Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals (Institute of Food Technologists Series)

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals Yoshinori Mine, Eunice Li-Chan, and Bo Jiang EDI...

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Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals Yoshinori Mine, Eunice Li-Chan, and Bo Jiang EDITORS

A John Wiley & Sons, Inc., Publication

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals

The IFT Press series reflects the mission of the Institute of Food Technologists—to advance the science of food contributing to healthier people everywhere. Developed in partnership with Wiley-Blackwell, IFT Press books serve as leading-edge handbooks for industrial application and reference and as essential texts for academic programs. Crafted through rigorous peer review and meticulous research, IFT Press publications represent the latest, most significant resources available to food scientists and related agriculture professionals worldwide. Founded in 1939, the Institute of Food Technologists is a nonprofit scientific society with 22,000 individual members working in food science, food technology, and related professions in industry, academia, and government. IFT serves as a conduit for multidisciplinary science through leadership, championing the use of sound science across the food value chain through knowledge sharing, education, and advocacy. IFT Book Communications Committee Syed S. H. Rizvi Casimir C. Akoh Barry G. Swanson Christopher J. Doona Ruth M. Patrick Mark Barrett Heather Troxell Karen Nachay IFT Press Editorial Advisory Board Malcolm C. Bourne Dietrich Knorr Theodore P. Labuza Thomas J. Montville S. Suzanne Nielsen Martin R. Okos Michael W. Pariza Barbara J. Petersen David S. Reid Sam Saguy Herbert Stone Kenneth R. Swartzel

A John Wiley & Sons, Inc., Publication

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals Yoshinori Mine, Eunice Li-Chan, and Bo Jiang EDITORS

A John Wiley & Sons, Inc., Publication

Edition first published 2010 © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Editorial Office 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services, and for information about how to apply for permission to reuse the copyright material in this book, please see our Website at www.wiley.com/wiley-blackwell. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Blackwell Publishing, provided that the base fee is paid directly to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate system of payments has been arranged. The fee code for users of the Transactional Reporting Service is ISBN-13: 978-0-8138-1311-0/2010. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks, or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Bioactive proteins and peptides as functional foods and nutraceuticals / edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. – 1st ed. p. ; cm. Includes bibliographical references and index. ISBN 978-0-8138-1311-0 (alk. paper) 1. Proteins in human nutrition. 2. Functional foods. I. Mine, Yoshinori. II. Li-Chan, Eunice. III. Jiang, Bo, 1962– [DNLM: 1. Dietary Proteins–metabolism. 2. Peptides–metabolism. 3. Dietary Proteins–analysis. 4. Nutritional Physiological Phenomena. 5. Peptides–analysis. QU 55.4 B615 2010] TX553.P7B65 2010 612.3′98–dc22 2009054220 A catalog record for this book is available from the U.S. Library of Congress. Set in 10 on 12 pt Times by Toppan Best-set Premedia Limited Printed in Singapore Disclaimer The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation warranties of fitness for a particular purpose. No warranty may be created or extended by sales or promotional materials. The advice and strategies contained herein may not be suitable for every situation. This work is sold with the understanding that the publisher is not engaged in rendering legal, accounting, or other professional services. If professional assistance is required, the services of a competent professional person should be sought. Neither the publisher nor the author shall be liable for damages arising herefrom. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. 1

2010

Titles in the IFT Press series • Accelerating New Food Product Design and Development (Jacqueline H. Beckley, Elizabeth J. Topp, M. Michele Foley, J.C. Huang, and Witoon Prinyawiwatkul) • Advances in Dairy Ingredients (Geoffrey W. Smithers and Mary Ann Augustin) • Biofilms in the Food Environment (Hans P. Blaschek, Hua H. Wang, and Meredith E. Agle) • Calorimetry and Food Process Design (Gönül Kaletunç) • Food Ingredients for the Global Market (Yao-Wen Huang and Claire L. Kruger) • Food Irradiation Research and Technology (Christopher H. Sommers and Xuetong Fan) • Foodborne Pathogens in the Food Processing Environment: Sources, Detection and Control (Sadhana Ravishankar, Vijay K. Juneja, and Divya Jaroni) • High Pressure Processing of Foods (Christopher J. Doona and Florence E. Feeherry) • Hydrocolloids in Food Processing (Thomas R. Laaman) • Improving Import Food Safety (Wayne C. Ellefson, Lorna Zach, and Darryl Sullivan) • Microbial Safety of Fresh Produce: Challenges, Perspectives and Strategies (Xuetong Fan, Brendan A. Niemira, Christopher J. Doona, Florence E. Feeherry, and Robert B. Gravani) • Microbiology and Technology of Fermented Foods (Robert W. Hutkins) • Multiphysics Simulation of Emerging Food Processing Technologies (Kai Knoerzer, Pablo Juliano, Peter Roupas, and Cornelis Versteeg) • Multivariate and Probabilistic Analyses of Sensory Science Problems (Jean-François Meullenet, Rui Xiong, and Christopher J. Findlay • Nanoscience and Nanotechnology in Food Systems (Hongda Chen) • Natural Food Flavors and Colorants (Mathew Attokaran) • Nondestructive Testing of Food Quality (Joseph Irudayaraj and Christoph Reh) • Nondigestible Carbohydrates and Digestive Health (Teresa M. Paeschke and William R. Aimutis) • Nonthermal Processing Technologies for Food (Howard Q. Zhang, Gustavo V. Barbosa-Cànovas, V.M. Balasubramaniam, Editors; C. Patrick Dunne, Daniel F. Farkas, James T.C. Yuan, Associate Editors) • Nutraceuticals, Glycemic Health and Type 2 Diabetes (Vijai K. Pasupuleti and James W. Anderson) • Organic Meat Production and Processing (Steven C. Ricke, Michael G. Johnson, and Corliss A. O’Bryan) • Packaging for Nonthermal Processing of Food (J. H. Han) • Preharvest and Postharvest Food Safety: Contemporary Issues and Future Directions (Ross C. Beier, Suresh D. Pillai, and Timothy D. Phillips, Editors; Richard L. Ziprin, Associate Editor) • Processing and Nutrition of Fats and Oils (Ernesto M. Hernandez, and Afaf Kamal-Eldin) • Processing Organic Foods for the Global Market (Gwendolyn V. Wyard, Anne Plotto, Jessica Walden, and Kathryn Schuett) • Regulation of Functional Foods and Nutraceuticals: A Global Perspective (Clare M. Hasler) • Resistant Starch: Sources, Applications and Health Benefits (Yong-Cheng Shi and Clodualdo Maningat) • Sensory and Consumer Research in Food Product Design and Development (Howard R. Moskowitz, Jacqueline H. Beckley, and Anna V.A. Resurreccion) • Sustainability in the Food Industry (Cheryl J. Baldwin) • Thermal Processing of Foods: Control and Automation (K. P. Sandeep) • Trait-Modified Oils in Foods (Frank T. Orthoefer and Gary R. List) • Water Activity in Foods: Fundamentals and Applications (Gustavo V. Barbosa-Cànovas, Anthony J. Fontana Jr., Shelly J. Schmidt, and Theodore P. Labuza) • Whey Processing, Functionality and Health Benefits (Charles I. Onwulata and Peter J. Huth)

Contents

Preface, ix Contributors, xi Part 1. Introduction, 3 1.

Biologically Active Food Proteins and Peptides in Health: An Overview, 5 Yoshinori Mine, Eunice C.Y. Li-Chan, and Bo Jiang

Part 2. Functions of Biologically Active Proteins and Peptides, 13 2.

Anti-inflammatory/Oxidative Stress Proteins and Peptides, 15 Denise Young and Yoshinori Mine

3.

Antioxidant Peptides, 29 Youling L. Xiong

4.

Antihypertensive Peptides and Their Underlying Mechanisms, 43 Toshiro Matsui and Mitsuru Tanaka

5.

Food Protein–Derived Peptides as Calmodulin Inhibitors, 55 Rotimi E. Aluko

6.

Soy Protein for the Metabolic Syndrome, 67 Cristina Martínez-Villaluenga and Elvira González de Mejía

7.

Amyloidogenic Proteins and Peptides, 87 Soichiro Nakamura, Takanobu Owaki, Yuki Maeda, Shigeru Katayama, and Kosuke Nakamura

8.

Peptide-Based Immunotherapy for Food Allergy, 101 Marie Yang and Yoshinori Mine

9.

Gamma-Aminobutyric Acid, 121 Bo Jiang, Yuanxin Fu, and Tao Zhang

10.

Food Proteins or Their Hydrolysates as Regulators of Satiety, 135 Martin Foltz, Mylene Portier, and Daniel Tomé

Part 3. Examples of Food Proteins and Peptides with Biological Activity, 149 11.

Health-Promoting Proteins and Peptides in Colostrum and Whey, 151 Hannu J. Korhonen vii

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Contents

12.

Functional Food Products with Antihypertensive Effects, 169 Naoyuki Yamamoto

13.

Secreted Lactoferrin and Lactoferrin-Related Peptides: Insight into Structure and Biological Functions, 179 Dominique Legrand, Annick Pierce, and Joël Mazurier

14.

Bioactive Peptides and Proteins from Fish Muscle and Collagen, 203 Nazlin K. Howell and Chitundu Kasase

15.

Animal Muscle-Based Bioactive Peptides, 225 Jennifer Kovacs-Nolan and Yoshinori Mine

16.

Processing and Functionality of Rice Bran Proteins and Peptides, 233 Rashida Ali, Frederick F. Shih, and Mian Nadeem Riaz

17.

Bioactive Proteins and Peptides from Egg Proteins, 247 Jianping Wu, Kaustav Majumder, and Kristen Gibbons

18.

Soy Peptides as Functional Food Materials, 265 Toshihiro Nakamori

19.

Bioactivity of Proteins and Peptides from Peas (Pisum sativum, Vigna unguiculata, and Cicer arietinum L), 273 Bo Jiang, Wokadala C. Obiro, Yanhong Li, Tao Zhang, and Wanmeng Mu

20.

Wheat Proteins and Peptides, 289 Hitomi Kumagai

Part 4. Recent Advances in Bioactive Peptide Analysis for Food Application, 305 21.

Peptidomics for Bioactive Peptide Analysis, 307 Icy D’Siva and Yoshinori Mine

22.

In silico Analysis of Bioactive Peptides, 325 Marta Dziuba and Bartłomiej Dziuba

23. Flavor-Active Properties of Amino Acids, Peptides, and Proteins, 341 Eunice C.Y. Li-Chan and Imelda W.Y. Cheung 24. Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides, 359 Idit Amar-Yuli, Abraham Aserin, and Nissim Garti Index, 383

Preface

Functional proteins and peptides are now an important category within the nutraceuticals food sector. A growing body of scientific evidence in the past decade has revealed that many food proteins and peptides exhibit specific biological activities in addition to their established nutritional value. Bioactive peptides present in foods may help reduce the worldwide epidemic of chronic diseases that account for 58 million premature deaths annually. This book aims to compile current science-based advances on biologically active food proteins and peptides for health promotion and reduction of risk of chronic diseases. The book is comprised of four parts: (1) “Introduction,” (2) “Functions of Biologically Active Proteins and Peptides,” (3) “Examples of Food Proteins and Peptides with Biological Activity,” and (4) “Recent Advances in Bioactive Peptide Analysis for Food Application.” The book considers fundamental concepts and structure-activity relations for the major classes of nutraceutical proteins and peptides. The international team from industry and academia also contributed current applications and future opportunities within the nutraceutical proteins and peptides of rapidly growing fields in food and nutrition research

and conveys the state of the science to date. We are grateful to all the stellar internationally renowned authors for their state-of-the-art compilation of recent rapid developments in this field, which made the publication of this book possible. We believe that this book deserves a broad readership in the disciplines of food science, nutrition, pharmaceuticals, cosmetics, nutraceutical/functional foods, biochemistry, and biotechnology. This book could also be useful as a comprehensive reference book by senior undergraduate and graduate students, as well as by the nutraceutical and pharmaceutical industries. We wish to thank all the contributors for sharing their expertise throughout our journey. We also thank the reviewers for giving their valuable comments on improving the contents of each chapter. All these professionals are the ones who made this book possible. We thank members of the production team at Wiley-Blackwell for their time, effort, advice, and expertise. Yoshinori Mine Eunice C.Y. Li-Chan Bo Jiang

ix

Contributors

Rashida Ali Division of Food Research H.E.J. Research Institute of Chemistry International Centre for Chemical and Biological Sciences University of Karachi Karachi-75270, Pakistan Rotimi E. Aluko University of Manitoba Department of Human Nutritional Sciences Winnipeg, Canada R3T 2N2 [email protected] Phone: 204-474-9555; Fax: 204-474-7593 Idit Amar-Yuli Casali Institute of Applied Chemistry Givat Ram Campus The Hebrew University of Jerusalem Jerusalem 91904, Israel Abraham Aserin Casali Institute of Applied Chemistry Givat Ram Campus The Hebrew University of Jerusalem Jerusalem 91904, Israel Imelda W.Y. Cheung The University of British Columbia Food Nutrition & Health Program 2205 East Mall Vancouver, BC, Canada V6T 1Z4

Icy D’Siva Department of Food Science University of Guelph Guelph, ON, Canada N1G 2W1 Bartłomiej Dziuba University of Warmia and Mazury Faculty of Food Science Chair of Industrial and Food Microbiology Pl. Cieszynski 1 10-712 Olsztyn-Kortowo, Poland Marta Dziuba University of Warmia and Mazury Faculty of Food Science Chair of Food Biochemistry Pl. Cieszynski 1 10-712 Olsztyn-Kortowo, Poland Martin Foltz Unilever R&D Vlaardingen Olivier van Noortlaan 120 3133 AT Vlaardingen The Netherlands Yuanxin Fu State Key Laboratory of Food Science and Technology Jiangnan University 1800 Lihu Avenue Wuxi, Jiangsu 214122, China

xi

xii

Contributors

Nissim Garti Casali Institute of Applied Chemistry Givat Ram Campus The Hebrew University of Jerusalem Jerusalem 91904, Israel [email protected] Phone: 972-2-658-6574/5; Fax: 972-2-652-0262 Kristen Gibbons Department of Agricultural, Food and Nutritional Science University of Alberta Edmonton, AB, Canada T6G 2P5 Elvira González de Mejía Department of Food Science & Human Nutrition Division of Nutritional Sciences University of Illinois at Urbana-Champaign 228 Edward R Madigan Laboratory, 1201 W. Gregory Drive Urbana, IL 61801 [email protected] Phone: 217-244-3196; 217-244-3198 Nazlin K. Howell Professor of Food Biochemistry University of Surrey Faculty of Health and Medical Sciences Guildford, Surrey GU2 7XH [email protected] Phone: +44 1483 686448 Bo Jiang State Key Laboratory of Food Science and Technology Jiangnan University Jiangsu, 214122, China [email protected] Phone: +86-510-85329055; Fax: +86-510-85919625 Chitundu Kasase University of Surrey Faculty of Health and Medical Sciences Guildford, Surrey GU2 7XH, UK

Shigeru Katayama Department of Bioscience and Biotechnology Shinshu University 8304 Minamiminowamura Ina, Nagano 399-4598, Japan Hannu J. Korhonen MTT Agrifood Research Finland Biotechnology and Food Research FIN-31600 Jokioinen, Finland [email protected] Phone: +358-3-41883271; Fax: +358-3-41883244 Jennifer Kovacs-Nolan Department of Food Science University of Guelph Guelph, ON, Canada N1G 2W1 Hitomi Kumagai College of Bioresource Sciences Nihon University [email protected] Phone/Fax: +81-466-84-3946 Dominique Legrand Unité de Glycobiologie Structurale et Fonctionnelle Université des Sciences et Technologies de Lille UMR No. 8576 du CNRS / IFR 147 F-59655 Villeneuve d’Ascq Cedex, France [email protected] Phone: 33-3-20-43-44-30; Fax: 33-3-20-43-65-55 Yanhong Li State Key Laboratory of Food Science and Technology Jiangnan University 1800 Lihu Avenue Wuxi, Jiangsu 214122, China Eunice C.Y. Li-Chan The University of British Columbia Food Nutrition & Health Program 2205 East Mall Vancouver, BC, Canada V6T 1Z4 [email protected] Phone: 1-604-822-6182; Fax: 1-604-822-5143

Contributors

Yuki Maeda Department of Bioscience and Biotechnology Shinshu University 8304 Minamiminowamura Ina, Nagano 399-4598, Japan Kaustav Majumder Department of Agricultural, Food and Nutritional Science University of Alberta Edmonton, AB, Canada T6G 2P5 Cristina Martínez-Villaluenga Department of Food Science & Human Nutrition Division of Nutritional Sciences University of Illinois at Urbana-Champaign 228 Edward R Madigan Laboratory, 1201 W. Gregory Drive Urbana, IL 61801 Toshiro Matsui Faculty of Agriculture Graduate School of Kyushu University Fukuoka, 812-8581, Japan [email protected] Phone: +81-92-642-3012 Joël Mazurier Unité de Glycobiologie Structurale et Fonctionnelle Université des Sciences et Technologies de Lille UMR No. 8576 du CNRS / IFR 147 F-59655 Villeneuve d’Ascq Cedex, France Yoshinori Mine Department of Food Science University of Guelph Guelph, ON, Canada N1G 2W1 [email protected] Phone: 519-824-4120 x52901; Fax: 519-824-6631 Wanmeng Mu State Key Laboratory of Food Science and Technology Jiangnan University 1800 Lihu Avenue Wuxi, Jiangsu 214122, China

xiii

Toshihiro Nakamori Fuji Oil Co., Ltd, 1 Sumiyoshi-Cho Izumisano-Shi, Osaka 598-8540, Japan [email protected] Phone: +81-297-52-6322; Fax: +81-297-52-6320 Kosuke Nakamura The Japan Health Sciences Foundation 13-4 Nihonbashi Kodenma-cho Chuo-ku, Tokyo 103-0001, Japan Soichiro Nakamura Department of Bioscience and Biotechnology Shinshu University 8304 Minamiminowamura Ina, Nagano 399-4598, Japan Wokadala C. Obiro State Key Laboratory of Food Science and Technology Jiangnan University 1800 Lihu Avenue Wuxi, Jiangsu 214122, China Takanobu Owaki Department of Bioscience and Biotechnology Shinshu University 8304 Minamiminowamura Ina, Nagano 399-4598, Japan Annick Pierce Unité de Glycobiologie Structurale et Fonctionnelle Université des Sciences et Technologies de Lille UMR No. 8576 du CNRS / IFR 147 F-59655 Villeneuve d’Ascq Cedex, France Myléne Potier AgroParisTech Life Sciences and Health 16 rue Claude Bernard 75005 Paris, France

xiv

Contributors

Mian Nadeem Riaz United States Department of Agriculture (USDA)-ARS-SRRC New Orleans, LA 70124 Frederick F. Shih English Biscuit Manufacturers (Private) Limited Korangi Industrial Area Karachi, Pakistan Mitsuru Tanaka Faculty of Agriculture Graduate School of Kyushu University Fukuoka, 812-8581, Japan Daniel Tomé AgroParisTech Life Sciences and Health 16 rue Claude Bernard 75005 Paris, France Jianping Wu Department of Agricultural, Food and Nutritional Science University of Alberta Edmonton, AB, Canada T6G 2P5 [email protected] Phone: 780-492-6885; Fax: 780-492-4346

Youling L. Xiong University of Kentucky Department of Animal and Food Sciences Room 206 W.P. Garrigus Bldg. Lexington, KY 40546-0215 [email protected] Phone: 859-257-3822 Naoyuki Yamamoto R&D Center Calpis Co., Ltd. 11-10, 5-Chome, Fuchinobe, Sagamihara, Kanagawa 229 Japan [email protected] Marie Yang Department of Food Science University of Guelph Guelph, ON, Canada N1G 2W1 Denise Young Department of Food Science University of Guelph Guelph, ON, Canada N1G 2W1 Tao Zhang State Key Laboratory of Food Science and Technology Jiangnan University Wuxi, Jiangsu 214122, China

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals

Part 1 Introduction

Chapter 1 Biologically Active Food Proteins and Peptides in Health: An Overview Yoshinori Mine, Eunice C.Y. Li-Chan, and Bo Jiang

A growing body of scientific evidence in the past decade has revealed that many food proteins and peptides exhibit specific biological activities in addition to their established nutritional value (Mine and Shahidi 2006; Hartmann and Meisel 2007; Tripathi and Vashishtha 2006; Yalcin 2006; Möller et al. 2008). Bioactive peptides have been found in enzymatic protein hydrolysates and fermented dairy products, but they can also be released during gastrointestinal digestion of proteins (Meisel 2005; Korhonen and Pihlanto 2007a, 2007b; Gobbetti et al. 2007; Hartmann and Meisel 2007). Bioactive peptides may help reduce the worldwide epidemic of chronic diseases that account for 58 million premature deaths annually. Functional proteins and peptides are an important category within the nutraceuticals food sector currently valued at $75 billion/ year. Nevertheless, several challenges should be addressed to allow sustained growth within this sector. Some earlier health benefits claimed for protein nutraceuticals were based on in vitro models or limited clinical trials leading to equivocal findings (Möller et al. 2008). Technological and fundamental problems remain in relation to large-scale production, compatibility with different food matrices, gastrointestinal stability, bioavailability, and long-term safety (Murray and FitzGerald 2007; Mine 2007). Research into consumer perception and Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

legislation is also necessary. Nutritionists, biomedical scientists, food scientists, and technologists are working together to develop improved systems for discovery, testing, and validation of nutraceutical proteins and peptides with increased potency and therapeutic benefits. Bioactive peptides and proteins are being developed that positively impact on body function and human health by alleviating conditions such as coronary (ischemic) heart disease, stroke, hypertension, cancer, obesity, diabetes, and osteoporosis. Screening novel bioactive sources using high-throughput ohmics technologies, specific disease biomarkers, and comprehensive clinical trials will facilitate the development of nutraceutical proteins and peptides for a further range of health conditions (Gilani et al. 2008; Mine et al. 2009; Boelsma and Kloek 2009). This book aims to compile current science-based advances on biologically active food proteins and peptides for health promotion and reduction of the risk of chronic diseases. The book is comprised of four parts: (1) Introduction, (2) Functions of Biologically Active Proteins and Peptides, (3) Examples of Food Proteins and Peptides with Biological Activity, and (4) Recent Advances in Bioactive Peptide Analysis for Food Application. Chapter 1 summarizes aims and scope as well as overall highlights of this book. Chapters 2 and 3 highlight antioxidative and anti-inflammatory proteins and peptides. Oxidative stress is a biological state that occurs when a cell’s antioxidant capacity is overwhelmed by reactive oxygen species (ROS), causing a redox imbalance. Oxidative stress and 5

6

Part 1 Introduction

inflammation are often related to chronic diseases involving the cardiovascular, neurological, and gastrointestinal systems. Proteins and peptides have been found not only to contribute to the body’s energy supply and growth but also to influence specific biological activities such as oxidative stress and inflammation. As our understanding of the efficacy and mechanism of action of antioxidative stress and anti-inflammatory proteins and peptides increases, so will the growing interest in their prophylactic, preventive, and therapeutic uses. Recent advances in peptide research have led to the accumulation of mounting evidence that many endogenous peptides have the biological function to stabilize radicals and neutralize other nonradical oxidizing species. Furthermore, this biological function has been demonstrated by in vitro digests of proteins. With an improved understanding of the structure-function relationship, it is now possible to develop antioxidant peptides and peptide mixtures through enzymatic or microbial hydrolysis of common food proteins or by means of chemical synthesis. While the question of whether peptides can act as antioxidants no longer remains, it is still a big challenge to identify the fate of dietary antioxidant peptides and their exact biological activity once entering the circulation system and crossing the cell membrane. The production cost as well as the potential allergenicity of antioxidant peptides must also be assessed in the continuing effort to develop such novel antioxidants to complement the human body’s natural defense system, including antioxidant enzymes, vitamins, and nonprotein compounds. Chapter 4 reviews the updated antihypertensive mechanism as well as the development of antihypertensive food products. The efficacy of peptide intake for borderline hypertensives is evidentially developed on the basis of extensive intervention trials, but such an effective foods for specified health use (FOSHU) produce has led to the question of whether the antihypertensive effect of peptides is achieved only by angiotensin-converting enzyme (ACE) inhibition or suppression of the renin-angiotensin system, like therapeutic ACE inhibitory drugs. Peptide research on this topic has become one of the growing fields in preventative medicinal chemistry,

since clinical evidence demonstrated the efficacy of peptide intake for improving the treatment of hypertension. Chapter 5 presents food protein–derived bioactive peptides as inhibitors for calmodulin (CaM), a protein that plays important roles in maintaining physiological functions of cells and body organs. Current knowledge indicates that CaM-binding peptides can be produced through the enzymatic hydrolysis of food proteins followed by separation and purification of cationic peptides. Inhibition of CaMdependent enzymes seems to be dependent mostly on the level of basic (positively charged) amino acid residues for short-chain (<7-mer) peptides, whereas long-chain (16- to 25-mer) peptides interact with CaM mostly through hydrophobic interactions. Structural changes occur during peptide binding, leading to an increased unfolding of CaM such that the protein loses its ability to effectively interact with and activate target metabolic enzymes. It is evident that food protein–derived CaM-binding peptides constitute a potential source of ingredients that may be used to formulate therapeutic diets against human chronic diseases. However, the ultimate utility of these cationic peptides will depend on their oral bioavailability and in vivo inhibition of target metabolic reactions. Chapter 6 describes the potential for soy protein to address the problem of metabolic syndrome (MS). Diets with increased protein and reduced carbohydrates have been shown to improve body composition and lipid and lipoprotein profiles, reducing blood pressure and playing a part in regulation of glucose and insulin homeostasis, all of which are factors associated with treatment of metabolic syndrome and obesity. Soy protein is a promising dietary component that needs to be further evaluated in regards to its long-term effect on adipogenesis and potential reduction of blood pressure. This will allow determination of the recommended intake of soy protein to reduce the risk of metabolic syndrome in humans from different backgrounds and environments. Attention must also be paid to the technological processes in order to optimize the protein quality, its bioavailability, and the main protein components that will produce the most biologically active peptides.

