

a

Levels are presented per reference amount customarily consumed (RACC), per labeled serving, or, for foods with small serving sizes, per 50 g. b See 21 CFR 101.13(m) for the definition of a main dish product. c See 21 CFR 101.13(l) for the definition of a meal-type product. Source: FDA, 2008a. U.S. Code of Federal Regulations (CFR). Title 21—Food and Drugs (Food and Drug Administration). Washington, DC: U.S. Government Printing Office (GPO). Available at http://www.access.gpo.gov/nara/cfr/cfrtable-search.html#page1 (See Table at the end of the reference list for CFR sections.); FDA, 2008b. Guidance for industry: Substantiation for dietary supplement claims made under Section 403(r) (6) of the Federal Food, Drug, and Cosmetic Act. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN). Available at http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/ GuidanceDocuments/DietarySupplements/ucm073200.htm

other nutrients at levels that are associated with undesirable effects on human health (e.g., cholesterol), then a disclosure statement (“See Nutrition Information for Cholesterol Content”) is required to appear immediately adjacent to the nutrient content claim (see Table 28.3 for nutrients requiring a disclosure statement) (21 CFR 101.13) (FDA, 2008a). 28.4.2.2 Applicable Regulations, Scientific Requirements, and Guidance Documents The Nutrition Labeling and Education Act of 1990 (NLEA) permits the use of nutrient content claims that are made in accordance with FDA’s authorizing regulations. Compositional requirements for nutrient content claims are specifically defined in 21 CFR 101.13 (FDA, 2008a).

28.4.3

STRUCTURE/FUNCTION CLAIMS

28.4.3.1 Definition and Scope Structure/function claims describe the role of a nutrient or dietary ingredient in the maintenance of normal structure or function in humans, the mechanism by which a nutrient or dietary ingredient acts to maintain structure or function, or the general well-being associated with the consumption of a nutrient or dietary ingredient (FDA, 2003). Examples of structure/function claims include the following: “calcium is important in the formation and maintenance of bones and teeth,” “iodine aids in the normal function of the thyroid gland,” and “iron is necessary for red blood cell formation and for carrying oxygen to body tissues.” Structure/function claims cannot, either implicitly or explicitly, make reference to a disease or a disease state; however, they may make reference to a disease or nutrient deficiency, such as scurvy or pellagra, provided the prevalence of the nutrient deficiency disease in the United States is included in the claim wording (e.g., “X% of Americans are deficient in iron, which is associated with reduced risk of certain types of anemia. X product is a good source of iron”). 28.4.3.2 Applicable Regulations, Scientific Requirements, and Guidance Documents Regulatory procedures for the use of structure/function claims on dietary supplement/nutraceutical labels, which have been better delineated than for functional food ingredients, can be found in the

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Dietary Supplement Health and Education Act of 1994 (DSHEA) and in 21 CFR 101.93 (FDA, 2008a). In brief, it is the responsibility of the manufacturer to ensure that structure/function claims are accurate, truthful, and not misleading, as structure/function claims are not preapproved by the FDA (2003). Structure/function claims on dietary supplements must include a disclaimer that the FDA has not evaluated the claim and that the dietary supplement is not intended to “diagnose, treat, cure, or prevent any disease” (FDA, 2003). Manufacturers of dietary supplements must submit a notification to the FDA that includes the text of the structure/function claim no later than 30 days after marketing the dietary supplement. Several guidance documents relevant to structure/function claims have been released by the FDA, including a Small Entity Compliance Guide (http://www.fda.gov/Food/GuidanceCompliance RegulatoryInformation/GuidanceDocuments/DietarySupplements/ucm103340.htm) and a guidance document on the scientific substantiation of dietary supplement claims (http://www.fda.gov/Food/ GuidanceComplianceRegulatoryInformation/GuidanceDocuments/DietarySupplements/ucm073200. htm) (FDA, 2002, 2008b). Because these documents are guidance documents, they are nonbinding; however, their contents are important in that they provide insight into the type of scientific evidence that is expected by the FDA for the substantiation of structure/function claims. According to the guidance document on the substantiation of structure/function claims, human studies are required to support efficacy (FDA, 2008b). Other types of studies, including animal studies, in vitro studies, testimonials and other anecdotal evidence, meta-analyses, and review articles, while useful in providing background information, on their own would be insufficient to support a structure/function claim (FDA, 2008b). Among human studies, causality is best demonstrated by intervention studies. While observational studies are useful in supporting a cause-and-effect relationship, the demonstration of a cause-and-effect relationship solely from observational studies is challenging. The gold-standard study design for assessing the relationship between a dietary substance and a specific outcome is the randomized, double-blind, parallel-group, placebo-controlled trial (FDA, 2008b).

28.4.4 HEALTH CLAIMS 28.4.4.1 Definition and Scope Health claims describe the role of a food ingredient in reducing the risk of a disease or healthrelated condition (FDA, 2003). The FDA oversees all health claims made on foods. Unlike nutrient content claims and structure/function claims, health claims must be reviewed and approved by the FDA. 28.4.4.2 Applicable Regulations, Scientific Requirements, and Guidance Documents In the United States, health claims are grouped into one of three categories: (i) authorized health claims, (ii) health claims based on authoritative statements, and (iii) qualified health claims. (i) Authorized health claims: Authorized health claims may be used on the labeling of a food, so long as use of the claim is authorized by an FDA regulation. The scientific and regulatory requirements for authorized health claims are detailed in the 1997 NLEA, the Dietary Supplement Act of 1992, and the DSHEA of 1994 (FDA, 2003). Authorized health claims must be supported by credible scientific evidence from methodologically robust human intervention and observational studies, with a preference for the former study type (FDA, 2009a). 21 U.S.C. 343(r)(3)(B)(i) permits the FDA to authorize a health claim if the agency “determines based on the totality of the publicly available evidence (including evidence from well-designed studies conducted in a manner which is consistent with generally recognized scientific procedures and principles), that there is significant scientific agreement among experts qualified by scientific training and experience to evaluate such claims, that

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TABLE 28.4 Authorized Health Claims Food Component/Dietary Supplement Ingredient Calcium, vitamin D Dietary lipids (fat) Dietary saturated fat and cholesterol Dietary noncariogenic carbohydrate sweeteners Fiber-containing grain products, fruits, and vegetables Folic acid Fruits and vegetables Fruits, vegetables, and grain products that contain fiber, particularly soluble fiber Sodium Soluble fiber from certain foods Soy protein Stanols/sterols

Disease Risk/Health Outcome

Regulation

Osteoporosis Cancer Coronary heart disease Dental caries Cancer Neural tube defects Cancer Coronary heart disease

21 CFR 101.72 21 CFR 101.73 21 CFR 101.75 21 CFR 101.80 21 CFR 101.76 21 CFR 101.79 21 CFR 101.78 21 CFR 101.77

Hypertension Coronary heart disease Coronary heart disease Coronary heart disease

21 CFR 101.74 21 CFR 101.81 21 CFR 101.82 21 CFR 101.83

Note: Shaded rows indicate those claims which are currently being re-reviewed by the Agency.

the claim is supported by such evidence” (FDA, 2009a). Although the significant scientific agreement (SSA) standard was originally applied to food health claims, it was subsequently adopted and applied to health claims for dietary supplements/nutraceuticals, as per 21 CFR 101.14(c) (FDA, 2008a, 2008b, 2009a). Detailed information on the meaning of the SSA standard, on the evidence that is considered “credible” in the scientific substantiation of a health claim, and on the agency’s process for evaluating the scientific support for a health claim can be found in the guidance document Evidence-Based Review System for the Scientific Evaluation of Health Claims (FDA, 2009b). Currently, there are 12 NLEAauthorized health claims (Table 28.4). (ii) Health claims based on authoritative statements: Health claims that are based on an authoritative statement from a scientific body of the U.S. Government or the National Academy of Sciences may be authorized by the FDA following FDA notification of such claims. This route of claim authorization is permitted by the Food and Drug Administration Modernization Act of 1997 (FDAMA, 1997). According to the FDAMA, a scientific body is defined as “a scientific body of the United States with official responsibility for public health protection or research directly related to human nutrition”; examples of U.S. scientific bodies that are provided in the FDAMA include the National Institutes of Health (NIH) and the Centers for Disease Control and Prevention (CDC). The guidance document Notification of a Health Claim or Nutrient Content Claim Based on an Authoritative Statement of a Scientific Body lists additional U.S. scientific bodies as appropriate sources for authoritative statements, including the Surgeon General within the Department of Health and Human Services, and the Food and Nutrition Service, the Food Safety and Inspection Service, and the Agricultural Research Service within the Department of Agriculture (FDA, 1998). As for authorized claims, scientific support for claims based on authoritative statements must come from methodologically robust human (primarily intervention) studies, with the overall scientific support meeting the SSA standard (FDA, 1998). The FDAMA does not permit health claims based on authoritative statements for dietary supplements. Information that is to be submitted in a notification to the FDA is detailed in the aforementioned guidance document (FDA, 1998). If successful, an FDAMA claim may be used 120 days following FDA receipt of the notification. Currently, there are six FDAMA claims (Table 28.5).

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TABLE 28.5 FDAMA Claims Food Component/Dietary Supplement Ingredient

Disease Risk/Health Outcome

Supporting Authoritative Body

Choline Fluoride

N/A (nutrient content claim) Dental caries

Potassium Saturated fat, trans fat, cholesterol Substitution of saturated fat with unsaturated fat Whole grain foods

High blood pressure, stroke Heart disease Heart disease Heart disease, certain cancers

NAS, NRC, and IOM CDC, Surgeon General, and Public Health Service NAS, NRC, and IOM DGA NAS NAS

Source: Adapted from FDAMA, 1997. Food and Drug Administration Modernization Act of 1997 (FDAMA) Claims. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN). Available at http://www.fda.gov/RegulatoryInformation/Legislation/FederalFoodDrugandCosmeticActFDCAct/ SignificantAmendmentstotheFDCAct/FDAMA/default.htm Notes: CDC, Center for Disease Control; DGA, Dietary Guidelines for Americans; FDAMA, Food and Drug Administration Modernization Act (1997); IOM, Institute of Medicine; N/A, not applicable; NAS, National Academy of Sciences; NRC, National Research Council.

(iii) Qualified health claims: Qualified health claims are supported by emerging scientific evidence, but do not meet the SSA standard (FDA, 2003). FDA’s 2003 Consumer Health Information for Better Nutrition Initiative provides for the use of qualified health claims on foods (FDA, 2003). Qualified health claims are different from authorized and FDAMA health claims in that qualifying language must be used to communicate to the consumer the level of scientific evidence in support of the health claim (e.g., “Some scientific evidence suggests that lycopene may reduce the risk of X cancer. However, the FDA has determined that this evidence is limited and not conclusive”). All qualified health claim petitions must be submitted to and reviewed by the FDA; the FDA uses its enforcement discretion in evaluating and ranking the totality of evidence in support of the health claim. Qualified health claim petitions are made available for review and comment by interested stakeholders. If the FDA review of a qualified health claim petition is successful, a letter of enforcement discretion is issued to the petitioner, which summarizes the results of the review process and the prescribed claim wording for the qualified health claim. Although this letter is sent to the petitioner, the qualified health claim may be used by other companies, so long as the conditions of the claim are observed. If the FDA review of the qualified health claim petition is unsuccessful, the petitioner is sent a letter of denial, which outlines FDA’s review process and the reasons why the qualified health claim was found to not be scientifically substantiated. Several guidance documents on the subjects of qualified health claims have been prepared by the FDA, including Interim Procedures for Qualified Health Claims in the Labeling of Conventional Human Food and Human Dietary Supplements (FDA, 2003), FDA’s Implementation of “Qualified Health Claims”: Questions and Answers (FDA, 2006), and Evidence-Based Review System for the Scientific Evaluation of Health Claims (FDA, 2009b). The Appropriations Act of 2008, which was signed by George Bush in 2007, contained a clause (H.R. 2764) in which Congress expressed concerns over the qualified health claims area, and in which Congress urged the FDA not to devote funds to the review of qualified health claim petitions for conventional foods. According to H.R. 2764 of the Appropriations Act, Congress has requested a report from the Government Accountability Office (GAO) on qualified health claims (http://www.foodlabels.com/

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archive/2008_02.htm) (Food Consulting Company, 2008). Currently, there are no qualified health claim petitions open for comment, and the last qualified health claim petition was submitted to the FDA on April 28, 2006.

28.4.3

CLAIMS AS THEY RELATE TO CROPS GENETICALLY MODIFIED FOR OUTPUT TRAITS

As reviewed in the preceding sections, there are three broad categories of health claims: nutrient content claims, structure/function claims, and health claims. ILSI has recently published a guidance document on the safety evaluation of crops genetically modified for output traits in animals (ILSI, 2007); however, no guidance has been provided on the efficacy evaluation of these crops, or foods derived from them, in humans. Structure/function claims and health claims are required to be substantiated by data from methodologically robust human intervention studies; in contrast, nutrient content claims may be supported by compositional data only. According to 21 CFR 101.9(g) (2), each sample for nutrient analysis should consist of 12 subsamples (consumer units), one taken from each of 12 different randomly chosen shipping cases, to be representative of a lot (FDA, 2008a). Analyses should be conducted according to an appropriate method, as given in the Official Methods of Analysis of the AOAC International, 15th Ed. (AOAC, 2000), or as stipulated in 21 CFR 101.9(c) (FDA, 2008a). From an industry perspective, crops genetically modified for output traits may benefit from nutrient content claims, particularly comparative claims (e.g., contains X% more essential amino acids than a regular potato). For structure/function and health claims, scientific support must come from human studies, particularly well-controlled human intervention studies. For some crops genetically modified for output traits, industry can “ride on the coat tails” of claims already approved. In fact, given the high cost of developing a GM crop, it is likely that the existing types of preapproved claims will drive the development of nutritionally enhanced foods derived from biotechnology, rather than the nutritionally enhanced crop driving the claim. For example, a qualified health claim already exists for long-chain omega-3 fatty acids and reduced risk of coronary heart disease, and a crop that has been genetically modified to contain increased levels of long-chain omega-3 fatty acids can use the same claim. Likewise, crops that have been genetically modified to contain increased levels of vitamins/minerals or increased bioavailability of vitamins/minerals can use structure/function claims that are already scientifically substantiated. In instances where nutritionally enhanced GM food contains a novel output trait for which scientific data supporting a health claim are lacking, human intervention studies will be required to support efficacy and to confirm the absence of safety concerns. The randomized, double-blind, placebo-controlled study is considered the gold-standard intervention study in the demonstration of a cause-and-effect relationship; however, it has been criticized as having limited applicability in the study of nutrition, given the limited ability to blind subjects to treatment, which is often a whole food. With GM crops, this limitation can be overcome, given that, in most cases, the non-GM counterpart crop can be administered as a true “placebo.” GM crops can also have a positive impact on the nutritional status and health of individuals (Table 28.1). Although the majority of GM output traits continue to be directed toward uses in domestic animals, industrial uses related to biofuel production, and nutritionally enhanced foods for developing and third-world countries where nutritional inadequacy is endemic, crops genetically modified for output traits are continually being developed and targeted to health-conscious people who are seeking optimal nutrition.

REFERENCES Akeley, R.V., Mills, W.R., Cunningham, C.E., and Watts, J., 1968. Lenape: A new potato variety high in solids and chipping quality. Am. Potato J. 45: 142–145. Aktories, K. and Just, I., (Eds), 2000. Bacterial protein toxins. In: Handbook of Experimental Pharmacology, Vol. 145. Berlin: Springer.