Chapter 1 Biologically Active Food Proteins and Peptides in Health

Chapter 7 deals with amyloidogenic proteins and peptides. To date, many different naturally occurring proteins and polypeptides have been recognized to be amyloidogenic in humans. These precursor proteins, clearly differing in their biochemical function and in their three-dimensional structure, may become prone to undergoing an irreversible transition from their native conformation into highly ordered amyloid fibrils. The extracellular deposition of amyloid fibrils in tissues is the basis of a wide range of human diseases, including neurodegenerative disorders of the central nervous system and various forms of systemic and cutaneous amyloidosis. Under certain conditions, soluble proteins and protein fragments (peptides) self-assemble into β-sheet-rich structures (cross-β conformation) and form amyloids. These amyloids can cause serious disorders known as amyloidosis such as Alzheimer ’s disease, hemodialysis-associated disease, and bovine spongiform encephalopathies, which are characterized by the transformation of soluble proteins/peptides into aggregated fibrils in different organs and tissues. Recent reliable discovery of amyloidforming sequences in amyloidogenic proteins should have a great impact on the development of antiamyloid agents. Food allergy is classically defined as an adverse reaction to food components resulting from an overt response mediated by the immune system. Its prevalence has been estimated at 2–4% of adults and 6–8% of children in Western countries. These figures would represent approximately twice those reported only a decade ago. Chapter 8 summarizes recent advances on peptide-based immunotherapy (PIT) for food allergy. Peptide-based immunotherapy has been praised as a promising strategy for food allergy by a number of reviews. While PIT investigations carried out with inhalant allergens have reached clinical settings with encouraging results, there is currently little information on the curative potential of PIT in food-induced allergies. However, recent PIT investigations using murine models of food allergy point the way toward promising avenues. Of the various strategies proposed, an immunotherapy based on the use of T-cell epitopecontaining peptides has been, to date, the object of

7

most investigations. Elucidation of the mechanisms underlying PIT has guided research into further understanding of allergic responses, which may lead the way toward the design of more efficient immunotherapeutic approaches. Gamma-aminobutyric acid (GABA), a four-carbon nonprotein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic cells. Chapter 9 highlights the recent updated applications of GABA for food and human health. GABA was discovered first in potatoes more than half a century ago and subsequently in rat brains. GABA has many biological functions such as neurotransmission and induction of hypotensive, diuretic, and tranquilizer effects. GABA production by various microorganisms has also been reported using bacteria, fungi, and yeast. Food intake in man is determined by both physiological and psychological factors. All factors are centrally received, organized, and integrated balancing both short-term and long-term energy intake with energy expenditure. The regulation of energy intake is determined by both the energy content and the energy source (fat, carbohydrate, and protein) of the meal, each of them having unique effects on satiation and satiety regulatory mechanism. Chapter 10 describes food-derived peptides as regulators of satiety. This chapter centers on protein-induced satiety, in particular focussing on mechanisms and regulation of protein-induced satiety. The evidence derived from studies with different experimental models with animals and humans is given, emphasis on the underlying biochemical and neural mechanisms. Chapter 11 deals with health-promoting proteins and peptides in colostrum and whey. The high nutritional value of bovine milk proteins is widely recognized. Also, the multiple functional properties of major milk proteins are well characterized and exploited by various industries. Over the last 2 decades, milk proteins have attracted growing scientific and commercial interest as a source of biologically active molecules having distinct characteristics. The best characterized bioactive bovine whey proteins include immunoglobulins (Igs), lactoferrin (Lf), lactoperoxidase (LP), and growth

8

Part 1 Introduction

factors. They occur in colostrum in much higher concentrations than in milk, reflecting their importance to the health of the neonate. These native proteins are known to exert a wide range of physiological activities, for example, enhancement of nutrient absorption, stimulation of cell growth, enzymatic activity, inhibition of enzyme activity, modulation of the immune system, and defense against microbial infections. At present, milk proteins are considered the most important source of bioactive peptides. Over the last decade a great number of peptide sequences with different bioactivities have been identified in various milk proteins. The best-characterized milk peptides exhibit antihypertensive, antithrombotic, antimicrobial, antioxidative, immunomodulatory, and opioid activities. Chapter 12 reviews antihypertensive peptides originating from fermented milk products and enzymatic hydrolysis of food proteins, such as milk casein whey proteins and fish meat. Most antihypertensive peptides with proven effects on spontaneously hypertensive rats have angiotensin I–converting enzyme inhibitory activities. Clinical experiences for these antihypertensive peptides were also reviewed to discuss the potential of antihypertensive peptides for high blood pressure. Studies reporting an in vivo mechanism, based mainly on animal studies, were also described. The benefits of antihypertensive peptides with ACE inhibitory activities generated from food proteins were introduced. Some of the peptides with antihypertensive effects in spontaneously hypertensive rats (SHR) demonstrated significant efficacies in humans. In Japan, some ACE inhibitory peptides with proven significant antihypertensive effects in humans have been approved as FOSHU products (functional food products) with health claims concerning high blood pressure. Taking into account the mild effects of ACE inhibitory peptides, which have no adverse effects, they may be considered to be ideal food-derived natural functional ingredients to keep blood pressure within the normal range. Chapter 13 summarizes our knowledge of the structure and biological functions of lactoferrin and of peptides derived from it. Lactoferrin is an iron-binding glycoprotein of the transferrin family

that is expressed in most biological fluids and is a major component of the innate immune system of mammals. Its protective effect ranges from direct antimicrobial activities against a large panel of microorganisms including bacteria, viruses, fungi, and parasites to anti-inflammatory and anticancer activities. Chapter 14 focuses on bioactive peptides and proteins from fish muscle and collagen. As marine fish supplies are threatened due to excessive fishing and poor management, the need to obtain fish protein from sustainable sources like aquaculture and improved processing has increased. Post-harvest losses, as discards at sea, deterioration during storage, and wastage during processing, are substantial. Therefore utilizing and upgrading waste from fish processing into valuable food products is a major goal. Like mammalian muscle proteins, fish myofibrillar proteins display excellent gelation and emulsifying properties. In the last decade, fish collagen from skin and bones has been used to produce gelatin. This chapter reviews hydrolysates and peptides from fish muscle and collagen and their ACE inhibitory and antioxidant activity that may lead to their applications as bioactive ingredients in functional foods, conventional foods, and neutraceuticals. Muscle-based bioactive peptides have been shown to possess many health benefits and are therefore also promising candidates as nutraceutical or functional food ingredients. Since meat and meat products are important in the diet in most developed countries, functional meat products would contribute to human health. The biological activities of muscle-based peptides are summarized in Chapter 15. Further studies will be required to demonstrate the clear benefits of muscle-derived bioactive peptides and their potential nutraceutical applications to benefit human health. Chapter 16 describes recent technology and value-added utilization of rice bran proteins (RBPs) and their peptides for human health promotion. Cereals are the most widely grown crops of the world and the kernel or caryopsis consists of bran, embryo, and endosperm. The bran is composed of fibers (pentosans, hemicelluloses, β-glucans,

Chapter 1 Biologically Active Food Proteins and Peptides in Health

cellulose, lignin, and glucofructans), ash, enzymes, vitamins, and storage proteins. The various fractions of rice bran such as oil, carbohydrates, and dietary fibers (β-glucans, phytates, tannins), and RBPs have demonstrated their ability in prevention of diseases. Some of them are found to have potent anticancer and antioxidant activity. Some of the second-generation by-products of rice bran, that is, RBP concentrates and isolates, show tremendous potentials for future industrial utilization in view of their multifactorial functionality, including their medicinal value. The residual proteins being of high molecular weight and with considerable water-binding capacity may be used as hydrocolloids, which have demonstrated health benefits in improving lipid profile, controlling hyperglycemia, and lowering glycemic index of foods. Chapter 17 summarizes recent advances of bioactive egg proteins and peptides. Nature forms an avian egg not for human food but to produce a chick. Birds invest in the reproductive process up front, transferring all the nutrients and energy needed to allow the embryo to reach the point of hatching. This concentration of highly available nutrients also makes the egg an ideal nutritional food source; eggs are one of the few foods that are consumed throughout the world. Recent studies have uncovered various roles of egg proteins that go well beyond their basic nutrient value. Indeed, egg proteins may gain increasing recognition in the future in human health, as well as in disease prevention and treatment. However, this value can only be captured through the development of industrially viable technologies that are essential for successfully harvesting valuable egg components for commercial use. Lysozyme and antibody technologies are two such examples of extracted components. Eggs contain more than 60 various types of proteins. The presence of biologically active proteins, especially minor egg proteins, has raised interest in developing novel agents from eggs against chronic diseases, such as cancer. The potential for developing novel bioactive peptides deserves further study. Chapter 18 focuses on soy peptides as functional food materials. Soy protein is one of the vegetable proteins examined

9

extensively for lipid-lowering effect in humans and in experimental animals. Although soy protein contains a certain amount of bioactive peptides that have distinct physiological activities in lipid metabolism, it is not clear that peptides are responsible for these effects. This chapter describes the nutritional benefits and the biochemical function of soy peptides. Soy peptides prepared from soy protein could well serve as an excellent nutritional supplement with improved absorption properties. The ingestion of soy peptides decreases the muscle damage induced after exercise and shows double effectiveness as compared with soy protein. On the other hand, it was revealed that soy peptides stimulate hypolipidemic events and accelerate antiobesity effects. It is important to conduct more research on active components in the future. Soy peptides may have the ability to prevent some lifestyle diseases. Chapter 19 presents bioactivity of proteins and peptides from peas (Pisum sativum, Vigna unguiculata, and Cicer arietinum L). Legumes are important sources of dietary proteins. There has been increased interest in their beneficial effects in terms of several health claims that are professed. This chapter reviews recent studies on the nutraceutical/pharmaceutical/therapeutic biological activities of proteins and peptides from legumes commonly referred to as peas (chick peas, Cicer arietinum; garden/green peas, Pisum sativum; and cow peas, Vigna unguiculata). Chapter 20 introduces wheat proteins and peptides. World wheat production accounts for about 30% of world cereal production, being about 587 million tons in 2006, according to the annual report of the United States Department of Agriculture (USDA) Foreign Agricultural Service. Wheat production and consumption are the highest in the European Union (EU) countries, followed by China and India. Wheat is a major crop that is consumed all over the world in the form of various products such as pasta, bread, cakes, and cookies. The quality of wheat products is affected by protein content because the viscoelastic property of gluten allows dough to expand during heating and provides firm texture to pasta after boiling. This characteristic of gluten is mainly attributed to the elastic property of

10

Part 1 Introduction

polymeric glutenins and the plasticizing property of gliadins. Glutelins and gliadins each account for about 40–46% of total protein, having unique amino acid sequences that are rich in glutamine and proline residues. Although the amount of albumin is not high, it contains amylase inhibitor that inhibits amylase activity and retards carbohydrate digestion, preventing the increase in postprandial hyperglycemia. However, amylase is resistant to proteolysis by digestive enzymes and may be a cause of allergies such as baker ’s asthma. On the other hand, gliadins sometimes become a cause of wheat-dependent exercise-induced anaphylaxis (WDEIA) and coeliac disease. As epitopes in gliadins for WDEIA and coeliac disease are comprised of glutamine-rich tandem repeat motifs, the cleavage or modification of the motifs is effective to reduce their allergenicity. Some peptides, such as Ile-Ala-Pro from wheat abgliadin and Ile-Val-Tyr from wheat germ, prevent hypertension by inhibiting ACE. Wheat proteins have various functions. Science needs to be developed to maximize their favorable functions, such as prevention of diabetes and hypertension, and to minimize their adverse functions, such as allergenicity. Peptidomics can be defined as the comprehensive multiplex analysis of endogenous peptides contained within a biological sample under defined conditions to describe the multitude of native peptides in a biological compartment. Peptidomics or “peptide proteomics” refers to the analysis of all the peptides of a cell or tissue (peptidome), while proteomics refers to the analysis of the proteins (proteome). Chapter 21 presents an overview of the advances in peptidomics and applications of peptidomics for discovering novel bioactive proteins and peptides in food and nutrition research. Chapter 22 highlights in silico analysis of bioactive peptides. Research conducted by numerous scientific centers around the world has contributed to the identification and description of a vast number of biologically active peptides isolated from the body tissues and fluids of many organisms, including bacteria, fungi, plants, and animals. Biologically active peptides are the recommended ingredients of functional food, that is, food designed with the aim of generating the desired functional and biological

properties. The emergence of new peptides from various sources necessitated the creation of a database and the establishment of additional criteria for assessing the value of proteins as precursors of bioactive peptides. The BIOPEP database of proteins and bioactive peptides was developed in 2003. Bioinformatics methods applied in biotechnology and biochemistry are becoming increasingly popular due to short waiting times for results, low testing costs, the option of recording results in the form of text files, and, consequently, the recoverability of results, and they continue to be improved as new advances are made in the field of information technology. With the increasing demand for incorporating functional proteins and peptides into food ingredients, natural health products, and dietary supplements to enhance health, it is crucial to examine the flavor-active properties of these components that may influence their acceptance by consumers. Three of the five basic taste modalities that are currently recognized, namely sweet, salty, and umami, have historically been associated with foods that contain nutrients important for human health and well-being—energy containing carbohydrates (sweet), essential minerals (salty), and amino acids (umami). Chapter 23 explores the flavor-active properties of amino acids, peptides, and proteins that must be considered for successful incorporation and acceptance of components with biologically active properties as ingredients in natural health products, nutraceuticals, and functional foods. The protein and peptide therapeutics have become an important class of drugs due to advancement in molecular biology and technology. However, an effective and convenient delivery of these drugs in the body remains a challenge. Over the last 10 years, extensive studies have been carried out to improve their delivery. Yet there are still a number of limitations due to their intrinsic physicochemical and biological properties, including poor permeation through biological membranes (due to large molecular size), short half-life, physical and chemical instability, enzymatic catalysis, aggregation, adsorption, bio-incompatibility, and immunogenicity. Chapter 24 presents the difficulties in delivery of proteins

Chapter 1 Biologically Active Food Proteins and Peptides in Health

and peptides and discusses the liquid crystal–based controlled delivery systems that were produced and examined to improve delivery. In summary, the collection of chapters in this book, written by an international team from industry and academia, provides a comprehensive overview of the fundamental concepts, mechanisms, and ongoing research needs, as well as current and prospective applications, of the major categories of biologically active proteins and peptides with potential for significant benefits to human health. It is hoped that the knowledge and insights gained from these chapters will pave the way to realizing the tremendous opportunities within this rapidly growing field of bioactive proteins and peptides from food sources as nutraceuticals and functional foods.

References Boelsma E, Kloek J. 2009. Lactotripeptides and antihypertensive effects: A critical review. Br J Nutr 101(6):776–786. Gilani GS, Xiao C, Lee N. 2008. Need for accurate and standardized determination of amino acids and bioactive peptides for evaluating protein quality and potential health effects of foods and dietary supplements. J AOAC Intl 91(4):894–900. Gobbetti M, Minervini F, Rizzello, CG. 2007. Bioactive peptides in dairy products. In: YH Hui, ed., Handbook of Food Products Manufacturing: Health, Meat, Milk, Poultry, Seafood, and Vegetables, 489–517. Hoboken, NJ: John Wiley & Sons.

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Hartmann R, Meisel H. 2007. Food-derived peptides with biological activity: From research to food applications. Curr Opin Biotechnol 18(2):163–169. Korhonen H, Pihlanto A. 2007a. Technological options for the production of health-promoting proteins and peptides derived from milk and colostrum. Curr Pharm Des 13(8):829–843. Korhonen H, Pihlanto A. 2007b. Bioactive peptides from food proteins. In: YH Hui, ed., Handbook of Food Products Manufacturing: Health, Meat, Milk, Poultry, Seafood, and Vegetables, 5–38. Hoboken, NJ: John Wiley & Sons. Meisel H. 2005. Biochemical properties of peptides encrypted in bovine milk proteins. Curr Medical Chemistry 12:1905– 1919. Mine Y. 2007. Egg proteins and peptides in human healthchemistry, bioactivity and production. Curr Pharm Des 13:875–884. Mine Y, Miyashita K, Shahidi F. 2009. Nutrigenomics and Proteomics in Health and Diseases: Food Factors and Gene Interactions. Ames, IA: Wiley-Blackwell. Mine Y, Shahidi F. 2006. Nutraceutical Proteins and Peptides in Health and Disease. New York: CRC-Taylor & Francis. Möller NP, Scholz-Ahrens KE, Roos N, Schrezenmeir J. 2008. Bioactive peptides and proteins from foods: Indication for health effects. Eur J Nutr 47(4):171–182. Murray BA, FitzGerald RJ. 2007. Angiotensin converting enzyme inhibitory peptides derived from food proteins: Biochemistry, bioactivity and production. Curr Pharm Des 13(8):773–791. Tripathi V, Vashishtha B. 2006. Bioactive compounds of colostrum and its application. Food Reviews Intl 22:225–244. Yalcin AS. 2006. Emerging therapeutic potential of whey proteins and peptides. Curr Pharm Des 12:1637–1643.

Part 2 Functions of Biologically Active Proteins and Peptides

Chapter 2 Anti-inflammatory/Oxidative Stress Proteins and Peptides Denise Young and Yoshinori Mine

Contents 1. Oxidative Stress, 15 1.1. Endogenous Antioxidative Stress Mechanisms, 16 1.2. Exogenous Protein/Peptide Antioxidants, 16 1.3. Antioxidative Stress Food Factors, 16 2. Antioxidant Proteins and Peptides, 16 3. Antioxidative Stress Proteins and Peptides, 17 3.1. Egg Yolk Peptides, 17 3.1.1. Phosvitin Phosphopeptides, 17 3.2. Milk Protein, 18 3.3. Plant Proteins/Herbal Medicine, 18 3.3.1. Gardenia jasminoides Glycoprotein, 18 4. Oxidative Stress and Inflammation, 18 4.1. Inflammation, 18 4.1.1. Cell Signaling Mechanisms Associated with Inflammation, 19

1. Oxidative Stress Oxidative stress is a biological state that occurs when a cell’s antioxidant capacity is overwhelmed by reactive oxygen species (ROS), causing a redox imbalance. Reactive oxygen species are a type

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

5. 5.1. 5.1.1. 5.1.2. 5.2. 5.2.1. 5.2.2. 5.2.3. 5.2.4. 5.3. 5.3.1. 5.4. 5.4.1. 5.4.2. 6. 7.

Anti-inflammatory Proteins and Peptides, 19 Egg Proteins, 19 Lysozyme, 19 Ovotransferrin, 19 Milk Peptides, 19 Bovine Casein, 19 Glycomacropeptide/κ-caseinoglycopeptides, 21 Lactoferrin, 21 Proteose Peptone-3, 22 Soy, 22 Trypsin Inhibitors, 22 Plant Proteins/Herbal Medicine, 23 Ulmus davidiana Nakai Glycoprotein, 23 Rhus verniciflua Stokes Glycoprotein, 23 Conclusion, 23 References, 23

of free radical that is formed with oxygen. Free radicals are chemical substances that contain one or more unpaired orbital electrons and are therefore unstable and liable to react with other molecules to form more stable compounds with a lower energy state. In an attempt to achieve this stable state, ROS react with proteins, lipids, and DNA, which can result in damage and inactivation of cellular components such as enzymes, membranes, and DNA (Halliwell and Cross 1994; Valko et al. 2004). As such, ROS and oxidative stress as a whole have been 15

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Part 2 Functions of Biologically Active Proteins and Peptides

suggested to participate in the initiation and/or propagation of chronic diseases such as cardiovascular and inflammatory diseases, cancer, and diabetes (Valko et al. 2004). ROS can be produced on a regular basis during oxidative metabolism and in more potent levels during inflammation. During oxidative metabolism, electrons are lost from the electron transport chain and combine with oxygen, resulting in the formation of superoxide anion (Oi− 2 ) (Boveris and Chance 1973). At the time of inflammation, innate immune cells produce and release ROS to destroy invading microorganisms (Rosen et al. 1995). Environmental factors such as tobacco smoke, ultraviolet radiation, overexertion during exercise, and the consumption of alcohol and certain foods can also result in the generation of ROS (Bunker 1992; Powers and Jackson 2008). The consumption of antioxidants and foods that influence endogenous antioxidative stress mechanisms can play a role in limiting the proliferation of ROS, thereby re-establishing a stable redox balance.

1.1. Endogenous Antioxidative Stress Mechanisms Aerobic organisms can increase production of biochemical antioxidants such as glutathione (GSH) and induce endogenous antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) to inactivate oxidants, forming instead biologically inert products (Cimino et al. 1997; Halliwell 1990). GSH (γ-glutamylcysteinylglycine) is the major nonenzymatic regulator of redox homeostatis and is ubiquitously present in all cell types (Meister and Anderson 1983). It can directly scavenge free radicals or act as a substrate for GPx and glutathione S-transferase (GST) during detoxification of hydrogen peroxide, lipid hydroperoxides and electrophilic compounds. Glutathione is synthesized in two sequential adenosine triphosphate– dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS) (Griffith 1999).

1.2. Exogenous Protein/ Peptide Antioxidants Antioxidant activities of peptides are defined by characteristics such as linoleic acid peroxidation, reducing activity, free radical scavenging, activeoxygen quenching, metal-ion chelation, and peroxynitrite scavenging. In essence it encompasses all the physical, nongenetic interactions that can occur between the antioxidative compound and the oxidants. The purpose of these exogenous antioxidants is to neutralize the antioxidant or prevent/minimize further oxidative reactions.

1.3. Antioxidative Stress Food Factors Recently, food-derived compounds like curcumin and flavonoids and olive oil biophenols have been shown to upregulate intracellular GSH synthesis (Biswas et al. 2005; Myhrstad et al. 2002) and increase GPx and GR antioxidant enzyme activities (Yang et al. 2005). Similarly, antioxidative stress peptides include peptides associated with the induction of endogenous antioxidants such as glutathione and the phase 2 enzymes. These antioxidant defense systems present in mammalian cells evolved to protect cells against oxidative and electrophilic damage. SOD catalyzes the dismutation of Oi− 2 to form hydrogen peroxide (H2O2). Since H2O2 is cellpermeable and toxic, it is quickly converted to water (H2O) by catalase or GPx. GPx oxidizes the enzyme cofactor GSH to oxidized glutathione (GSSG) during the decomposition of H2O2. GSSG can be reduced to GSH by GR. GSH is also required as a cofactor for GST in the detoxification of organic hydroperoxides and electrophiles. The coordinate actions of these molecules are highly effective in oxidant and electrophilic cellular detoxification, therefore the induction/upregulation of these oxidants and enzymes by bioactive peptides is an area of growing interest.

2. Antioxidant Proteins and Peptides Although antioxidant peptides are not the focus of this chapter, a few examples are given in broad

Chapter 2

Anti-inflammatory/Oxidative Stress Proteins and Peptides

detail to highlight the differences between antioxidant and antioxidative stress peptides. Soy peptides have been found to have increased antioxidant activities compared to intact proteins (Chen et al. 1998). The enzymatic digests of the soy storage proteins β-conglycinin and glycinin, had three to five times higher radical-scavenging activities compared to the unhydrolyzed proteins. During hydrolysis the unfolding of the protein structure exposes amino acids previously “hidden” within the parent protein. Amino acids such as His, Tyr, Trp, Met, and Lys have known potent antioxidant activity, and the exposure of these amino acid R groups in peptides increases the oxidant quenching potential (Saito et al. 2003). Alteration of the enzyme, temperature, and sample preparation conditions during peptide manufacture can affect the types of peptides and their subsequent antioxidant potential. Soy protein hydrolysates prepared from native and heated soy protein isolates using different enzymes had varying degrees of hydrolysis (1.7–20.6%) and antioxidant activity (28–65%) (Peña-Ramos and Xiong 2002). It is important to note soy proteins and soy protein isolates contain nonprotein components such as isoflavones, saponins, phytic acid, and dietary fibers, which possess bioactive antioxidant properties. Therefore unless these nonprotein components are extracted and removed prior to protein use and processing, the antioxidant properties of soy protein/peptide preparations may be due to the biological activities of these compounds. In addition to

17

soy peptides, enzymatic hydrolysates from wheat germ protein (Zhu et al. 2006) and alpha- and betalactoglobulin (Hernández-Ledesma et al. 2005) have been identified as possessing antioxidant and free radical–scavenging activities. Antioxidant proteins and peptides have also been identified in egg (Sakanaka and Tachibana 2006), potato (Wang and Xiong 2005), and gelatin (Park et al. 2005). A detailed account of other antioxidants can be found in Elias’s review (Elias et al. 2008) of antioxidative proteins and peptides.

3. Antioxidative Stress Proteins and Peptides Proteins and peptides with antioxidative stress properties can be derived from a variety of food sources including egg, milk, and plant. Table 2.1 summarizes these bioactive components and their mechanism of action.

3.1. Egg Yolk Peptides 3.1.1. Phosvitin Phosphopeptides Hen egg yolk phosvitin is a highly phosphorylated protein with 10% phosphorus (Taborsky 1983) monoesterified to 57.5% serine (Ser) residues (Allerton and Perlmann 1965). Oligophosphopeptides prepared from egg yolk phosvitin with 35% phosphate retention

Table 2.1. Antioxidative stress food proteins and peptides. Sources

Proteins/Peptides

Test Conditions

Oxidative Stress Properties

References

Egg yolk

Phosvitin phosphopeptides

H202 stimulation of Caco-2 cells

Katayama et al. 2006 Katayama et al. 2007

Milk

Whey protein and casein Gardenia jasminoides glycoprotein

DMH-induced intestinal tumors in rats Glucose/glucose oxidase-induced and hypoxanthine/xanthine oxidase-induced cytotoxicity and apoptosis in NIH/3T3 cells DSS-induced mouse colitis

↓ IL-8, ↓ MDA ↑ GSH, ↑ GR ↑γ-GCS (activity and mRNA), ↑ GST, ↑ CAT ↑ GSH ↓ redox sensitive signal mediators, ↓ protein kinase C, ↓ NF-κB

Lee et al. 2006

↑ SOD, ↑ CAT, ↑ GPx

Oh and Lim 2006

Plant

McIntosh et al. 1995

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Part 2 Functions of Biologically Active Proteins and Peptides

have enhanced calcium- and iron-binding abilities (Taborsky 1983; Jiang and Mine 2000). Phosvitin phosphopeptides (PPPs) prepared from hen egg yolk phosvitin by partial alkaline dephosphorylation and tryptic hydrolysis were shown to have antioxidative stress activity (Katayama et al. 2006, 2007). Caco-2 cells were pretreated with PPPs for 2 hours followed by a 6-hour H2O2 stimulation. A highly phosphorylated HPLC phosvitin fraction, PPP-3, inhibited H2O2-induced interleukin-8 (IL-8) secretion, suppressed the formation of the lipid peroxidation biomarker malondialdehyde, increased intracellular glutathione levels, and elevated glutathione reductase, gamma-glutamylcysteine synthetase (γ-GCS), GST, and catalase activities. γ-GCS-HC (heavy chain) mRNA was also significantly elevated by PPP-3, indicating this transcriptional upregulation is directly related to the increase in GSH levels during oxidative stress conditions. It was noted that PPPs with high phosphorus content exhibited higher protective activities than those lacking phosphorus, but there was a lack of antioxidative stress activity associated with phosphoserine. This suggests that the presence of phosphorus alone is not responsible for the antioxidative stress activities, but molecular size and amino acid composition may also be important.

3.2. Milk Protein Whey protein– and casein-fed rats had significantly higher liver concentrations of glutathione during dimethylhydrazide (DMH)-induced intestinal tumor development compared with soybean- or red meat– fed rats (McIntosh et al. 1995). Whey proteins such as β-lactoglobulin, serum albumin, and lactoferrin contain high levels of cysteine and glutamylcysteine, which are precursors for glutathione synthesis. This increased production of glutathione may be important in providing protection to the rats, as noted by the decreased tumor incidence and burden in whey- and casein-fed animals.

3.3. Plant Proteins/Herbal Medicine 3.3.1. Gardenia jasminoides Glycoprotein Gardenia jasminoides Ellis (GJE) has traditionally been used as a food additive and herbal medicine (Chang

and But 1987). Glycoprotein isolated from GJE fruits is an effective oxygen radical scavenger and is able to inhibit the redox-sensitive signal mediators, protein kinase C (PKC) and nuclear factor-κB, in NIH/3T3 cells (Lee et al. 2006). In a mouse colitis model where GJE was orally administered for 10 days followed by 7 days of dextran sodium sulfate (DSS) supplementation, GJE augmented SOD, CAT, and GPx activities (Oh and Lim 2006). Without GJE treatment, DSS-induced colitis resulted in a large infiltration of leukocytes in the inflamed colon mucosa and subsequent lowering of SOD, CAT, and GPx antioxidant activities. Even in the absence of DSS supplementation, GJE significantly increased SOD and GPx activities compared to negative (DSS-absent) controls.

4. Oxidative Stress and Inflammation Reactive oxygen molecules are often present during the inflammation process. During the course of inflammation, macrophages and neutrophils that contain the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex generate superoxide radicals and hydrogen peroxide to aid in the destruction of foreign agents (Rosen et al. 1995). While ROS play a role in host immune defense, they can also damage biological macromolecules like lipids, proteins, and DNA. However, in healthy individuals this risk is small in comparison to the huge benefit the body receives in containing and clearing an infection in a short period of time. Inflammation becomes problematic when it is uncontrollable, recurring, and chronic as in the case of inflammatory bowel disease.

4.1. Inflammation When host pattern-recognition receptors (PRRs) such as toll like receptors (TLRs) bind bacterial lipopolysaccharide (LPS), the transcription factor nuclear factor-kappa B (NF-κB) is activated. NF-κB activation leads to the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-8, cyclo-oxygenase-2 (COX-2), and interleukin-1β

Chapter 2

Anti-inflammatory/Oxidative Stress Proteins and Peptides

(IL-1β), as well as adhesion molecules (DeForge et al. 1993; Rogler and Andus 1998; Yamamoto et al. 2003). Pro-inflammatory cytokines like TNFα, IL-1, and IL-8 mediate the immune response by recruiting immune cells like neutrophils to the site of injury and stimulate cells to produce more cytokines and other inflammatory molecules, thereby increasing the response exponentially. In the nearby tissue and blood vessel vicinity, vasodilation and an increase in cell adhesion molecules assist in the extravasation of monocytes, neutrophils, and polymorphonuclear cells to the site of injury. Monocytes not only differentiate into macrophages upon entry into tissue and engulf bacteria but also, along with neutrophils, generate ROS, which can result in destruction of both bacteria and host tissue. 4.1.1. Cell Signaling Mechanisms Associated with Inflammation ROS, bacterial cell wall LPS, and the pro-inflammatory cytokine TNF-α can trigger the activation of multiple signaling pathways, including the phosphorylation cascades leading to the activation of mitogen activated protein kinase (MAPK) and NF-κB (Aggarwal 2003; Tak and Firestein 2001). LPS binding to TLR-4 and TNF-α to the TNF receptor activate the Ikappa B kinase (IKK)–NF-κB pathway and the three MAPK pathways: ERK 1/2, JNK, and p38. These pathways in turn activate a variety of transcription factors that include NF-κB and activator protein-1 (AP-1) (Dalton et al. 1999; Morel and Barouki 1999). AP-1 binding sites are present in the promotor regions of a large number of pro-inflammatory cytokine and adhesion molecule genes and play a role in regulating the expression of γ-GCS, the key enzyme in the synthesis of GSH (Rahman et al. 1998). Hydrogen peroxide is able to permeate the cell membrane and act as a cell-signaling molecule by oxidizing the thiol moiety of sulfhydryl-containing proteins involved in signaling transduction pathways (Dalton et al. 1999; Morel and Barouki 1999; Arrigo 1999). Both NF-κB and AP-1 are redox-sensitive and are activated during oxidative stress and inflammation. Inhibiting their expression and resulting activity are key to limiting the expression of oxidative stress and inflammatory mediators.

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5. Anti-inflammatory Proteins and Peptides Proteins and peptides from egg, milk, soy, and plant sources have been shown to have anti-inflammatory properties. In vitro and in vivo studies of these bioactive proteins and peptides are summarized in Table 2.2.

5.1. Egg Proteins 5.1.1. Lysozyme A recent study by Lee et al. (2009) found hen egg lysozyme (HEL) supplementation attenuated physiological markers of inflammation; reduced the colon expression of proinflammatory cytokines TNF-α, IL-6, IFN-γ, IL-8, and IL-17; and increased the expression of antiinflammatory IL-4 and TGF-β in a porcine model of chemical colitis. The increase in TGF-β and Foxp3 mRNA expression points to the possibility that HEL may help in restoring gut homeostatis through the activation of T regulatory cells. Milk lysozyme was also postulated to possess a new anti-inflammatory activity through its inhibition of the hemolytic activity of serum complement when tested within the levels present in normal and inflamed breast milk samples (Ogundele 1998). 5.1.2. Ovotransferrin Ovotransferrin is an egg white protein well known for its antibacterial activity (Valenti et al. 1983). In chickens, it is often present in large amounts as an acute phase protein during inflammation and infections. It has immunomodulating effects on chicken macrophages and heterophil-granulocytes (Xie et al. 2002) and has the ability to inhibit proliferation of mouse spleen lymphocytes (Otani and Odashima 1987).

5.2. Milk Peptides 5.2.1. Bovine Casein κ-casein is a phosphorylated glycoprotein comprised of a C-terminal fragment, glycomacropeptide (GMP). Intact κ-casein and its pancreatin and trypsin digests significantly inhibited the proliferation of mouse spleen lymphocytes and rabbit Peyer ’s patch cells in the presence of Salmonella typhimurium LPS, concanavalin A

Table 2.2. Anti-inflammatory food proteins and peptides. Sources

Proteins/Peptides

Test Conditions

Egg

Lysozyme

DSS-induced colitis in pigs

Ovotransferrin

LPS- and PHA-stimulated mouse spleen lymphocytes

Bovine κ-casein and κ-casein digests

LPS-, Con A-, PHA-, and PW-stimulated mouse spleen and rabbit Peyer ’s patch cells; PHA-induced PBMC Phorbol 12,13-dibutyrate (plus calcium ionophore)-stimulated T-cells LPS-, Con A-, and PHA-stimulated murine splenocytes; LPS-stimulated DC LPS- and PHA-induced spleen and Peyer ’s patch cells LPS-stimulated monocytes/ macrophages, spleen cells LPS-induced DC PHA-induced mouse splenocytes TNBS-induced colitis in rats

Milk

Glycomacropeptide/ κ-caseinoglycopeptides

Lactoferrin

Endotoxemia and bacteremia in mice TNBS-, DSS- and carrageenan-induced inflammation in rats Influenza virus infection in mice Zymosan-induced skin inflammation LPS-stimulated adjuvant arthritis rat model LPS-stimulated monocytes

Soy

Proteose peptone-3 Lysozyme

LPS-stimulated PBMC with liposomal bLf LPS-induced murine spleen cells Normal and inflamed breast milk samples

Kunitz trypsin inhibitor

LPS-stimulated gingival fibroblasts; LPS-induced lethality in mice

Bowman-Birk trypsin inhibitor

LPS-induced macrophages DSS-induced colitis in mice Active UC patients

Plant

Ulmus davidiana Nakai glycoprotein Rhus verniciflua Stokes glycoprotein

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LPS-treated RAW 264.7, HT-29, and HCT-116 cells, and DSS-treated A/J mouse; DMH- and DMH/DSS-treated mice LPS-stimulated macrophages

Anti-inflammatory Properties

References

↓ TNF-α, ↓ IL-6, ↓ IFN-γ, ↓ IL-8, ↓ IL-17, ↑ IL-4, ↑ TGF-β, ↑ Foxp3 Inhibits lymphocyte proliferation

Lee et al. 2009

Inhibits lymphocyte proliferation

Otani and Hata 1995; Sutas et al. 1996

↓ T-cell proliferation

Pessi et al. 2001

↓ TNF-α, ↓ IL-10, ↓ IL-12, ↓ IL-6, ↓ IL-1β Inhibits lymphocyte proliferation Secretion of IL-1ra

Mikkelsen et al. 2005

↓ IL-1β ↓ IL-2 receptor expression ↓ IL-1, ↓ iNOS mRNA, ↓ IL-17 ↓ TNF-α, ↓ IL-6 ↓ TNF-α, ↓ IL-1β, ↓ IL-6, ↑ IL-4, ↑ IL-10 ↓ infiltrating leukocytes ↓ TNF-α-producing spleen cells ↓ TNF-α, ↑ IL-10 ↓ TNF-α, ↓ IL-1β, ↓ IL-6, ↓ IL-8 protein and mRNA, ↓ NF-κB ↓ TNF-α Inhibits proliferation Inhibits hemolytic activity of serum complement ↓ TNF-α, ↓ IL-6, ↓ IL-1β, ↓ ERK1/2, JNK and p38 MAPK, ↓ NF-κB ↓ NO, ↓ prostaglandin E2 ↓ in histopathological inflammation ↓ disease activity index ↓ NF-κB and AP-1, ↓ activities of iNOS, COX-2, MMP-9, ↓ TNF-α, ↓ IL-6 ↓ PKC, ↓ NF-κB, ↓ AP-1, ↓ iNOS, ↓ COX-2

Otani and Odashima 1987

Otani et al. 1992 Monnai and Otani 1997; Otani and Monnai 1995 Mikkelsen et al. 2005 Otani et al. 1996 Daddaoua et al. 2005; Requena et al. 2008 Zimecki et al. 2006 Togawa et al. 2002a, 2002b; Zimecki et al. 1998 Shin et al. 2005 Hartog et al. 2007 Hayashida et al. 2004 Haversen et al. 2002

Ishikado et al. 2005 Mikkelsen et al. 2005 Ogundele 1998 Kobayashi et al. 2005a, 2005b Dia et al. 2008 Ware et al. 1999 Lichtenstein et al. 2008 Lee and Lim 2007a, 2007b, 2007c, 2007d, 2008 Oh et al. 2007

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Anti-inflammatory/Oxidative Stress Proteins and Peptides

(Con A), phytohemagglutinin (PHA), and pokeweed (PW) mitogen (Otani and Hata 1995). Immunosuppressive results were also found with pancreatin and trypsin digests of alpha s1-casein and beta-casein, but not the native proteins. Similar suppressive effects on mitogen-stimulated lymphocyte proliferation were obtained by Sutas et al. (1996) using pepsin- and trypsin-digested alpha s1-casein and beta-casein. Further Lactobacillus casei GGderived enzymatic hydrolysis of these digests along with similar κ-casein digests resulted in reduced proliferation as well. L. rhamnosus GG-degraded bovine casein was also found to suppress T-cell activation (Pessi et al. 2001). Mikkelsen et al. (2005) confirmed the inhibitory role of κ-casein in LPS-, Con A-, and PHA-stimulated murine splenocytes, and using an LPS-stimulated murine dendritic cell (DC) model, found κ-casein significantly inhibited production of TNF-α, IL-10, IL-12, IL-6, and IL-1β. 5.2.2. Glycomacropeptide/κ-caseinoglycopeptides It is difficult to address the immunosuppressive activity of κ-casein without mention of glycomacropeptide, since κ-casein’s inhibitory activity is attributable to its C-terminal fragment, GMP (Otani et al. 1992). GMP or casein macropeptide or kappa-caseinoglycopeptide (CGP) is a 64 amino acid peptide with varying amounts (0–5 U) of N-acetylneuraminic (sialic) acid. It is naturally produced as a result of chymosin-digested milk κ-casein in the stomach (Brody 2000) or can be prepared from rennin-digested κ-casein and from whey protein concentrate (Otani et al. 1995). Bovine CGP (residues 106–169) inhibited LPSand PHA-induced proliferation in spleen and Peyer ’s patch cells (Otani et al. 1995). CGP fractions with varying N-acetylneuraminic (NANA) residues were found to differentially affect cells according to the mitogenic stimulant. More NANA residues increased the inhibitory effect on PHA-induced proliferation, but only 2 NANA residues were necessary to maximally decrease LPS-induced proliferation. The addition of neuraminidase and chymotrypsin to the fractions resulted in a reduction of inhibitory activity; therefore, both carbohydrate and polypeptide

21

portions are necessary to maintain CGP’s bioactivity. In LPS-stimulated lymphocytes and monocytes, CGP induced an IL-1 receptor antagonist (IL-1ra)like component while inhibiting proliferation (Monnai and Otani 1997; Otani and Monnai 1995). This inhibition was completely overcome by the addition of anti-IL-1ra antibody. Interestingly, in a study by Mikkelsen et al. (2005), various concentrations of κ-casein and GMP significantly reduced IL-1β levels in LPS-induced DC. The inhibition of IL-1β complements the increased secretion of IL-1ra and supports GMP’s immunosuppressive mechanism of action. IL-1ra and IL-1β both competitively bind to the IL-1β receptor, but increased IL-1ra prevents IL-1β signaling, thereby modulating the inflammatory cascade. In PHA-induced lymphocytes, anti-IL-1ra antibody addition also reduced the degree of suppression conferred upon by CGP (Otani et al. 1996). In this study CGP preferentially bound to mouse CD4+ T cells, suppressing the IL-2 receptor expression during PHA stimulation. The anti-inflammatory effect of GMP was tested and confirmed in rat trinitrobenzenesulfonic acid (TNBS) models of colitis (Daddaoua et al. 2005; Requena et al. 2008). GMP pretreatment prior to the induction of inflammation resulted in a decrease in physiological colitis parameters such as body weight loss, anorexia, colon damage, and colon weight to length ratio, as well as a reduction of colonic alkaline phosphatase activity, IL-1β, inducible nitric oxide synthase, and IL-17. TNBS-induced colitis is a CD4+ Th-1 type of inflammation that results in the infiltration of monocytes and lymphocytes. Since CGP exerts inhibitory effects by directly binding to cells, it is probable that CGP interacted with immune cells to bring about a decrease in inflammation. Studies of experimentally induced endotoxemia or bacteremia in BALB/c mice have also shown that GMP given intraperitoneally 24 hours prior to an intravenous injection of E. coli LPS significantly inhibited serum TNF-α and IL-6 (Zimecki et al. 2006). 5.2.3. Lactoferrin Lactoferrin (Lf) is an ironbinding glycoprotein and a member of the transferrin family. It is abundantly expressed and secreted

22

Part 2 Functions of Biologically Active Proteins and Peptides

in milk (Masson and Heremans 1971), exocrine secretions (Lonnerdal and Iyer 1995), and neutrophil secondary granules (Masson et al. 1969); and it plays an essential role in innate immune defense. Among its many functions, Lf has been shown in both in vivo and in vitro studies to inhibit the production of pro-inflammatory cytokines such as TNF-α and IL-1β (Crouch et al. 1992; Machnicki et al. 1993). It was first thought that Lf directly interacted with LPS lipid A to prevent its subsequent binding to the CD14 macrophage receptor (Appelmelk et al. 1994), thereby averting the inflammatory response. Yet it is now believed that Lf might interact with surface receptors on immune cells (Birgens et al. 1983; Legrand et al. 2006; Mazurier et al. 1989; Van Snick and Masson 1976) and influence the receptor-mediated inflammatory signaling pathways that affect cytokine expression and production. Bovine-milk derived lactoferrin (bLf) has been associated with attenuating the production of proinflammatory cytokines including TNF-α, IL-1β, and IL-6, as well as reducing physiological inflammatory parameters like disease activity index, macroscopic and histological scores, and myeloperoxidase activity in rat colonic tissue in both TNBS- and DSS-induced colitis (Togawa et al. 2002a, 2002b). There was also a significant induction of antiinflammatory IL-4 and IL-10 cytokines. Oral treatment of rats with bLf also decreased pro-inflammatory cytokine production in a rat model of carrageenaninduced inflammation (Zimecki et al. 1998). Orally administered bLf suppressed the number of infiltrating macrophages and neutrophils during an influenza virus infection in mice (Shin et al. 2005). A combination of glycine and bLf had a synergistic anti-inflammatory effect on zymosan-induced skin inflammation and an enhanced decrease in the number of TNF-α producing spleen cells (Hartog et al. 2007). Orally administered bLf also inhibited inflammation and nociception in an LPS-stimulated adjuvant arthritis rat model (Hayashida et al. 2004). Using THP-1 and Mono Mac 6 monocytic cell lines stimulated with E .coli LPS, Haversen (Haversen et al. 2002) demonstrated lower levels of TNF-α, IL-1β, IL-6, and IL-8 mRNA expression and their

corresponding proteins when bLf and human recombinant Lf (hLf) were provided before and after LPS addition. TNF-α was the cytokine most affected by Lf, and in fact, Lf was found to decrease the LPSinduced binding of NF-κB to the TNF-α promoter. bLf can be readily transported from the lumen to the plasma in piglets (Harada et al. 1999). After administration of bLf via an intragastric catheter, plasma concentrations of Lf increased significantly. Using immunostaining and immunofluorescence, Haversen (Haversen et al. 2002) found that Lf could be internalized into cells and detected in the nucleoli. This supports other studies in which Lf binds to human blood monocytes and monocytic cell lines via high- and low-affinity receptors and is taken up inside the cell (Birgens et al. 1983, 1984; Britigan et al. 1991; Ismail and Brock 1993). To date why Lf is taken in the cell and what happens to Lf afterward is not entirely clear; however, it may interfere with intracellular signaling pathways. In an effort to further enhance Lf immunosuppressive effects, bLf was encapsulated into an egg yolk phosphatidylcholine and phytosterol liposome for oral administration (Ishikado et al. 2005). Oral pretreatment of the liposomal bLf resulted in much higher anti-inflammatory effects on CC14-induced hepatic injury in rats and LPS-induced TNF-α production compared with nonliposomal bLf. Liposomalization did not influence the absorption of Lf to the venous blood or lymph. 5.2.4. Proteose Peptone-3 Proteose peptone-3 (PP3) is a sialic acid–containing protein found in cow’s milk (Coddeville et al. 1998). LPS-induced proliferation of murine spleen cells was inhibited by PP3 in a dose-dependent manner (Mikkelsen et al. 2005). Though studies with PP3 are limited, future studies may expand on its anti-inflammatory mechanisms.

5.3. Soy 5.3.1. Trypsin Inhibitors Soy-derived Kunitz trypsin inhibitor (KTI) and Bowman-Birk trypsin inhibitor (BBI) have recently been found to be potent anti-inflammatory agents. In gingival fibroblasts,

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Anti-inflammatory/Oxidative Stress Proteins and Peptides

soybean KTI was able to abrogate LPS-induced upregulation of TNF-α mRNA and protein expression in a dose-dependent manner, as well as reduce the induction of IL-6 and IL-1β proteins (Kobayashi et al. 2005b). The interference with TNF-α mRNA correlates with suppressed activation of ERK1/2 and p38 MAPK; and interestingly, KTI pretreatment blocked LPS-induced NF-κB activation. When soybean KTI and BBI were administered intraperitonally and orally to mice subjected to bacterial LPSinduced lethality, KTI significantly reduced lethality and decreased expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 (Kobayashi et al. 2005a). In addition, KTI inhibited LPSactivated ERK1/2, JNK, and p38 MAPK pathways in peritoneal macrophages. The researchers found no anti-inflammatory activity associated with BBI; however, Dia et al. (2008) found low concentrations of BBI inhibited nitric oxide production in LPSinduced RAW 264.7 macrophages by almost 50%, while prostaglandin E2 production was reduced by 45%. A BBI concentrate reduced histopathological signs of colon inflammation in DSS-induced mice when given during and after DSS induction (Ware et al. 1999). Similarly, a soy BBI concentrate administered to ulcerative colitis patients resulted in a lower disease activity index compared to those given a placebo (Lichtenstein et al. 2008). BBI’s potent inhibitory effect may be related to its ability to inhibit the catalytic activities of major proteases involved in inflammation such as cathepsin G (Larionova et al. 1993), elastase (Larionova et al. 1993), and chymase (Ware et al. 1997).

5.4. Plant Proteins/Herbal Medicine 5.4.1. Ulmus davidiana Nakai Glycoprotein Ulmus davidiana Nakai (UDN) is a deciduous tree used in oriental medicine for treating edema, mastitis, and gastric cancer. A glycoprotein isolated from UDN consisting of 78.65% carbohydrate and 21.35% protein (Lee et al. 2004) was recently found to have anti-inflammatory activity in LPS-treated RAW 264.7, HT-29, and HCT-116 cells, and DSStreated A/J mouse (Lee and Lim 2007a, 2007c, 2007d). Overall results show UDN glycoprotein

23

dose-dependently inhibited DNA binding activity of NF-κB and AP-1, activities of iNOS, COX-2, and matrix metalloproteinases-9 (MMP-9). In DMHtreated and DMH/DSS-treated mice, similar results were obtained with the consumption of UDN in addition to the inhibition of TNF-α and IL-6 (Lee and Lim 2007b, 2008). 5.4.2. Rhus verniciflua Stokes Glycoprotein Rhus verniciflua Stokes (RVS) has traditionally been used as an anti-inflammatory agent in Korea. A glycoprotein isolated from RVS fruits consisting of 38.75% carbohydrates and 61.25% protein was found to inhibit ROS production and protein kinase C translocation and to downregulate expression of NF-κB and AP-1 (Oh et al. 2007). In addition, expression of iNOS and COX-2 was inhibited.

6. Conclusion Oxidative stress and inflammation are often related to chronic diseases involving the cardiovascular, neurological, and gastrointestinal systems. Ingestion of bioactive food proteins and peptides are a relatively safe way in which to prevent or treat these diseases. Proteins and peptides have been found not only to contribute to the body’s energy supply and growth but also to influence specific biological activities such as oxidative stress and inflammation. As our understanding of the efficacy and mechanism of action of antioxidative stress and anti-inflammatory proteins and peptides increases, so will the growing interest in their prophylactic, preventive, or therapeutic uses.

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Otani H, Horimoto Y, Monnai M. 1996. Suppression of interleukin-2 receptor expression on mouse CD4+ T cells by bovine kappa-caseinoglycopeptide. Biosci Biotechnol Biochem 60(6):1017–1019. Otani H, Monnai M. 1995. Induction of an interleukin-1 receptor antagonist-like component produced from mouse spleen cells by bovine kappa-caseinoglycopeptide. Biosci Biotechnol Biochem 59(6):1166–1168. Otani H, Monnai M, Hosono A. 1992. Bovine κ-casein as inhibitor of the proliferation of mouse splenocytes induced by lipopolysaccharide stimulation. Milchwissenschaft 47(8): 512–515. Otani H, Monnai M, Kawasaki Y, Kawakami H, Tanimoto M. 1995. Inhibition of mitogen-induced proliferative responses of lymphocytes by bovine kappa-caseinoglycopeptides having different carbohydrate chains. J Dairy Res 62(2):349– 357. Otani H, Odashima M. 1987. Inhibition of proliferative responses of mouse spleen lymphocytes by lacto- and ovotransferrins. Food Agric Immunol 9:9193–9202. Park E, Murakami H, Mori T, Matsumura Y. 2005. Effects of protein and peptide addition on lipid oxidation in powder model system. J Agric Food Chem 53(1):137–144. Peña-Ramos EA, Xiong YL. 2002. Antioxidant activity of soy protein hydrolysates in a liposomal system. J Food Sci 67(8):2952–2956. Pessi T, Isolauri E, Sutas Y, Kankaanranta H, Moilanen E, Hurme M. 2001. Suppression of T-cell activation by Lactobacillus rhamnosus GG-degraded bovine casein. Intl Immunopharmacol 1(2):211–218. Powers SK, Jackson MJ. 2008. Exercise-induced oxidative stress: Cellular mechanisms and impact on muscle force production. Physiol Rev 88(4):1243–1276. Rahman I, Smith CA, Antonicelli F, MacNee W. 1998. Characterisation of gamma-glutamylcysteine synthetase-heavy subunit promoter: A critical role for AP-1. FEBS Lett 427(1):129–133. Requena P, Daddaoua A, Martinez-Plata E, Gonzalez M, Zarzuelo A, Suarez MD, Sanchez de Medina F, MartinezAugustin O. 2008. Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17. Br J Pharmacol 154(4):825–832. Rogler G, Andus T. 1998. Cytokines in inflammatory bowel disease. World J Surg 22(4):382–389. Rosen GM, Pou S, Ramos CL, Cohen MS, Britigan BE. 1995. Free radicals and phagocytic cells. FASEB J 9(2):200– 209. Saito K, Jin DH, Ogawa T, Muramoto K, Hatakeyama E, Yasuhara T, Nokihara K. 2003. Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry. J Agric Food Chem 51(12):3668–3674. Sakanaka S, Tachibana Y. 2006. Active oxygen scavenging activity of egg-yolk protein hydrolysates and their effects on lipid oxidation in beef and tuna homogenates. Food Chem 95(2):243–249.

Shin K, Wakabayashi H, Yamauchi K, Teraguchi S, Tamura Y, Kurokawa M, Shiraki K. 2005. Effects of orally administered bovine lactoferrin and lactoperoxidase on influenza virus infection in mice. J Med Microbiol 54:717–723. Sutas Y, Soppi E, Korhonen H, Syvaoja EL, Saxelin M, Rokka T, Isolauri E. 1996. Suppression of lymphocyte proliferation in vitro by bovine caseins hydrolyzed with Lactobacillus casei GG-derived enzymes. J Allergy Clin Immunol 98(1):216– 224. Taborsky G. 1983. Phosvitin. Adv Inorg Biochem 5:235–279. Tak PP, Firestein GS. 2001. NF-kappaB: A key role in inflammatory diseases. J Clin Invest 107(1):7–11. Togawa J, Nagase H, Tanaka K, Inamori M, Nakajima A, Ueno N, Saito T, Sekihara H. 2002a. Oral administration of lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance. J Gastroenterol Hepatol 17(12):1291–1298. Togawa J, Nagase H, Tanaka K, Inamori M, Umezawa T, Nakajima A, Naito M, Sato S, Saito T, Sekihara H. 2002b. Lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance. Amer J Physiol Gastrointest Liver Physiol 283(1):G187–195. Valenti P, Antonini G, Hunolstein Cv, Visca P, Orsi N, Antonini E. 1983. Studies on the antimicrobial activity of ovotransferrin. Intl J Tissue React 5(1):97–105. Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. 2004. Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem 266(1–2):37–56. Van Snick JL, Masson PL. 1976. The binding of human lactoferrin to mouse peritoneal cells. J Exp Med 144(6):1568– 1580. Wang LL, Xiong YL. 2005. Inhibition of lipid oxidation in cooked beef patties by hydrolyzed potato protein is related to its reducing and radical scavenging ability. J Agric Food Chem 53(23):9186–9192. Ware JH, Wan XS, Newberne P, Kennedy AR. 1999. BowmanBirk inhibitor concentrate reduces colon inflammation in mice with dextran sulfate sodium-induced ulcerative colitis. Dig Dis Sci 44(5):986–990. Ware JH, Wan XS, Rubin H, Schechter NM, Kennedy AR. 1997. Soybean Bowman-Birk protease inhibitor is a highly effective inhibitor of human mast cell chymase. Arch Biochem Biophys 344(1):133–138. Xie H, Huff GR, Huff WE, Balog JM, Rath NC. 2002. Effects of ovotransferrin on chicken macrophages and heterophilgranulocytes. Dev Comp Immunol 26(9):805–815. Yamamoto K, Kushima R, Kisaki O, Fujiyama Y, Okabe H. 2003. Combined effect of hydrogen peroxide induced oxidative stress and IL-1 alpha on IL-8 production in CaCo-2 cells (a human colon carcinoma cell line) and normal intestinal epithelial cells. Inflammation 27(3):123–128. Yang H, Lee M, Kim Y. 2005. Protective activities of stilbene glycosides from Acer mono leaves against H2O2-induced oxidative damage in primary cultured rat hepatocytes. J Agric Food Chem (10):4182–4186.

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Anti-inflammatory/Oxidative Stress Proteins and Peptides

Zhu K, Zhou H, Qian H. 2006. Antioxidant and free radicalscavenging activities of wheat germ protein hydrolysates (WGPH) prepared with alcalase. Process Biochemistry 41(6):1296–1302. Zimecki M, Artym J, Chodaczek G, Kocieba M, Rybka J, Skorska A, Kruzel M. 2006. Glycomacropeptide protects

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against experimental endotoxemia and bacteremia in mice. EJPAU 9(2). Zimecki M, Miedzybrodzki R, Szymaniec S. 1998. Oral treatment of rats with bovine lactoferrin inhibits carrageenaninduced inflammation; correlation with decreased cytokine production. Arch Immunol Ther Exp (Warsz) 46(6):361–365.

Chapter 3 Antioxidant Peptides Youling L. Xiong

Contents 1. Introduction, 29 2. Endogenous Antioxidant Peptides, 30 3. Antioxidant Activity of In Vitro Protein Digests, 32 4. Antioxidant Activity of Peptides in Food Systems, 34 5. Action Mechanisms of Antioxidant Peptides, 35

1. Introduction Oxidation is one of the main causes for diseases and pathogenesis in humans. For example, free radical attack on proteins, lipids, and nucleic acids results in cell damage and apoptosis and plays an important role in atherosclerosis, Alzheimer ’s disease, inflammatory bowel disease, and certain cancers (Berlett and Stadtman 1997). Essentially, all the cellular components and the specific constituents are susceptible to reactive oxygen species (ROS), for example, hydroxyl radicals (• OH), peroxyl radicals (• OOR), superoxide anion (O2−i), and peroxynitrite (ONOO−). The accumulation of protein carbonyl compounds, a result of oxidation, is believed to be a main mechanism of the aging process in humans (Stadtman 2006).