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Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403–410. AOAC, 2000. In: W. Horowitz (Ed.), Official Methods of Analysis of the Association of Official Analytical Chemists. Vol 2: Food Composition; Additives; Natural Contaminants, 17th edition. Arlington, VA: Association of Official Analytical Chemists (AOAC). Berg, P., Baltimore, D., Boyer, H.W., Cohen, S.N., Davis, R.W., Hogness, D.S., Nathans, D., Roblin, R., Watson, J.D., Weissman, S., and Zinder, N.D., 1974. Biohazards of recombinant DNA. Science 185: 3034. Berg, P., Baltimore, D., Brenner, S., Roblin, R.O., III, and Singer, M.F., 1975. Summary statement of the Asilomar Conference on recombinant DNA molecules. Proc. Natl. Acad. Sci. USA 72: 1981–1984. Codex, 2003. Alinorm 03/04: App III: Guideline for the Conduct of Food Safety Assessment of Foods Derived From Recombinant-DNA Plants. App IV, Annex on the Assessment ofPpossible Allergenicity. Joint FAO/WHO Food Standard Programme, Codex Alimentarius Commission, 25th Session, Rome, Italy, June 30–July 5, pp. 47–60. Constable, A., Jonas, D., Cockburn, A., Davi, A., Edwards, G., Hepburn, P., Herouet-Guicheney, C. et al., 2007. History of safe use as applied to the safety assessment of novel foods and foods derived from genetically modified organisms. Food Chem. Toxicol. 45(12): 2513–2525. Delaney, B., Astwood, J.D., Cunny, H., Conn, R.E., Herouet-Guicheney, C., MacIntosh, S., Meyer, L.S. et al., ILSI International Food Biotechnology Committee Task Force on Protein Safety, 2008. Evaluation of protein safety in the context of agricultural biotechnology. Food Chem. Toxicol. 46(Suppl. 2): S71–S97. EFSA, 2006. European Food Safety Authority Guidance document of the Scientific Panel on Genetically Modified Organisms for the risk assessment of genetically modified plants and derived food and feed. EFSA J. 9: 1–100. EFSA GMO Panel Working Group, 2008. Safety and nutritional assessment of GM plants and derived food and feed: The role of animal feeding trials. Food Chem. Toxicol. 46(Suppl. 1): S2–S70. Falkow, S., 1997. What is a pathogen? ASM News 7: 359–365. FAO, 2009. In: H. Greenfield, H. and D.A.T. Southgate (Eds), Food Composition Data: Production, Management and Use, 2nd edition, Rome: Food and Agriculture Organization. Available at http://www. fao.org/infoods/directory_en.stm FDA, 1992. Statement of policy: Foods derived from new plant varieties; Notice [Docket No. 92N-0139]. Federal Register (US) 57(104): 22983–23005. FDA, 1997. Substances generally recognized as safe; Proposed rule [21 CFR Parts 170, 184, 186, and 570; Docket No. 97N-0103]. Federal Register (US) 62(74): 18937–18964. FDA, 1998. Guidance for industry: Notification of a health claim or nutrient content claim based on an authoritative statement of a scientific body. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Nutrition, Labeling, and Dietary Supplements. Available at http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/ FoodLabelingNutrition/ucm056975.htm FDA, 2001. Premarket notice concerning bioengineered foods. 21 CFR Parts 192 and 592. Federal Register (US) 66(12): 4706–4738. FDA, 2002. Guidance for industry: Structure/function claims small entity compliance guide. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN). Available at http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/ DietarySupplements/ucm103340.htm FDA, 2003. Claims that can be made for conventional foods and dietary supplements. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Nutritional Products, Labeling, and Dietary Supplements. Available http://www.fda.gov/Food/ LabelingNutrition/LabelClaims/ucm111447.htm FDA, 2006. Guidance for industry: FDA’s implementation of “qualified health claims”: questions and answers: Final guidance. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Nutrition, Labeling, and Dietary Supplements. Available at http://www. fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/FoodLabelingNutrition/ ucm053843.htm FDA, 2008a. U.S. Code of Federal Regulations (CFR). Title 21—Food and Drugs (Food and Drug Administration). Washington, DC: U.S. Government Printing Office (GPO). Available at http://www.access.gpo.gov/nara/ cfr/cfr-table-search.html#page1 (See Table at the end of the reference list for CFR sections.) FDA, 2008b. Guidance for industry: Substantiation for dietary supplement claims made under Section 403(r) (6) of the Federal Food, Drug, and Cosmetic Act. College Park, MD: Food and Drug Administration

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(FDA), Center for Food Safety and Applied Nutrition (CFSAN). Available at http://www.fda.gov/Food/ GuidanceComplianceRegulatoryInformation/GuidanceDocuments/DietarySupplements/ucm073200.htm FDA, 2009a. List of completed consultations on bioengineered foods. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at http://www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?rpt= bioListing FDA, 2009b. Evidence-based review system for the scientific evaluation of health claims: Guidance for industry. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Nutrition, Labeling, and Dietary Supplements. Available at http://www.fda. gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/FoodLabelingNutrition/ ucm073332.htm FDAMA, 1997. Food and Drug Administration Modernization Act of 1997 (FDAMA) Claims. College Park, MD: Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN). Available at http://www.fda.gov/RegulatoryInformation/Legislation/FederalFoodDrugandCosmeticActFDCAct/ SignificantAmendmentstotheFDCAct/FDAMA/default.htm Food Consulting Company, 2008. Food labels qualified health claims in question (Food Label News, vol. 9, no. 2). Del Mar, CA. Available at http://www.foodlabels.com/archive/2008_02.htm Franklin, C.A., Nestmann, E.R., and Ritter, L., 1987. Biotechnology: Risk assessment and regulation of genetically engineered products. In: H.M. LeBaron, R.O. Mumma, R.C, Honeycutt, and J.H. Duesing (Eds), Biotechnology in Agricultural Chemistry (ACS Symposium Series 334). pp. 336–351. Washington, DC: American Chemical Society . Goodman, R.E., Hefle, S.L., Taylor, S.L., and van Ree, R., 2005. Assessing genetically modified crops to minimize the risk of increased food allergy: A review. Int. Arch. Allergy Appl. Immunol. 137: 153–166. Health Canada, 2008. Approved products (Examples of How Past Safety Assessments Proceeded). Ottawa, ON: Health Canada. Available at http://www.hc-sc.gc.ca/fn-an/gmf-agm/appro/index-eng.php ILSI, 2003. Best practices for the conduct of animal studies to evaluate crops genetically modified for input traits. Washington, DC: International Life Sciences Institute (ILSI). Available at http://www.ilsi.org/NR/ rdonlyres/4A2F7C13-B4AA-4BC9-AEDE-696B4B72E3C4/0/BestPracticesGuidelines.pdf ILSI, 2004. Ridley, W.P., Shillito, R.D., Coats, I., Steiner, H.-Y., Shawgo, M., Phillips, A., Dussold, P., and Kurtyka, L. Development of the International Life Sciences Institute Crop Composition Database. J. Food Composit. Anal. 17(3–4): 423–438. ILSI, 2007. Best practices for the conduct of animal studies to evaluate crops genetically modified for output traits. Washington, DC: International Life Sciences Institute (ILSI). Available at http://www.ilsi.org/ FoodBioTech/Pages/BestPracticesforAnimalFeedStudies.aspx Kessler, D.A., Taylor, M.R., Maryanski, H.J., Flamm, E.L., and Kahl, L.S., 1992. The safety of foods developed by biotechnology. Science 256(5065): 1747–1749, 1832. McHughen, A. and Smyth, S., 2008. US regulatory system for genetically modified [genetically modified organism (GMO), rDNA or transgenic] crop cultivars. Plant Biotechnol. J. 6: 2–12. Millstone, E., Brunner, E., and Mayer, S., 1999. Beyond ‘substantial equivalence’. Nature 401(6753): 525–526. Munro, I.C., McGirr, L.G., Nestmann, E.R., and Kille, J.W., 1996. Alternative approaches to the safety assessment of macronutrient substitutes. Regul. Toxicol. Pharmacol. 23(1, Part 2): S6–S14. Nestmann, E., Copeland, T., and Hlywka, J., 2002. The regulatory and science based evaluation of genetically modified food crops: A USA perspective. In: K. Atherton (Ed.), Genetically Modified Crops: Assessing Safety, pp. 1–44. New York: Taylor & Francis. OECD, 1993. Safety Evaluation of Foods Derived by Modern Biotechnology: Concepts and Principles. Paris, France: Organisation for Economic and Cooperative Development (OECD). OSTP, 1986. Coordinated framework for the regulation of biotechnology: Announcement of policy and notice for public comment. Federal Register (US) 51: 23302–23393. OSTP, 1992. Exercise of federal oversight within scope of statutory authority: Planned introductions of biotechnology products into the environment. Federal Register (US) 57: 6753–6762. Pariza, M.W., 1992. Foods of new biotechnology vs traditional products: Microbiological aspects. Food Technol. 46(3): 100–102. Pariza, M.W. and Johnson, E.A., 2001. Evaluating the safety of microbial enzyme preparations used in food processing: Update for a new century. Regul. Toxicol. Pharmacol. 33(2): 173–186. Pearson, W.R. and Lipman, D.J., 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85: 2440–2448.

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Table of CFR Sections Referenced (Title 21—Food and Drugs) Part

Section §

101—Food labeling

101.9 101.12 101.13 101.14 101.72 101.73 101.74 101.75 101.76 101.77 101.78 101.79 101.80 101.81

170—Food additives

101.82 101.83 101.93 170.3

Section Title Nutrition labeling of food Reference amounts customarily consumed per eating occasion Nutrient content claims: general principles Health claims: general requirements Health claims: calcium and osteoporosis Health claims: dietary lipids and cancer Health claims: sodium and hypertension Health claims: dietary saturated fat and cholesterol and risk of coronary heart disease Health claims: fiber-containing grain products, fruits, and vegetables and cancer Health claims: fruits, vegetables, and grain products that contain fiber, particularly soluble fiber, and risk of coronary heart disease Health claims: fruits and vegetables and cancer Health claims: Folate and neural tube defects Health claims: dietary noncariogenic carbohydrate sweeteners and dental caries Health claims: soluble fiber from certain foods and risk of coronary heart disease (CHD) Health claims: soy protein and risk of coronary heart disease (CHD) Health claims: plant sterol/stanol esters and risk of coronary heart disease (CHD) Certain types of statements for dietary supplements Definitions

Sun, S.S., 2008. Application of agricultural biotechnology to improve food nutrition and healthcare products. Asia Pac. J. Clin. Nutr. 17(Suppl. 1): 87–90. USDA, 2008. USDA National Nutrient Database for Standard Reference, Release 21 (Nutrient Data Laboratory Home Page). Beltsville, MD: U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), Beltsville Human Nutrition Research Center Nutrient Data Laboratory. Available at http://www. nal.usda.gov/fnic/foodcomp/Data/SR21/nutrlist/sr21w303.pdf Vetter, J.L., 1996. Food Laws and Regulations. Manhattan KS: American Institute of Baking (AIB). Zitnak, A. and Johnston, G.R., 1970. Glycoalkaloid content of B5141–6 potatoes. Am. Potato J. 47: 256–260.

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Food Biotechnology 29 Global Regulations and Urgency for Harmonization Dilip K. Ghosh and Peter Williams CONTENTS 29.1 29.2 29.3 29.4

Introduction .......................................................................................................................... 531 Categories of Foods Produced through Biotechnology ........................................................ 532 Regulation in General ........................................................................................................... 532 Regulation in Different Countries ........................................................................................ 533 29.4.1 Australia/New Zealand............................................................................................. 534 29.4.2 EU ............................................................................................................................. 534 29.4.3 USA........................................................................................................................... 535 29.4.4 Canada ...................................................................................................................... 536 29.4.5 Japan ......................................................................................................................... 536 29.5 Consumer Attitudes .............................................................................................................. 537 29.6 Political Consideration in the Development of Regulatory Policy ....................................... 537 29.7 Challenges for Industry, Regulators, and Consumers .......................................................... 538 29.8 Food Biotechnology Resources ............................................................................................ 539 29.9 Conclusions ........................................................................................................................... 539 References ...................................................................................................................................... 539

29.1 INTRODUCTION Biotechnology, or genetic engineering, means different things to different people. The simplest definition of biotechnology is “applied biology.” Another definition is “the use of living organisms to make a product or run a process” (Zinnen and Voichick, 1999). Government agencies and research entities refer to biotechnology as “the application of biological systems and organisms to the production of useful goods and services” (IFT, 2000). This definition encompasses applications in biology, genetics, and biochemistry to advance technical and industrial processes and techniques ranging from drug development to fish farming, forestry, crop development, fermentation, and oil spill clean up. Modern agricultural biotechnology includes genomics and bioinformatics, marker-assisted selection, micropropagation, tissue culture, cloning, artificial insemination, embryo transfer, and genetic engineering (http://www.cbd.int/). Various terms have been used for new crop varieties produced by biotechnology, including genetically modified (GM or GMO), genetically engineered (GE or GEO), transgenic, biotech, bioengineered, recombinant, and plants with novel traits (PNT). Throughout this chapter, the term GM or GMO will be used because of its simplicity and broad recognition. There is a vast array of scientific information both in favor of and against this technology, particularly when applied to food production. 531 © 2010 Taylor and Francis Group, LLC

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Supporters of modern agricultural biotechnology have suggested that the debate about GM food products has not recognized the potential benefits for human health and environmental problems, and the extensive scientific reviews that support the safety of the products. Others, however, have questioned the adequacy of scientific knowledge about this technology and its benefits, and raised concerns regarding its potential risks, claiming that regulatory decisions have been based more on politics than on science (http://www.twnside.org.sg/). Global debates are going on in both political and commercial domains about the status of biotechnology for international development and particularly its implications for food safety, impacts on biodiversity, ethical issues, trade related issues, and consumer concerns (McHughen, 2007). Meanwhile, the global area of approved GM crops is now over 90 million hectares with an annual growth rate of 11% (James, 2005). There is thus an urgent need for the development of policy and regulatory frameworks for a common regional position or harmonized models to assist governments and policy makers in decisions involving the regulation of GMOs and other emerging technologies.

29.2 CATEGORIES OF FOODS PRODUCED THROUGH BIOTECHNOLOGY Foods produced through modern biotechnology can be categorized as (WHO, 2000): • Foods consisting of or containing living/viable organisms (e.g., corn) • Foods derived from or containing ingredients derived from genetic modification (e.g., cornmeal containing protein or oil from GM soybeans) • Foods containing single ingredients or additives produced by GM microorganisms (e.g., colors, vitamins, or essential amino acids) • Foods containing ingredients processed by enzymes produced through GM microorganisms (e.g., high-fructose corn syrup produced from starch using the enzyme glucose isomerase, or cheese produced using the enzyme chymosin) Current research and development activities and future projects are targeted mainly at crops, livestock, fish, and microorganisms. Programs have focused on addressing the following: • • • • • • • • •

Biotic stresses—pest and disease resistance, virus resistance Enhanced nutritional value and altered composition of agricultural products Development of processing aids for foods and feeds Treatments for human infertility Production of vaccines and diagnostic tools to prevent diseases in domestic animals Tolerances to environmental stresses Repair of damaged organs and tissues and improved detection of diseases Bacteria capable of cleaning up oil spills Environmentally friendly biofuels

In this chapter we discuss only the regulatory framework related to foods.

29.3 REGULATION IN GENERAL Around the world, biotech crops and foods are regulated wherever they are grown and/or consumed. Experience has shown that the science of biotechnology is always changing, requiring more tailored, complex, and risk-based regulations. While developed countries have established their own national frameworks to deal with agrobiotechnology and biosafety, focusing primarily on domestic priorities and strategies, many developing countries have incompletely established regulatory regimes, mainly due to limited expertise and resources, and political interference. In addition to the

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national regulatory bodies, the Organization for Economic Cooperation and Development and the United Nations World Health Organization and Food and Agriculture Organization (FAO, 2006) have put forward statements of support and guidance for individual governmental bodies who are developing regulations to improve consistency throughout the global community. There is an urgent need for strengthened institutional capacity building in the regulatory agencies of developing member countries through regional and international cooperation.