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

5.1. 5.2. 6. 6.1. 6.2. 6.3. 7. 8.

Chemical Reactions, 35 Physical Interactions, 37 Preparation of Antioxidant Peptides, 38 Enzymatic Production, 38 Microbial Fermentation, 38 Chemical Synthesis, 39 Conclusion, 39 References, 39

The health of cells, tissues, and organs of humans under normal physiological conditions is maintained through a delicate antioxidation-oxidation balance. While oxidation is involved in the normal biological processes, including nutrient metabolism and detoxification, various antioxidants existing in the cells are essential for protecting tissues and organs from oxidative damage. The endogenous antioxidant systems include enzymes, for example, superoxide dismutase, catalase, and glutathione peroxidase, and various nonenzymatic compounds, for example, selenium, α-tocopherol, and vitamin C. In addition, amino acids, peptides, and proteins contribute to the overall antioxidant capacity of cells, thereby protecting the health of biological tissues. Examples of reported endogenous antioxidant amino acids are histidine, cysteine, methionine, and tyrosine (Marcuse 1960; Karel et al. 1966), and those of well-known antioxidant peptides are glutathione, carnosine, and anserine (Babizhayev et al. 1994). Although some proteins also exhibit antioxidant activity, their efficacy is generally low because 29

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many of the reactive amino acid residues and peptide segments are occluded in the native protein structure and inaccessible by reactive oxygen species. In an “ideal” biological system and a clean environment within which to live, the body’s endogenous antioxidants provide an adequate protection of health. However, the antioxidant-prooxidant balance changes with the progression of age and is also sensitive to environmental factors. The plasma and cellular antioxidant potential gradually diminishes with age (Rizvi et al. 2006), leading to an increased vulnerability of proteins to free radical attack. This explains why aging is accompanied by the accumulation of oxidized compounds, for example, protein carbonyls (Chakravarti and Chakravarti 2007). Furthermore, the efficiency of nutrient absorption, including antioxidants, decreases with aging, which would also contribute to decreased cellular antioxidant capacity (Elmadfa and Meyer 2008). On the other hand, environmental stress conditions, for example, pollutants, fatigue, excessive caloric intake, and a high fat diet, could greatly weaken the body’s immune system and make the body vulnerable to oxidative attack. Hence, dietary interventions to reinstate and reinforce the body’s antioxidant status have been widely regarded as a feasible and potentially effective means to promote human health. The market of dietary antioxidant supplements has expanded exponentially over the past 2 decades. It is filled with traditional antioxidants, for example, vitamin E and vitamin C, as well as less traditional, plant-derived antioxidant compounds known as phytochemicals and plant extracts, for example, lycopene, lutein, isoflavones, green tea extracts, and grape seed extracts. A new family of antioxidants that has emerged in recent years is antioxidant peptides and protein hydrolysates that contain mixed peptides. Antioxidant activity of protein hydrolysates was initially observed by Doan and Miller (1940), who reported that trypsin-treated milk had an improved oxidative stability. However, it was not until the beginning of this new millennium that the research on antioxidant peptides took a full spin, and the number of published studies of antioxidant peptides has since increased exponentially.

Unlike nonprotein antioxidants, peptidyl antioxidants tend to be multifunctional compounds that not only are capable of stabilizing free radicals and oxidation initiators but also may impart other bioactivities, including antihypertensive, anticancer, antimicrobial, immunomodulatory, and opioid activities. Thus, naturally occurring antioxidant peptides and those derived from protein hydrolysis are now considered as novel and potential dietary ingredients to promote human health. Indeed, a myriad of peptides and peptide mixtures prepared from enzymatic hydrolysis of plant proteins (soy, corn, potato, wheat, etc.) and animal proteins (whey, casein, meat, gelatin, etc.) exhibit strong antioxidant activity in food systems. Many of the protein in vitro digests also act as radical scavengers and prooxidant metal-ion chelators.

2. Endogenous Antioxidant Peptides The natural defense system of the human body and organs encompasses a number of antioxidant peptides. Examples of some well-studied endogenous antioxidant peptides are shown in Table 3.1, and the structures of several of them are illustrated in Figure 3.1. Glutathione, a tripeptide (γGlu-Cys-Gly), is probably the best-known example (Bray and Taylor 1994). The primary function of glutathione in cells is to serve as a nonenzymatic reducing agent to help maintain cysteine sulfhydryl groups in proteins in a reduced state. Glutathione protects cells from free radicals by acting as an electron donor. In effect, it reduces any disulfide bonds formed within cytoplasmic proteins to cysteine residues. Carnosine, a dipeptide with the sequence of βalanyl-histidine, and its related dipeptides anserine (β-alanyl-1-methylhistidine) and balenine (β-alanyl3-methylhistidine), have strong antioxidant and pH-buffering properties in muscle cells. These histidine-containing antioxidant dipeptides are implicated in a number of physiological functions, for example, neurotransmitter synthesis and inducing muscle enzymes, in addition to scavenging free radicals and sequestering prooxidative metal ions (Boldyrev and Johnson 2002; Kim et al. 2002). Both

Chapter 3 Antioxidant Peptides

Table 3.1. Examples of endogenous peptides that exhibit antioxidant activity in vitro. Peptide

Sequence

Reference

Glutathione Carnosine

γ Glu-Cys-Gly β-Ala-His

Anserine Belenine

β-Ala-1-CH3-His β-Ala-3-CH3-His

Melatonin

N-CH3CO-5-CH3Otryptamine Arg-2′,6′-(CH3)Tyr-Lys-Phe

Bray and Taylor 1994 Nagasawa et al. 2001; Boldyrev and Johnson 2002 Kim et al. 2002 Boldyrev and Johnson 2002 Hardeland 2005

SS31 peptide

Thomas et al. 2007

Figure 3.1. Chemical structures of glutathione, carnosine, and melatonin.

in vitro and in vivo studies of carnosine in oxidatively stressed rat muscle showed a remarkable inhibitory effect of the dipeptide on lipid and protein oxidation (Nagasawa et al. 2001). Recently, the biological role of melatonin as an antioxidant has received much attention. Melatonin,

31

N-acetyl-5-methoxytryptamine, is best known as a hormone involved in the regulation of the circadian rhythms (daily clock or periodicity). It has been demonstrated that melatonin can protect cells against oxidative damage as well as restore the expression of glucose transporter gene in hyperthyroid heart of the rat subjected to oxidative stresses (Ghosh et al. 2006). Melatonin is generally more effective than vitamin E and glutathione in neutralizing •OH, • OOR, O2−i, NO, singlet oxygen, and hydrogen peroxide. Furthermore, melatonin, along with epithalamin and epitalon from the pineal gland, possesses strong ROS-scavenging activity. Epithalamin from bovine brain and its active fragment epitalon (Ala-Glu-Asp-Gly) are able to stimulate melatonin production as well as the expression of antioxidant enzymes superoxide dismutase and ceruloplasmin (Kozina et al. 2007). Since many of the melatonin metabolites and degradation products are also powerful antioxidants, the processing through which melatonin and its derivatives react with ROS and reactive nitrogen species has been referred to as “the free radical scavenging cascade” (Tan et al. 2007). Cyclo(His-Pro), an endogenous cyclic dipeptide present in the central nervous system and in several body fluids and the gastrointestinal tract, has a neuroprotective role. In vitro studies on rat pheochromocytoma PC12 cells have demonstrated that cyclo(His-Pro) enhances the cellular antioxidant capacity as well as the expression of small heat shock proteins (Minelli et al. 2008). There is evidence that cyclo(His-Pro) has a role in ameliorating diabetes. Interestingly, low concentrations (pmol/g levels) of cyclo(His-Pro) are detected in a number of foods, for example, shrimp, ham, and bread (Hilton et al. 1992); in tuna, the level can be as high as 2 μmol/g. Because of its cyclic structure, cyclo(His-Pro) is resistant to digestive enzymes, and therefore could be absorbed and confer its biological and possibly therapeutic functions. Oxidative stress is implicated in cerebral ischemia/reperfusion injury, and the process involves massive production of ROS. A family of small antioxidant peptides has the ability to penetrate the cell

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membrane and minimize ischemia damage; of these, SS31 has received considerable attention. SS31, with the peptide sequence of Arg-2′,6′-dimethylTyr-Lys-Phe-NH2, can lower the concentration of cellular free radicals and has been shown to inhibit low-density lipoprotein (LDL) oxidation and lipid peroxidation (Zhao et al. 2004). Its role of protecting against cerebral ischemia-induced injury is attributed to its ability to downregulate CD36, a scavenger receptor localized in lipid rafts of plasma membrane, resulting in a diminished CD36 prooxidant activity (Cho et al. 2007). In addition, SS31 has been shown to reduce islet cell apoptosis, increase islet cell yield, and improve post-transplantation function (Thomas et al. 2007). Amyloid β peptide (Aβ), an amphipathic peptide composed of 39–42 amino acid residues, is a pathological marker of Alzheimer ’s disease due to its plaque-forming ability (Butterfield et al. 2007). When solubilized, Aβ forms free radicals and induces lipid and protein oxidation. Aβ has a strong affinity for transition metal ions, for example, Cu2+; and the Aβ-Cu2+ complex in excess of Cu2+ acts as a prooxidant. However, Aβ(25–35), an 11-residue fragment of Aβ, which possesses much of the biological activity of Aβ, exhibits antioxidant activity (Walter et al. 1997). Small-angle x-ray diffraction shows that Aβ(25–35) intercalates into the liposome membrane hydrocarbon core, where it inhibits lipid peroxidation. In fact, it has been reported that lipid peroxidation is inhibited when Cu2+ is bound to two molar equivalents of the Aβ(1–42) peptide, suggesting that Aβ itself possesses antioxidant activity under certain specific physiological conditions (Hayashi et al. 2007).

3. Antioxidant Activity of In Vitro Protein Digests Many food proteins possess antioxidant peptide sequences and structural domains; the active fragments are released during the gastrointestinal (GI) digestion process. The digestion by GI enzymes in effect is a natural production process for antioxidant peptides, differing from bioactive peptides produced commercially which are subjected to further degra-

dation and hence, inactivation, after oral intake. Many of the oligopeptides (2-10 amino acids) are believed to be transportable through the absorption channel in the upper GI. The presence of antioxidant peptide segments in proteins may help explain why dietary protein intake can promote animal and human health beyond the normal nutritional benefits exerted. For example, feeding rats the extract of Douchi (a traditional Chinese fermented soybean product that contains numerous antioxidant peptides) was found to enhance the superoxide dismutase activity in liver and kidney, catalase activity in liver, and glutathione peroxidase activity in kidney (Wang et al. 2008). Not surprisingly, the extract also lowered lipid peroxidation in liver. A number of studies have linked ROS, generated in the digestion process, to GI diseases. For example, dietary iron-induced hydroxyl radicals were shown to cause oxidative damage to GI tract mucosa of experimental animals, leading to gastric ulcers and inflammatory intestinal disorders (Carrier et al. 2001; Srigiridhar et al. 2001). Similarly, nitric oxide radicals were found to inflict oxidative GI inflammation (Dedon and Tannenbaum 2004). Therefore, even without being absorbed, resistant antioxidant peptides generated by the gastric and pancreatic proteases are expected to protect the health of the epithelial linings of the GI tract. Several in vitro digestion studies have produced conclusive evidence that peptides generated from certain food proteins by human digestive enzymes under physiological conditions possess strong antioxidant activity. Partially hydrolyzed zein (from corn) by alcalase exhibits a complex antioxidant pattern during the process of further degradation by sequential pepsin and pancreatin treatments in vitro. The digestion process generates mixed peptides with the majority being comprised of 2–4 amino acid residues (Zhu et al. 2008). The reducing power of zein increased two-fold following pancreatin digestion when compared with nondigested protein. The 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid radical (ABTS+ •) scavenging activity was decreased by 27% after pepsin treatment but was fully recovered upon subsequent pancreatin digestion (Figure 3.2). The ability of zein to sequester

Chapter 3 Antioxidant Peptides

33

Figure 3.2. Reducing, metal chelation, and ABTS−• scavenging activity of zein in vitro digests. Adapted from Zhu et al. 2008.

Cu2+ was reduced by pepsin digestion but was re-established following pancreatin treatment. It was hypothesized that limited peptide bond cleavage rendered zein a relatively open structure with a reduced capability of trapping radicals or binding metal ions. However, when zein was further degraded by pancreatin, the resulting active peptide fragments became a dominant force to stabilize these oxidizing agents (Zhu et al. 2008). The antioxidant activity demonstrated by in vitro digests of zein (1–8 mg/mL) was comparable to or exceeded that of 0.1 mg/mL of ascorbic acid or BHA. The results suggested that dietary zein could promote the health of the human digestive tract. Shellfish is another potential source for antioxidant peptide production in the GI system. The GI digests of oyster (Crassostrea gigas) yielded an active 1.6 kDa peptide with the amino acid sequence of Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-GluLys-Gln-Glu (Qian et al. 2008). This peptide was shown to inhibit lipid peroxidation and neutralize hydroxyl and superoxide radicals. The antioxidant

property lent itself to the protection of DNA against radical-induced damage. In another study, a low molecular weight peptide (1.59 kDa) with potent antioxidant activity was isolated from the GI digest of mussel (Mytilus coruscus) muscle protein (Jung et al. 2007). This peptide, with a sequence of LeuVal-Gly-Asp-Glu-Gln-Ala-Val-Pro-Ala-Val-CysVal-Pro, exhibited higher protective activity against lipid peroxidation than ascorbic acid and αtocopherol at comparable dosage levels. The antioxidant activity of the peptide was attributed to its strong • OH scavenging ability. Moreover, the purified peptide could quench superoxide and carboncentered radicals, but those activities were weaker than for ascorbic acid. Antioxidant peptides have also been isolated from hydrolysates with individual GI digestive enzymes. A 16-amino acid peptide (1.8 kDa) showing strong antioxidant activity was isolated from peptic hydrolysate of hoki (Johnius belengerii) frame protein (Kim et al. 2007b). The protein hydrolysate inhibited lipid peroxidation more effectively

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than α-tocopherol and efficiently quenched different free radicals, including •OH and O2−i . It also reduced peroxide-induced cytotoxicity on human embryonic lung fibroblasts as well as protected DNA from radical damage.

4. Antioxidant Activity of Peptides in Food Systems While clinical evidence of the health benefits of antioxidant peptides remains limited but is accumulating, a large number of recent studies have been published on the evaluation of protein-derived antioxidants in food systems. Through using food model systems, these studies have provided insight into the mechanism of food quality protection and often also shed light on the mechanism of human health protection by antioxidant peptides. Various antioxidant peptides have been produced from common food protein sources. Essentially, the hydrolysates of both plant and animal proteins all contain antioxidant peptides. Because not all peptides are antioxidative and some are actually prooxidative, the specific peptide composition of a protein hydrolysate determines its overall antioxidant potential. Therefore, the type of protein substrate, the hydrolytic enzyme used, and the condition under which peptides are released from a given protein (pH, ionic strength, temperature, and preheat treatment) can affect the type and efficiency of antioxidant peptide production (Peña-Ramos and Xiong 2001). In fact, several intact polypeptides naturally present in food materials (e.g., lactoferrin and albumin in milk) have the ability to inhibit lipid peroxidation due to their metal-binding properties. It is now established that many peptides and protein hydrolysates of plant and animal origins possess antioxidant activity, for example, peptides derived from soy protein (Chen et al. 1998), whey protein (Peña-Ramos and Xiong 2001; Elias et al. 2006), casein (Kim et al. 2007a), corn protein (Kong and Xiong 2006; Li et al. 2008), potato protein (Wang and Xiong 2005; Pihlanto et al. 2008), wheat protein (Wang et al. 2007), gelatin (Mendis et al. 2005), egg protein (Davalos et al. 2004; Xu et al. 2007), and muscle protein (Saiga et al. 2003;

Thiansilakul et al. 2007; Raghavan and Kristinsson 2008). Because unsaturated lipids in food systems are the most common targets of free radicals, the assessment of antioxidant activity of peptides and protein hydrolysates is usually carried out in model systems that represent bulk oil or lipid emulsions, or directly in food products (i.e., in situ). The addition of enzyme-hydrolyzed whey and zein hydrolysates significantly inhibited the peroxidation as well as thiobarbituric acid reactive substances (TBARS) formation in a phosphatidyl choline liposome system (Peña-Ramos and Xiong 2001; Kong and Xiong 2006). Peptides having molecular weights less than 5 kDa and rich in histidine and hydrophobic amino acid residues were of particularly high activity. Statistical modeling with multivariate analysis showed that both the peptide size and the amino acid distribution were important in the overall expression of antioxidant activity (Peña-Ramos et al. 2004). β-Lactoglobulin exhibited antioxidant activity in Brij-stabilized oil-in-water emulsions; however, its chymotryptic hydrolysate was more effective in inhibiting lipid peroxidation (Elias et al. 2006). Tyrosine and methionine present in the antioxidant peptide fragments, which had strong radicalscavenging activity, played a major role in the overall antioxidant activity of the protein hydrolysate. In addition to inhibiting lipid peroxidation, peptides prepared from enzymatic protein hydrolysis are capable of preventing oxidative modification of intact proteins. For example, the presence of potato protein hydrolysate reduced amino acid side chain damage and structural changes in myofibrillar proteins exposed to an •OH-generating oxidizing system (Wang and Xiong 2008). The antioxidant activity of several protein hydrolysates and peptide mixtures has been tested in situ, some already used as ingredients in commercial food processing. Whey, casein, soy, potato, and yolk protein hydrolysates have been shown to inhibit lipid oxidation in muscle foods (Peña-Ramos and Xiong 2003; Diaz and Decker 2005; Sakanaka and Tachibana 2006; Wang and Xiong 2005). Because of their high water solubility, the antioxidant

Chapter 3 Antioxidant Peptides

peptides could readily sequester prooxidative Fe2+ and Cu2+ and stabilize soluble radicals dissolved in the aqueous phase as well as present at the interface. Antioxidant caseinophosphopeptides, derived from tryptic digestion of casein, have been used in breakfast cereal, bread, pastry, chocolate, juice, tea, and mayonnaise (FitzGerald 1998).

5. Action Mechanisms of Antioxidant Peptides 5.1. Chemical Reactions The action modes of antioxidant peptides have been extensively investigated and they are illustrated in Figure 3.3. In general, antioxidant peptides are capable of acting as a radical scavenger, a proton donor, and metal-ion chelator. Antioxidant peptides are generally short peptides (2-10 amino acid residues), and the amino acid sequence is a determinant factor of the efficacy. Furthermore, the presence of certain particular amino acid residues, notably histidine, tyrosine, tryptophan, methionine, cysteine, and proline, is significantly correlated with radical quenching activity of peptides (Saito et al. 2003;

35

Peña-Ramos et al. 2004; Elias et al. 2006; Nguyen et al. 2006; Hernandez-Ledesma et al. 2007). The radical-scavenging property of peptides is assessed by a number of methods, for example, the colorimetric binding assays for synthetic 2,2 ′ - azinobis(3 - ethylbenzothiazoline - 6 - sulfonic acid) (ABTS+ •) and 1,1-diphenyl-2-picrylhydrazyl (DPPH •) radicals, and electron spin resonance (ESR) microscopy for the detection of radicals. Peng et al. (2009) separated an antioxidant whey hydrolysate into four peptide fractions using size exclusion chromatography, of which the fraction of 0.1–2.8 kDa exhibited the strongest scavenging effects on DPPH •, • OH, and O2−i radicals (Figure 3.4). In an oxidizing environment where unsaturated lipids are also present, active peptides are more inclined to donate electrons than lipids, and they form much more stable radicals or nonreactive polymers through radical-radical condensation. The structure-function relationship of antioxidant peptides has been explored. Because peptides exhibiting antioxidant activity tend to be small molecules, they do not possess a higher order structure than the peptide sequence, that is, the primary structure. Therefore, much of the research is focused on how

Figure 3.3. Schematic representation of chemical and physical mechanisms of antioxidant peptides to inhibit oxidative processes. (1) Metal chelation; (2) radical scavenging; (3) physical hindrance (shielding; repulsion).

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Part 2 Functions of Biologically Active Proteins and Peptides

Figure 3.4. Gel filtration of whey protein hydrolysate (top) and ESR spectrum (bottom) of •OH signals for samples containing different peptide fractions (I–IV). Adapted from Peng et al. 2008.

the order of amino acids along the peptide chain, the chain length, and the predominance of particular amino acids in the peptides affect the peptides’ ability to inhibit lipid oxidation. Table 3.2 lists some examples of numerous antioxidant peptides that have been reported in the literature. These peptides are either derived from enzymatic or microbial hydrolysis or synthesized chemically. Chen et al. (1995) isolated six peptides with antioxidant activity from enzyme hydrolyzed soy βconglycinin. The peptide with the sequence of

Leu-Leu-Pro-His-His showed a strong inhibition of lipid peroxidation, and the His-His segment played an essential role in the noted antioxidant activity of the peptide. The activity of the dipeptide His-His was further improved by the addition of leucine or proline to the N-terminus (Chen et al. 1996). Peptides with the Pro-His-His sequence had the greatest synergism with lipid-soluble antioxidants, for example, tocopherols and butylated hydroxyanisole (BHA). These peptides have the ability to chelate transitional metal ions and quench active oxygen species,

Chapter 3 Antioxidant Peptides

37

Table 3.2. Examples of exogenous peptides that exhibit antioxidant activity in vitro and in model systems. Source

Sequence

Reference

β-Conglycinin (soy) Albumin (rice) Yellowfish sole (fermented) Mussel (fermented) Milk (fermented) β-Lactoglobulin (milk) Synthetic (albumin) Synthetic Synthetic Synthetic

Leu-Leu-Pro-His-His Asp-His-His-Gln Arg-Pro-Asp-Phe-Asp-Leu-Glu-Pro-Pro-Tyr His-Phe-Gly-Asp-Pro-Phe-His Val-Leu-Pro-Val-Pro-Gln-Lys Trp-Tyr-Ser-Leu-Ala-Met-Ala Asp-Ala-His-Lys Phe-His-Lys-Ala-Leu-Tyr Lys-Arg-Glu-Ser Pro-His-His

Chen et al. 1995 Wei et al. 2007 Jun et al. 2004 Rajapakse et al. 2005 Rival et al. 2000 Hernandez-Ledesma et al. 2007 Bar-Or et al. 2001 Nguyen et al. 2006 Navab et al. 2005 Saito et al. 2003

including •OH. The overall antioxidant activity is attributed to the collective effects of these chemical actions. Saito et al. (2003) constructed a comprehensive tripeptide library using a solid-phase synthesis procedure, and the antioxidant activity of the resulting peptides was evaluated. Of the 108 peptides containing either histidine or tyrosine residues, two tyrosine-containing tripeptides showed higher activity than those of two His-containing tripeptides in the peroxidation of linoleic acid. Tyr-His-Tyr showed strong synergistic effects with phenolic antioxidants. Of the 114 synthesized tripeptides containing proline or histidine, Pro-His-His exhibited the greatest antioxidant activity. Substitution of other amino acid residues for either the N-terminus or C-terminus of the tripeptide did not significantly alter their antioxidative activity. Furthermore, cysteine-containing tripeptides showed a strong peroxynitrite-scavenging activity. Tripeptides containing tryptophan or tyrosine residues at the C-terminus had strong radical-scavenging activities, but weak peroxynitritescavenging activity. Sequestering metal-ion catalysts is another important mechanism by which peptides inhibit oxidative processes (Wang and Xiong 2005; Nguyen et al. 2006). In cells and living tissue, transitional metal ions act as initiators to produce radical and nonradical oxygen species; and •OH generated in a Fenton-like process is a classic example. Stabilization of metal prooxidants through sequestering inhibits the radical production. The endogenous dipeptide

carnosine in muscle tissue has a strong Fe2+ and Cu2+ binding power, which is attributed to the histidine residue (Boldyrev and Johnson 2002). Amino acid residues containing phosphorylated hydroxyl side chain groups (serine and threonine) and carboxyl groups (glutamic acid and aspartic acid) are also metal-ion binders. Because transitional metal ions promote lipid hydroperoxide decomposition, metal binding by peptides would partially inhibit the propagation of lipid peroxidation that forms reactive oxygen radicals. Binding of metal ions by peptides may also change the redox cycling capacity, which is important for some metal-catalyzed oxidation. Using ferritin (a serum polypeptide) as an example, the less reactive ferric ion (Fe3+) is localized in the polypeptide’s cavity, where it nucleates and aggregates to form ferric hydroxide core unable to be converted to the more reactive ferrous species (Fe2+) (Arosio and Levi 2002). The disruption of iron redox equilibrium leads to a diminishment of free Fe2+, thereby preventing the decomposition of hydroperoxides.

5.2. Physical Interactions In lipid droplet and emulsion systems, peptides could inhibit lipid peroxidation by serving as a physical barrier, that is, a membrane (Figure 3.3). Because peptides are surface active components, they can partition at the oil-water interface, forming a thick membrane or coating to prevent direct contact of lipids and radicals and other oxidizing

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Part 2 Functions of Biologically Active Proteins and Peptides

compounds. It was reported that the formation of a thick protein membrane (whey protein) barrier instead of a nonprotein film (Tween 20) resulted in an improved oxidative stability of emulsified Menhaden oil (Donnelly et al. 1998). Furthermore, under acidic conditions, protonated amino groups could repel cationic prooxidants (for example, Fe2+ and Cu2+) thereby inhibiting the initiation of lipid peroxidation (Kellerby et al. 2006). It is not clear whether lipid micelles in the digestive tract and in the body circulation system are protected by antioxidant peptides through such physical hindrances. The efficacy of the protective fat globule membrane could be further enhanced by the construction of a multilayer interface where cationic protein membrane (the inner layer) formed at low pH may be reinforced by the deposit of another layer of an anionic polymer, for example, a polysaccharide (Pallandre et al. 2007).

6. Preparation of Antioxidant Peptides 6.1. Enzymatic Production Enzymatic digestion is by far the most efficient and reliable method to produce peptides with target functionalities, including antioxidant activity. A broad range of antioxidant peptides and peptide mixtures (hydrolysates) have been produced from soy, corn, potato, peanut, milk, whey, egg, and meat proteins. The antioxidant efficacy of protein hydrolysates and peptides depends on the source of proteins, the protein substrate pretreatment, the type of proteases used, and the hydrolysis conditions applied. Both pure and crude enzymes can be used to produce antioxidative peptides. However, to reduce the production cost, crude enzyme mixtures are preferred. Because protein hydrolysate is a heterogeneous mixture that can contain both antioxidant and prooxidant peptides, it is desirable to fractionate the peptide mixture to remove oxidizing components. Separation and purification of antioxidant peptides from protein digests are commonly accomplished through size-exclusion and membrane filtration chromatography, which can be operated

with automation. There are continuing efforts to develop efficient, selective column chromatography methods that can replace batch operations for desalting. Polarity-based solvent extraction is also a feasible technique to separate and isolate antioxidant peptides. For peptides with different positive and negative charge distributions, an ion-exchange column can be useful. A membrane system to produce and separate antioxidant peptides from whey protein has been described by Cheison et al. (2007). In this system, an enzymatic membrane reactor fitted with either a 10- or 3- kDa nominal molecular weight cutoff tangential flow filter membrane is reportedly successful in obtaining active peptide fractions with high product recovery rates.

6.2. Microbial Fermentation The production of antioxidant peptides through direct microbial fermentation rather than using purified enzymes is an integral part of healthy food production in many countries. Natto and tempeh are fermented soybean products that contain antioxidant peptides by the action of fungal proteases (Sheih et al. 2000; Iwa et al. 2002). The type, amount, and activity of the peptides produced depend on the specific cultures used, and Rhizopus spp. are some of the commonly used cultures. Douchi, also a soybean product fermented by fungal cultures (e.g., Aspergillus spp.), contains antioxidant peptides released by microbial enzymes. As reported by Wang et al. (2008), the rats that were fed antioxidant douchi extracts showed significantly elevated antioxidant enzyme activity in liver and kidney, when compared with control rats. Lipid peroxidation in both organs of douchi-fed rats was also suppressed. Furthermore, antioxidant peptides can be produced from fermented meats. A hepta-peptide with the sequence of His-Phe-Gly-Asp-Pro-Phe-His and having a strong radical-scavenging activity was isolated from fermented marine blue mussel (Mytilus edulis) (Rajapakse et al. 2005). Many strains of lactic acid bacteria (LAB) are known for their antioxidant activity. LAB-fermented goat milk has been shown to lower oxidative stress and alleviate atherogenicity in humans (Kullisaar

Chapter 3 Antioxidant Peptides

et al. 2003). LAB cultures used in yogurt fermentation are able to stabilize ROS and inhibit lipid peroxidation (Lin and Yen 1999). In order to identify and isolate the specific antioxidant LAB strains, Virtanen et al. (2007) screened 25 LAB strains using a milk whey fermentation model system. Of them, Leuconostoc mesenteroides ssp. Cremoris strains, Lactobacillus jensenii (ATCC25258), and Lactobacillus acidophilus (ATCC 4356) exhibited the highest activity in scavenging radicals and inhibiting lipid peroxidation. Peptides with high radicalscavenging activity were mostly in the 4–20 kDa size range.

6.3. Chemical Synthesis The therapeutic roles exhibited by many peptides have led to the attempt to synthesize bioactive peptides to remedy certain pathological conditions related to oxidation. Oral administration of a chemically synthesized 4 amino acid peptide (Lys-ArgGlu-Ser) lowered LDL peroxidation, alleviated inflammation, and reduced atherosclerosis in apoEnull mice (Navab et al. 2005). Interestingly, changing the order of the peptide sequence to Lys-Glu-Arg-Ser resulted in the loss of all biological activity, suggesting a specific structure-function relationship. Negative stain electron microscopy demonstrated the formation of an organized peptide-lipid structure. Larger peptides that are synthesized to contain amphipathic helixes can also function to suppress LDL peroxidation and reduce inflammation (Reddy et al. 2006). Peptides containing the active Pro-HisHis fragment were synthesized, and all showed remarkable inhibition of lipid peroxidation. A series of synthesized histidine-containing peptides with sequences present in human paraoxonase 1 (an enzyme associated with HDL) have been shown to strongly inhibit oxidation of lipoproteins (Nguyen et al. 2006). The antioxidant actions of these peptides were attributed to their metal-ion chelation and radical-scavenging capabilities based on the Cu2+ binding and peroxyl radical-quenching tests. The presence of tyrosine and cysteine residues was essential for the elicitation of the antioxidant activity. A synthetic histidine-containing analog of

39

the human albumin N-terminus fragment (Asp-AlaHis-Lys) is also highly effective in inhibiting the Cu2+-induced formation of ROS (Bar-Or et al. 2001). Saito et al. (2003) have built an antioxidant tripeptide library; of all synthesized tripeptides, Tyr-HisTyr and Pro-His-His were particularly effective in stabilizing radical and nonradical oxygen species, including peroxynitrite and lipid peroxide.

7. Conclusion Recent advances in peptide research have led to the accumulation of mounting evidence that many endogenous peptides are capable of stabilizing radicals and neutralizing other nonradical-oxidizing species. Furthermore, this biological function has been demonstrated by in vitro digests of proteins. With an improved understanding of the structurefunction relationship, it is now possible to develop antioxidant peptides and peptide mixtures through enzymatic or microbial hydrolysis of common food proteins or by means of chemical synthesis. While the question of peptides acting as antioxidants no longer remains, it is still a big challenge to identify the fate of dietary antioxidant peptides and their exact biological activity once entering the circulation system and crossing the cell membrane. The production cost as well as the potential allergenicity of antioxidant peptides must also be assessed in the continuing effort to develop novel antioxidants to complement the human body’s natural defense system, including antioxidant enzymes, vitamins, and nonprotein compounds.

8. References Arosio P, Levi S. 2002. Ferritin, iron homeostasis, and oxidative damage. Free Rad Biol Med 33:457–463. Babizhayev MA, Seguin MC, Gueyne J, Evstigneeva RP, Ageyeva EA, Zheltukhina GA. 1994. L-Carnosine (β-alanylL-histidine) and carcinine (β-alanylhistamine) act as natural antioxidants with hydroxyl-radical-scavenging and lipid-peroxidase activities. Biochem J 304:509–516. Bar-Or D, Rael LT, Lau EP, Rao NK, Thomas GW, Winkler JV, Yukl RL, Kingston RG, Curtis CG. 2001. An analog of the human albumin N-terminus (Asp-Ala-His-Lys) prevents formation of copper-induced reactive oxygen species. Biochem Biophys Res Commun 284:856–862.

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Berlett BS, Stadtman ER. 1997. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272(33):20313– 20316. Boldyrev A, Johnson P. 2002. Carnosine and related compounds: Antioxidant dipeptides. In: P Johnson, A Boldyrev, eds., Oxidative Stress at Molecular, Cellular and Organ Levels, 101–114. Trivandrum, India: Research Signpost. Bray TM, Taylor CG. 1994. Enhancement of tissue glutathione for antioxidant and immune functions in malnutrition. Biochem Pharmacol 47:2113–2123. Butterfield DA, Reed T, Newman SF, Sultana R. 2007. Roles of amyloid β-peptide-associated oxidative stress and brain protein modifications in the pathogenesis of Alzheimer ’s disease and mild cognitive impairment. Free Rad Biol Med 43:658–77. Carrier J, Aghdassi E, Platt I, Cullen J, Allard JP. 2001. Effect of oral iron supplementation on oxidative stress and colonic inflammation in rats with induced colitis. Aliment Pharmacol Ther 15:1989–1999. Chakravarti B, Chakravarti DN. 2007. Oxidative modification of proteins: Age-related changes. Gerontology 53:128–139. Cheison SC, Wang Z, Xu SY. 2007. Preparation of whey protein hydrolysates using a single- and two-stage enzymatic membrane reactor and their immunological and antioxidant properties: Characterization by multivariate data analysis. J Agric Food Chem 55:3896–3904. Chen HM, Muramoto K, Yamauchi F. 1995. Structural analysis of antioxidative peptides from soybean β-conglycinin. J Agric Food Chem 43:574–578. Chen HM, Muramoto K, Yamauchi F, Fujimoto K, Nokihara K. 1998. Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein. J Agric Food Chem 46:49–53. Chen HM, Muramoto K, Yamauchi F, Nokihara K. 1996. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. J Agric Food Chem 44:2619–2623. Cho S, Szeto HH, Kim E, Kim H, Tolhurst AT, Pinto JT. 2007. A novel cell-permeable antioxidant peptide, SS31, attenuates ischemic brain injury by down-regulating CD36. J Biol Chem 282:4634–4642. Davalos A, Miguel M, Bartolome B, López-Fandiño R. 2004. Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis. J Food Prot 67:1939–1944. Dedon PC, Tannenbaum SR. 2004. Reactive nitrogen species in the chemical biology of inflammation. Arch Biochem Biophy 423(1):12–22. Diaz M, Decker EA. 2005. Antioxidant mechanisms of caseinophosphopeptides and casein hydrolysates and their application in ground beef. J Agric Food Chem 52:8208–8213. Doan FJ, Miller GM. 1940. Trypsin. An antioxygen for milk. Milk Plant Monthly 29:42. Donnelly JL, Decker EA, McClements DJ. 1998. Iron-catalyzed oxidation of Menhaden oil as affected by emulsifiers. J Food Sci 63:997–1000.

Elias RJ, Bridgewater JD, Vachet RW, Waraho T, McClements DJ, Decker EA. 2006. Antioxidant mechanisms of enzymatic hydrolysates of β-lactoglobulin in food lipid dispersions. J Agric Food Chem 54:9565–9572. Elmadfa I, Meyer A. 2008. Body composition, changing physiological functions and nutrient requirements of the elderly. Ann Nutr Metab 52(Suppl 1):2–5. FitzGerald RJ. 1998. Potential uses of caseinophosphopeptides. Intl Dairy J 8:451–457. Ghosh G, De K, Maity S, Bandyopadhyay D, Bhattacharya S, Reiter RJ, Bandyopadhyay A. 2006. Melatonin protects against oxidative damage and restores expression of GLUT4 gene in the hyperthyroid rat heart. J Pineal Res 42:71–82. Hardeland R. 2005. Antioxidative protection by melatonin: Multiplicity of mechanisms from radical detoxification to radical avoidance. Endocrine 27:119–130. Hayashi T, Shishido N, Nakayama K, Nunomura A, Smith MA, Perry G, Nakamura M. 2007. Lipid peroxidation and 4-hydroxy-2-nonenal formation by copper ion bound to amyloid-β peptide. Free Rad Biol Med 43:1552–1559. Hernandez-Ledesma B, Amigo L, Recio I, Bartolome B. 2007. Ace-inhibitory and radical-scavenging activity of peptides derived from β-lactoglobulin f(19-25). Interactions with ascorbic acid. J Agric Food Chem 55:3392–3397. Hilton CW, Prasad C, Vo P, Mouton C. 1992. Food contains the bioactive peptide, cyclo(His-Pro). J Clin Endocrinol Metab 75:375–378. Iwa K, Nakaya N, Kawasaki Y, Matsue H. 2002. Antioxidative function of natto, a kind of fermented soybeans: Effect on LDL oxidation and lipid metabolism in cholesterol-fed rats. J Agric Food Chem 50:3597–3601. Jun SY, Park PJ, Jung WK, Kim SK. 2004. Purification and characterization of an antioxidative peptide from enzymatic hydrolysate of yellowfish sole (Limanda aspera) frame protein. Eur Food Res Intl 219:20–26. Jung WK, Qian ZJ, Lee SH, Choi SY, Sung NJ, Byun HG, Kim SK. 2007. Free radical scavenging activity of a novel antioxidative peptide isolated from in vitro gastrointestinal digests of Mytilus coruscus. J Med Food 10:197–202. Karel M, Tannenbaum SR, Wallace DH, Maloney H. 1966. Antioxidation of methyl linoleate in freeze-dried model systems. III. Effects of added amino acids. J Food Sci 31:892–896. Kellerby S, McClements DJ, Decker EA. 2006. Role of proteins in oil-in-water emulsions on the stability of lipid hydroperoxides. J Agric Food Chem 54:7879–7884. Kim GN, Jang HD, Kim CI. 2007a. Antioxidant capacity of caseinophosphopeptides prepared from sodium caseinate using Alcalase. Food Chem 104:1359–1365. Kim KS, Choi SY, Kwon HY, Won MH, Kang TC. 2002. Carnosine and related dipeptides protect human ceruloplasmin against peroxyl radical-mediated modification. Mol Cells 13:498–502. Kim SY, Je JY, Kim SK. 2007b. Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame

Chapter 3 Antioxidant Peptides

protein by gastrointestinal digestion. J Nutr Biochem 18:31– 38. Kong B, Xiong YL. 2006. Antioxidant activity of zein hydrolysates in a liposome system and the possible mode of action. J Agric Food Chem 54:6059–6068. Kozina LS, Arutjunyan AV, Khavinson VK. 2007. Antioxidant properties of geroprotective peptides of the pineal gland. Arch Gerontol Geriatr 44(Suppl 1):213–216. Kullisaar T, Songisepp E, Mikelsaar M, Zilmer K, Vihalemm T, Zilmer M. 2003. Antioxidative probiotic fermented goats’ milk decreases oxidative stress-mediated atherogenicity in human subjects. Br J Nutr 90:449–456. Li X, Han L, Chen L. 2008. In vitro antioxidant activity of protein hydrolysates prepared from corn gluten meal. J Sci Food Agric 88:1660–1666. Lin MY, Yen CL. 1999. Reactive oxygen species and lipid peroxidation product-scavenging ability of yoghurt organisms. J Dairy Sci 82:1629–1634. Marcuse R. 1960. Antioxidative effect of amino acids. Nature 186:886–887. Mendis E, Rajapakse N, Kim SK. 2005. Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate. J Agric Food Chem 53:581–587. Minelli A, Bellezza I, Grottelli S, Galli F. 2008. Focus on cyclo(His-Pro): History and perspectives as antioxidant peptide. Amino Acids 35:283–289. Nagasawa T, Yonekura T, Nishizawa N, Kitts DD. 2001. In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine. Mol Cell Biochem 225:29–34. Navab M, Anantharamaiah GM, Reddy ST, Hama S, Hough G, Frank JS, Grijalva VR, Ganesh VK, Mishra VK, Palgunachari MN, Gogelman AM. 2005. Oral small peptides render DHL anti-inflammatory in mice and monkeys and reduce atherosclerosis in ApoE null mice. Cir Res 97:524–532. Nguyen SD, Jeong TS, Kim ME, Sok DE. 2006. Broad-spectrum antioxidant peptides derived from His residue-containing sequences present in human paraoxonase 1. Free Rad Res 40:349–358. Pallandre S, Decker EA, McClements DJ. 2007. Improvement of stability of oil-in-water emulsions containing caseinate-coated droplets by addition of sodium alginate. J Food Sci 72:E518– 524. Peña-Ramos EA, Xiong YL. 2001. Antioxidative activity of whey protein hydrolysates in a liposomal system. J Dairy Sci 84:2577–2583. Peña-Ramos EA, Xiong YL. 2003. Whey and soy protein hydrolysates inhibit lipid oxidation in cooked pork patties. Meat Sci 64:259–263. Peña-Ramos EA, Xiong YL, Arteaga GE. 2004. Fractionation and characterization for antioxidant activity of hydrolyzed whey protein. J Sci Food Agric 84:1908–1918. Peng X, Xiong YL, Kong B. 2009. Antioxidant activity of peptide fractions from whey protein hydrolysates as measured by electron spin resonance. Food Chem 113(1):196–201.

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Pihlanto A, Akkanen S, Korhonen HJ. 2008. ACE-inhibitory and antioxidant properties of potato (Solanum tuberosum). Food Chem 109:104–112. Qian ZJ, Jung WK, Byun HG, Kim SK. 2008. Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas, against free radical induced DNA damage. Bioresource Technol 99:3365–3371. Raghavan S, Kristinsson HG. 2008. Antioxidative efficacy of alkali-treated tilapia protein hydrolysates: A comparative study of five enzymes. J Agric Food Chem 56:1434–1441. Rajapakse N, Mendis E, Jung WK, Je JY, Kim SK. 2005. Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Res Intl 38(2):175–182. Reddy ST, Anantharamaiah GM, Navab M, Hama S, Hough G, Grijalva V, Garber DW, Datta G, Fogelman AM. 2006. Oral amphipathic peptides as therapeutic agents. Exp Opin Invest Drugs 15:13–21. Rival SG, Fornaroli S, Boeriu CG, Wichers HJ. 2000. Caseins and casein hydrolysates. 1. Lipoxygenase inhibitory properties. J Agric Food Chem 48:287–294. Rizvi SI, Jha R, Maurya PK. 2006. Erythrocyte plasma membrane redox system in human aging. Rejuv Res 9:470– 474. Saiga A, Tanabe S, Nishimura T. 2003. Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. J Agric Food Chem 51:3661–3667. Saito K, Jin DH, Ogawa T, Muramoto K, Hatakeyama E, Yasuhara T, Nokihara K. 2003. Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry. J Agric Food Chem 51:3668–3674. Sakanaka S, Tachibana Y. 2006. Active oxygen scavenging activity of egg-yolk protein hydrolysates and their effects on lipid oxidation in beef and tuna homogenates. Food Chem 95:243–249. Sheih IC, Wu HY, Lai YJ, Lin CF. 2000. Preparation of high free radical scavenging tempeh by a newly isolated Phizopus sp. R-69 from Indonesia. Food Sci Agric Chem 2:35–40. Srigiridhar K, Nair KM, Subramanian R, Singotamu L. 2001. Oral repletion of iron induces free radical mediated alterations in the gastrointestinal tract of rat. Mol Cell Biochem 219:91–98. Stadtman ER. 2006. Protein oxidation and aging. Free Rad Res 40:1250–1258. Tan DX, Manchester LC, Terron MP, Flores LJ, Reiter RJ. 2007. One molecule, many derivatives: A never-ending interaction of melatonin with reactive oxygen and nitrogen species? J Pineal Res 42:28–42. Thiansilakul Y, Benjakul S, Shahidi F. 2007. Compositions, functional properties and antioxidative activity of protein hydrolysates prepared from round scad (Decapterus maruadsi). Food Chem 103:1385–1394. Thomas DA, Stauffer C, Zhao K, Yang H, Sharma VK, Szeto HH, Suthanthiran M. 2007. Mitochondrial targeting with antioxidant peptide SS-31 prevents mitochondrial depolarization,

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reduces islet cell apoptosis, increases islet cell yield, and improves posttransplantation function. J Amer Soc Nephrol 18:213–222. Virtanen T, Pihlanto A, Akkanen S, Korhonen H. 2007. Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. J Appl Microbiol 102:106–115. Walter MF, Mason PE, Mason RP. 1997. Alzheimer ’s disease amyloid β peptide 25-35 inhibits lipid peroxidation as a result of its membrane interactions. Biochem Biophys Res Commun 233:760–764. Wang D, Wang L, Zhu F, Zhu J, Chen XD, Zou L, Saito M, Li L. 2008. In vitro and in vivo studies on the antioxidant activities of the aqueous extracts of Douchi (a traditional Chinese salt-fermented soybean food). Food Chem 107:1421– 1428. Wang JS, Zhao MM, Zhao QZ, Jiang YM. 2007. Antioxidant properties of papain hydrolysates of wheat gluten in different oxidation systems. Food Chem 101:1658–1663.

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Chapter 4 Antihypertensive Peptides and Their Underlying Mechanisms Toshiro Matsui and Mitsuru Tanaka

Contents 1. Introduction, 43 2. Hypertension and the Renin-Angiotensin System, 44 3. Design of ACE Inhibitory Peptides, 44 4. Antihypertensive Mechanism of Small Peptides, 46 4.1. Inhibition of the Renin-Angiotensin System by ACE Inhibitory Peptides, 46

1. Introduction Peptides, which are condensed amino acids, have been well documented as physiologically functional compounds in nature. In this chapter, we review their updated antihypertensive mechanism as well as the development of antihypertensive food products, because small peptides including di- and tripeptides are favorably absorbed through intestinal peptide transporter PepT1. In Japan, the number of hypertensives and high-normal hypertensives (systolic blood pressure [SBP] > 135 mmHg and/or diastolic blood pressure [DBP] > 85 mmHg) was ca. 55 million in 2006 (Japanese Ministry of Health, Labour and Welfare). In the case of essential hypertension, which afflicts >90% of all hypertensions, the onset followed by

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

4.2. Regulation of Vascular Events by Dipeptides, 47 4.3. Relaxation of Vascular Constrictive Events by Dipeptides, 48 5. Future Prospects, 51 6. References, 53

cardiovascular disease, renal dysfunction, or peripheral vascular disease is closely associated with lifeand/or food-style, including excess high-salt intake (Kawano et al. 2007). Namely, disruption of Na+-K+ balance or increase in fluid volume as well as increasing vascular resistance will lead to promotion of blood pressure (BP). The most beneficial improvement for essential/borderline hypertension is, therefore, said to be one that achieves modification of food-style and/or moderate exercise. In addition to such lifestyle modification treatments, peptide research has become one of the growing fields for preventing-medicinal chemistry, since some clinical evidence shows the efficiency of peptide intake for improving hypertension disease. In Japan, evidencebased foods with health claim (FOSHU, or foods for specified health use) have been developed and accepted by the Japanese Ministry of Health, Labour and Welfare (http://www/mhlw.go.jp/). For FOSHU products effective for modulating BP in borderline hypertensives, seven products (tryptic hydrolysate of casein, Katsuobushi [dried bonito] oligopeptide, 43

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Part 2 Functions of Biologically Active Proteins and Peptides

Table 4.1. Antihypertensive FOSHU products. Functional Ingredient

Active Peptide

Daily Intake

Tryptic hydrolysate of casein

Phe-Phe-Val-Ala-Pro-PhePro-Glu-Val-Phe-Gly-Lys Leu-Lys-Pro-Asn-Met Leu-Lys-Pro Ile-Tyr

20 g

Val-Pro-Pro Ile-Pro-Pro Val-Tyr

VPP equiv. : 2.53 mg IPP equiv. : 1.52 mg 4g

Katsuobushi (dried bonito) oligopeptide Aqueous extract from Mycoleptodo-noides aitchisonii Lactobacillus helveticus— fer-mented milk (sour milk) Sardine peptide a

1.5 g 1g

Reduc-tion of SBP/DBPa (mmHg)

Reference

4.6/6.6

Sekiya et al. 1992

11.7/6.9

Fujita et al. 2001

9.4/6.7

Tsuchida et al. 2001

12.5/6.2

Kajimoto et al. 2001

9.3/5.2

Kawasaki et al. 2000

SBP: systolic blood pressure; DBP: diastolic blood pressure.

aqueous extract from Mycoleptodonoide s aitchisonii, Lactobacillus helveticus-fermented milk [sour milk], sardine peptide, seaweed peptides, and sesame peptides) are available in the local market, most of which involve the inhibition of angiotensin I–converting enzyme (ACE) by small peptides (Table 4.1). The efficacy of peptide intake for borderline hypertensives is evidentially developed on the basis of extensive intervention trials, but such an effective FOSHU medication led us to a potential question of whether the antihypertensive effect of peptides is achieved only by ACE inhibition or suppression of the renin-angiotensin system, like therapeutic ACE inhibitory drugs.

2. Hypertension and the Renin-Angiotensin System Arterial BP regulation is mainly achieved by diverse metabolic systems: pressor and depressor hormonal systems and nerve systems (Sealey and Laragh 1990). Among these, the renin-angiotensin (-aldosterone) system is thought to be one of the predominant pressor systems, widely occurring in not only the circulatory blood system but also in diverse organs such as brain, lung, aorta, and kidney. In this system, angiotensinogen from the liver is primarily cleaved by renal renin to produce angiotensin (Ang) I that is a decapeptide. Ang I is then converted by the action of ACE (EC 3.4.15.1, dicarboxypepti-

dase) to potent vasoconstrictor Ang II by cleaving the dipeptide His-Leu from the C-terminal of Ang I. The major physiological role of Ang II is to exert a vasoconstrictive effect at the vessel wall via the binding of Ang II to Ang II receptor (AT1) (Millatta et al. 1999). Ang II also participates in the increment of extracellular fluid volume through a stimulation of adrenal aldosterone release, by which renal tubular sodium reabsorption is enhanced. Thus, it has been recognized that the circulatory reninangiotensin system plays a crucial role in the development and maintenance of hypertension. Updated research (Bader and Ganten 2008) also helps us to understand the importance of the local renin-angiotensin system in regulating BP promotion, where new candidates of (pro)renin and ACE2, an Ang (1-7) producing enzyme, involve the local (kidney, heart, vessel, or brain) BP regulation system like autocrine/paracrine hormone (Figure 4.1).

3. Design of ACE Inhibitory Peptides In order to prevent the pathogenesis of hypertension or to treat an elevated BP, suppression of Ang II production via inhibition of ACE activity would be of great benefit, because both renin-angiotensin and kinin-kallikrein systems involve ACE action. In addition, taking into consideration that ACE has four functional amino acid residues of Tyr, Arg, Glu, and Lys at the active site, and three hydrophobic

Chapter 4 Antihypertensive Peptides and Their Underlying Mechanisms

45

Renin-Angiotensin System (RAS) Prorenin

Angiotensinogen A i t i Renin

Angiotensin(1-9)

(Pro)renin receptor

Angiotensin I ACE2

ACE

ACE

Angiotensin(1-7)

Angiotensin II ACE2

Mas-R

AT2-R

Antihypertensive events

AT1-R

Hypertensive events

Figure 4.1. Updated circulatory renin-angiotensin system.

binding subsites, favorable blockade of ACE action would be achieved by small peptides or peptidic inhibitors having high affinity with active sites. The finding that the transporter-recognized di- and tripeptide length is expressed at the intestine (PepT1) (Minami et al. 1992; Vig et al. 2006) also allows us to investigate the possible functionality of small peptides after absorption. From this point of view, many ACE inhibitory peptides have been designed and identified from natural proteins (Matsui and Matsumoto 2006); as a therapeutic ACE inhibitory drug, captopril or enalapril, was designed on the basis of a basal structure of Ala-Pro or Phe-Ala-Pro, respectively (Hooper 1991). This is the reason why small peptides are targeted for developing antihypertensive food through ACE inhibitory action. ACE inhibitory peptides (>400) reported so far provide the evidence that small peptides with hydrophobic and aromatic amino acid residues such as Tyr, Phe, Trp, and Pro at the C-terminal have a potent ability to inhibit ACE activity with an IC50 value of < 100 μmol/L (Matsui and Matsumoto 2006). A successful human study was reported using sardine muscle hydrolysate by alkaline protease showing an ACE inhibitory power of IC50 value

of 0.018 mg-protein/mL. After a 4-week administration of the sardine hydrolysate (4 g/day) to 29 borderline hypertensive volunteers (systolic BP/ diastolic BP, peptide-drink: 146.4/90.5 mmHg; placebo: 145.5/92.3 mmHg), a significant BP reduction (systolic BP/diastolic BP: 9.3/5.2 mmHg) was observed in the peptide-drink group, whereas there was no BP change in the placebo group (Kawasaki et al. 2000). The human volunteer study provided some useful information on the BP-lowering effect of natural antihypertensive food: no rebound phenomena and no side effects, such as dry cough, which are often observed when ACE inhibitory drugs are taken. Therefore, these findings evidentially reveal that the intake of antihypertensive FOSHU products prepared from natural food resources is of great benefit for regulating or improving an elevated BP. It seems likely that FOSHU products containing ACE inhibitory peptides possess a similar extent of BP-lowering power (Table 4.1). Contrary to the prevalence, there was a substantially great difference in ACE inhibitory activity between therapeutic drug (e.g., captopril: IC50 = 0.021 μmol/L) and peptides (e.g., Leu-Arg-Pro: IC50, 0.27 μmol/L). Some reports (FitzGerald et al.

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Table 4.2. Reported antihypertensive peptides from natural proteins in spontaneously hypertensive rats.

Peptide

Source

Tyr-Pro Leu-Lys-Pro Ile-Pro-Pro Val-Tyr

αs1-casein Dried bonito β-casein Sardine muscle

IC50a (μmol/L) 720 0.32 5 26

Decrease in SBP(mmHg)/ dose(mg/kg) −32.1/101 −16/92 −28.3/0.33 −43.4/104

a

Concentration of inhibitor required to inhibit 50% of the ACE activity. 1 Yamamoto et al. 1999. 2 Fujita and Yoshikawa 1999. 3 Nakamura et al. 1995. 4 Matsui et al. 2004.

2004) have also revealed the controversies between in vivo antihypertensive effect and in vitro ACE inhibitory activity of peptides. As summarized in Table 4.2, there seems to be no relationship between the BP-lowering effect of a given ACE inhibitory peptide and its IC50 value. In this regard, it remains unclear whether the antihypertensive effect of ACE inhibitory peptides is elicited only due to ACE inhibition; and, as can be seen in the later sections, we will find the potential involvement of small peptides in local BP regulation systems.