29.4 REGULATION IN DIFFERENT COUNTRIES A useful summary of and links to regulations covering GM foods around the world is available from the Government of Canada (2008). Carter and Gruère (2006) and Gruère (2007) have also summarized the GM regulations from over 25 different regions according to their characteristics (Table 29.1). At an international level, harmonization efforts are led by the Codex Alimentarius Commission, the Cartagena Protocol on Biosafety (CPB), and the World Trade Organization (WTO). While internationally harmonized guidelines for safety approval have been finalized by Codex Alimentarius (2003) there is no clear consensus on labeling requirements for GM food, some of which may be inconsistent with WTO, and there is an increasing risk of conflicts between CPB and WTO. Developed countries differ in their general approach to regulations, with most GM producers and exporters having more relaxed regulations, while many importers tend to have stricter marketing and labeling regulations.

TABLE 29.1 Characteristics of GM Regulations in Different Country Groups Group

Food Safety Approval Regulations

Labeling Regulations

Specificity

1

Process-based, mandatory

Stringent mandatory. Includes derived products

2

Process-based, mandatory Process-based, mandatory

Stringent mandatory. Includes derived products Product-based, mandatory

4

Substantial equivalence, mandatory

Voluntary for substantially equivalent

5% threshold for labeling

5

Mandatory

7

Mandatory (in place or pending) Mandatory (in place or pending) Considering mandatory

Intention to require labeling No clear position

“Pragmatic” productbased labeling Slow regulatory process Wait-and-see approach

8

No

No

GM free

3

6

Traceability requirements, 0.9% threshold No traceability, low threshold Labeling exceptions, 1–5% threshold

Countries EU, East Europe

Brazil, China, Russia, Switzerland, Norway Australia, Japan, Korea, Saudi Arabia, Thailand, Taiwan USA, Canada, Argentina, Hong Kong, Philippines, South Africa Indonesia, Chile, Ecuador, Vietnam India, Kenya Bangladesh, most African countries Zimbabwe, Zambia

Source: Based on Sheldon, I.M., 2002; Eur. Rev. Agric. Econom. 29: 155–176; Carter, C.A. and Gruère, G.P., 2006. In: R.E. Just, J.M. Alston, and D. Zilberman (Eds), Regulating Agricultural Biotechnology—Economics and Policy, pp. 459–480. New York: Springer Science + Business; Gruère, G.P., 2007. Environmental and Production Technology Division (EPTD) Discussion Paper 147. Washington DC: International Food Policy Research Institute. Available at http://www.ifpri.org/sites/default/files/publications/eptdp147.pdf. Accessed on September 2008.

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Below are summaries of the regulations in a selection of developed countries or regions, illustrating the variety of approaches currently in place.

29.4.1

AUSTRALIA/NEW ZEALAND

In both Australia and New Zealand foods are regulated by a joint trans-Tasman agency called Food Standard Australia New Zealand (FSANZ, http://www.foodstandards.gov.au). This means that, with a few exceptions, foods approved for sale in Australia are also able to be sold in New Zealand, and vice versa. Australia has some of the most stringent regulations in the world concerning biotechnology, particularly when it is used in the production of crops and food. Biotechnology is currently regulated by a number of different bodies depending on the intended use of the GMO or GM product. Some national government regulators include: • Food Standards Australia New Zealand (FSANZ)—regulating food standards and labeling • Office of the Gene Technology Regulator (OGTR)—regulating most aspects of GMOs in Australia, including research, manufacture, production, experimental trials, commercial release, and importation • Therapeutic Goods Administration (TGA)—regulating therapeutic goods and human gene therapy • Australian Pesticides and Veterinary Medicines Authority (APVMA)—regulating agricultural and veterinary chemicals • Australian Quarantine and Inspection Service (AQIS)—regulating the import and export of GM products and GMOs As of August 2008, FSANZ had approved 34 GM food commodities, which consist of varieties of GM corn, cotton, canola, sugar beet, soy, and potato. Although there is approval for the commercial growing of GM canola, only GM cotton, from which cottonseed oil is derived, is currently grown commercially in Australia. It is mandatory that all packaged and bulk food must be labeled if it contains DNA or proteins derived from a GM organism, or has altered characteristics that are due to genetic modification even if no GM material is present in the product (such as oil derived from GM canola which has a different fatty acid profile) (http://www.nzfsa.govt.nz/consumers/gm-ge/ index.htm). The few exemptions from labeling requirements are as follows: • If a food ingredient unintentionally contains less than 1% GM ingredient • If a food flavoring is GM but makes up less than 0.1% of the food • Food prepared in restaurants, cafes, and takeaways does not have to be labeled If the food is GM or is a species new to New Zealand, and is to be grown in New Zealand, then an approval for conditional release or full release from the Environmental Risk Management Authority of New Zealand (http://www.ermanz.govt.nz) is required. “New” in this context means an organism that has not been grown or present in New Zealand previously, or is a GM organism. Importation of a new food (such as a variety of GM maize) may also require an appropriate Import Health Standard from Biosecurity New Zealand (http://www.biosecurity.govt.nz) before it can be imported.

29.4.2

EU

In Europe, European Community (EC) law provides the core regulatory framework on GM food. The general principles are set out in Regulation (EC) No. 178/2002, laying down the general principles and requirements of food law, establishing the new European Food Safety Authority (EFSA)

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(http://www.efsa.europa.eu/) and setting out procedures in matters of food safety (General Food Law). Specific regulations on GM food came into force in April 2004 governing GM food and feed (EC No. 1829/2003), and traceability and labeling (EC No. 1830/2003). These new regulations take GM foods out of the scope of regulations on novel foods. Premarket approval of GM foods is required. National authorities have to accept an application and forward it to EFSA for scientific review (within a 6-month timeframe). By mid-2008, EFSA had adopted opinions on around 30 GMO applications with another 35 first-time GMO applications under consideration by the GMO Panel and are at various stages in their completion. Opinions from EFSA are passed to the Commission to draft a proposal granting or refusing authorization, which has to be approved by a majority of member states. European legislation requires the labeling of all food produced from GM organisms, regardless of whether there is any detectable genetically modified DNA or protein in the final food, although unintentional contamination levels of no more than 0.9% in conventional products is permitted provided this presence is technically unavoidable. Generally speaking, prepackaged products consisting of or containing GMOs must be labeled that “This product contains genetically modified organisms.” In the case of non-prepackaged products offered to the final consumer, these words must appear on, or in connection with, the display of the product. The regulations also require labeling of GM feed and the General Food Law requires traceability at all stages of production, processing, and distribution (EFSA, 2008).

29.4.3

USA

The majority of the world’s biotechnologically modified crops are grown in the United States. A number of crops with improved quality and/or quantity of nutrients have reached the field trial stage and are advancing through the regulatory approval process before commercialization (ILSI, 2004). Since 1987 more than 10,000 field tests of GE organisms has been authorized and approved (USDA BRS Factsheet, February 2006). Three federal agencies share the regulatory oversight of biotechnology in the United States. Regulation is coordinated among the U.S. Department of Agriculture (USDA), Environmental Protection Agency (EPA), and Food and Drug Administration (FDA) depending on the intended use of the products. Throughout the regulatory process, each agency provides several opportunities for public comment. The Biotechnology Regulatory Service (BRS), of the Animal and Plant Health Inspection Service (APHIS) within the USDA, regulates field testing, movement, and importation of biotech crops and seed, including the assessment of the agricultural and environmental safety of newly developed varieties during field testing and prior to commercialization. The EPA regulates the safe use of pesticides in agriculture, including the pest-protection properties of plants developed using biotechnology. The FDA has primary responsibility for ensuring the safety and appropriate labeling of biotechnologically derived plant foods and feeds (FDA, 2001a, 2001b). During a consultation with developers of bioengineered foods, the FDA evaluates safety and nutritional information provided by the developers, including information on the altered nutrients, significantly different composition, allergenic proteins, new antibiotic resistant markers, or levels of toxicants significantly higher than those found naturally in edible varieties of the same food (American Dietetic Association, 1999). The FDA does not require labeling to indicate whether a food or food ingredient is a bioengineered product, just as it does not require labeling to indicate which conventional breeding techniques were used in developing a food plant. Rather, any significant differences in the food itself have to be disclosed on the label. Currently BRS is considering broadening its regulatory scope beyond GE organisms that may pose a plant pest risk to include GE plants that may pose a noxious weed risk, as well as GE organisms that may be used as biological control agents. Potential regulation changes currently under review include the development of a risk-based, tiered permitting program, which would allow BRS to focus more time and resources on new or unfamiliar GE traits, such as pharmaceutical and industrial crops.

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29.4.4 CANADA In Canada, biotechnology allows for the development of new food products under the umbrella name of “novel foods” through a variety of scientific tools and techniques. Health Canada establishes science-based regulations, guidelines, and public health policies for these novel foods. As with other novel foods, the main concern in regulating biotechnology products is the health and safety of Canadians, animals, and the environment. Novel foods are defined in Canada as (http://www.hc-sc.gc.ca/fn-an/legislation/guide-ld/nf-an/ guidelines-lignesdirectrices-eng.php): • Foods resulting from a process not previously used for food • Products that have never been used as a food • Foods that have been modified by genetic manipulation, also known as GM foods, GE foods, or biotechnology-derived foods To date, over 70 novel and GM foods have been approved for sale in Canada, such as GM corn, genetically altered soybeans, potatoes resistant to the Colorado potato beetle and omega-3-enhanced pork, etc. (http://www.hc-sc.gc.ca/fn-an/gmf-agm/appro/index-eng.php). Health Canada and the Canadian Food Inspection Agency (CFIA) advise industry manufacturers to follow regulatory directives and guidelines to get approval for the commercialization of novel foods, novel feeds, and for environmental release. Companies must supply evaluators with thorough and detailed information about their novel food products before they can receive approval to sell or advertise them in Canada. The premarket safety assessment of all biotechnology-derived foods is conducted by Health Canada to demonstrate that a novel food is safe and nutritious, and includes reviews of: • Molecular biological data in relation to the genetic change • Nutritional information about the novel food and its major constituents, such as fats, proteins, carbohydrates, and minor constituents, such as minerals and vitamins compared to a nonmodified food of the same type • Potential for production of new toxins in the food • Potential for causing allergic reactions • Potential for any unintended or secondary effects • Key nutrients and toxicants • Microbiological and chemical safety of the food Currently in Canada food labeling is mandatory and includes all GM foods, with both Health Canada and CFIA sharing the responsibility for these policies under the Food and Drugs Act.

29.4.5

JAPAN

Japan’s food self-sufficiency is relatively low and it imports GM maize, soybeans, and canola in large quantities for feed and oil production. In 2000 Japan introduced regulations defining the authorization procedure for GM food. The safety of GM food is assessed under the Food Sanitation Law and assessments are conducted by an Expert Panel of the Biotechnology Subcommittee. Final recommendations of the Food Sanitation Committee are passed to the Minister of Health, Labor and Welfare for gazettal. This regulatory process usually takes about one year. There is a GM labeling regulation in Japan, although the regulation does not cover processed foods in which modified DNA cannot be detected—such as oils or soy sauce—and also does not cover animal feeds. Moreover, the threshold level for unintended GM contamination is set at a higher level than many countries—up to 5%. This level was decided pragmatically after considering the actual conditions of imported crops. Food processors and retailers in Japan have typically avoided products with

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GM labels. As in the EU, most GM products are used for animal food but, unlike the EU, many highly processed products derived from GM ingredients (e.g., soybean oil) are sold without labels.

29.5 CONSUMER ATTITUDES Widespread science illiteracy means that most consumers’ knowledge about the details of biotechnology is quite poor (McHughen, 2007), but consumer attitudes to GM food have been very influential in the development of biotechnology regulations. Research indicates that there is some evidence of cross-cultural differences; however, public attitudes tend to be mostly negative in developed countries. There is societal cynicism about the adequacy of current regulations, and a preference for accountable regulatory structures (Miles et al., 2005). The use of new biotechnologies to produce novel medicines is rather less controversial than for food, but while the public does recognize the potential benefits of GM foods, the acceptability is contingent on whether the specific benefits are actually desired by consumers, and on who is perceived to be the recipient of the benefits (food companies or consumers). However, consumer views are not uniform. The extent to which people are motivated to eat foods they consider “natural” has a strong negative effect on views of GM, while consumers who are motivated to find foods that are convenient are more likely to have a positive accepting attitude to GM (Lockie et al., 2005). There are also geographical differences in attitudes: European and UK consumers are generally focused on the unknown risks associated with GM products, while Americans are more indifferent to the risks and benefits (Moon and Balasubramanian, 2004; IFIC, 2007). Surveys of consumers in the United States and Europe indicate a relative lack of trust in the government, media, grocery stores, and the food industry in relation to GM foods, while scientists, consumer advocacy groups, environmental organizations, and farmers are relatively more trusted (Lang and Hallman, 2005). By contrast, in developing countries there is a more positive perception of GM foods stemming from the more urgent need for food availability, and also a higher trust in government regulation (Curtis et al., 2004). It has been pointed out that consumer views in surveys do not always translate into behavior and that, in reality, supposed concerns about GM foods are no more important in purchasing decisions than issues such as artificial preservatives or pesticides in food (Biotechnology Australia, 2005). A “right-to-know” provision, which allows consumers to make informed choices between GM and non-GM foods, can potentially mitigate negative perceptions. In an effort to address perceived risks of biotech foods, different countries have adopted markedly different approaches. Public demand for costly segregation is evident in the EU, but is less pronounced in the United States. Labeling regulations in Europe (and most other jurisdictions) are mainly based on product (what is in the final food) rather than process (whether GM modification has been used) characteristics. In contrast, evidence suggests that consumer perception is driven more by process characteristics (Grunert et al., 2001).

29.6

POLITICAL CONSIDERATION IN THE DEVELOPMENT OF REGULATORY POLICY

In striking contrast to the initial acceptance of GM food in the United States, the history of vehement public opposition in Europe led the EU to suspend the introduction of GM crops pending legislation that has taken many years to finalize. To some extent the trans-Atlantic debate seems to have been based on arguments about food safety and trade restrictions, and issues of labeling and patent protection. However it has been suggested that the opposition in Europe to American GM imports was determined by different cultural values reflecting different sensitivities to dread and unknown risk (Finucane, 2002). There were also many social and cultural reasons for the opposition in Europe, including sensational and negative media coverage, widespread support for small farms and open spaces, and a general opposition to foreign producers and their products (Falk et al., 2002).

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The reluctant European approach was considered by the United States to constitute a barrier to trade. In 2003, the United States, Canada, and Argentina initiated legal proceedings against the EU with the WTO, relying on the Agreement on Sanitary and Phytosanitary Measures to complain about the prohibition on the importation of GM foods. That agreement gives members the right to take measures to protect health, even if these constitute a barrier to trade, but the necessity must be scientifically established and nondiscriminatory. In this case, the EU authorities were unable to refute evidence presented by the United States that GM food was safe and in 2006 ruled that the moratorium by six EU members on imports of GM food and crops was illegal.

29.7

CHALLENGES FOR INDUSTRY, REGULATORS, AND CONSUMERS

It has been claimed that the existing regulatory approaches in the United States and elsewhere— which require the use of precise and predictable techniques in the use of new biotechnologies (e.g., recombinant DNA)—elicits a discriminatory and excessive regulatory regime and violates the principle of proportionality, which holds that the degree of regulatory scrutiny should be commensurate with the degree of risk posed by the product (Miller et al., 2008). In fact the amount of scrutiny applied to field trials and the commercialization of new plant varieties may be inversely proportional to risk. Carefully crafted and predictable recombinant DNA-modified organisms are the most regulated, and field trials of such plants cost 10–20 times more compared to identical plants modified by conventional techniques. One of the particular challenges for regulators has been the demand for the precautionary principle (PP) to be applied in the risk assessments of GM foods (Soule, 2002). Under the PP, regulators are asked to anticipate harm before it occurs or before there is adequate scientific evidence on which to make a full assessment of likely damage. The PP, which grew out of concerns about potential environmental damage, lowers the burden of proof for adverse effects, but it does not make it obsolete (CEC, 2000). A recent decision of the European Court is significant because it ruled that novel foods containing transgenic proteins may still be considered substantially equivalent to existing foods (Fernandez, 2006). It emphasized that assessments have to be based on real perceived risk, not hypothetical risk. It ruled that the PP allows restrictive measures on the grounds that there is uncertainty about risks, but only “if the risks, although the reality and extent thereof has not been fully demonstrated by conclusive scientific evidence, appears nevertheless adequately backed up by the scientific data available.” While the new EU legislation is now in place and the moratorium on GM foods in Europe has been lifted, some individual member states still strive to block authorization of GM crops, often through local laws on nature conservation and the coexistence between GM agriculture and organic farming (van den Daele, 2007). This raises troubling questions for governments. A ban on GM crops is likely to be insignificant as a precautionary measure, yet relying solely on conventional agriculture is unlikely to help address more urgent current issues of soil erosion, salinity, water eutrophication, and loss of biodiversity through high-yield crops. Nonetheless the PP remains a contentious area of debate, and one that is obscured by the prevalent policy language. There are assumptions that “science-based regulation” can find an expert objective basis for decisions, that precautions and socio-ethical issues remain external to risk assessment, and that risk assessment can be functionally separated from risk management (Levidow et al., 2005). It is likely that regulatory decisions about GM products will continue to encounter legitimacy problems into the future because they fundamentally suffer from the great burden placed on science as the basis for societal choices about GM products. At the same time, there will continue to be calls from industry for a less burdensome risk assessment process for the commercialization of transgenic crops, with proposals that the regulatory emphasis should be on the phenotypic rather than the genomic characteristics, once a gene or trait has been shown to be safe (Bradford et al., 2005).

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539

FOOD BIOTECHNOLOGY RESOURCES

• United States Department of Agriculture (USDA, http://www.usda.gov/wps/portal/ usdahome) • Environmental Protection Agency (EPA, http://www.epa.gov/) • Food and Drug Administration (FDA, http://www.fda.gov/) • United States Regulatory Agencies Unified Biotechnology (http://usbiotechreg.nbii.gov/) • Society of Toxicology Position Paper: The Safety of Genetically Modified Foods Produced Through Biotechnology. Available at http://www.toxicology.org/ai/gm/gm_food.asp. Adapted September 25, 2002 • American Dietetic Association, 2006. Position paper of the American Dietetic Association: Agricultural and food biotechnology. J. Am. Diet. Assoc. 106: 285–293 • IOM (Institute of Medicine and National Research Council), 2004. Safety of Genetically Engineered Foods. Approaches to Assessing the Unintended Health Effects. Washington DC: National Academies Press • International Service for the Acquisition of Agri-Biotech Applications (ISAAA): Global Status of Commercialized Biotech/GM Crops: 2005. Available at http://www.isaaa.org/ • Food Standards Australia New Zealand, 2005. Safety assessment of genetically modified foods. Canberra: FSANZ. Available at http://www.foodstandards.gov.au/_srcfiles/GM%20 Foods_text_pp_final.pdf. Accessed on September 2, 2008)

29.9

CONCLUSIONS

Strong safety standards for agriculture and food are legitimate principles in international law, but diverse approaches to regulation in different countries create uncertainty for business and place heavy administrative burdens on both food companies and regulators. At all stages in the food chain food producers who may come in touch with traces of GM material may be required to adhere to stricter labeling and traceability requirements than other business operators. There have now been useful moves to harmonize the risk assessment processes internationally through the work of Codex, but there is still much work to be done to find a common and less burdensome process for risk management.

REFERENCES American Dietetic Association, 1999. Statement on biotechnology before the Food and Drug Administration. ADA: Washington DC. Available at http://ucbiotech.org/resources/reports/ADA.PDF. Accessed on October 5, 2005. Biotechnology Australia, 2005. What you really need to know about what the public really thinks about GM foods. Available at http://www.biotechnology.gov.au/assets/documents/bainternet/GMFood20052005111 5160254%2Epdf. Accessed on September 1, 2008. Bradford, K.J., Van Deynze, A., Gutterson, N., Parrott, W., and Strauss, S.H., 2005. Regulating transgenic crops sensibly: Lessons form plant breeding, biotechnology and genomics. Nat. Biotech. 23: 439–444. Carter, C.A. and Gruère, G.P., 2006. International approval and labelling regulations of genetically modified food in major trading countries. In: R.E. Just, J.M. Alston, and D. Zilberman (Eds), Regulating Agricultural Biotechnology—Economics and Policy, pp. 459–480. New York: Springer Science + Business. CEC, Council of the European Communities. 2000. Communication from the Commission on the precautionary principle. Available at http://ec.europa.eu/dgs/health_consumer/library/pub/pub07_en.pdf. Accessed on August 3, 2008. Codex Alimentarius, 2003. Principles for the risk assessment of foods derived from modern biotechnology. CAC/GL 44-2003. Available at http://www.codexalimentarius.net/download/standards/10007/CXG_ 044e.pdf. Accessed on September 3, 2008. Curtis, K.R., McCluskey, J.J., and Wahl, T.I., 2004. Consumer acceptance of genetically modified food products in the developing world. Agri. Bio. Forum 7: 7–75.

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EFSA, European Food Safety Authority, 2008. Food safety—from the farm to the fork. Available at http://ec. europa.eu/food/food/biotechnology/index_en.htm. Accessed on September 2, 2008. Falk, M.C., Chassy, B.M., Harlander, S.K., Hoban, T.J., McGloughlin, M.N., and Akhlaghi, A.R., 2002. Food biotechnology: Benefits and concerns. J. Nutr. 132: 1384–1390. FAO, Food and Agriculture Organization, 2006. Policy and regulation of biotechnology in food production. Available at ftp://ftp.fao.org/unfao/bodies/arc/24arc/J6967E.pdf. Accessed on August 3, 2008. Fernandez, R.R., 2006. Monsanto and the requirement of real risks in GM food regulation. Loy L.A. Int’l. & Comp. L. Rev. 28: 335–350. Finucane, M.L., 2002. Mad cows, mad corn and mad communities: The role of socio-cultural factors in the perceived risk of genetically modified food. Proc. Nutr. Soc. 61: 31–37. FDA, Food and Drug Administration, 2001a. Premarket notice concerning bioengineered foods: Proposed rule. Federal Register 66: 4706. FDA, Food and Drug Administration, 2001b. Draft guidance for industry: Voluntary labeling indicating whether foods have or have not been developed using bioengineering: Notice of availability. Federal Register 66: 4839. Available at http://www.cfsan.fda.gov/~dms/biolabgu.html. Accessed on September 1, 2008. Government of Canada, 2008. Biotech and Government—a world view. Regulation of biotechnology (General). Available at http://www.bioregulations.gc.ca/english/view.asp?x=732. Accessed on September 2, 2008. Gruère, G.P., 2007. An analysis of trade-related international regulations of genetically modified food and their effects on developing countries. Environmental and Production Technology Division (EPTD) Discussion Paper 147. Washington DC: International Food Policy Research Institute. Available at http://www.ifpri. org/sites/default/files/publications/eptdp147.pdf. Accessed on September 2008. Grunert, K.G., Lahteenamaki, L., Nielsen, N.A., Poulsen. J.B., Ueland, O., and Astrom, A., 2001. Consumer perceptions of food products involving genetic modifications—results from a qualitative study in four Nordic countries. Food Qual. Pref. 12: 527–542. IFIC, International Food Information Council, 2007. Food biotechnology: A study of US consumer attitudinal trends. Available at http://www.ific.org/research/biotechres.cfm. Accessed on September 1, 2008. IFT, Institute of Food Technologists, 2000. IFT expert report on biotechnology and foods. Food Technol. 54: 124. Available at http://members.ift.org/NR/rdonlyres/3B1B6245-CAC7-4B83-91E6-2C6F18C7A041/0/ report.pdf. Accessed September 2, 2008. ILSI, International Life Science Institute, 2004. Nutritional and safety assessments of food and feeds nutritionally improved through biotechnology. Compr. Rev. Food Sci. Technol. 3: 38–104. James, C., 2005. Global status of commercialised biotech. GM crops. ISAAA Brief No 34. Ithaca, New York: International Service of the Acquisition of Agri-Biotech Application. Available at http://www.isaaa.org/ Resources/publications/briefs/34/download/isaaa-brief-34-2005.pdf. Accessed on September 2, 2008. Lang, T. and Hallman, W.K., 2005. Who does the public trust? The case of genetically modified food in the United States. Risk Anal. 25: 1241–1252. Levidow, L., Carr, S., and Wield, D., 2005. European Union regulation of agri-biotechnology: Precautionary links between science, expertise and policy. Sci. Pub. Pol. 32: 261–276. Lockie, S., Lawrence, G., Lyons, K., and Grice, J., 2005. Factors underlying support or opposition to biotechnology among Australian food consumers and implications for retailer-led food regulation. Food Policy 30: 399–418. McHughen, A., 2007. Public perceptions of biotechnology. Biotechnol. J. 2: 1105–1111. Miles, S., Ueland, Ø., and Frewer, L.J., 2005. Public attitudes to genetically modified food. Brit. Food J. 107: 246–262. Miller, H.I., Morandini, P., and Ammann, K., 2008. Is biotechnology a victim of anti-science bias in scientific journals? Trends Biotechnol. 26: 122–125. Moon, W. and Balasubramanian, S.K., 2004. Public attitudes toward agribiotechnology: The mediating role of risk perceptions on the impact of trust, awareness and outrage. Rev. Agric. Econom. 26: 186–208. Office of Science and Technology Policy, 1992. Coordinated framework for regulation of biotechnology. Federal Register 51: 23302. Sheldon, I.M., 2002. Regulation of biotechnology: Will we ever ‘freely’ trade GMOs? Eur. Rev. Agric. Econom. 29: 155–176. Soule, E., 2002. Assessing the precautionary principle in the regulation of genetically modified organisms. Int. J. Biotechnol. 4: 18–33. USDA BRS Factsheet. APHIS biotechnology: Permitting progress into tomorrow. February 2006. Available at: https://scholarworks.iupui.edu/handle/1805/819.

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van den Deale, W., 2007. Legal framework and political strategy in dealing with the risks of new technology: The two faces of the precautionary principle. In: H. Somsen (Ed.), The Regulatory Challenge of Biotechnology, pp. 118–155. Cheltenham: Edward Elgar Publishing Ltd. WHO, World Health Organization, 2000. Turning the tide of malnutrition: Responding to the challenge of the 21st century. Available at http://who.int/nut/documents/nhd_brochure.pdf. Accessed on October 10, 2005. Zinnen, T. and Voichick, J., 1994. Biotechnology and Food. North Central Regional Extension Publication No. 569. Madison, WI: Cooperative Extension Publications, University of Wisconsin-Extension.

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Part VI Future of Biotechnology

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Strategies for 30 Future the Development of Biotechnology-Enhanced Functional Foods and Their Contribution to Human Nutrition Dilip K. Ghosh, Francis C. Lau, and Debasis Bagchi CONTENTS 30.1 Introduction ......................................................................................................................... 545 30.2 Biotechnology in Food and Nutrition Business ..................................................................546 30.3 Consumers and Regulation .................................................................................................546 30.4 Conclusion .......................................................................................................................... 547 References ...................................................................................................................................... 547

30.1

INTRODUCTION

Biotechnologies have evolved over the past 20 years from intellectually intriguing biosciences into diversifying industries which produce useful biologicals from biocatalytic reactions to genetically modified bacteria, fungi, viruses, and plant, mammalian, and insect cells. Some advancements modify the genetic composition and expression of genes; others accelerate and adjust metabolic processes. In bioengineering sectors in particular, significant developments have been achieved in the expansion from laboratory to factory scale, and in technologies for the isolation, purification, and sterilization of end products. The creation of reliable systems of product quality and process control has been equally successful. Earlier research and development processes of extracting, screening, and chemically modifying natural biochemical substances targeted the identification of disease-causing mechanisms and their prevention or cure. Today’s molecular modeling and combinatorial biochemistry aim to design and synthesize more effective diagnostics, prophylactics, and therapeutics tools. In the past, an organic chemist might hope to synthesize 50–100 new compounds in a year, but today with the help of computer-assisted modern biochemistry and software a company can generate several thousand molecules and screen them against a target protein every 6 months. Rapid biological screenings are extensively employed, using membranes from human or animal organ cells grown in tissue culture. Diagnostic processes including immunogenicity have been accelerated by the introduction of molecular modeling and wide arrays of microchip analyses. Drugs synthesized by GM organisms include vaccines, immune regulators, substances to control cardiovascular 545 © 2010 Taylor and Francis Group, LLC

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disorders, and various hormones; they are unique examples of the application of modern biotechnology. Recent addition of “omics” tools to the bioscience lexicon include “Genomics”—the study of genomes and DNA nucleotide sequences; “Proteomics”—related to specific proteins produced by genomes; “Metabolomics”—the influence of gene expression on metabolites; “Transcriptomics”— profiling gene expressions using DNA/RNA microassays. These have paved the way for an unparallel understanding of the molecular functioning of the human body that will ultimately transform medical and nutritional practices.

30.2

BIOTECHNOLOGY IN FOOD AND NUTRITION BUSINESS

Selective breeding, fermentation, and enzyme production have been used for centuries to produce foods and medicines, whereas new biotechnologies such as genetic engineering and cloning allow direct transfer of genetic material between species, and accelerated reproduction within species. The recent development of nanotechnology and its limited application has revolutionized the entire chain of food and nutrition, particularly in the field of delivery and packaging systems. Reported benefits of the new biotechnologies include reduced environmental damage and improvements in animal welfare, farm productivity, product quality, and human health, but there still seems to be a hesitancy in the adoption of such technologies by farmers and by food companies. According to United Nations (UN) projections, the world’s population will grow from 6.1 billion in 2000 to 8 billion in 2025 and 9.4 billion in 2050; most (93%) of the increase will take place in developing countries. This rapid population growth in developing countries creates major challenges for governments regarding food and nutrition security. According to current World Health Organization estimates, more than 3 billion people worldwide, especially in developing countries, are malnourished, lacking essential nutrients. Despite a little progress over the past few decades, malnutrition still imposes a high cost on a country’s population due to impaired physical and cognitive abilities and the reduced ability to work. Since supplements are expensive and difficult to distribute widely, the Food and Agriculture Organization of the UN would like to see more nutrientrich foods introduced into these countries. Biofortification of staple crops through modern biotechnology can potentially help in alleviating malnutrition in developing countries. Several genetically modified crops, including rice, potatoes, oilseeds, and cassava, with elevated levels of essential nutrients (such as vitamin A, iron, zinc, protein and essential amino acids, and essential fatty acids), reduced levels of antinutritional factors (such as cyanogens, phytates, and glycoalkaloid), and increased levels of factors that influence bioavailability and utilization of essential nutrients (such as cysteine residues), are advancing through field trial stage and regulatory processes toward commercialization. The successful introduction of these biofortified crops would have a significant impact in reducing malnutrition and the risk of chronic disease in developing countries (Sarwar et al., 2007).

30.3 CONSUMERS AND REGULATION An Australian study on consumer attitudes to biotechnology has shown that consumers have mixed, but on average, less than positive attitudes toward biotechnologies (Lockie et al., 2005). Many consumers in the United States and elsewhere are unaware of the widespread use of biotechnology, the potential advantages of the genetic techniques, and the safety and regulatory procedures used before a product is approved for commercial use (Levy and Derby, 2000). There is no regulatory scheme for functional foods per se, but functional food products are clearly subject to federal regulation. If a functional food product is marketed for a therapeutic purpose (e.g., to treat a disease), it will be subject to regulation as a “drug.” If a product is subject to regulation as a “food,” it may be further classified as a conventional food, dietary supplement, food for special dietary use (including infant formula), or medical food, again depending on its intended use and other factors.