4. Antihypertensive Mechanism of Small Peptides 4.1. Inhibition of the Renin-Angiotensin System by ACE Inhibitory Peptides Genetically defined Tsukuba-Hypertensive Mouse (THM) was used to demonstrate the antihypertensive mechanism of small peptides, in which the pathogenesis of hypertension is restrictively determined by an enhanced renin-angiotensin system induced by human renin and angiotensinogen genes. Therefore, a BP lowering of THM would refer to a suppression of enhanced human renin-angiotensin system. As a result, a single oral administration of Val-Tyr, a predominant ACE inhibitor in sardine hydrolysate to 11 weeks THM (0.1 mg/g), was proven to cause a long-lasting BP reduction up to 9

hours (systolic BP9h: 120.7 ± 2.3 mmHg) (Matsui et al. 2003). This preferable result demonstrates that the antihypertensive effect induced by ACE inhibitory peptides closely correlates with human reninangiotensin system. In contrast to these findings, there was no difference in plasma ACE activity between control and sample groups at 6 hours, while a transient inhibition was observed at 1 hour after the administration. A transient ACE inhibition of Val-Tyr was also observed in spontaneously hypertensive rats (SHR) and human studies (Kawasaki et al. 2000; Matsui et al. 2004). For ACE activity at diverse organs, a long-term suppression of ACE activity was observed in the kidney and aorta, while no change was observed in plasma ACE activity (Figure 4.2). These results suggested that both organs were targeting tissues of Val-Tyr. In other words, increasing plasma dipeptide has the power to suppress the pressor Ang II production via ACE inhibitory action transiently, but an alternative antihypertensive action occurring at the local tissues should be involved in the long-lasting BP-lowering effect of dipeptide. A similar long-lasting BP-lowering effect of ACE inhibitors was observed in an acute SHR administration study of spirapril as an ACE inhibitory drug (Okunishi et al. 1991) and 5-caffeoylquinic acid as an acetylcholine receptor mediator (Suzuki et al. 2002). A prolonged effect of the latter was reported to be due to slow metabolism of it to the candidate compound, ferulic acid, in the circulatory system. On the contrary, the effect induced by spirapril was reported to result from its long-lasting ACE inhibition at the aorta. Additionally, the finding that a young THM had already developed hypertension partly due to a remarkable vascular hypertrophy by excess vascular Ang II production led us to speculation that Val-Tyr or antihypertensive peptide might suppress an enhanced vascular renin-angiotensin system. The findings that (1) plasma Val-Tyr level at 1 hour was as little as in normotensives (12 mg dose of Val-Tyr: Cmax; 1.9 ± 0.1 pmol/mLplasma) (Matsui et al. 2002), much lower than that of captopril (Cmax at 25 mg dose; 603 pmol/mLplasma) (Jankowski et al. 1995), and (2) in vitro ACE inhibitory activity of Val-Tyr (IC50; 26 μmol/L)

Chapter 4 Antihypertensive Peptides and Their Underlying Mechanisms

47

Plasma 50 ACE activity (mU/mL plas sma)

% Reduction of SBP

0

40 30 20 10 0

##

0

1

-10

6

Time (h) ## ##

Aorta

##

-20

Kidney 5

1

3

6

Time after administration (h)

9

4

＊＊

＊＊

3 2

ACE activity A (mU/mg protein)

0

ACE activity A (mU U/mg protein)

5

4 3 2

1

1

0

0 0

1 Time (h)

6

0

＊

＊

1

6

Time (h)

Figure 4.2. Change in systolic blood pressure (A) and ACE activities (B) of 18-week-old spontaneously hypertensive rats after a single oral administration of 10 mg/kg Val-Tyr (䊉) or control (䊊). Each value is expressed as mean ± SEM (n = 5). ##p < 0.01 compared with the control group. *p < 0.05, **p < 0.01 compared with 0 hours.

was much weaker than that of captopril (IC50; 0.021 μmol/L), also suggest little contribution of plasma Val-Tyr increment to long-lasting BPlowering effect.

4.2. Regulation of Vascular Events by Dipeptides Antihypertensive peptide may play a potential role in regulating the vascular function, though the mechanism at the aorta remains unclear. Thus, our next trial was to demonstrate the latent function in the cell line experiment. Human vascular smooth muscle cell (VSMC) was used, since the aorta was reportedly one of the accumulated tissues of some ACE inhibitory peptides and directly responsible for vasoconstriction tone. In a VSMC proliferation experiment in 5% FBS-SmBM using Val-Tyr (IC50; 26 μmol/L),

Ile-Trp (IC50; 2.0 μmol/L), and Ile-Val-Tyr (IC50; 0.48 μmol/L), Val-Tyr was found to show a marked antiproliferation action in serum-stimulated VSMC growth (46% decrease in the cell number at 1 mmol/L Val-Tyr) (Matsui et al. 2005). Although Ile-Val-Tyr also caused a slight decrease in the growth to 82% of control, Ile-Trp was no longer an antiproliferative peptide, being provided a conflicting result with its ACE inhibitory potentials. Taking into consideration the signal-transduction pathway via AT1-receptor, an Ang II stimulation experiment was then conducted. In the presence of the peptides at a concentration of 1 mmol/L, a potent suppression of the WST-8 incorporation into Ang II–stimulated VSMC for Val-Tyr was observed, with a reduction to ca. 65% of the control (Figure 4.3). Captopril as well as Ile-Trp and Ile-Val-Tyr did not show any influence on the incorporation

48

Part 2 Functions of Biologically Active Proteins and Peptides

Bay K 8644-stimulation

200

200

180

180

160 140

＊＊

120 100 80 60 40 20

160 140 120

＊

＊＊

＊＊

100 80 60 40 20 0

0 Control Captopril

ACE inhibitory activity (µmol/L)

Incorporation (% of unstimu ulated)

Inco orporation (% % of unstimula ated)

Ang II-stimulation

Ile-Trp Ile-Val-Tyr Val-Tyr

2.0

0.48

Control Verapamil Val-Tyr

Paxillin

Paxillin + Val-Tyr

26

Figure 4.3. Effect of small peptides on angiotensin (Ang) II- or Bay K 8644-induced VSMC proliferation. VSMCs at a density of 1 × 104 cells/well were treated for 48 hours with 1 μmol/L Ang II in the presence of either 1 mmol/L peptides, 1 μmol/L captopril (ACE inhibitor), 1 μmol/L verapamil (Ca2+ channel inhibitor), or 1 μmol/L paxillin (K+ channel blocker). *p < 0.05 and **p < 0.01 compared with control by Tukey-Kramer’s t-test.

at a concentration of 1 μmol/L, which strongly suggested that the antiproliferative effect induced by Val-Tyr was not a result of aortic ACE inhibition. A similar suppression effect of the incorporation by Val-Tyr was observed, irrespective of the presence of AT1-receptor selective antagonist (losartan) or nonselective AT-receptor antagonist (saralasin) (see Matsui et al. 2005, indicating that Val-Tyr had no antagonistic effect against Ang II–related receptors). When Bay K 8644 acting as a mitogen through voltage-gated L-type Ca2+ channel stimulation was used, 1 mmol/L Val-Tyr significantly inhibited the increasing incorporation stimulated by 1 μmol/L Bay K 8644, like that which 1 μmol/L verapamil (therapeutic L-type Ca2+ channel blocker) inhibited (Figure 4.3). In contrast, the presence of paxillin (K+ channel blocker) did not affect the inhibition by Val-Tyr. This provided evidence that the antiproliferation by Val-Tyr would be in part due to inhibition of extracellular Ca2+ influx into VSMC by blocking voltage-gated L-type Ca2+ channel, not by stimulating K+ channel. So far, no study has been reported

on bioactive small peptides responsible for blocking voltage-gated L-type Ca2+ channel.

4.3. Relaxation of Vascular Constrictive Events by Dipeptides Either vascular constriction or relaxation closely relates with VSMC actions, which are critical for hypertension disease including vascular or arteriosclerotic lesions (Dzau 2001). VSMC proliferation, hypertrophy, or migration is induced by Ang II and/ or norepinepherine stimulation; among these Ang II that acts as an autocrine/paracrine mediator at the renin-angiotensin system plays a prominent role in the pathogenesis of vascular lesions via cell proliferation (Mehta and Griendling 2007). Thus, the application of dipeptides having voltage-gated L-type Ca2+ channel blocking action, like AT1receptor antagonist (Miura et al. 2003) or Ca2+ channel blocker (Stepien et al. 1997), for the treatment of vascular lesion-related diseases including hypertension would be highly appreciated.

Chapter 4 Antihypertensive Peptides and Their Underlying Mechanisms

p < 0.05

Consttrictive tens sion (% % of Controll)

140 120 100 80 60 40 20 0

Peptide (1 mmol/L) Control

Ile-Tyr

Tyr-Val

Val-Tyr

Val-Tyr

Figure 4.4. Vascular relaxation effect of 1 mmol/L Ile-Tyr, Tyr-Val, and Val-Tyr in 18-week-old SHR thoracic aorta rings constricted by 30 mmol/L KCl. Val-Tyr was subjected to endothelium-intact and –denuded vasoconstrictive experiments. EC: endothelium.

On the basis of the finding that Val-Tyr would be a Ca2+ channel blocker, an ex vivo vasoconstrictive experiment using rat aorta rings was primarily conducted. As a result, Val-Tyr exerted a significant vascular relaxation effect in SHR thoracic aorta rings in an endothelium- and ACE inhibition-independent manner (Figure 4.4) (Tanaka et al. 2006). The effective and beneficial effect of Val-Tyr in constricted aorta rings was also reported and demonstrated by Vercruysse et al. (2008). Regarding bioactive peptides with respect to vascular functions, some reports to date have investigated their vascular responses. Akpaffiong and Taylor (1998) reported on the effect of glutathione on relaxation of SHR aorta rings, in which the tetrapeptide potentiated acetylcholineinduced relaxation in an endothelium-dependent manner. However, the vascular relaxation through improving antioxidant systems may be restrictive to peptides with antioxidant activity like glutathione, but a possibility that other peptides are also involved in endothelium-dependent relaxation can be excluded. An interesting study on vascular responses of bioactive peptides was reported on dipeptide carnosine (beta-Ala-His) (Ririe et al. 2000), in which 1 mmol/L carnosine exerted ca. 10% relaxation effect in Sprague-Dawley (SD) rat aorta rings, com-

49

parable with the effect of 1 mmol/L Val-Tyr (Figure 4.4). The relaxation mechanism of carnosine was reported to be due to its suppression of cyclic GMP production in VSMC (Ririe et al. 2000), but any crucial role of carnosine in VSMC signaling pathways was not clarified. Peptides from ovalbumin, kappa-casein, egg white protein and rubisco were also reported to be a peptidic vasodilator: Ala-AspHis-Pro-Phe from ovalbumin (Matoba et al. 1999), Met-Ala-Ile-Pro-Pro-Lys-Lys from kappa-casein (Miguel et al. 2007a), and Lys-Ala-Asp-His-Pro and Tyr-Pro-Ile from egg white protein (Miguel et al. 2007b) were clarified as endothelium-dependent vasoactive peptides. Thus, it seems likely that peptides have an ability to regulate the vascular response, although the structural properties required for the effect remain unclear. To clarify the structure-vasoactivity relationship of peptides, a first attempt was performed using 62 synthetic di- and tripeptides in SD rat aorta rings (Tanaka et al. 2008). As summarized in Table 4.3, N-terminal amino acids of tryptophanyl dipeptides would make an important contribution to vasorelaxation action in thoracic aorta and would play an alternative crucial role in exerting a vasorelaxation effect. Among the screened peptides, Trp-His evoked the most potent vasorelaxation effect with an EC50 value of 3.4 mmol/L. As can be seen in Figure 4.5, Trp-His relaxed the 50 mmol/L KCl-constricted aorta rings in an endotheliumindependent manner, as Val-Tyr did. As mentioned above, carnosine had an endothelium-dependent vasorelaxation power, in which it stimulated the soluble guanylate cyclase/cyclic GMP relaxation pathways (Ririe et al. 2000). Arg-Ala-Asp-His-Pro from egg white proteins also acted as an endothelium-dependent vasodilator, through the stimulation of endothelial NO production pathways via B1 bradykinin receptor (Miguel et al. 2007b). In contrast to the endothelium-dependent vasorelaxation action of longer peptides, the endothelium-independent vasorelaxation action of Trp-His would be closely associated with some events in vascular smooth muscle layer, apart from endothelium signaling talks including the renin-angiotensin system. Interestingly, a competitive inhibition study with Ca2+ channel

50

Part 2 Functions of Biologically Active Proteins and Peptides

Table 4.3. Screening of vasorelaxant peptidesa of 50 mmol/L KCl-constricted aortic ring.1 Å@ X-Tyr X=

Val-X X= X-Phe X=

X-Trp X=

Peptidea

Relaxation (% of KCl Constriction)

Ala Phe Gly His Ile-Phe Ile-Leu Ile-Val Lys Leu-Ile Pro Ser Trp-Ile Trp Tyr-Ile Tyr-Val

Å—b Å— Å— Å— Å— Å— Å— Å— Å— Å— Å— Å— Å— <10 Å—

Phe His

Å— Å—

Ile Gly Pro Trp Ala Val-His Ile-His Trp-Ile His-Ile

Å— Å— Å— <10 Å— Å— Å— Å— <10

Phe Ile-Val Ile

Å— <10 Å—

Å@ X-Trp X=

Trp-X X=

Peptide

Relaxation (% of KCl Constriction)

Lys Leu-Ile Val-Ile Val Tyr-Ile Trp

Å— <10 Å— Å— <10 Å—

His Val Leu Ala Gly Ser Met Glu

97 11 26 <10 Å— Å— Å— Å—

Asp Lys Ile

Å— Å— Å—

Asn Arg Gln Thr

<10 Å— Å—

Others Ala-Pro Phe-Pro His-Ala His-Pro Ile-His Arg-Pro Phe-Ile Phe-His Trp

Å— Å— Å— Å— <10 Å— <10 Å— Å—

a

Concentration of peptide was set at 5.0 mmol/L. No relaxation. 1 Tanaka et al. 2008. b

blockers revealed that the Trp-His induced relaxation was not attenuated in the presence of nifedipine (dihydropyridine [DHP]-type Ca2+ channel blocker). In contrast, a significant attenuation of the effect was observed in the presence of verapamil (phenyl alkylamine [PAA]-type Ca2+ channel blocker) (Figure 4.6). This strongly suggested that Trp-His would affect the extracellular Ca2+ influx via an L-type Ca2+ channel. Photo-affinity analyses

demonstrated that nifedipine binds to the extracellular side of α1 subunit of Ca2+ channel protein, while verapamil favorably binds to the intracellular side (Kurokawa et al. 1997; Yamakage and Namiki 2002). Verapamil (molecular weight of 454) belongs to a PAA-type drug, having two di-methoxyphenyl groups at the end of the molecule as well as cyano group and amine in the middle. Hockerman et al. (1997) have clarified that the direct binding of basic

Chapter 4 Antihypertensive Peptides and Their Underlying Mechanisms

51

Endothelium (+) ACh PE

0.5 1.0 2.0

3.0 3.5

KCl

4.0

Wash

5.0

Wash

PE

0.2 g

Trp-His (mmol/L) 60 min

Endothelium (-) ACh PE

W h Wash

0.5 1.0

2.0 3.0

PE 3.5

KCl

4.0

5.0

Wash

02g 0.2 60 min

Trp-His (mmol/L)

Figure 4.5. Vascular relaxation profiles of Trp-His in endothelium-intact (+) or endothelium-denuded Sprague-Dawley (SD) rat aorta rings. Trp-His was added to endothelium-intact or –denuded aorta rings in a cumulative manner. Removal of endothelium was confirmed by no relaxation induced by 100 μmol/L acetylcholine (ACh) in 1 μmol/L phenylephrine (PE)-constricted aorta rings.

nitrogen of PAA to Glu from domains III and IV is essential in eliciting effective Ca2+ channel blocking action. This indicates that an ionization of nitrogen of PAA would be important for blocking action at the intracellular channel site. The reported pKa of verapamil was around 8.8, indicating that an ionized form of verapamil is dominant at the physiological condition (pH 7.4). According to this structural information on PAA-type blockers, the developed Ca2+ channel blocking action of Trp-His might be associated with its structural similarity with PAA. In a recent peptide-induced vasorelaxation study (Erdmann et al. 2006), it was found that Met-Tyr

derived from the sardine hydrolysate relaxed the vascular constriction in a different vascular regulating mechanism from Trp-His or Val-Tyr, by which endothelium heme oxygenase-1 was activated to increase CO production, followed by the stimulation of soluble guanylyl cyclase/cyclic GMP vasorelaxation pathway.

5. Future Prospects So far, there are many reports regarding peptidefunctionality (Hartmann and Meisel 2007). Some peptides showed vasorelaxation, antioxidative

52

Part 2 Functions of Biologically Active Proteins and Peptides

Figure 4.6. Schematic representation of Trp-His binding site to a voltage-gated L-type Ca2+ channel in VSMC. Vasorelaxation profile of Trp-His in the presence (䊐) or absence (䊏) of nifedipine (2.5 nmol/L) or verapamil (30 nmol/L).

(Erdmann et al. 2006; Zhu et al. 2006; HernandezLedesma et al. 2005), antithrombotic (Chabance et al. 1995), and hypocholesterolemic effects (Nagaoka et al. 2001; Morikawa et al. 2007); and others showed mineral binding, (Walker et al. 2006) as well as antimicrobial (McCann et al. 2006) and immunomodulatory effects (Takahashi et al. 1994; Mine and

Kovacs-Nolan 2006; Meisel 2005; Horiguchi et al. 2005). Given these diverse functionalities and the present findings on antihypertensive actions, the intake of peptides would be of benefit for maintaining our homeostasis, and further studies should be performed to overwhelm any discrepancy between in vitro and in vivo phenomena of bioactive peptides.

Chapter 4 Antihypertensive Peptides and Their Underlying Mechanisms

6. References Akpaffiong MJ, Taylor AA. 1998. Antihypertensive and vasodilator actions of antioxidants in spontaneously hypertensive rats. Amer J Hypertens 11:1450–1460. Bader M, Ganten D. 2008. Update on tissue renin-angiotensin systems. J Mol Med 86:615–621. Chabance B, Jolles P, Izquierdo C, Mazoyer E, Francoual C, Dronet L, Fiat AM. 1995. Characterization of antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion. Brit J Nutr 73:583–590. Dzau JV. 2001. Tissue angiotensin and pathobiology of vascular disease: A unifying hypothesis. Hypertension 37:1047–1052. Erdmann K, Grosser N, Schipporeit K, Schroder H. 2006. The ACE inhibitory dipeptide Met-Tyr diminishes free radical formation in human endothelial cells via induction of heme oxygenase-1 and ferritin. J Nutr 136:2148–2152. FitzGerald JR, Murray AB, Walsh JD. 2004. Hypotensive peptides from milk proteins. J Nutr 134:980–988S. Fujita H, Yamagami T, Ohshima K. 2001. Effects of an ACEinhibitory agent, katsuobushi oligopeptide, in the spontaneously hypertensive rat and in borderline and mildly hypertensive subjects. Nutr Res 21:1149–1158. Fujita H, Yoshikawa M. 1999. LKPNM: A prodrug-type ACEinhibitory peptide derived from fish protein. Immunopharmacology 44:123–127. Hartmann R, Meisel H. 2007. Food-derived peptides with biological activity: From research to food applications. Curr Opin Biotechol 18:163–169. Hernandez-Ledesma B, Davalos A, Bartolome B, Amigo L. 2005. Preparation of antioxidant enzymatic hydrolysates from alphalactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS. J Agric Food Chem 53:588–593. Hockerman GH, Peterson BZ, Johnson BD, Catterall WA. 1997. Molecular determinants of drug binding and action on L-type calcium channels. Annu Rev Pharmacol Toxicol 37:361–396. Hooper NM. 1991. Angiotensin converting enzyme: Implications from molecular biology for its physiological functions. Intl J Biochem 23:641–647. Horiguchi N, Horiguchi H, Suzuki Y. 2005. Effect of wheat gluten hydrolysate on the immune system in healthy human subjects. Biosci Biotechnol Biochem 69(12):2445–2449. Jankowski A, Skorek A, Krzysko K, Zarzycki KP, Ochocka JR, Lamparczyk H. 1995. Captopril: Determination in blood and pharmacokinetics after single oral dose. J Pharm Biomed Anal 13:655–660. Japanese Ministry of Health, Labour and Welfare. http://www. mhlw.go.jp/. Kajimoto O, Nakamura Y, Yada H, Moriguchi S, Hirata H, Takahashi T. 2001. Hypotensive effects of sour milk in subjects with mild or moderate hypertension. J Jpn Soc Nutr Food Sci 54:347–354. Kawano Y, Ando K, Matsuura H, Tsuchihashi T, Fujita T, Ueshima H. 2007. Report of the Working Group for Dietary Salt Reduction of the Japanese Society of Hypertension: (1)

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Rationale for salt restriction and salt-restriction target level for the management of hypertension. Hypertens Res 30:879–886. Kawasaki T, Seki E, Osajima K, Yoshida M, Asada K, Matsui T, Osajima Y. 2000. Antihypertensive effect of Valyl-Tyrosine, a short chain peptide derived from sardine muscle hydrolyzate, on mild hypertensive subjects. J Hum Hypertens 14:519– 523. Kurokawa J, Adachi-Akahane S, Nagao Taku. 1997. 1,5benzothiazepine binding domain is located on the extracellular side of the cardiac L-type Ca2+ channel. Mol Pharmacol 51:262–268. Matoba N, Usui H, Fujita H, Yoshikawa M. 1999. A novel antihypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR mesenteric artery. FEBS Lett 452:181–184. Matsui T, Hayashi A, Tamaya K, Matsumoto K, Kawasaki T, Murakami K, Kimoto K. 2003. Depressor effect induced by dipeptide, Val-Tyr, in hypertensive transgenic mice is due, in part, to the suppression of human circulating renin-angiotensin system. Clin Exp Pharmacol Physiol 30:262–265. Matsui T, Imamura M, Oka H, Osajima K, Kimoto K, Kawasaki T, Matsumoto K. 2004. Tissue distribution of antihypertensive dipeptide, Val-Tyr, after its single oral administration to spontaneously hypertensive rats. J Pept Sci 10:535–545. Matsui T, Matsumoto K. 2006. Antihypertensive peptides from natural resources. In: THM Khan, A Ather, eds., Lead Molecules from Natural Products: Discovery and New Trends, 255–271. Amsterdam: Elsevier. Matsui T, Tamaya K, Seki E, Osajima K, Matsumoto K, Kawasaki T. 2002. Val-Tyr as a natural antihypertensive dipeptide can be absorbed into the human circulatory blood system. Clin Exp Pharmacol Physiol 29:204–208. Matsui T, Ueno T, Tanaka M, Oka H, Miyamoto T, Osajima K, Matsumoto K. 2005. Antiproliferation action of an angiotensin I-converting enzyme inhibitory peptides, Val-Tyr, via an L-type Ca2+ channel inhibition in cultured vascular smooth muscle cells. Hypertens Res 28:545–552. McCann BK, Shiell JB, Michalski PW, Lee A, Wan J, Roginski H, Coventry JM. 2006. Isolation and characterisation of a novel antibacterial peptide from bovine aS1-casein. Intl Dairy J 16:316–323. Mehta KP, Griendling KK. 2007. Angiotensin II cell signaling: Physiological and pathological effects in the cardiovascular system. Amer J Physiol–Cell Physiol 292:C82–97. Meisel H. 2005. Biochemical properties of peptides encrypted in bovine milk proteins. Curr Med Chem 12:1905–1919. Miguel M, Alvarez Y, López-Fandiño R, Alonso JM, Salaices M. 2007b. Vasodilator effects of peptides derived from egg white proteins. Regul Pept 140:131–135. Miguel M, Manso AM, López-Fandiño R, Alonso JM, Salaices M. 2007a. Vascular effects and antihypertensive properties of kappa-casein macropeptide. Intl Dairy J 17:1473–1477. Millatta JL, Abdel-Rahmanb ME, Siragy MH. 1999. Angiotensin II and nitric oxide: A question of balance. Regul Pept 81:1–10.

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Minami H, Morse EL, Adibi SA. 1992. Characteristics and mechanism of glutamine-dipeptide absorption in human intestine. Gastroenterology 103(1):3–11. Mine Y, Kovacs-Nolan J. 2006. New insights in biologically active proteins and peptides derived from hen egg. World’s Poultry Sci J 62:87–96. Miura S, Saku K, Karnik SS. 2003. Molecular analysis of the structure and function of the angiotensin II type 1 receptor. Hypertens Res 26:937–943. Morikawa K, Ishikawa K, Kanemaru Y, Hori G, Nagaoka S. 2007. Effects of dipeptides having a C-terminal lysine on the cholesterol 7α-hydroxylase mRNA level in HepG2 cells. Biosci Biotechnol Biochem 71(3):821–825. Nagaoka S, Futamura Y, Miwa K, Awano T, Yamauchi K, Kanamaru Y, Kojima T, Kuwata T. 2001. Identification of novel hypocholesterolemic peptides derived from bovine milk betalactoglobulin. Biochem Biophys Res Commun 281:11–17. Nakamura Y, Yamamoto N, Sakai K, 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. Okunishi T, Kawamoto Y, Kurobe Y, Oka K, Ishii K, Tanaka M, Miyazaki M. 1991. Pathogenic role of vascular angiotensinconverting enzyme in the spontaneously hypertensive rat. Clin Exp Pharmacol Physiol 18:649–659. Ririe GD, Roberts RP, Shouse NM, Zaloga PG. 2000. Vasodilatory actions of the dietary peptide carnosine. Nutrition 16:168–172. Sealey JE, Laragh JH. 1990. The renin-angiotensin-aldosterone system for normal regulation of blood pressure and sodium and potassium homeostasis. In: JH Laraph, BM Brenner, eds., Hypertension: Pathophysiology, Diagnosis, and Management, 1287–1317. New York: Raven Press. Sekiya S, Kobayashi Y, Kita E, Imamura Y, Toyama S. 1992. Antihypertensive effects of tryptic hydrolysate of casein on normotensive and hypertensive volunteers. J Jpn Soc Nutr Food Sci 45:513–517. Stepien O, Iouzalen L, Herembert T, Zhu D, Marche P. 1997. Amlodipine and vascular hypertrophy. Intl J Cardiol 62(Suppl 2):S79–84.

Suzuki A, Kagawa D, Ochiai R, Tokimitsu I, Saito I. 2002. Green coffee bean extract and its metabolites have a hypotensive effect in spontaneously hypertensive rats. Hypertens Res 25:99–107. Takahashi M, Moriguchi S, Yoshikawa M. 1994. Isolation and characterization of oryzatensin: A novel bioactive peptide with ileum-contracting and immunomodulating activities derived from rice albumin. Biochem Mol Biol Intl 33:1151–1158. Tanaka M, Matsui T, Ushida Y, Matsumoto K. 2006. Vasodilating effect of di-peptides in thoracic aortas from spontaneously hypertensive rats. Biosci Biotechnol Biochem 70(9):2292– 2295. Tanaka M, Tokuyasu M, Matsui T, Matsumoto K. 2008. Endothelium-independent vasodilation effect of di- and tripeptides in thoracic aorta of Sprague–Dawley rats. Life Sci 82:869–875. Tsuchida T, Masuko K, Osada H, Sato T. 2001. Effect of an aqueous extract from Mycoleptodonoides aitchisonii on human blood pressure. Jpn Pharmacol Thers 29:899–906. Vercruysse L, Morel N, Camp JV, Szust J, Smagghe G. 2008. Antihypertensive mechanism of the dipeptide Val-Tyr in rat aorta. Peptides 29:261–267. Vig SB, Stouch RT, Timoszyk KJ, Quan Y, Wall AD, Smith LR, Faria NT. 2006. Human PEPT1 pharmacophore distinguishes between dipeptide transport and binding. J Med Chem 49:3636–3644. Walker G, Cai F, Shen P, Reynolds C, Ward B, Fone C, Honda S, Koganei M, Oda M, Reynolds E. 2006. Increased remineralization of tooth enamel by milk containing added casein phosphopeptide-amorphous calcium phosphate. J Dairy Res 73:74–78. Yamakage M, Namiki A. 2002. Calcium channels—basic aspects of their structure, function and gene encoding. Can J Anaesth 49:151–164. Yamamoto N, Maeno M, Takano T. 1999. Purification and characterization of an antihypertensive peptide from a yogurt-like product fermented by Lactobacillus helveticus CPN4. J Dairy Sci 82:1388–1393. Zhu K, Zhou H, Qian H. 2006. Antioxidant and free radical scavenging activities of wheat germ protein hydrolysates (WGPH) prepared with alcalase. Process Biochem 41:1296–1302.