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The production of pharmaceuticals with recombinant DNA technology (also known as genesplicing or genetic modification) has enjoyed significant success over a quarter-century, with minimal controversy. However, the use of recombinant DNA-modified plants for food, feed, and environmental applications has not fared so well. These shortcomings have helped to generate “The Big Lie”—that recombinant DNA technology applied to agriculture and food production is unproven, unsafe, untested, unregulated, and unwanted. These misconceptions, in turn, have given rise to unwarranted opposition and tortuous, distorted public policy (Miller et al., 2008; Sun, 2008). We need to tackle this issue globally with the help of academics, regulators, industries, consumers, and policy makers.

30.4

CONCLUSION

It has been argued that biotechnology as such is neither good nor bad, and has the potential to alleviate or aggravate the impact of agriculture on the environment, to improve human and animal nutrition or to pose danger to human or animal health. The challenge is thus to develop, supply, and manage biotechnology for the benefit of humankind and the environment. The following suggestions have been proposed to tackle this challenge. • Promote research and market development for life sciences and biotechnology applications and the knowledge-based bioeconomy • Foster competitiveness, knowledge transfer, and innovation from the science base to industry • Encourage informed societal debates on the benefits and risk of life sciences and biotechnology • Ensure a sustainable contribution of modern biotechnology to agriculture • Improve the implementation of the legislation and its impact on competitiveness

REFERENCES Levy, A.S. and Derby, B.M., 2000. Report on Consumer Focus Groups on Biotechnology. Washington, DC: Center for Food Safety and Nutrition, Food and Drug Administration. Lockie, S., Lawrence, G., Lyons, K., and Grice, J., 2005. Factors underlying support or opposition to biotechnology among Australian food consumers and implications for retailer-led food regulation. Food Policy 30: 399–418. Miller, H.I., Morandini, P., and Ammann, K., 2008. Is biotechnology a victim of anti-science bias in scientific journals? Trends Biotechnol. 26: 122–125. Sarwar, G. and Nasim A., 2007. Impact of foods nutritionally enhanced through biotechnology in alleviating malnutrition in developing countries. J. AOAC Int. 90: 1440–1444. Sun, S.S., 2008. Application of agricultural biotechnology to improve food nutrition and healthcare products. Asia Pac. J. Clin. Nutr. 17: 87–90.

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Index A AA. See Arachidonic acid (ARA) AA2-otG. See 2-O-a-d-glucopyranosyl-lascorbic acid (AA2-otG) ABC. See ATP-binding cassette (ABC) Abiotic stresses. 148 Abnormal regulations. 258 ABR. See Auditory brainstem response (ABR) ABTS + •. See 2,2-azinobis-3-ethylbenzothiazoline6-sulfonic acid (ABTS + •) Acarbosc, 271 ACE. See Angiotensin converting enzyme (ACE) ACE inhibitory peptides. 29, 30, 32 Acetic acid, 265. 268 Acetobacter, 268 formulation, 267 Gluconobacter, 268 metabolic pathways, 267 Acetyl-5-methoxytryptamine. See N-Melatonin (MT) Acetylglucosamine, 133 N-, 132 GlmS catalyzes, 133 producer strain. 133-134 production, 132, 133 supplements, 133 Acetylglucosamine N-(GlcNAc). 131. 132 chitin, 133 E.coii. 133

producer strain, 133-134 production, 133 UDP, 132 Acctyl-L-carnitinc (LC), 115-116 GSH and GABA levels increases, 116 mitochondrial p-oxidation. 116 nerve regeneration in rats. 116 observations. 116 Acctyloxy-4-trimelhylammonio-butanoat. See 3-Acctyl-L-carnitinc (LC) Active hcxosc correlated compound (AHCC), 51 alleviating outcome, 54 body weights. 56 drug interaction, 52, 53 granulocytopenia mouse model, 55 hindlimb-unloading murine model, 56 ingredient composition, 52 manufacturing process. 51-52 metabolism profile. 53 NK cell activity, 56 oral administration. 55 research results, 53 safety assessment, 52, 53 surgical wound infection model, 56 WNV, 56 Active ingredients. 30, 33. 41 active peptides, 30, 31 development, 33

Acyl-CoA synthetase, 108 Adenosine triphosphate (ATP). 128, 206,272 Adiponectin, 99,324 Adjuvants, 241,244, 284 ADP-glucose pyrophosphorylase (AGPase). 73 African horse sickness (AHS), 242 virus (AHSV), 242 AGAMOUS. 73 Age-related hearing loss (AHL). 113-115 Aging, 114 AHL, 113-114 cochlear pathology, 113 free radical theory, 120 hair cells loss. 113 SG neurons, 113 AGPase. See ADP-glucose pyrophosphorylase (AGPase) Agricultural productivity, 198,205 Agrobacterium-mediated gene transfer, 141, 144 AHCC. See Active hcxosc correlated compound (AHCC) AHL. See Age-related hearing loss (AHL) AHS. See African horse sickness (AHS) AHSV. See African horse sickness—virus (AHSV) AHV-1. See Alcephaline herpesvirus-1 (AHV-1) ALA. See a-linolenic acid (ALA) Ala-Gin. See t-Alanyl-L-glutamine (Ala-Gin) D-Alaninc-D-alaninc (D-Ala-D-Ala) ligasc, 120 Alanine aminotransferase (ALT), 455 L-AIanyl-L-glutamine (Ala-Gin). 129 Bacillus subtilis. 129 i.-glutamine. 129 mutations, 130 producer strain, 130 synthesize. 129 usage, 129 Alcephaline herpesvirus-1 (AHV-1). 243 Alfalfa mosaic viruses, 143, 160 Alkaline phosphatase (ALP), 455 Allele-specific inhibition, 189 Allergens elimination, 168 food allergy, 168 gene-silenced soybean. 168 proteomic analysis, 168 ALP. See Alkaline phosphatase (ALP) a-amylase gene. 142 oc-amylase inhibitor. 141. 208 a,-antiirypsin (al AT), 208 a,-AT. See a-antitrypsin (a,AT) a-D-galactosylsucrosc See Raffinosc a-glucosylation enzyme, 38-39 a-linolenic acid (ALA). 12. 164,422 a-lipoic acid (LA). 115 DHLA, 115 endogenous antioxidants. 115 isomers, 115 oxidative stress models, 115 a-MenG. See Menthyl a-d-glucopyranoside (a-MenG)

549

Index

550

a-tocopherol. 117,421. See Vitamin E (VE); Antioxidants extraction, 421,422 sources. 422 ALS. See Amyotrophic lateral sclerosis (ALS) ALT. See Alanine aminotransferase (ALT) AmAl. See Amaranth seed albumin (AmAl) Amaranth seed albumin (AmAl). 159 AmDFR and Mi ANS transgenic lines, 74 Amyotrophic lateral sclerosis (ALS), 285 Anesthesia. 429 cell death, 431

effect, 435 Angiotensin converting enzyme (ACE). 29. 343, 351 Animal and Plant Health Inspection Service (APHIS). 535 Animal biotechnology. 220 agricultural applications. 232 applications. 232 avian influenza virus, 246 cloning. 225-226 development, 221 disease susceptibility, 246

human genetic diseases. 233 immunological disorders, 245-246 industrial applications, 233 knockout technology, 227

medical applications, 232-233 recombinant protein production. 236 risks. 245 stem cells in cloning, 245 technology involved. 225 transgenesis, 226 TSEs. 246 uncertainties, 245 vaccine production. 238 veterinary vaccines. 240 xenotransplantation, 233 Animal cell culture, 220, 221 aseptic conditions, 222 growth, 220 media formulation. 221-222 organ culture. 223 organotypic culture. 223 tissue culture, 223 Antenna, 290. See also Photosynthesis Anthocyanins. 63.66,81, 184, 188 acylation, 83-84 bioavailability, 84-85 composition. 63 Anthranilate synthase (AS), 161 Antifatigue efTect, 96. 97.98 fatigue effect, 97 forced swimming mouse model, 96,97 Antifungal genes. 392 Anti-inflammatory effect, 57-58 NO production. 57 suppression of NO synthesis. 57 TNBS induced model, 57 Antinutricnts, 17, 168 factors, 149,546 GLPA, 17 KBNTIpal-1,17 phytic acid foods. 17

Antioxidants, 16, 455 activity, 62,63,65,68, 70 p-carotcne, 452

C.chinensis nanopartides. 456 curcuminoids. 455 dietary, 456 enzymes, 456 S.mittiorrhizo, 456 Spirulina platensis, 424

supplementation, 120 Antioxidant, cochlear protection, 119-120 cochlear hair cell loss. 120 CR, 121 free radical theory of aging. 120

LA, 121 LC. 121 mitochondria role, 120 mtDNA, 120 ROS. 120 supplementation, 120 Antiscnsc technology. 72 gene silencing, 168-169 APHIS. See Animal and Plant Health Inspection Service (APHIS) Apoptosis, 256 bioactive food components. 258 death receptor-mediated pathway, 257 dietary agents, 257-258 functional foods. 251 milochondrial-mcdiatcd pathway, 257 natural dietary components. 256 nutraceuticals. 252 pathways in cells, 256 APVMA. See Australian Pesticides and Veterinary Medicines Authority (APVMA) APX. See Ascorbatc peroxidase (APX) AQIS. See Australian Quarantine and Inspection Service (AQIS) ARA. See Arachidonic acid (ARA) Arachidonic acid (ARA), 43. 106. 286,422 Arbulin, 42 Archetypal technology, 8 Area under the plasma concentration curves (AUC), 82 Arginine dciminasc pathway. 366 AS. See Anthranilate synthase (AS) AS ot-subunit of rice gene (OASAID), 161 Ascorbate peroxidase (APX). 73 Ascorbic acid. See Vitamin C (VC) L-ascorbic acid glucosidcs, 40 AA2-0tG preparation, 41 enzymatic glycosylations. 41 provitamin C. 40 Asp. See Asparagine (Asp) Asparagine (Asp), 6,158 Aspartate aminotransferase (AST), 455 ASP-1 protein, 159-160 AST. See Aspartate aminotransferase (AST) Astaxanthin. 43. 291. 321. 416. 418. See also Carolenoids; Chlorophylls; Functional foods antioxidative activity. 322 atopic dermatitis, 323 benefits. 323 biological purpose. 292

brain health, 323-324

Index Chlorella vulgaris, 418 C0 2 flow rates. 420 concentrations. 43 ethanol, 420 extraction, 419 formation, 292-293 Haematococcus. 292, 294. 322. 418 light-induced formation, 293 metabolic syndrome, 324-326 Phaffia rhodozyma, 418 producing organisms, 291-292 production flow scheme, 319 quality assurance system, 320 raw material manufacturing, 318 SFA, 325 source. 322 structure, 322 temperature effect. 420 TFA. 325 two-stage extraction, 421 VFA. 325 Astragalus sinicus. See Legume ATP. See Adenosine triphosphate (ATP) ATP-binding cassette (ABC), 83 AUC. See Area under the plasma concentration curves (AUC) Auditory brainstem response (ABR), 115 Australian Pesticides and Veterinary Medicines Authority (APVMA), 534 Australian Quarantine and Inspection Service (AQIS), 534 Autotrophic nutrition, 294 Auxin analogue. See 1-naphthalcncacctic acid (NAA) Axcnic. 306 2. 2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS+^), 474 Azuki bean, 139

B Bacillus subtilis. 332,427.498 C0 2 effect, 427,428 var.natto, 333, 339 Bacillus thuringiensis (BT), 206-207. 498 corn. See Insect-resistant corn cotton. 210 crystal protein, 207 fermentation process, 206 lepidoptcran-type toxins isolation, 206 parasporal crystals, 206 strains, 207 Bacterial morphology. 498 Bacterial spore. See Spore Bacterial vaccines, 241 DNA recombinant, 241 inactivated, 241

live, 241 Bactcrins, 241. See also Bacterial vaccines Basic local alignment search tool (BLAST), 515 BE. See Branching enzyme (BE) Berries, 63, 391. See also Vilis vinifera anthocyanins, 63 antioxidant activity, 63

551 biochemical changes. 391 blueberry, 63 breeding, 63.64 cell wall genes, 392 ch kinases, 392 compositional analysis, 391 cllagic acid, 63 nutritional values. 63.64 PR proteins. 392 proanthocyanidins. 63 quality traits, 64 strawberry, 63 VvPGIPl, 392 P-carotcnc, 291, 416.452. See also Anlioxidants crossover range. 420 Dunaliella salina, 291 formation, 291 p-glucan, 294 [J-glycosidase, 396 encoding genes, 3% lactic acid bacteria, 396 S.cerevisiae, 396 BHV-1.&* Bovine herpesvirus 1 (BHV-l) Bialaphos, 203 Bifidobacteria, 486,488. See also Lactic acid bacteria (LAB) acid stress, 366 acid tolerance, 371 ammonia production, 366 anticarcinogens. 490 Bacillus bifidus. 486 bile stress, 367 clinical cases, in, 493 diarrhea, against, 489 humans, in, 488 probiotic cultures. 487 scavenging activity, 365 taxonomy. 487 Bifidobacterium longum SBT-(2828), 489 Bilberry anthocyanins, 468 Bile salts hydrolases (BSH). 367 Bioactivc components, 30. 36.61. 258 compounds, 63 Bioactive peptides process, 29 ACE inhibitory peptides, 29, 30, 31, 32 active peptides, 30,31 calpis sour milk production, 32 casoxin. 32 CPP. 33 dipeptide. 32 globin peptide isolation, 33 muscle hydrolysatc, 33 Mycoleptodonoides aitchisonii, 32 opioid peptides, 32 soy protein digest. 32 SPI-H. 32-33 tetrapeptides, 31 Bioavailability, 82, 86, 185 crop plants, 166-168

flavonoids, 82 ingested food factors. 85 minerals and vitamins. 165 NK. 338

552 BioTcrtilizcrs. 200 nitrogen fixers, 200 phosphate-solubilizers, 201 uses, 200 Biofortification, 5-8 alliums consumption, 11 archetypal technology, 8 cereal enrichment, 10 epidemiology, 11 ferritin, 9 GR technology, 8 IR(68144),9 iron deficiency, 8-9 micronutricnts, 10 phytochemicals, 10-11 staple foods, 5 VAD,8 vitamin A, 8 zinc, 10 Bioinformatics, 160,514,515 Bioinsecticides, 206 Biomass components, 412,436 ot-tocopherol extraction, 421 carotenoids extraction, 416 extraction, 412 lipids extraction, 422 modifier, 415 nutraceuticals extraction, 416 pressure and temperature, 415 SCFE mechanism, 413 variables affecting SCFE, 415 waste removal, 425 Biomass fuels preparation, 106 Biosynthetic, 265 branch, 158 genes identification, 190 pathway, 181,182, 183 Biosynthetic activities, novel, 190 genes identification, 190 glycosy (transferase enzymes, 190 IFS, 190 stilbenes production. 190 STS, 190 Biotechnology agricultural, 198,531 animal, 220 biotech crops generation, 213 "blight-proof" potato, 213 cloning of Dolly, 226 crops generation, 208,210,213 definition, 4,531 equipment, 414 in fruit quality, 71-75 GM crops, 6-7,212 microalgal, 280-307 miRNA, 256 modern, 4,5 in nutritional genomics, 252 oil and fat industry, 104 in pest control, 205-210 potato crops, 213 "reductionist" approach, 400 regulatory bodies, 212,507-527,532-539,547 Regulatory Service (BRS), 535

Index salt-tolerant tomatoes, 213 scope, 4-5,62, 191,258,532 TF genes, 191 third-generation, 213 transgenic mice. 229,230 Vaccines, 238 Biotechnology-derived foods, 532. See also Food biotechnology animal based food, 17-22 edible oil, 104-112 food claims in US, 518 GM crops safety evaluation, 527 GRAS,508,509 health claims, 524 nutrient content claims, 521-523 plant based food, 5-17 prcmarkct approval, 508,509 regulation of. 508 structure/function claims, 523 winemaking, 389-402 Biotransformation, 82-83 Blackcurrant anthocyanins, 86,468 Black gram. 139, 141 phosphoinothricin, 141 regeneration, 141 somatic embryogenesis, 141 stable genetic transformation, 141 BLAST See Basic local alignment search tool (BLAST) "Wight-proof" potato, 213 Blood flow improvement, 96 effect on NO production. 96 thermograph measurement, 96,97 Blood polyphenol concentration, 95 Blueberry, 64 anthocyanins, 66 Fragaha virginiana glattca, 68 FRAP average values, 65 fruit traits, 64 genotypes variation, 64-65 heritability, 67-68 nutritional values, 67 phytochemical composition, 65 phytochemical traits, 68 RP-HPLC chromatogram, 66 total ACY, 67 Blue-green algae, 200 Bluetongue virus (BTV), 242 BMAA. See b-N-methylamino-1-alanine (BMAA) BMI. See Body mass index (BMI) B-N-methylamino-1-alanine (BMAA), 285 Body fat accumulation, 108-109,110 Body fat ratio reduction, 110,111 Body mass index (BMI), 108,325,339 Botrytis cinerea, 392,393 Bovine herpesvirus 1 (BHV-1). 242 Bovine pancreatic trypsin inhibitor (BPTIX 208 Bovine spongiform encephalopathy (BSE), 246 Bovine virus diarrhoea virus (BVDV). 242 BPTI. See Bovine pancreatic trypsin inhibitor (BPTI) Branching enzyme (BE), 36, 37 Brassica napus. See Rapeseed Breeding, 62-64,232 BRS. See Biotechnology—Regulatory Service (BRS) BSE. See Bovine spongiform encephalopathy (BSE)

Index BSH. See Bile salts hydrolases (BSH) BT. See Bacillus thuringiensis (BT) BTV. See Bluetongue virus (BTV) BVDV. See Bovine virus diarrhoea virus (BVDV)

C Calcium/calmodulin-dependent protein kinase (CCaMK), 199 Caloric restriction (CR), 114, 119. 183 age-related diseases, 114 average lifespan, 114 maximum lifespan. 114 observations. 119 results. 114 Calpis sour milk. 32 Calvin cycle. See Photosynthesis—dark reactions CAM. See Complementary and alternative medicine (CAM) Canadian Food Inspection Agency (CF1A), 536 Canadian oil. low acid (canola), 13. 14. 162 Candida cylindrcea, 43 Canning. 497. See also High-pressure treatment (HPT) autoclave, 499 cooked foods, 501 survival spores after, 500 Canola. See Canadian oil, low acid (canola) Canola™. See Low-erucic rapeseed (Canola™) Carbon dioxide. 426 pressure-temperature phase diagram, 408 properties, 408 Cardiovascular diseases (CVDs), 3,11-12, 18 Carnitine binding, 108 Carotenoids, 287-288 astaxanthin. 291, 292. 293, 416 beta carotene (J-, 291,416 biological function, 290

biomass productivity, 293 chemical nature. 288-290 extraction, 416.418. 419 humans. 287 hydrocarbon. 288 lycopene, 416 nutraceutical formula, 289 photosynthesis, 288 phytocne, 288 phytoenesynthase. 288 primary. 288. 322 secondary, 289, 290,322 solubility in SC-C02-cthanol, 420 temperature effect, 419 two-stage extraction, 421 vegetable oils. 421 Cartagena Protocol on Biosafety (CPB). 533 Casein phosphopeptides (CPPs), 33 Casoxin, 32 Ca-SP. See Ca-Spirulan (Ca-SP) Ca-Spirulan (Ca-SP), 294 Cassava, 168-169 CAT. See Catalase (CAT) Catalase (CAT). 456 Cathecol O-methyl transferase (COMT), 82 CBD. See Convention on Biological Diversity (CBD) CBE. See Chocolate equivalent (CBE)

553 CCaMK. See Calcium/calmodulin-dcpcndcnt protein kinase (CCaMK) CDC. See Centers for Disease Control and Prevention (CDC) CDNA. See complementary DNA (cDNA) CDP-choline. SeeCytidine 5'-diphosphate choline (CDP-cholinc) Center for Food Safety and Applied Nutrition (CFSAN), 518 Centers for Disease Control and Prevention (CDC). 525 CFIA. See Canadian Food Inspection Agency (CFIA) CFSAN. See Center for Food Safety and Applied Nutrition (CFSAN) CFU. See Colony forming unit (CFU) CGA. See Chlorogcnic acid (CGA) CGF. See Chlorella-growth factor (CGF) CGMP. See current Good Manufacturing Practice (cGMP) CGTase. See Cyclodextrin glucanotransferase (CGTase) Chalcone, 187 Chalcone isomerase (CHI), 186 Chalconc reductase (CHR). 186 Chalcone synthase (CHS), 182, 183 Chaperonc proteins, 366, 369. 370 CHD. See Coronary heart diseases (CHD) Chemical pesticides replacement, 206 Chemotherapy, side effects. 53,53. 54 ChFadBl. See Cuphea hooker iana CHI. See Chalconc isomerase (CHI) Chickpea, 140 against insect. 140 Allium sativum. 141 a-amylase inhibitor. 141 genetic transformation, 141 plant regeneration, 140 Chilinase gene, 143, 392 Chlorclla, 280. 281-282 growth factor (CGF), 294 pyrenoidosa, 418,419 radical scavenging. 424 Chlorogenic acid (CGA), 183-184, 187 Chlorophylls. 288. 290 Chloroplast transformation. 171 Chocolate equivalent (CBE), 42 Cholera toxin B subunit (CTB). 170 Cholesterol oxidase (CO), 207 CHR. See Chalcone reductase (CHR) CHS. See Chalconc synthase (CHS) Cicer arietinum L. See Chickpea CIMMYT. See International Maize and Wheat Improvement Center, the (CIMMYT) Citric acid, 266 Aspergillus. 266 metabolic pathways, 267 Penicillium glaucum, 266 production. 266 source. 266 Classical strain improvement (CSI), 266. 269. 270 Classical swain fever vaccine (CSFV). 242 Climacteric fruits, 71 Cloning, 225-226. See Somatic cell nuclear transfer (SCNT). See also Transgenic technology Clonogcnic assay. 225 Clostridium difficile, 489 CMP. See Cytidine 5'-monophosphate (CMP) CO. See Cholesterol oxidase (CO)

Index

554 Coat protein gene. 143 Cochlear, 119 hair cell loss, 120 pathology. 113,114 Coexpression, 160, 162-163 CogHealth, 324 Collagen hydrolysis method, 130-131 Colony formation assay. See Clonogenic assay Colony forming unit (CFU). 57. 339 Common bean, 143. 167. See Phaseolus vulgaris Phaseolus acutifolius transformation, 143 regeneration, 143 susceptibility, 143 Competitive exclusion, 363 Complementary and alternative medicine (CAM). 51 Complementary DNA (cDNA), 252, 392 clones isolation, 72-73 interferons production, 236-237 Compressed C0 2 . See Dense C0 2 Computerized tomography (CT). 325 COMT. See Cathecol O-methyl transferase (COMT) Convention on Biological Diversity (CBD), 4 Conventional breeding. 5.9.71 Cooking oils, healthy, 13 acyl-CoA synthetase, 108 carnitine binding. 108 fatty acid metabolism. 107. 108 fatty acids, 106-107 LCT absorption. 107 LCT/MCT nutritional characteristics, 108 MCT digestion, 107 MCT fat accumulation, 108-109 Corn, 14 Coronary heart diseases (CHD), 16 Cotton. 163 Cotyledonary nodes, 142 Coumarins. 183 Cowpea, dry, 139, 142 genetic transformation, 142 storage pests, 142 Cowpea Trypsin Inhibitor Gene (CpTI). 207-208 CPB. See Cartagena Protocol on Biosafety (CPB) CPPs. See Casein phosphopeptides (CPPs) CpTl gene, 207-208. See Cowpea Trypsin Inhibitor Gene (CpTI) CR. See Caloric restriction (CR) Creutzfeld-Jacob disease. 246 Crop plants, 157,210 amino acid balance. 157 enrichment, 157-158 genetic engineering, 158 Lys content improvement, 158-159 Met content improvement, 159-160 problems, 200 Trp content improvement. 161 Crass protection, 368,372 Crossover effect. 410,420 CrtB. See Psy Crtl. See Phytocnc desaturase CrtY. See Lycopene-p-cyclase Crystal protein. 207 CSFV. See Classical swain fever vaccine (CSFV) CSI. See Classical strain improvement (CSI) CT. See Computerized tomography (CT)

CTB. See Cholera toxin B subunit (CTB) CTP. See Cytidinc 5'-triphosphate (CTP) Cuphca hookcriana, 162. 163 Curcuminoids. 455 Current Good Manufacturing Practice (cGMP), 510 CVDs. See Cardiovascular diseases (CVDs) CY. See Cyclophosphamide (CY) Cyanogen ic glucoside synthesis, 169 Cyclodextrin glucanotransfcrasc (CGTase), 36 Cyclophosphamide (CY), 55 Cys. See Cysteine (Cys) Cysteine (Cys), 157 Cytidine 5'-diphosphate choline (CDP-choline), 134 administration, 134 Corynebacterium ammoniagenes> 135 CTP accumulation. 135 ischemia, 134 production, 134-135 uses. 134 Cytidine 5'-monophosphate (CMP), 134 Cytidinc 5'-triphosphatc (CTP), 135 Cytotoxicity. 56. 223 assay. 224-225 limitations. 224 metabolism, 224 pharmacokinetics, 224 tissue and systemic responses, 224

D Dainippon Ink and Chemicals Inc. (D1C), 301 Dark respiration. 296. See also Photosynthesis DASH. See Dietary-approaches to stop hypertension (DASH) DC. See Dendritic cells (DC) Death receptor-mediated pathway, 257 Dendritic cells (DC), 55 Dense C02,425,475-476. See also Nonthermal inactivation method advantages, 436, 475 applications, 436-437 Bacillus subtilis. 427 conditions affecting inactivation, 432 enlrainer, 434 enzymes recovery, 437 inactivation mechanism, 425-427,475 microbial disruption. 432 microbial structural alterations. 435 pH effect, 428 pressure, 432-433 spore inactivation, 427 temperature, 433 treatment equipment. 431 Deoxyxylulose 5-phosphate (DXP), 288 Desertification. 457-458 DGLA. See Dihomo-7-linolenic acid (DGLA) DHA. See Docosahcxaenoic acid (DHA) DHDPS. See Dihydrodipicolinate synthase (DHDPS) DHLA. See Dihydrolipoic acid (DHLA) Diacylglycerol production. 42 Diazotrophic microorganisms, 200 DIC. See Dainippon Ink and Chemicals Inc. (DIC) Diet, 3

Index Dietary agents, 257-258 approaches to stop hypertension (DASH), 349 control, 110

fibers, 35 ligands, 167 Reference Intake (DRI), 185 Supplement Health and Education Act (DSHEA), 284.524 supplements, 4, 52, 53.252. 524 Digested soy peptides (SPI-H), 32 Digestibility, 139 Dihomo-y-linolcnic acid (DGLA). 44 Dihydrodipicolinatc synthase (DHDPS), 158 Dihydrolipoic acid (DHLA), 115 Dimethylformamide (DMF), 416 3-(4-5-Dimethylthiazol-2yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfonyl)2-tetrazoIium (MTS), 225 3-(4, 5-Dimcthylthiazol-2-y 1)-2, 5-diphcnyltctrazolium bromide (MTT), 225 Dipeptide. 32, 129 U-DiphenyI-2-picrylhydrazyl (DPPH»). 474 5'-Diphosphate-N-acetylglucosamine (UDP-GlcNAc). 132 DMF. See Dimethylformamide (DMF) DNA chip. 252 Docosahexaenoic acid (DH A), 12,43.55, 106. 164. 282. 315 Docosapentaenoic acid (DPA), 422 Double-blind randomized trial. 55 Down regulation, 190 DPA. See Docosapentaenoic acid (DPA) DPPH*. See l.l-diphenyl-2-picrylhydrazyl (DPPH*) DRI. See Dietary Reference Intake (DRI) Drug interaction AHCC metabolism profile, 53 cisplatin' antitumor effect, 53, 54

dietary supplements in CAM, 52, 53 safely assessment and. 52-53 techniques to study, 224-225 Dry heat. 499 Dry weight (DW), 165. 283 DSHEA. See Dietary Supplement Health and Education Act (DSHEA) DW. See Dry weight (DW) DXP. See Deoxyxylulose 5-phosphate (DXP) Dye exclusion assay. 224 Dye uptake assay. 224 E EAA. See Epoxyaconitic acid (EAA) EC. See Epicatechin (EC): European Community (EC) ECG. See Epicatechingallate (ECG) Edible oil, 104, 164 application, 109 fatty acid composition, 105

generation. 104 genetically modified crops. 104 herbicide-resistant soybean, 104, 105 insect-resistant corn, 105 MLCToil, 109 oleic acid-rich oil, 105

555 Ostrinia nubiiaiis. 105 rape seed. 105 roundup. 104 soybean. 105 Edible vaccine, 169 cereal crop seeds, 170 chloroplast transformation technology, 171 lettuce leaves, 171 Molecular Pharming™. 169-170 plant expression. 169 plant systems benefits, 169 plant-based mucosal vaccine, 170 rice-based cholera vaccine, 170 soybean seeds, 170-171 surface protein antigen. 170 therapeutic proteins, 170 EFA. See Euglobulinfibrinolyticactivity (EFA) EFSA. See European Food Safety Authority (EFSA) EGases. See E-type endo-0-1,4-glucanase (EGases) EGCG. See Epigallocatcchingallatc (EGCG) EHV. See Equine herpesvirus vaccine (EHV) Eicosapentaenoic acid (EPA), 12, 106, 282, 416 producing organisms. 423 production, 164-165

recovery, 423 Electrospray ionization mass spectrometry (ESI-MS), 272 Ellagic acid, 63 Elm. 163 ELT. See Euglobin lysis time (ELT) Embryonic stem (ES) cell, 256 Embryonic stem cell method, 228,230 EMS. See Ethylmcthancsulfonatc (EMS) Endogenous antioxidants, 115 Endomctabolomc, 401 Endospore. See Spore 5-enoyl-pyruvylshikimate 3-phosphate synthase (EPSPS). 203 gene, 203 Entraincrs, 420,434 Environmental Protection Agency (EPA), 508, 535 Enzymatic glycosylations. 41 EPA. See Eicosapentanoic acid (EPA); Environmental Protection Agency (EPA) Epicatechin (EC), 37,94 Epicatechingallate (ECG), 94 Epigallocatechin gallate (EGCG). 38,94 acctylation. 85 taste sensitivities. 39 EPO. See Erythropoietin (EPO) Epoxyaconitic acid (EAA). 272. See also Hydroxycitric acid (HCA) EPSPS. See 5-cnoyl-pyruvylshikimalc 3-phosphatc synthase (EPSPS) Equine herpesvirus vaccine (EHV), 240, 242 Erucicacid. 13 Erythropoietin (EPO). 237-238 production, 237 therapeutic uses, 237 ES cell. See Embryonic stem (ES) cell ESI-MS. See Electrospray ionization mass spectrometry (ESI-MS) Essential nutrients, 546 EST. See Expressed sequence tag (EST)