Chapter 5 Food Protein–Derived Peptides as Calmodulin Inhibitors Rotimi E. Aluko

Contents 1. Introduction, 55 2. Milk Protein–Derived Peptides, 56 3. Flaxseed Protein–Derived Peptides, 58

1. Introduction Recent trends in the functional foods and nutraceuticals industry have shown that bioactive peptides obtained from food proteins could play important roles in the prevention and therapeutic management of human diseases. The use of food proteins as substrates for the production of bioactive peptides is a suitable approach to formulating nutraceutical and functional foods that can be used against diseases without the need for a doctor ’s prescription. A potential target for food protein–derived bioactive peptides is calmodulin (CaM), a protein that plays important roles in maintaining physiological functions of cells and body organs (Cho et al. 1998). CaM consists of 148 amino acid residues and is a 16.7 kDa calcium-binding protein that plays multifunctional roles in the translation of intracellular messages (Klee and Vanaman 1982). Some of the functions of CaM include calcium-dependent cell division, cell proliferation, and neurotransmission (Veigel et al. 1984; Rasmussen and Means 1987). However, excessive levels of CaM could pose

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

4. Pea Protein–Derived Peptides, 62 5. Conclusion, 64 6. References, 64

serious problems within the body and may lead to the development of chronic diseases such as cancer, cardiac hypertrophy, and Alzheimer ’s disease (Hait and Lazo 1986; O’Day and Myre 2004; Obata et al. 2005). Therefore, bioactive inhibitors are designed to prevent or treat diseases that arise from excessive levels of CaM. At physiological pH, CaM is an acidic protein with a net negative charge; inhibitors are designed to have net positive charges in order to increase their affinity toward CaM (O’Neil and DeGrado 1990). This was demonstrated by the fact that a reduction in the number of net positively charged groups through acetylation led to decreased affinity and inhibitory effect of insect venom peptides toward CaM (Barnette et al. 1983). Calcium is required for activation of CaM and binding to calcium results in structural changes in the protein that leads to increased exposure of hydrophobic patches (Zhang et al. 1995). Therefore, in addition to a requirement for net positive charges at physiological pH, the presence of exposed hydrophobic amino acid residues could also enhance binding of inhibitory molecules to CaM. The main mechanism by which CaM influences cellular reactions is activation of various enzymes, including phosphodiesterases, protein kinases, and the isozymes that make up the group of nitric oxide synthases. CaM-dependent protein kinase II 55

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Part 2 Functions of Biologically Active Proteins and Peptides

(CaMKII) is responsible for the phosphorylation of various cellular proteins that participate in critical metabolic reactions (Soderling and Stull 2001; Praseeda et al. 2004). A unique feature of CaMKII is that it requires calcium and CaM only for an initial autophosphorylation of the enzyme’s two main 50and 60-kDa polypeptide units (Lai et al. 1986). Once phosphorylated, the enzyme becomes active and no longer requires calcium and CaM. It is believed that the initial autophosphorylation of CaMKII is a critical point during cellular proliferation and can lead to pathological conditions such as cancer and cardiac hypertrophy if this initial step is not properly regulated within the cell. In pathological conditions where the level of CaM becomes elevated, the use of agents such as bioactive peptides that bind to and block/decrease the interactions of CaM with CaMKII could serve as a therapeutic approach toward management of chronic diseases. Phosphodiesterase I is a CaM-dependent enzyme (CaM-PDE) that breaks down cyclic adenosine monophosphate (cAMP) within the cell matrix (Jiang et al. 1996). cAMP has been shown to have negative effects on cell proliferation and cell function through a mechanism that involves irreversible G1 arrest that is followed by cytolysis (Jiang et al. 1996). The induction of apoptotic cell death by cAMP has been shown to occur in normal and cancerous cells (Kizaki et al. 1990). Excessive levels of CaM-PDE cause the depletion of cellular cAMP and prevent physiological regulation of cell death, which can then lead to uncontrolled cell proliferation as seen in cancer pathology. Therefore, inhibition of CaM-PDE activity could elevate cellular levels of cAMP to restore controlled cell division and provide therapeutic relief during cancer. CaM-binding peptides obtained from food proteins could serve as suitable ingredients in the formulation of therapeutic products for the prevention and management of cancer development and metastasis. Nitric oxide synthases (NOS) are a group of CaM-dependent enzymes that are responsible for the production of nitric oxide from arginine (Alderton et al. 2001). Nitric oxide is a well-known cellular messenger that participates in neurotransmission, vasodilation, immune response, and smooth-muscle

contraction-relaxation, among several other functions (Alderton et al. 2001). However, excessive levels of nitric oxide could lead to development of pathological conditions such as acute and chronic neurodegenerative disorders that lead to stroke, Alzheimer ’s disease, Parkinson’s, migraine headaches, convulsion, and pain (Huang et al. 1999; Di Giacomo et al. 2003; Mühl and Pfeilschifter 2003). There are three main isozymes of NOS: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS), each with its own distinct physiological role. Whereas nNOS and eNOS are constitutive enzymes that require CaM binding only prior to activation of target enzymes, iNOS is permanently bound to CaM (Salerno et al. 1997). Therefore, CaM inhibitors are usually more effective against the constitutive NOS isozymes, since activation of target enzymes could be prevented through prevention of effective interactions between CaM and enzyme proteins. nNOS participates mostly in neurotransmission pathways, while eNOS has been implicated in vasodilatory activities and in the production of tumor necrosis factor-a (TNFa) by human monocytes or macrophages (Cho et al. 1998; Mühl and Pfeilschifter 2003). Therefore, eNOS could be regarded as a pro-inflammatory agent, and inhibitory compounds could provide therapeutic benefits during disease conditions. The aim of this review is to discuss the current state of knowledge with regard to food protein– derived peptides and protein hydrolysates that have been shown to inhibit the in vitro activities of three CaM-dependent metabolic enzymes: CaM-PDE, CaMKII, and NOS.

2. Milk Protein–Derived Peptides The initial works on food protein–derived CaM peptide inhibitors were carried out in Japan using α-casein as the substrate for proteolysis and CaMdependent phosphodiesterase (CaM-PDE) as the target enzyme for testing potency of peptides (Kizawa et al. 1995, 1996; Kizawa 1997). Casein was digested with pepsin followed by separation of the digest on a carboxymethyl Sephadex C-26

Chapter 5 Food Protein–Derived Peptides as Calmodulin Inhibitors

57

Protein Add protease Ultrafiltration

Low molecular weight permeates

Protein hydrolysate

Load onto a cation-exchange column Wash column to remove unbound peptides

Eluate (discard) Column retentate (bound peptides) Elute with salt or pH gradient Eluate (cationic peptide fractions) Test for calmodulin-binding properties Isolate and purify individual peptides from active fractions

Figure 5.1. Typical protocol involved during production of food protein–derived calmodulin-binding peptides.

Table 5.1. Calmodulin-binding peptides isolated from peptic hydrolysate of bovine casein and their potency against phosphodiesterase I.* Peptide Sequence LKKISQRYQKFALPQY VYQHQKAMKPWIQPKTKVIPYVRY VYQHQKAMKPWIQPKTKVIPYVRYL AMKPWIQPKTKVIPYVRYL KPWIQPKTKVIPYVRYL

IC50 (μM)

Position on the αs2-Casein Chain

65.0 7.0 2.6 3.2 1.1

f164-179 f183-206 f183-207 f189-207 f191-207

*Adapted from Kizawa et al. 1996.

cation-exchange column, which was eluted with ammonium carbonate in ammonium acetate gradient buffer. The bound fraction was collected and further purified on an HPLC reverse-phase column from which three main peptides were obtained and used for CaM-PDE inhibition assays. A general strategy for the production of CaM-binding peptides from food proteins is shown in Figure 5.1. The principal factor is the ability to obtain peptides that bind to a negatively charged column matrix at conditions similar to physiological pH values. Bound peptides are then eluted to obtain a positively charged group of peptides with a high potential to interact with CaM, a protein with net negative charges at physiological pH.

Table 5.1 shows the amino acid sequences, locations on casein primary structure, and inhibitory activities of the three peptides purified from pepsin hydrolysate of casein. The shortest peptide (16-mer) was the least active, while the 17-, 19-, 24-, and 25-mer peptides were the most active. The 16-mer peptide had fewer bulky aromatic amino acid residues (hydrophobic character), which could be responsible for the lower inhibitory property when compared to the 24- and 25-mer peptides that have higher levels of hydrophobic amino acids (Kizawa et al. 1995, 1996). Thus, it is evident from these earlier works that the content of hydrophobic amino acid residues is an important determinant of the inhibitory capacity

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Part 2 Functions of Biologically Active Proteins and Peptides

of food protein–derived CaM-binding peptides. However, Kizawa and co-workers also emphasized the importance of helix structure configuration of the casein peptides as an important determinant of their effectiveness in binding to CaM (Kizawa et al. 1996). Kizawa et al. (1996) also showed that the C-terminal residues of the casein peptides were the determinant group responsible for effective interaction with and inhibition of CaM activities. It was shown that under nondenaturing gel electrophoresis conditions, protein bands composed of CaM-casein peptide complexes were detected and provided evidence of the protein-ligand interactions (Kizawa et al. 1996). In contrast, earlier reports (Barnette et al. 1983; Comte et al. 1983) have suggested that the density of positively charged amino acid residues is an important determinant of the degree of potency of CaM-binding peptides. However, it should be noted that the long chain (16- to 25-mer) nature of the casein peptides could limit their oral bioavailability because of the potential for degradation in the gastrointestinal tract (GIT) by digestive proteases. Therefore, recent works have focused on producing short-chain (<7-mer) CaM-binding peptides from pea and flaxseed proteins in order to increase the potential to escape digestion in the GIT and be absorbed intact into the bloodstream for transport into target organs.

3. Flaxseed Protein–Derived Peptides Flaxseed is recognized as an excellent source of omega-3 fatty acids as well as bioactive phenolic compounds. In addition to these properties, however, flaxseed proteins have desirable levels of hydrophobic and positively charged amino acids that could enhance the production of CaM-binding peptides through enzymatic hydrolysis. Flaxseed proteins were hydrolyzed with alcalase, and the resultant peptide-rich digest was recovered as a supernatant after centrifugation of the reaction mixture (Omoni and Aluko 2006a). The supernatant was passed through a 1-kDa ultrafiltration membrane to isolate low molecular weight peptides in the permeate, which was then freeze-dried. The freeze-dried permeate was dissolved in ammonium acetate buffer,

adjusted to pH 7.0 using ammonium hydroxide, and fractionated using a column packed with SPSepharose, a strong cation-exchanger; bound peptides were eluted with a linear gradient of ammonium carbonate in ammonium acetate buffer. The flaxseed permeate was fractionated into two main cationic fractions on the SP-Sepharose column. Fraction I had a very high level of arginine (∼36%) at more than one-third of the total amount of amino acids but low levels of histidine (2%) and lysine (∼5%). Fraction II also had a high level of arginine (28% w/w) in addition to high levels of histidine (10%) and lysine (11%). Thus, positively charged amino acid residues accounted for half of the total level of amino acids in fraction II and about 42% in fraction I (Omoni and Aluko 2006a). Activity of nNOS was reduced in the presence of fractions I and II, with greater effects seen with fraction II. Potency of the flaxseed peptides was reduced as the concentration of CaM increased, indicating that the inhibitory effects of the peptides were manifested through interactions with CaM molecule and not direct interaction with nNOS (Omoni and Aluko 2006a). The mode of action of the flaxseed peptides was mostly through noncompetitive inhibition of CaM-dependent nNOS (Figure 5.2). This means that the binding site of the flaxseed protein–derived peptides on CaM is different from the binding site of nNOS. It could also mean that upon interacting with the peptides, CaM undergoes structural changes that reduce its ability to activate nNOS. Fraction II had a lower Ki value than fraction I, which indicates stronger affinity of fraction II for CaM (Omoni and Aluko 2006a). Moreover, catalytic efficiency of nNOS was higher in the absence of the inhibitory peptides, suggesting a peptidemediated reduction in the rate of enzyme catalysis. Structural changes in nNOS proteins as monitored by intrinsic fluorescence spectroscopy were monitored using tryptophan emission intensity. Due to the absence of tryptophan residues in CaM, the observed fluorescence data represented the structural properties of nNOS. When CaM was added to nNOS, there was an increase in tryptophan emission intensity (Table 5.2), suggesting a partial unfolding of the enzyme protein into a catalytic conformation;

Chapter 5 Food Protein–Derived Peptides as Calmodulin Inhibitors

Figure 5.2. Kinetics of the inhibition of neuronal nitric oxide synthase by flaxseed protein–derived cationic peptide fractions. Vo is the initial velocity, which is the net decrease in absorbance at 340 nm after a 30-minute reaction time. (A) Fraction I (42% content of positively charged amino acid residues) peptide concentrations (mg/ml): 0.0, 䊐; 1.5, 䊏; 2.5, 䊊; 3.5, 䊉. (B) Fraction II (50% content of positively charged amino acid residues) peptide concentrations (mg/ml): 0.0, 䊐; 0.25, 䊏; 0.75, 䊊; 1.0, 䊉. From Omoni and Aluko 2006a. Reproduced with copyright permission of Springer Publishers.

this is probably the ideal structure required for catalysis. Addition of flaxseed peptide fractions led to further increases in tryptophan fluorescence accompanied by a red shift in wavelength of maximum emission (Table 5.2), which is consistent with protein unfolding and interactions of the tryptophan residues with a hydrophilic environment. In this regard, fraction II was more effective than fraction

59

I in causing protein unfolding in nNOS (Table 5.2). In fact at a relatively high level of fraction II, there was a reduction in fluorescence intensity, indicating some structural adjustment (probably folding) that moved the tryptophan residues into the enzyme protein core. Such an effect could be the result of excessive binding of water molecules by the highly charged peptides such that the enzyme molecule is deprived of appropriate solvation and hence formation of a more closed structure. It is evident, therefore, that the peptides with high density of positively charged groups as shown for fraction II of the flaxseed peptides could have substantial effects on enzyme protein structure accompanied by reduction in catalytic efficiency. The effect of the flaxseed protein–derived peptides on structural and catalytic properties of eNOS was also investigated. Unlike nNOS, the inhibitory effect of fraction I peptides against eNOS was completely overcome at 50 nM CaM concentration (Omoni and Aluko 2006b). In contrast, fraction II retained its inhibitory property in the presence of 50 nM CaM level and with greater potency; IC50 values range from 3.2 to 14.51 mg/ml for fraction I when compared to 0.32–1.06 for fraction II. eNOS was inhibited mostly through a mixed type of enzyme inhibition, which suggests that the peptides inhibited catalysis by binding to CaM alone or the CaM/eNOS complex. Inhibition constants (Ki) were 2.13 and 0.01 mg/ml for fraction I and II, respectively (Omoni and Aluko 2006b). When compared to the Ki values obtained for nNOS, it is evident that the flaxseed peptides have greater affinity for eNOS (low Ki) than nNOS (high Ki). An implication of the results is that when used as a therapeutic product, the flaxseed peptides will affect eNOS to a greater extent than the nNOS. The effects of flaxseed peptides on CaMdependent eNOS protein molecule seem to be different from those observed for nNOS, though structural changes were greater in the presence of fraction II than fraction I (Table 5.2). Since CaM contains no tryptophan residue, the changes obtained at 295 nm represent the effects of peptides on eNOS protein molecules. The flaxseed peptides did not produce any substantial change in the wavelength of

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Part 2 Functions of Biologically Active Proteins and Peptides

Table 5.2. Effect of flaxseed protein-derived cationic peptide fractions on the intrinsic fluorescence of Ca2+/CaM-dependent nitric oxide synthases.* λmax (nm) Treatment None CaM CaM + 12.5 μg Fraction Ib CaM + 25 μg Fraction I CaM + 1.25 μg Fraction IIc CaM + 2.5 μg Fraction II CaM + 5 μg Fraction II

Fmax/Foa

nNOS

eNOS

nNOS

eNOS

331 330 334 337 333 334 329

331 334 333 331 333 330 330

— 1.07 1.62 1.96 1.22 2.08 0.73

— 0.99 0.97 1.22 0.92 0.94 0.85

*Adapted from Omoni and Aluko 2006a, 2006b. a Excitation wavelength of 295 nm; Fmax and Fo (arbitrary units) represent the maximum fluorescence intensity of nNOS or eNOS in the presence and absence of peptides, respectively. b First eluted fraction from SP-Sepharose column and contained 42% (w/w) of positively charged amino acid residues. c Second eluted fraction from SP-Sepharose column and contained 50% (w/w) of positively charged amino acid residues.

maximum emission, which shows that the microenvironment of the tryptophan residues in eNOS was not affected (Table 5.2). However, fluorescence intensity was greatly affected by peptides depending on the NOS isoform. At 5 μg/ml peptide concentration, the fluorescence intensity of CaM/nNOS complex was about 50% of the value obtained for CaM/eNOS complex, indicating a more open structure for the latter (Omoni and Aluko 2006a, 2006b). The Fmax/Fo value (ratio of fluorescence intensity in the presence and absence of peptides) provides a numerical indication of the structural changes that occur when proteins bind to ligands. A value of 1.0 indicates no effect of ligand on protein structure, while a higher value indicates protein unfolding that leads to a loose structural conformation with greater numbers of exposed aromatic groups. In contrast, if the Fmax/Fo ratio is less than 1.0, then there is protein folding into a more globular structure that shields the aromatic group from the environment. Overall, CaM/eNOS complexes with flaxseed peptides produced Fmax/Fo values that were less than 1, whereas similar nNOS complexes had values that were greater than 1.0, except at 5 μg/ml peptide concentration (Omoni and Aluko 2006a, 2006b). Therefore, it is evident that the flaxseed peptides

were more active against eNOS because of the more globular structural conformation that results from the interactions. It is possible that the globular structure shields the active site of eNOS from the substrates, which leads to reduced catalysis. In contrast, nNOS structural changes in the presence of flaxseed peptides seem to be mostly of the protein unfolding type, as shown by the >1.0 Fmax/Fo values (Omoni and Aluko 2006a). Pea protein–derived peptides also produced increases in Fmax/Fo values for CaMKII interactions (Li and Aluko 2005), which are similar to the values obtained for nNOS in the presence of flaxseed peptides. Thus it is reasonable to suggest that protein folding (increased globular conformation) may be a more effective way for inhibitory agents to modulate the catalytic activities of NOS isoforms and CaMKII. Apart from the effects on enzyme activities and protein structure, the flaxseed peptides were also investigated for their ability to modulate the structure of CaM alone (Table 5.3). Similar to the results obtained for the pea protein–derived peptides (Li and Aluko 2005), the flaxseed peptides induced protein unfolding (Fmax/Fo > 1.0) when added to CaM, which is also consistent with previous reports. Therefore, it is possible that one of the multiple

Chapter 5 Food Protein–Derived Peptides as Calmodulin Inhibitors

ways through which the peptides inhibited CaMdependent enzymatic reactions is excessive unfolding such that the CaM protein molecule loses its ability to effectively bind to and activate target enzymes. This is further evident in the results that showed changes in the wavelength of maximum emission depending on the peptide concentration. When compared to the active form of CaM where the tyrosine residues had maximum emission at 315 nm, all the peptide concentrations (with the exception of 25 μg/ml of fraction I) produced emissions at wavelengths of between 338 and 349 nm, indicating a red shift that is typically observed during protein unfolding (Table 5.3). Secondary structure of CaM as measured by circular dichroism (CD) showed that helical conforma-

Table 5.3. Effect of flaxseed protein–derived peptides on the intrinsic fluorescence of calmodulin.* Treatment none Ca2+ 12.5 μg Fraction Ib 25 μg Fraction I 1.25 μg Fraction IIc 2.5 μg Fraction II 5 μg Fraction II

λmax (nm)

Fmax/Foa

302 315 342 305 349 343 338

— 1.60 1.75 2.75 9.73 4.61 1.86

*Adapted from Omoni and Aluko 2006b. Excitation wavelength of 275 nm; Fmax and Fo (arbitrary units) represent the maximum fluorescence intensity of CaM in the presence and absence of Ca2+ or peptides, respectively. b First eluted fraction from SP-Sepharose column and contained 42% (w/w) of positively charged amino acid residues. c Second eluted fraction from SP-Sepharose column and contained 50% (w/w) of positively charged amino acid residues. a

61

tion (62%) increased in the presence of calcium when compared to 49% helical conformation for CaM alone (Table 5.4). The CD result confirms previous reports from far-UV data (LaPorte et al. 1980; Malencik and Anderson 1982) that have showed the need for higher α-helix content if CaM is to effectively modulate activity of target enzymes. The αhelix content of CaM was reduced to ∼54% when peptides were added to the calcium/CaM complex (Table 5.4), confirming that structural change in CaM protein molecule is a possible mechanism through which peptide-mediated inhibition of CaMdependent enzymatic reactions can occur. Also important is the fact that addition of calcium reduced the proportion of random structures in CaM but peptides had the opposite effect (Table 5.4), which indicates that a certain degree of orderly protein conformation is necessary for CaM to activate target enzymes. The near-UV data of CaM reflects the tertiary conformation, which was changed by calcium and peptides as shown by the intensities obtained for phenylalanine and tyrosine. In general, fraction II of the flaxseed peptides produced greater effect on phenylalanine intensity when compared to fraction I (Omoni and Aluko 2006b). Similar results were also obtained upon addition of pea protein–derived peptides to CaM (Li and Aluko 2005), which again confirms the fluorescence data that point to structural changes in CaM structure as a possible mechanism for the peptide-induced modulation of CaM activities. The results also confirm that increased levels of positively charged amino acid residues contribute to greater interactions between the food protein–derived peptides and CaM, ultimately

Table 5.4. Effect of flaxseed protein–derived peptides on secondary structure fractions of calmodulin.* Treatment None Ca2+ 0.5 mg/ml Fraction Ia 0.5 mg/ml Fraction IIb

α-Helix (%)

β-Sheet (%)

β-Turn (%)

Random (%)

49 62 53 54

13 11 28 21

15 13 4 6

24 16 19 19

*Adapted from Omoni and Aluko 2006b. First eluted fraction from SP-Sepharose column and contained 42% (w/w) of positively charged amino acid residues. b Second eluted fraction from SP-Sepharose column and contained 50% (w/w) of positively charged amino acid residues. a

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translating to decreased ability of CaM to modulate target enzymes.

4. Pea Protein–Derived Peptides Li and Aluko (2005) have produced CaM-binding peptides through alcalase-dependent hydrolysis of pea proteins. Pea protein isolate was hydrolyzed for 6 hours at pH 9.0 and 50°C using 4% (w/w) alcalase. Enzyme reaction was stopped by addition of HCl and the reaction mixture was centrifuged to obtain a supernatant that is rich in peptides. The supernatant was passed through a 1 kDa molecular weight cut-off ultrafiltration membrane, and the permeate that contained low molecular weight peptides was isolated and freeze-dried. In order to isolate desirable CaM-binding peptides, the 1 kDa permeate was fractionated on an SP-Sepharose column into two main fractions that contained positively charged peptides. Fraction I, which was eluted earlier from the column, was more abundant in terms of quantity of peptides when compared to fraction II that eluted later from the column. The two peptide fractions differed in amino acid composition, especially the levels of positively charged residues (lysine and arginine), which were ∼8% and 41% (w/w) for fraction I and II, respectively (Li and Aluko 2005). This is expected since peptides that have a high density of positively charged groups will reside longer on the cation-exchange column. Consequently, the two fractions also differed in their inhibitory effects toward CaMKII. Since the levels of hydrophobic amino acid residues were similar for both fractions, it seems that differences in the CaMKII inhibitory effects of fractions I and II were due mainly to the effects of charge density. Fraction I with less contents of the positively charged lysine and arginine residues had less inhibitory effects toward CaMKII when compared to fraction II that has 5–6 times the levels of those amino acids. The inhibitory concentration that reduced CaMKII activity by 50% (IC50) was at least 10 times lower for fraction II, which indicates higher potency than fraction I (Table 5.5). The IC50 for fraction I was very susceptible to CaM concentration, with values increasing substantially when the level of CaM was increased

Table 5.5. Inhibition (IC50 values) of CaM-dependent protein kinase II activity by pea protein–derived cationic peptide fractions at varying CaM levels.* CaM (μg/ml) 5 15 25 35

Fraction I (mg/ml)a

Fraction II (mg/ml)b

2.78 4.00 5.20 22.98

0.24 0.30 0.37 0.40

*Adapted from Li and Aluko 2005. a First eluted fraction from SP-Sepharose column and contained 10% (w/w) content of basic amino acids residues. b Second eluted fraction from SP-Sepharose and contained 43% (w/w) content of basic amino acid residues.