556 Estcri Heat ion. 85,86 Esters, volatile. 73 Ethylmethanesulfonate (EMS), 293 E-type endo-P-l, 4-glucanase (EGases), 72 EU. See European Union (EU) Euglobin lysis time (ELT), 342 Euglobulin fibrinolytic activity (EFA), 342 European Community (EC). 534 European Food Safety Agency (EFSA). 280 European Food Safety Authority (EFSA), 534 European Union (EU), 243, 280,450 Exomctabolome, 401 Expressed sequence tag (EST). 149,245 Extensive screening. 131 F FA. See Fatty acid (FA); Folic acid (FA) FAO. See Food and Agriculture Organization (FAO) FASTA. See FAST-AI1 (FASTA) FAST-AU (FASTA). 515 Fatty acid (FA), 11-12,42 classification, 107 composition, 105 Fatty acid, long-chain, 107 FDA. See Food and Drug Administration (FDA); Food and Drug Agency (FDA) FDAM A. See Food and Drug Administration Modernization Act (FDAMA) FDP. See Fibrin and fibrinogen degraded products (FDP) Federal Food Drug and Cosmetic Act (FFDCA), 508 Feline leukaemia virus (FeLV), 243 FcLV. See Feline leukaemia virus (FcLV) Fermentation. 20. 132. 390. See also Winemaking acetic acid, 268 biotechnological strategies. 399 citric acid, 266 gluconic acid, 266 organic acids produced, 266 phcnolypcs, 397 process, 206 products, 332 propionic acid, 268 yeast, 397 Fermented milk, 351 antihypcrtension, 354. 356 containing GABA (FMG), 351.354. See also Functional food LAB. 351 L-glutamic acid, 351, 352 long-term FMG dose, 355 single FMG dose, 355 Fcrmcntcrs. 281, 306-307 axenic, 306 steaming. 306 Ferric reducing antioxidant power (FRAP), 64. 65.69.70 Ferritin (pfe) genes, 9 expression. 167 rice plants. 167 Fertilizers. 198 genetic complementation technique. 199-200 multipronged strategies, 198 nif gene, 199

Index nitrogen-fixers, 198-199 nod genes isolation, 199 phosphorus. 200 FFDCA. See Federal Food Drug and Cosmetic Act (FFDCA) Fibrin andfibrinogendegraded products (FDP). 342 Fibrin degradation unit, 341 Fibrinolysis. 333, 343 Field bean. See Vicia faba Flash decompression, 429 Flashing light effect. See intermittent illumination Flavan-3-ols acylation. 84 Flavanols and proanthocyanidins, 94 Flavone synthase (FNS), 186 Flavones, 184. 187 Flavonoids. 81,424-425 anthocyanins. 81 bioavailability. 82 isoflavones. 81 structural variation, 183 Flavonoids bioavailability, 82 anthocyanins acylation, 83-84 biotransformation, 82-83 EGCG acetylation. 85 esterification, 85. 86 factors affecting, 85 food absorption, 82, 83, 84, 85, 86 hydrophilic nature, 85 MDR, 83. 86 piperine. 86 protein binding. 84 sugar conjugation. 83 of tea polyphenols, 84 Flavonoids glycosylation, 36, 37 a-glucosylation enzyme. 38-39 catechin, 39 catechins transglycosylation, 38 EGCG taste sensitivities, 39 glycosides, 40 hesperidin gtucosides, 38 isoquercitrin glucosides, 38 rutin glucosides, 38 Flavonol, 184, 187 synthase (FLS). 190 Floral dip method. 144 FLS. See Flavonol-synthase (FLS) 5-Flurouracil (5-FU). 54 Fluxomics, 401 FMG. See Fermented milk-containing GABA (FMG) FNS. See Flavone synthase (FNS) Folic acid (FAX 117-118 deficiency, 118 folate. 117-118 human studies, 118 neural tube defects, 118 red blood cells production. 117 Food allergy. 168 Food and Agriculture Organization (FAO), 14. 533 Food and Drug Administration (FDA), 13,96,105,231, 284, 450, 469, 508,535 Food and Drug Administration Modernization Act (FDAMA). 525 claims. 525-526

Index Food biotechnology Australia regulations. 534 Canada regulations, 536 challenges for regulators, 538 consumer attitudes, 537 EU regulations, 534-535 Japan regulations, 536 New Zealand regulations, 534 political consideration. 537-538 resources. 539 US regulations, 535 Food for Specified Health Use (FOSHU), 29, 358 active ingredients. 30 approval seal. 30 approved peptides, 32 diacylglycerol, 42 health claims, 30 oligosaccharides. 33 Food ingredients absorption, 82 Food preservation methods, 497 autoclaving, 499 canning. 497 dry heat, 499 HPT, 498 intermittent sterilization, 499 nonthermal inactivation method, 497 pasteurization, 498 Food Standard Australia New Zealand (FSANZ). 534 Forced swimming mouse model. 96.97 FOSHU. See Food for Specified Health Use (FOSHU) Fractional extraction. See Two-step pressure gradient operation Fractionation, 437 FRAP. See Ferric reducing antioxidant power (FRAP) Fresh weight (FW), 159 Fructo-oligosaccharides production, 34 Fruit juices, 465 clarification, 469 enzymatic maceration, 466-467 extraction. 469 haze-active proteins, 468-469 hydrolytic enzymes. 466 optimization, 466-469 pectinex maceration, 467-468 PPO loss. 469 premium, 469 preservation, 469-478 Fruit quality AGAMOUS, 73 AGPasc small subunii, 73 AmDFR and MiANS transgenic lines, 74 antisense technology, 72 carbohydrate content, 73 cDNA clones isolation. 72-73 climacteric fruits. 71 complex mix of compounds, 73 conventional breeding, 71 EGascs, 72 expansin expression. 72 MADS-box genes, 73 mRNA populations, 72 ripening, 71-72 tissue softening, 72

557 transgenic approaches, 71, 73 volatile esters formation, 73 Fruits nutritional quality improvement, 70,71 nutritional values, 63, 64 Fruits and vegetables. 61 antioxidant activity, 62 breeding approach. 62 consumption, 61 phytochemical effect, 61 FSANZ. See Food Standard Australia New Zealand (FSANZ) 5-FU. See 5-flurouracil (5-FU) Fumonisin, 211 Functional foods, 4,29,251,258, 362,451. See also Nanotcchnology foods apoplosis, 256 AR A, 43-44 astaxanthins. 43 biological systems, 253 CBE, 42 development, 186 DGLA production, 44 DH A. 43.44 diacylglycerol production. 42 discrimination of. 259 diseases, 256 by fermentation, 43 genes regulation. 252. 253 miRNA, 252-253 MLCT production. 42 palm oil mid fraction. 42 polyunsaturated FA (PUFA), 43 post-translational modification, 253 relationship among, 259 strategy, 252 triacylglycerols. 43 Functional foods, animal-based. 17-18 dairy foods, 19 HLZ expression, 20 HLZmilk,2l increasing SCD activity. 21 in vitro meat, 18 meat with FA profile. 18-19 milk for lactose-intolerant population. 19-20 milk with FA profile, 21 milk with lysozyme. 20-21 post-harvest treatment, 19 skim milk fermentation, 20 Functional foods, plant-based. 5 biofortification. 5. 8 micronutrient deficiency. 5 rice, 5 FW. See Fresh weight (FW) C GABA. See y-aminobutyric acid (GABA) GAD. See Glutamate decarboxylase (GAD) Galacto-oligosaccharides (GOS), 33. 34 y-aminobutyric acid (GABA). 350 antihypertensive effect, 354-356 hypertension, 350. 357 hypotensive effect, 357-358

558 y-aminobutyric acid (GABA) (Continued) LAB, 351, 352. 354 long-term administration. 355 placebo-controlled trial. 354-356 production, 351, 354 single oral dose. 355 temperature and pH, 353 y-glutamylcystcinc (y-GC) y-glutamyl-L-cysteinylglycine. See also Glutathione (GSH) y-linolenic acid (GLA), 106, 164.416.423 Arthrospira maxima, 423 DGLA, 44 health effects, 106 medical applications. 423 poduction, 164 Spirit Una species. 423 GAO. See Government Accountability Office (GAO) Garcinia mangostana. See Mangos teen GC. See Guanine and Cytosine (GC); y-glutamylcysteine (Y-GC) GE. See Genetically engineered (GE) Gene overexpression, 191 Gene regulation. 252, 253 Gene silencing, 164, 168 Gene transfer efficiency, 144 Agrobacterium-mtd'iaicd gene transfer, 144 floral dip, 144 genetic transformation in pulse crops. 147 microprojectile bombardment. 144 nontissue culture, 144 pulse crop transformation, 145-146 transgenic plants, 144,147 transgenic pulse crops, 147 Vigna aconitifotia, 144 Generally recognized as safe (GRAS), 132. 267. 284, 509 chemistry and manufacturing. 510 food products regulation, 510 HOSU.513 intended uses, 511 safety, 511, 517-518 safety of novel proteins, 514 substantial equivalence. 512 Genesplicing. See Recombinant DNA technology Genetic complementation technique. 199-200 Genetic engineering, 4,20, 166 advantage, 158 fatly acid composition improvement, 162 LC-PUFA content improvement. 164-165 low-lactose milk production. 20 Lys content improvement, 158-159 MCFA content improvement, 162-163 Met content improvement, 159-160 mineral bioavailability improvement. 166-168 oleic acid content improvement, 164 phenylpropanoid improvement. 190-192 saturated fatty acid content improvement. 163-164 staple crops. 6-7 strategies, 205-206 Trp content improvement, 161 vitamin content improvement, 165-166 Genetic manipulations, 199 Genetic modification (GM). 183. See Recombinant DNA technology

Index Genetic transformation, 141, 142, 147 Genetically engineered (GE), 531 organism (GEO), 531 Genetically modified (GM). 531 crop. 5, 104, 140. 147-148, 158, 210. 212.450.508 organism (GMO), 531 regulations, 533 Genome shuffling, 269, 270 homologous recombination, 270 improved acid tolerance, 268 Lactobacillus, 269 principle, 268 strain improvement vs.. 270 Streptomyces sp, 269, 273 Genomics, 401,546 agriculture. 531 nulrigcnomics, 185,252, 258 progress in. 171 Genotoxic compounds. 490 Genotypes variation, 64-65 Gcntiobiosc, 35 GEO. See Genetically engineered—organism(GEO) Gcrminant, 499 GLA. See y-linolenic acid (GLA) GlcNAc. See N-acetylglucosamine (GlcNAc) GlcN-6-P. See Glucosamine-6-phosphate (GlcN-6-P) GlclfA. See Glucuronic acid (GlcUA) GlmS. See Glucosamine synthase (GlmS) Global warming. 457 Globin peptide isolation, 33 GLP. See Good Laboratory Practice (GLP) GLPA. See Goldhull low phytic acid (GLPA) Gluconic acid, 266 Aspergillus niger, 268 Gluconobacter oxydans, 268 metabolic pathways, 267 source, 266 2-O-a-d-glucopyranosyl-L-ascorbic acid (AA2-aG), 402 Glucosamine synthase (GlmS). 133 Glucosamine-6-phosphate (GlcN-6-P), 133 acetyltransfera.se (GN A1), 133 Glucuronic acid (GlcUA). 131 Glufosinate resistance. See Phosphinothricin Glulamate decarboxylase (GAD). 351 L-GIutamine. 129 Glutathione (GSH). 115, 128 Glutathione peroxidase (GPx). 456 Glutathione synthetase (GSHII), 128 Glycine max. See Soybean Glycosylated products cc-MenG preparation, 41 arbutin, 42 L-ascorbic acid glucosides. 40 flavonoids glycosylation, 36. 37 Mogroside V preparation. 41. 42 stevioside preparation, 41-42 Glyphosatc resistance. 202-203 detoxifying glyphosatc. 203 EPSPS enzyme. 203 GM. See Genetically modified (GM or GMO) GM crop. See Genetically modified (GM)-crop GM foods. See Food biotechnology GMO. See Genetically modified-organism (GMO) GMP. See Good Manufacturing Practice (GMP)

Index GM traits. 167-168, 169 GNA1. See GlcN-6-P-acetyltransferase (GNAI) Golden Potato. 166 Golden Rice (GR), 8,166 Goldhull low phytic acid (GLPA), 17 Good Laboratory Practice (GLP), 52,95 Good Manufacturing Practice (GMP). 317 quality assurance system, 320 requirements to achieve, 320 GOS. See Galacto-oligosaccharides (GOS) Gossypium hirsutum. See Cotton Government Accountability Office (GAO), 526 GPx. See Glutathione peroxidase (GPx) GR. See Golden rice (GR) Grain legumes face, 148 Granulocytopenia mouse model. 55 Grape cell-wall. See also Berries associated polysaccharides and proteins, 391 genetic and biochemical changes, 391 pathogcncsis-associatcd changes, 392-393 Grapevine. See Vitis vinifera GR AS. See Generally recognized as safe (GRAS) Gray rot fungus. See Botrytis cinerea Green gram, 139, 142 a-amylasc gene transfer. 142 regeneration, 142 green manuring, 200 GSH. See Glutathione (GSH) GSHII. See Glutathione synthetase (GSHII) Guanine and Cytosine (GC\ 486

H HA. See Hyaluronan: Hyaluronic acid (HA) HACCP. See Hazard analysis and critical control points (HACCP) Hazard analysis and critical control points (HACCP), 285 HBCD. See Highly branched cyclic dextrin (HBCD) HCA. See Hydroxycitric acid (HCA): Hydroxycinnamic acid (HCA) HCC cell. See Hepatocellular carcinoma (HCC)cell Heat shock proteins (Hsp) 370. See also Chapcronc proteins Heat-labile cnterotoxin subunil B (LTB), 170 Helianthus annuus. See Sunflower Hepatitis, 455 C.chine nsis, 455 cytokines. 455 drugs, 455 Hepatocellular carcinoma (HCC) cell, 254 HEPES. See 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES) Herbicide. 204 mechanism, 205 resistant plants, 104, 105, 204, 211 tolerance limitations. 204, 205 Herbicide resistance, 201 biotech crops, 208,210 commercial use, 208. 209 genetic engineering. 202 GM crops, 212 insect-protected crops, 209

559 no-till farming. 202 parasitic witchweed. 202 Hcspcridin, 38 preparation, 38 Heterotrophic growth, 295 nutrition, 294 HGM-CSF. See human granulocyte macrophage-colony simulating factor (hGM-CSF) HHPs. See High hydrostatic pressures (HHPs) High hydrostatic pressures (HHPs). 472 antioxidants. 473. 474 carotenoid recovery, 472 microbial activity reduction, 473 plant foods, 473 High performance liquid chromatography (HPLC). 64, 272. 336.472 High-fat diet, 98 High-hydrostatic pressure treatment (HPT), 426 Highly branched cyclic dextrin (HBCD), 36 High-oleic canola (NatrconTM), 162 High-oleic sunflower (SunolaTM), 162 High-pressure treatment (HPT). 498 cooked foods. 501 dry heat, 499 food quality, 500-502 hurdle technology, 499 spore inactivation, 499-500 survival spores after. 500 Hindlimb-unloading murine model. 56 History of safe use (HOSU), 513 HLZ. See Human lysozyme (HLZ) HNL. See Hydroxynitrile lyase (HNL) Horizontal gene transfer prevention, 148 HOSU. See History of safe use (HOSU) HPLC. See High-performance liquid chromatography (HPLC) HPT. See High-hydrostatic pressure treatment (HPT): High-pressure treatment (HPT) HPV. See Human papillomavirus (HPV) HQT. See Hydroxycinnamoyl-CoA quinatc:hydroxycinnamoyl transferase (HQT) Hsp. See Heat shock proteins (Hsp) Human granulocyte macrophage-colony simulating factor (hGM-CSF), 257 Human lysozyme (HLZ), 20 Human papillomavirus (HPV). 238 Human studies. 118 FA effect, 118 supplementation, 118 Humulin, 236 Hyaluronan. See Hyaluronic acid (HA) Hyaluronic acid (HA), 131, 133 extraction, 132 fermentation method. 132 producer strain. 132. 133 UDP-GlcNAc. 132 Hydrodynamic cavitation, 477 Hydroquinone-O-P-d-glucopyanoside. See Glycosylated products—Arbutin Hydroxycinnamic acid (HCA), 181 Hydroxycinnamoyl-CoAquinalc:hydroxycinnamoyl transferase (HQT). 186

Index

560 Hydroxycitric acid (HCA). See also Citric acid bioactivities, 271 genome shuffling, 272.273 microbial production, 270 plant-occurring organic acid, 270-271 stereoisomers, 271 4-(2-Hydroxycthyl)-l-pipcrazinccthancsulfonicacid (HEPES). 221 4-hydroxy-3-methoxybenzaldehyde. See Vanillin Hydroxynitrile lyase (HNL). 169 Hydroxyproline, 130, 131 collagen hydrolysis method, 130-131 hurdles in construction, 131 2-oxoglutaratc production, 131 physiological function. 130 producer strain, 132 proline 4-hydroxylase, 131 L-proline hydroxylation, 131 uses, 130 Hypertension, 343, 349 DASH. 349 fermented milk, 351 GABA. 350 LAB, 351 placebo-controlled trial, 354-356 Hypoallergenic food production, 16 Lactobacillus plantarn mt 16 reduce allergenicity, 16-17 soybean. 16 transgene-induced gene, 16

I IAA. See Indole acetic acid (IAA) IAF. See International Accreditation Forum (IAF) ICAR. See Indian Council for Agricultural Research (ICAR) ICT. See Immobilized cell technology (ICT) IFS. See Isoflavone synthase (IFS) IFST. See Institute of Food Science and Technology (IFST) He. See Isoleucine (He) ILSI. See International Life Sciences Institute (ILSI) Imidazolinones resistance, 204 ALS, 204 herbicide mechanisms, 205 herbicide-resistant plants. 204 transferring mutant genes. 204 Immature cotyledons. 142 Immobilized cell technology (ICT), 362 advantages, 375 cell entrapment, 374 cell immobilization matrices, 375 effects, 376 encapsulation. 377-380 physiology changes, 376 Immolina. 294. See also Ca-Spirulan (Ca-SP) Immune modulation, 363,491 Immunomodulating action, 54, 55 double-blind randomized trial. 55 total DC, 55 IMO. See Isomalto-oligosaccharides (IMO) Indian Council for Agricultural Research (ICAR). 15 Indole acetic acid (IAA), 161 Inducible NO synthase (iNOS) Inflammation assay, 225

Influenza, 239 INFOODS. See International Network of Food Data Systems (INFOODS) iNOS. See inducible NO synthase (iNOS) Input traits, 518. See also Output traits Insect resistance, 206 corn, 4-5, 105 genetically modified crop plants, 210 GM crops, 212 plant insecticidal genes. 209 transgenic crop plants. 206 transgenic plants. 209 Insect-protected crops, 209 Institute for One World Health (iOWH), 20 Institute of Food Science and Technology (IFST), 450 Insulin, 236 a-amylase. 271 TNF-a. 324-325 Interactomics, 401 Interferons, 236 applications, 237 production. 236 Saccharomyces ereviciae. 237 types. 236 Interleukines, 491 Intermittent illumination, 316 Intermittent sterilization, 499 International Accreditation Forum (IAF), 285 International Life Sciences Institute (ILSI), 513 International Maize and Wheat Improvement Center, the (CIMMYT), 14 International Network of Food Data Systems (INFOODS), 513 International Rice Research Institute (IRRI), 9 Intestinal absorption improvement. 85 Intestinal uptake. 83 Intracellular pH (pHi), 366 Ionizing radiation. 478 Ionone, 288 iOWH. See Institute for One World Health (iOWH) IPR See Isopentenyl diphospate (IPP) IPT. See Isopentenyl transferase (IPT) Iron. 8-10 bioavailability. 166. 167

deficiency. 8-9. 166 ferritin, 9 IR68144, 9 micronutricnts. 10 IRRI. See International Rice Research Institute (IRRI) Ischemia. 134 Isoflavone synthase (IFS), 186 Isoflavones, 81 isoflavonoids, 184-185, 189 Isoleucine (He), 159 Isomalto-oligosaccharides (IMO), 33, 34 Isomers, 115 Isopentenyl diphospate (IPP). 288 Isopentenyl transferase (IPT). 15 Isoquercitrin glucosides composition, 38

j Japan Bio Science Laboratory Co. Ltd. (JBSL), 335 Japanese Health Food Standards Association (JIHFS). 320

Index Japan Health Food and Nutrition Association (JHFA). 337 Japan Natto Kinase Association (JNKA), 337 JBSL. See Japan Bio Science Laboratory Co. Ltd. (JBSL) JHFA. See Japan Health Food and Nutrition Association (JHFA) JIHFS. See Japanese Health Food Standards Association (JIHFS) JNKA. See Japan Natto Kinase Association (JNKA)

K KAS. See 3-kctoacyl-acyl-carricr protein synthase (KAS) Kaybonnet (KBNT). 17 KBNT. See Kaybonnet (KBNT) 3-Kctoacyl-acyI-carricr protein synthase (KAS), 162 Kidney bean, 139. See also Common bean Knockout technology. 227

L LA. See Linoleic acid (LA): ot-lipoic acid (LA) LAB. See Lactic acid bacteria (LAB) Labeled chromium uptake assay. 224 Lactic acid. 267. 269 Lactic acid bacteria (LAB), 351, 362. 425.492. See also Lactobacilli; Probiotic bacteria ammonia production, 366 argininc dciminasc pathway, 366 P-glycosidase, 396 clinical cases, in, 493 GABA concentrations. 354 L-glutamic acid. 354, 353 pH regulation. 428 scavenging activity, 365 Lactobacilli, 369,486. See also Lactic acid bacteria (LAB) acid stress. 366 anticarcinogens. 490 chaperone proteins. 366, 369 cross protection, 368 diarrhea, against, 489 heat stress/cold stress, 367,368 heterologous expression. 370-371 L.acidophilus. 487-488 L.casei, 492 L.rhamnosus GG. 492 microaerophilic, 365 osmotic stress, 369 probiotic cultures, 486 QacT, 369 safety. 494 serum cholesterol reduction. 491 sublethal stress. 371-373 Lactose malabsorption, 489-490 Lactulose synthesis, 33-34 LB-WT. See Wild-type Lactobacillus (LB-WT) LC. See Acetyl-L-carnitine (LC) LC-PUFA. See Longchain polyunsaturated fatty acid (LC-PUFA) LCT. See Long-chain triglyceride (LCT); Long-chain triacylglycerol (LCT)

561 LD. See Lethal dose (LD) LDL. See Low density lipoprotein (LDL) Lectins. 208 Legume. 161 Lens culinaris. See Lentil Lentil. 139 Lethal dose (LD), 95 Lifespan, 119 Lifespan, maximum. 114 Lignans. 182. 183, 187 Lignins. 182, 183 Lima bean. 139 Linola™. See Low-linolenic linseed (Linola™) Linoleic acid (LA). 164 Lipid, 286. 422 autotrophic microalgal yield, 287 classes, 287 Crypthecodinium, 286 EPA, 423 GLA. 423 modification, 287 Pavlova viridis. 287 peroxide (LPO), 97 production, 286 PUFA.422 Schizochytrium, 286 Ulkenia.286 Longchain polyunsaturated fatty acid (LC-PUFA), 164 Long-chain triacylglycerol (LCT). 107 absorption, 107 digestion, 107 nutritional characteristics. 108 Long-chain triglycerides (LCT), 162 Long-duration crops. 149 Long-term safety, 109 Low density lipoprotein (LDL), 117. 160,467 Low-crucic rapesced (Canola™)« 162 Low-linolcnic linseed (Linola™), 162 low-molecular weight conversion, 91-92 LPO. See Lipid—peroxide (LPO) LTB. See Heat-labile enterotoxin subunit B (LTB) Lung cancer cells, 254 lct-7 over expression, 254 Lutein. 418 Lychee polyphenols. 92,93 absorption in human. 95 antifatigue effect, 96, 97,98 beauty effects, 99 blood flow improvement. % composition, 93 flavanols and proanthocyanidins. 94 functions. 96 level in blood, 95 oligonol, 92. 93-95 safety assessments, 95,96 visceral fat reduction, 98 Lycopene. 289. 416,418 P-cyclasc, 165 solvents. 420 Lys. See Lysine (Lys) Lysine (Lys), 98,99,157. See Adipokine production

562 M Macronutricnts modification canola oil extraction. 14 erucic acid, 13 healthier cooking oils, 13-14 healthier FA profile, 12 oils, I I stearic acid rich oils, 12-13 Thalassiosira pseudonana, 12 MADS-box genes. 73 Maize line LY038. 158 Major histocompatibility complex (MHC), 241 Malondialdchydc (MDA). 456 Man, Rogosa, and Sharpe agar (MRS), 375 Manduca scxta. See Tobacco hornworm Manganese superoxide dismutase (MnSOD). 324 Manganese-dependent catalase (MnKat), 370 Mangosteen, 163 Manihot esculenta Crantz. See Cassava Mannoprotcins, 396,397, 399 beneficial properties. 399

encoding genes. 399 dor-forming strains, 399 recombinant strain, 400 S.cerevhiae, 399 Marine oils, 452 Marker genes, 147 G M crops, 147-148

nptll gene, 147 transformants selection. 147 MARs. See Matrix attachment regions (MARs) Matrix attachment regions (MARs), 231 MCFAs. See Medium-chain fatty acids (MCFAs) MCT. See Medium-chain triglyceride (MCT); Medium-chain triacylglycerol (MCT) MDA. See Malondialdchydc (MDA) MDR. See Multiple-drug resistance (MDR) Media, 222 artificial, 222 composition. 221 formulation, 221-222 natural. 222 serum-free, 222 Medicago sativa. See Alfalfa Medium and long-chain triacylglycerol (MLCT), 42, 104 body fat accumulation, 109, 110 body fat ratio reduction. 110. I l l dietary control. 110 energy expenditure. 110. I l l healthy subjects, 110 induced changes, 111

oil. 109 production, 42 smoking point improvement, 110 transesterification. 109 Medium-chain fatty acids (MCFAs). 106-107, 162 acyl-CoA synthetase, 108 application in foods, 109 carnitine binding, 108 long-term safety. 109 MCT, 109 metabolism. 107. 108

Index Medium-chain triacylglycerol (MCT), 107, 109 BMI. 108 body fat accumulation, 108-109 digestion, 107 disadvantages. 109 fat accumulation, 108-109 nutritional characteristics, 108 Medium-chain triglyceride (MCT), 162 Melatonin (MT), 118 Parkinson's disease, 118 uses, 118 Menthyl a-D-glucopyranoside (a-MenG), 41 Messenger ribonucleic acid (mRNA). 253. 392 populations, 72 Met. See Methionine (Met) Metabolic engineering, 172. 181, 186 bioavailability. 185 DRI, 185 focus on antioxidant properties. 185 functional foods development, 186 mclabolomics, 185 OMICS groups, 185 phcnylpropanoids, 182, 186. 187-189 proteomics. 185-186 targets. 185 transcriptomics. 185 Metabolic syndrome, 324-326 adiponectin, 324 astaxanthin. 325 glycohcmoglobin (HbAlc), 324, 325 tumor necrosis factor (TNF), 324-325 Metabolomics. 185, 401. 546 metabolic engineering, 172 role, 185 Metallothionein-like (rgMT) genes, 9 Mclhicillin-rcsistant Staphylococcus aureus (MRSA). 55 Methionine (Met). 157 synthetic. 15-16 Met-sink manipulations, 160 Mel-source manipulation, 160 MHC. See Major histocompatibility complex (MHC) MHLW. See Ministry of Health, Labor and Welfare (MHLW) MHW. See Ministry of Health and Welfare ( M H W ) Microalgae. 280.314 biomass production, 281 carotenoid production, 287 Chlorella growth factor, 294 composition, 286 lipid contents. 286 lipid production. 286 molecular biology. 281 nutraceutical carotenoids, 289 nutraceuticals,281,281,307 product formation, 281 production costs, 283 respiration types. 296 use of. 280 Microalgae mass culture, 314. See also Microalgal production methods commercial cultivation, 315 hurdles. 315 photobioreactor. 315 productivity, 315

Index Microalgal nutraceuticals, 281, 281, 307 carotenoids configuration formula. 289 Chlorella, 281, 282 Crxpthecodinium, 281 Dunaliella, 281, 282 existing markets for, 282 Haematococcus. 281 Schizochytrium, 281-282 Spirulina.2&\.2V2 Microalgal production methods, 294 autotrophic growth, 295 biomass production systems, 299 growth rate and light intensity, 297-299 heterotrophic growth. 299 open ponds, 300 photobioreactors, 301, 306 photosynthesis efficiency, 297 Microalgal products. See also Microalgal nutraceuticals food quality assurance, 283 HACCP. 285 health concerns. 285 microalgal nutraceutical production, 281, 307 production costs. 283 quality by self-regulation. 285 regulation, 284 Microbial inactivation, 426,427,470, 476 acidification, 428 buffer solution. 435 cell disruption. 432 cell membrane modification, 429 cell wall lipids extraction. 429 C0 2 effect. 429 complete inactivation, 431 continuous processes. 431 curve, 430 en trainer, 434 enzyme inactivity, 429 inactivation rate, 430 intracellular substances leakage, 430 lag phase. 430 major stages. 427-428 mechanism. 427 media composition effects, 434 morphological and structural alterations, 435 N2 prcssurization. 428 pressure. 432-433 repeated pressure release. 435 temperature. 433-434 Microencapsulation, 377,451 alginate matrix, 377 gel particles, 377-378 microcapsules, 379-380 spray-coating. 378-379 Microfluidics, 453-454 Microinjection method. 228 Micronutrient deficiency, 5 Microprojcctile bombardment method. 144 Middle latency response (MLR). 119 Minerals, 165 approaches, 166-167 bioavailability improvement, 166 dietary ligands, 167 ferritin gene expression, 167 fortification routes, 165

563 GM traits. 167-168 iron bioavailability, 166, 167 zinc deficiency, 167 Ministry of Health and Welfare (MHW). 338 Ministry of Health, Labor and Welfare (MHLW), 29,345 miRNA,251.252,253.255 advantage, 256 applications, 256

functional foods. 251 let-7 over-expression. 254 nutraceuticals, 252 regenerative medicines, 256 regulation, 253-254. 256, 258 relationship. 259 role. 254.255 tumor suppressors. 254 Mitochondria, 120 (5-oxidation, 116

DNA(mtDNA), 114 mediated pathway, 257 mtDNA deletions, 120 Mixotrophic. 295 MLCT. See Medium and long-chain triacylglycerols (MLCT) MLR. See Middle latency response (MLR) MnKat. See Manganese-dependent catalase (MnKat) MnSOD. See Manganese superoxide dismutase (MnSOD) Model legume genomics translation, 149 EST projects, 149 whole-genome sequencing report, 149 Model plants, 158 Mogrosidc V.41.42 Molecular weight (MW). 32. 335 Monounsaturated FAs (MUFAs), 12, 107 Moth bean. 139. 141 DNA transfer, 141 plant regeneration. 141 protoplasts. 141 mRNA. See messenger ribonucleic acid (mRNA) MRS. See Man. Rogosa, and Sharpe agar (MRS) MRSA. See Melhicillin-resistant Staphylococcus aureus (MRSA) MT. See Melatonin (MT) mtDNA. See Mitochondria—DNA (mtDNA) MTS. See 3-(4-5-dimcthylthiazol-2yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfonyl)2-tetrazolium (MTS) MTT. See 3-(4.5-dimethylthiazol-2-yl)-2. 5-diphenyltetrazolium bromide (MTT) MUFAs. See Monounsatu rated FAs (MUFAs) Multiple age-associated diseases, 117 anticancer agents. 53. 54 biosynthetic steps. 182, 191 drug resistance (MDR), 83 Must, 389 P-glycosidase, 396

Botrytis cincrca-dcrivcd glucans, 393 pectin polymers. 393 polysaccharide hydrolases, 393 MW. See Molecular weight (MW)

564 N NAA. See 1-Naphthalcncacctic acid (NAA) Nanocapsules. 454 Nanonization. 454 Chinese herbal medicines, 454 functional foods, 451 vitamin C particles, 453 Nanoparticlc formulation, 454 antioxidant. 455 curcuminoids, 455 Cuscuta chinensis. 455 hepatq>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. ^e«/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