(Table 5.5). This demonstrates that the observed inhibitory effects were due mainly to the ability of pea protein–derived peptides to bind with CaM and inhibit activation of CaMKII. With an increase in CaM concentration, inhibition of CaMKII by fraction I peptides was severely reduced, suggesting that the peptides probably compete with the enzyme for binding onto CaM. By contrast, the inhibition of CaMKII activity by fraction II was not substantially influenced by the level of CaM present (Table 5.2). Therefore, it is possible that the affinity between fraction II peptides and CaM was very strong because of the high levels of lysine and arginine, and increasing the levels of CaM could not easily displace the peptides from the enzyme-binding site on CaM molecule. Proof of strong interactions between fraction II and CaM was obtained from the inhibition constant (Ki), which was 0.019 mg/ml when compared to the value of 0.47 mg/ml for fraction I (Li and Aluko 2005). A lower value of Ki indicates higher affinity of the peptides for CaM and consequently less activation of CaMKII. Both fractions inhibited CaM in a competitive way, suggesting that enzyme binding sites on CaM were occupied by the peptides. Km values were significantly higher (p < 0.05) for fraction II, which reinforces the fact that CaMKII activity was inhibited to a much greater extent by high density of positively charged groups than by a lower density. In order to further determine the molecular basis for the inhibition of CaMKII by pea protein–derived

Chapter 5 Food Protein–Derived Peptides as Calmodulin Inhibitors

peptides, the tertiary structure of enzyme protein was measured using intrinsic and extrinsic fluorescence as well as CD (Li and Aluko 2006). Apo-CaM (which lacks calcium) has very low intrinsic fluorescence, indicating folding of protein structure such that most of the phenylalanine and tyrosine residues are located away from the surface of the protein. However, holo-CaM (containing bound calcium) had an almost four-fold increase in intrinsic fluorescence, which indicates that interactions with calcium caused partial unfolding of the CaM molecule. Addition of pea protein–derived peptides to the calcium-CaM complex resulted in an even higher increase in intrinsic fluorescence, suggesting greater unfolding of protein structure (Li and Aluko 2006). Similar trends were also obtained when the intrinsic fluorescence of the enzyme-calcium-CaM complex was studied (Table 5.6). In both instances, there was no significant difference in the wavelength of maximum fluorescence intensity, which indicates that the microenvironment of tryptophan residues in CaMKII did not change in the presence of bound ligands (Table 5.6). The authors concluded that it is possible that the initial binding of calcium causes an optimum CaM protein unfolding such that it can bind to and activate target enzymes through exposed hydrophobic Table 5.6. Effect of pea protein-derived cationic peptide fractions on the intrinsic fluorescence of Ca2+/CaM-dependent protein kinase (CaMKII).* Sample CaMKII CaMKII + 10 μg Fraction Ib CaMKII + 20 μg Fraction I CaMKII + 40 μg Fraction I CaMKII + 10 μg Fraction IIc CaMKII + 20 μg Fraction II CaMKII + 40 μg Fraction II

λmax (nm)

Fmax/Foa

325 325 325 325 325 325 325

— 0.63 0.79 1.33 0.82 0.90 5.25

*Adapted from Li and Aluko 2006. Excitation wavelength of 295 nm; Fmax and Fo (arbitrary units) represent the maximum fluorescence intensity of CaMKII in the presence and absence of peptides, respectively. b First eluted fraction from SP-Sepharose column and contained 10% (w/w) content of basic amino acid residues. c Second eluted fraction from SP-Sepharose and contained 43% (w/w) content of basic amino acid residues. a

63

residues. Binding of peptides seemed to have caused excessive CaM protein unfolding such that CaMKII could not be activated. Therefore, excessive protein unfolding could be a mechanism by which pea protein–derived peptides reduce the affinity of CaM for its target enzymes. This was evidenced by the fact that intrinsic fluorescence of CaM was much higher in the presence of the more potent pea protein– derived peptide fraction II than in the presence of fraction I (Li and Aluko 2006). ANS (8-anilino-1-naphthalene sulfonic acid) is a hydrophobic probe that is used to estimate the exposure of aromatic residues in proteins. Fluorescence intensity of ANS is increased upon interactions with aromatic amino acid residues and could provide an estimation of the level of protein unfolding to complement information obtained from intrinsic fluorescence measurements. ANS fluorescence was increased substantially (from 171 to 247 arbitrary units) when added to CaM in the presence of calcium. Slight increase (from 247 to 256 arbitrary units) in ANS fluorescence was obtained when cationic pea peptide fraction I was added to the calcium-CaM complex, which is consistent with increased exposure of aromatic amino acid residues (Li and Aluko 2006). There was also a noticeable blue shift (from 502 to 494 nm) in the wavelength of maximum fluorescence emission upon interaction of CaM with the pea peptide fractions. This is an indication that the ANS molecules become bound to more hydrophobic environments that were exposed as a result of peptide-dependent CaM protein unfolding. Tertiary structure measurements with CD indicate that the presence of calcium led to increased intensities of bands associated with phenylalanine and tyrosine, which is an indication of molecular rearrangements within the CaM molecule. Addition of pea protein–derived peptides led to more pronounced intensity of these bands, with fraction II having a stronger effect than fraction I (Li and Aluko 2006). Though there was no change in the wavelength of the band transitions, the increased intensity indicates that structural modulation of CaM was caused by the pea protein–derived peptide fractions. This structural modulation was greater

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when peptides were present than when calcium alone was present. Results from the pea protein– derived peptides were similar to those obtained when the tertiary structure of CaM was studied in the presence of flaxseed protein–derived peptides. When combined with data from fluorescence spectroscopy, it is evident that the pea protein–derived peptides bind to CaM to induce structural changes that prevent normal functioning of CaM and associated metabolic enzymes. Therefore, pea protein– derived cationic peptides may be useful in the formulation of functional foods and nutraceuticals aimed at preventing or management of diseases that are dependent on CaM activity.

5. Conclusion The current stage of knowledge indicates that CaMbinding peptides can be produced through the enzymatic hydrolysis of food proteins followed by separation and purification of cationic peptides. Inhibition of CaM-dependent enzymes seems to be dependent mostly on the level of basic (positively charged) amino acid residues for short-chain (<7mer) peptides, whereas long-chain (16- to 25-mer) peptides interact with CaM mostly through hydrophobic interactions. From kinetic experiments, it is evident that inhibition of CaM-dependent enzymes can occur through various mechanisms that involve interactions of peptides with active sites as well as nonactive sites of CaM. Structural changes occur during peptide binding leading to an increased unfolding of CaM such that the protein loses its ability to effectively interact with and activate target metabolic enzymes. The enzymes behave differently in the presence of inhibitory peptides; eNOS assumes a more globular conformation while nNOS and CaMKII have more open and unfolded structures. However, either conformational change leads to reduced enzyme activity, though the peptideinduced increase in globular structure seems to be more effective than the open structure. Overall it is evident that food protein–derived CaM-binding peptides constitute a potential source of ingredients that may be used to formulate therapeutic diets against human chronic diseases.

However, the ultimate utility of these cationic peptides will depend on their oral bioavailability and in vivo inhibition of target metabolic reactions.

6. References Alderton WK, Cooper CE, Knowles RG. 2001. Nitric oxide synthases: Structure, function and inhibition. Biochem J 357(3):593–615. Barnette MS, Daly R, Weiss B. 1983. Inhibition of calmodulin activity by insect venom peptides. Biochem Pharmacol 32(19):2929–2933. Cho MJ, Vaghy PL, Kondo R, Lee SH, Davis JP, Rehl R, Heo WD, Johnson JD. 1998. Reciprocal regulation of mammalian nitric oxide synthase and calcineurin by plant calmodulin isoforms. Biochemistry 37(45):15593–15597. Comte M, Maulet Y, Cox JA. 1983. Ca2+-dependent high-affinity complex formation between calmodulin and melittin. Biochem J 209(1):269–272. Di Giacomo C, Sorrenti V, Salerno L, Cardile V, Guerrera F, Siracusa MA, Avitabile M, Vanella A. 2003. Novel inhibitors of neuronal nitric oxide synthase. Exp Biol Med 228(5):486– 490. Hait WN, Lazo JS. 1986. Calmodulin: A potential target for cancer chemotherapeutic agents. J Clin Oncol 4:994–1012. Huang H, Martasek P, Roman LJ, Masters BSS, Silverman RB. 1999. Nω-Nitroarginine-containing dipeptide amides. Potent and highly selective inhibitors of neuronal nitric oxide synthase. J Med Chem 42(16):3147–3153. Jiang X, Li J, Paskind M, Epstein PM. 1996. Inhibition of calmodulin-dependent phosphodiesterase induces apoptosis in human leukemic cells. Proc Natl Acad Sci USA 93(10): 11236–11241. Kizaki H, Suzuki K, Tadakuma T, Ishimura Y. 1990. Adenosine receptor-mediated accumulation of cyclic AMP-induced T-lymphocyte death through internucleosomal DNA cleavage. J Biol Chem 265(9):5280–5284. Kizawa K. 1997. Calmodulin binding peptide comprising αcasein exorphin sequence. J Agric Food Chem 45(5):1579– 1581. Kizawa K, Naganuma K, Murakami U. 1995. Calmodulinbinding peptides isolated from κ-casein peptone. J Dairy Res 62(4):587–592. ———. 1996. Interactions of amphiphilic peptides derived from κs2-casein with calmodulin. J Dairy Sci 79(10):1728–1733. Klee CB, Vanaman TC. 1982. Calmodulin. Adv Protein Chem 35:213–221. Lai Y, Nairn AC, Greengard P. 1986. Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/ calmodulin-dependent protein kinase II. Proc Natl Acad Sci USA 83(12):4253–4257. LaPorte DC, Wierman BM, Storm DR. 1980. Calcium-induced exposure of a hydrophobic surface on calmodulin. Biochemistry 19(16):3814–3819.

Chapter 5 Food Protein–Derived Peptides as Calmodulin Inhibitors

Li H, Aluko RE. 2005. Kinetics of the inhibition of calcium/ calmodulin-dependent protein kinase II by pea protein–derived peptides. J Nutr Biochem 16(11):656–662. ———. 2006. Structural modulation of calmodulin and calmodulin-dependent protein kinase II by pea protein hydrolysates. Intl J Food Sci Nutr 57(3/4):178–189. Malencik DA, Anderson SR. 1982. Binding of simple peptides, hormones, and neurotransmitters by calmodulin. Biochemistry 21(14):3480–3486. Mühl H, Pfeilschifter J. 2003. Endothelial nitric oxide synthase: A determinant of TNFα production by human monocytes/ macrophages. Biochem Biophys Res Commun 310(3):677– 680. Obata K, Nagata K, Iwase M, Odashima M, Nagasaka T, Izawa H, Murohara T, Yamada Y, Yokota M. 2005. Overexpression of calmodulin induces cardiac hypertrophy by a calcineurindependent pathway. Biochem Biophys Res Commun 338(2): 1299–1305. O’Day DH, Myre MA. 2004. Calmodulin-binding domains in Alzheimer ’s disease proteins: Extending the calcium hypothesis. Biochem Biophys Res Commun 320(4):1051–1054. Omoni A, Aluko RE. 2006b. Effect of cationic flaxseed protein hydrolysate fractions on the in vitro structure and activity of calmodulin-dependent endothelial nitric oxide synthase. Mol Nutr Food Res 50(10):958–966.

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——— RE. 2006a. Mechanism of the inhibition of calmodulindependent neuronal nitric oxide synthase by flaxseed protein hydrolysates. J Amer Oil Chem Soc 83(4):335–340. O’Neil KT, DeGrado WF. 1990. How calmodulin binds its targets: Sequence independent recognition of amphiphilic αhelices. Trends Biochem Sci 15(2):59–64. Praseeda M, Mayadevi M, Omkumar RV. 2004. Interaction of peptide substrate outside the active site influences catalysis of CaMKII. Biochem Biophys Res Commun 313(3):845–849. Rasmussen CD, Means AR. 1987. Calmodulin is involved in regulation of cell proliferation. EMBO J 6(13):3961–3968. Salerno JC, Harris DE, Irizarry K, Patel B, Morales AJ, Smith SME, Martasek P, Roman LJ, Masters BSS, Jones CL, Weissman BA, Lane P, Liu Q, Gross SS. 1997. An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase. J Biol Chem 272(47):29769–29777. Soderling TR, Stull JT. 2001. Structure and regulation of calmodulin-dependent protein kinases. Chem Rev 101(8): 2341–2351. Veigel ML, Vanaman TC, Sedwick WD. 1984. Calcium and calmodulin in cell growth and transformation. Biochem Biophys Acta 738(1–2):21–48. Zhang M, Tanaka T, Ikura M. 1995. Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nature Struct Biol 2(9):758–767.

Chapter 6 Soy Protein for the Metabolic Syndrome Cristina Martínez-Villaluenga and Elvira González de Mejía

Contents 1. Introduction, 67 2. Pathogenesis of the Metabolic Syndrome, 69 3. Effect of Soy Protein in Weight Loss and Adiposity Reduction, 71 4. Effect of Soy Protein in Reduction of Caloric Intake, 74

1. Introduction The term “syndrome X,” known later as the metabolic syndrome (MS), was introduced by Reaven (1988) for the first time while discussing the role of insulin resistance in disease. The MS consists of a deadly quintet of factors, namely diabetes, centripetal obesity, hypertension, dyslipidemia (elevated triglycerides, dense low-density lipoproteins, and low high-density lipoproteins), and alterations in the thrombotic potential that are related to hyperinsulinemia and insulin resistance (IR) (Fulop et al. 2006; Hollander and Mechanick 2008). The definition of MS is still not standardized, and each professional association has its own definition and criteria of diagnosis (Table 6.1). Existing data suggest that the incidence of the MS is rising at an alarming rate (Cankurtaran et al. 2006). Regarding the prevalence of the syndrome, the MS appeared to affect 23% of the American

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

5. Role of Soy Protein in Glycemic Control and Insulin Resistance, 74 6. Effect of Soy Protein on Serum Lipids, 76 7. Effect of Soy Protein on Blood Pressure, 79 8. Conclusion, 80 9. References, 81

population (Ford et al. 2004) and 15% of the European population (Hu et al. 2004). As for Asians, the prevalence varied from 7% in Korea (Lee et al. 2004) to 30% in Iran (Azizi et al. 2003), 32% in India (Gupta et al. 2004), and 40% in middle-aged and elderly Chinese (Ye et al. 2007). Currently, the MS is a real epidemic not only affecting middleaged people but also adolescents and elderly (15– 20% over 70 years of age). In addition, it is important to note that the prevalence of the MS is highly agedependent (Fulop et al. 2006). The MS is an important public health problem that led the National Cholesterol Education Program (NCEP) to publish a series of clinical management guidelines (NCEP 2001). Recommendations include lifestyle changes, healthful eating, and increased physical activity as a first line of intervention in the prevention and therapy of obesity and the MS (Hollander and Mechanick 2008). Dietary advice represents a key element in improving lifestyle and generally aims to limit saturated fat intake, restrict energy intake, and stimulate energy expenditure. The main goal is to reduce fat mass and lower the prevalence of obesity, which has been associated with IR and the development of type 2 diabetes 67

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Table 6.1. Criteria for definitions and diagnoses of risk factors in the metabolic syndrome, according to the World Health Organization (WHO), National Cholesterol Education Program (NCEP), Adult Treatment Panel III (ATP III), and the International Diabetes Federation (IDF). Risk Factor

WHOa

NCEP ATP IIIb

IDFc

Criteria of diagnosis

Type 2 diabetes, impaired glucose tolerance plus two or more risk factors Waist-hip ratio >0.9 (male) or >0.85 (female) and/or body mass index >30 ≥150 <35 (male), <39 (female)

Any three risk factors

Increased waist circumference plus two or more risk factors Elevated waist circumference dependent of ethnicity

≥140/90 Impaired glucose tolerance, impaired fasting glucose, type 2 diabetes 30 mg albumin/g creatinine

≥130/85 ≥100

≥130/85 ≥100

—

—

Obesity

Triglycerides (mg/dL) High-density lipoprotein cholesterol (mg/dL) Hypertension (mmHg) Fasting plasma glucose (mg/dL) Microalbuminuria

Waist circumference >40 (male) or >35 (female) ≥150 <40 (male), <50 (female)

≥150 <40 (male), <50 (female)

Adapted from Hollander and Mechanick 2008. http://www.who.int/diabetesactiononline/diabetes/basics/en/index4.html (accessed November 2008). b http://www.americanheart.org/presenter.jhtml?identifier=4500 (accessed November 2008). c http://www.idf.org/webdata/docs/Diabetes_meta_syndrome.pdf (accessed November 2008). a

(International Diabetes Federation 2004). Therapeutic diets including high amounts of vegetables, fruits, legumes, whole grains, low-fat dairy foods, and low amounts of saturated fats and salt have been used in previous studies (Esposito et al. 2004; Azadbakht et al. 2005). Proteins are formed by amino acids and serve as the major structural component of muscle and other tissues in the body and are also used to produce hormones, enzymes, and hemoglobin. High-protein diets have become a method to enhance weight reduction (Johnston et al. 2004) due to feelings of satiety and reduction in caloric consumption (Eisenstein et al. 2002). For adults, a proper combination of vegetable proteins may provide similar benefits as proteins from animal sources. Interesting data exist on the health benefits associated with soy protein consumption (Hoffman and Falvo 2004). There are different processing technologies used to produce commercially available soy proteins (Figure 6.1). It is therefore imperative to consider how these processes affect the protein composition (Singh et al. 2008).

Soy protein is of particular interest because of its association with several health benefits, including weight loss and prevention of complications related to cardiovascular risk factors, as well as the improvement of lipid profile and glucose and insulin homeostasis (Sirtori and Johnson 2006; Velasquez and Bhathena 2007). The beneficial effects of soy protein are the result of its amino acid pattern, the biological activities of soy peptides, and nonprotein compounds such as isoflavones (Torres and Tovar 2007). Therefore, it has been hypothesized that the intake of soy protein may be associated with a decreased risk of MS. This proposition has been tested in animal studies (Dyrskog et al. 2005; Davis et al. 2005, 2007) and a few clinical trials (Azadbakht et al. 2007a, 2007b; Pan et al. 2008). Azadbakht et al. (2007a) found that 30 g/d of soy protein or soy nuts as a replacement for red meat used in the Dietary Approaches to Stop Hypertension diet significantly improved glycemic control and lipid profiles in postmenopausal women. Pan et al. (2008) found that habitual soy protein intake in 2,811 middle-aged and elderly Chinese (7.82 g/d)

Chapter 6 Soy Protein for the Metabolic Syndrome

Soybean Oil oil

Hulls Soybeans

Grinding

Cleaning Cracking

Soy Grits grits

Flaking

Soy chips Chips

Fat Oil extraction Extraction

Edible Defatted defatted Flakes flakes

Grinding and Sizing sizing Sugar removal Removal

Protein extraction Extraction

Blending

Drying

Protein precipitates Precipitates

Texturizing

Soy Protein protein Concentrate concentrate

Water

69

Soy flour Flour

Drying Screening

TSP products Products

Drying

Isolated Soy soy Protein protein

Figure 6.1. Typical industrial processing of soybeans (adapted from Singh et al. 2008). TSP = texturized soy protein.

tended to be associated with a reduced risk of MS in women but an elevated risk in men. In this study, soy protein intake was positively associated with hyperglycemia in men, whereas it was inversely associated with elevated blood pressure. This chapter addresses the state of scientific evidence regarding the effect of proteins, especially soy protein, on the different factors (central obesity, diabetes, dyslipidemia, and hypertension) that comprise the MS. In particular, the proposed mechanisms of action that could explain the impact of soy protein on the MS will be discussed.

2. Pathogenesis of the Metabolic Syndrome Even after decades of research, the pathogenesis of the MS is poorly understood because it is a multifac-

tor phenomenon including environmental, genetic, and epigenetic causes (Laakso and Kovanen 2006; Magliano et al. 2006). Dietary factors and physical activity, mental stress, and environmental toxicants can influence gene expression and have shaped the genome over several million years of human evolution (Pella et al. 2003). There is gene susceptibility to diseases, although environmental factors determine which susceptible individuals will develop the MS. Rapid changes in diet and lifestyle resulting from socioeconomic changes provide added stress, causing exposure of underlying genetic predisposition to chronic diseases such as type 2 diabetes, obesity, hypertension, coronary artery disease, and atherosclerosis. IR and abdominal obesity seem to be the most acceptable processes that may explain the MS including all or at least most of its components (Figure 6.2).

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Figure 6.2. Pathogenesis of the metabolic syndrome. BP = blood pressure; HDL = high-density lipoprotein; IL-1 = interleukin 1; IL-6 = interleukin 6; LDL = low-density lipoprotein; TG = triglycerides; TNF-α = Tumor necrosis factor α.

It is believed that obesity and inactivity promote IR (Abassi et al. 2002) and compensatory hyperinsulinemia. The accumulation of adipose tissue, particularly central visceral adiposity, likely incites the IR and proinflammatory state to develop the MS. Adipose tissue is not only an efficient energy reservoir but also a metabolically active endocrine organ in constant communication with other tissues, including the central nervous system. Adipose tissue exerts its systemic effects via a number of substances, including free fatty acids (FFA), which impair insulin signaling

in multiple organs, and several adipokines, including adiponectin, leptin, visfatin, and resistin (StehnoBittel 2008). Alterations in the concentrations of adipokines exacerbate the inflammatory state by upregulating inflammatory cytokines (i.e., tumor necrosis factor-α, TNF-α; and interleukin 6, IL-6) and contribute to disordered insulin signaling (Johnson and Weinstock 2006). So fat itself via these mediators may in fact be the pathologic impetus behind MS. The exact mechanism leading from obesity to inflammation is not clear, but there are several

Chapter 6 Soy Protein for the Metabolic Syndrome

hypotheses. One of these states that the larger number of enlarged adipocytes associated with obesity actually causes a local oxygen shortage, triggering a state of chronic hypoxia and local inflammation in the surrounding cells (Trayhurn and Wood 2005). Conversely, it is known that adipocytes themselves can stimulate inflammatory responses. Locally secreted adipokines such as monocyte chemoattractant protein 1 (MCP-1), resistin, leptin, and adiponectin attract pro-inflammatory macrophages into adipose tissue, where they encircle dying adipocytes (Horio 2007; Stehno-Bittel 2008). The macrophages release their own markers that further stimulate the inflammatory process (Kim et al. 2005). In addition, obesity often is accompanied by increased levels of FFA, which could activate proinflammatory responses in blood vessels, fat cells, and immune cells (Kim et al. 2005). Hence there was development of the concept of fat dysfunction, whereby pathologic disturbances in fat function (i.e., adiposopathy) contribute to familiar metabolic consequences including type 2 diabetes, dyslipidemia, hypertension, and cardiovascular disease (CVD) (Sattar et al. 2003; Bays et al. 2006). Therefore, food components with an impact in reduction of adiposity would be of great value either in the prevention or the improvement of the metabolic syndrome.

3. Effect of Soy Protein in Weight Loss and Adiposity Reduction Animal models have demonstrated that soy protein has a positive effect on weight and fat loss that was especially prominent when comparing casein-based diets to soy-based diets (Table 6.2). Some of these studies suggested that β-conglycinin fraction is responsible for the effect of soy protein in weight and fat loss (Moriyama et al. 2004; Koba et al. 2007; Martinez-Villaluenga et al. 2008). A recent epidemiological study examined the relation between soy consumption and body mass index (BMI) among 1,418 women in Hawaii (Maskarinec et al. 2008). Women consuming more soy during adulthood had a lower BMI. Studies investigating weight loss in overweight and obese humans suggest

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that soy as a source of dietary protein may be used to achieve significant weight loss on low-calorie diets, although there is no convincing evidence to show whether soy protein is better than other protein sources to achieve weight loss (Anderson and Hoie 2005; Anderson et al. 2007; Lukaszuk et al. 2007). There is one study concluding that supplementation with soy protein–rich foods does not enhance weight loss in overweight women (St-Onge et al. 2007). These authors explained their results by the fact that the amount of soy protein provided in the diets (25 g soy protein/d) was lower compared to other studies; therefore, they suggested that soy protein intakes >30 g/day may be necessary to assist in weight loss. Moreover, they reported that providing soy protein as a liquid meal may also have played a role in determining weight-loss success. Other studies also provided soy protein in the form of a liquid meal (Allison et al. 2003; Deibert et al. 2004; Anderson and Hoie 2005; Anderson et al. 2007; Lukaszuk et al. 2007; Konig et al. 2008). The liquid meal, a defined controlled portion, in the treatment group may have provided added benefits to the weight-loss plan and resulted in improved compliance relative to the control group, which did not receive a liquid formula diet. A metaanalysis of meal-replacement studies has found greater efficacy of liquid meal replacements for weight loss compared to dietary counseling alone (Heymsfield et al. 2003). It has been proposed that soy protein may reduce adiposity by modulating the expression of sterol regulatory element binding proteins (SREBPs), a family of transcription factors that control multiple genes involved in fatty acid and cholesterol synthesis (Figure 6.3). In rats, soy protein feeding was shown to reduce hepatic lipogenesis through the reduced expression of the hepatic SREBP-1 and mRNA level of lipogenic enzymes including fatty acid synthase (FAS), acetyl-Co A carboxylase, steroyl-CoA-desaturase-1, and Δ-5 and Δ-6 desaturases (Tovar et al. 2005; Xiao et al. 2006; Takahashi and Ide 2008). In addition, the soy protein diet may also ameliorate fatty liver through reduced clearance of very-low-density lipoprotein (VLDL) by the liver and increase hepatic activities of mitochondrial and peroxisomal β-oxidation (Gudbrandsen et al. 2006).

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Table 6.2. Effect of soy protein intake on body weight and adiposity reduction and metabolic parameters in recent animal studies. Duration (Weeks)

Model

Diet

Obese (KK yellow) mice (n = 10)

Casein vs. soy protein isolate (20% protein)

2

Obese (KK yellow) mice (n = 10)

Energy-restricted diets (20% protein, 30% fat) Casein vs. β-conglycinin vs. glycinin Casein vs. soy protein (20%)

2

Male Sprague Dawley rats (n = 10) Rats (n = 10)

Casein vs. soy protein (20% protein) Casein vs. soy protein in high-fat diet (% protein not specified)

2

Obese male ZDF rats (n = 10)

Casein vs. low or high isoflavone soy protein (23%)

11

Male rats (n = 10)

Soy protein isolate (SPI) β-conglycinin (CON) (20%) vs. casein (CAS)

4

Diet-induced obese rats (n = 8)

Low-fat diets (5% fat) and high-fat diets (25% fat) containing casein or soy protein (30%)

12

Rats with metabolic syndrome (n = 10)

36

25

Body Weight

Metabolic Parameters

References

Significant lower body weight and less adipose tissue (mesenteric, epididymal, and brown fat) in soy protein–fed group. Mice fed β-conglycinin diet had lower body weights and lower liver weights than corresponding groups fed casein diet. Soy protein–diet group (with either low or high isoflavone content) had significantly lower body and liver weight.

Plasma cholesterol, TG, FFA, and glucose levels were decreased more effectively by soy diet.

Nagasawa et al. 2002

Soy β-conglycinin–fed animals showed lower serum TG, insulin, and glucose levels.

Moriyama et al. 2004

Low soy protein diet exhibited better glycemic control. High isoflavone soy protein–fed rats had higher glucose and TG levels and lower plasma insulin. Not determined

Davis et al. 2005

Epididymal adipose tissue was lower in soy protein–fed rats. Reduction in epididymal adipocyte number and area and lower serum leptin and FFA concentration in rats fed soy protein. Body weight and liver adiposity was only significantly reduced in high isoflavone soy protein–fed group. Higher reduction in epididymal and perineal adipose tissues in SPI and CON-fed groups compared with CAS-fed group. Rats fed soy protein diet showed smaller adipocyte area, which was associated with a lower body fat content, significantly lower lipid droplet area in adipose tissue, and lower weight gain.

Soy protein–fed animals showed lower serum FFA levels.

Soy protein–fed animals showed lower fasting glucose, improved plasma insulin level, and improved dyslipidemia. Soy protein–fed animals showed lower serum TG and higher serum adiponectin levels. Soy protein–fed animals showed higher glucagon, lower serum leptin, cholesterol, and TG.

Morifuji et al. 2006 Torre-Villalvazo et al. 2006

Davis et al. 2007

Koba et al. 2007

Torre-Villalvazo et al. 2008

Chapter 6 Soy Protein for the Metabolic Syndrome

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Figure 6.3. Proposed molecular mechanism of soy protein action on adiposity and caloric intake reduction (adapted from Torres and Tovar 2007). CCK = Cholecystokinin; FFA = Free fatty acids; GLP-1 = Glucagon-like peptide 1; PPAR = Peroxisome proliferator activated receptor; SREBP = Sterol regulatory element binding protein; TG = Triglycerides; UCPs = Uncoupling proteins; VLDL = Very low density lipoproteins.

Histological analysis of epididymal adipose tissue from rats fed soy protein revealed that there were more adipocytes per area but they were smaller in size than those fed casein (Torre-Villalvazo et al. 2006, 2008). This indicates that soy protein prevents hypertrophy and stimulates adipocyte hyperplasia associated with high peroxisome proliferator activated receptor gamma (PPARγ) gene expression, preventing the release of excessive amounts of FFA to the circulation (Torres and Tovar 2007; Takahashi and Ide 2008). PPARγ ) is responsible for adipocyte differentiation, fatty acid storage, and sensitizing the body to insulin (Torres and Tovar 2007). Soy protein

may reduce hepatic lipotoxicity reducing adipocyte hypertrophy, increasing perilipin expression, and preventing the transfer of FFA to extra adipose tissues (Torre-Villalvazo et al. 2006, 2008). Moreover, soy protein feeding in rats decreased hepatic triacylglycerol levels and epididymal adipose tissue weight mediated by an increased activity and mRNA levels of hepatic and skeletal muscle enzymes involved in fatty acid oxidation, including carnitine palmitoyltransferase (CPT1), beta-hydroxyacylCoA dehydrogenase (HAD), acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase (Morifuji et al. 2006; Takahashi and Ide 2008). In addition,

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PPARγ and PPARα mRNA levels were also found to be elevated, suggesting that soy protein intake stimulates hepatic and skeletal muscle fatty acid oxidation by activating PPAR pathways leading to reduced accumulation of body fat in rats (Takahashi and Ide 2008; Torre-Villalvazo et al. 2008) and in male monkeys (Wagner et al. 2008). Moreover, brown adipose tissue was lower in rats fed a soy and high-fat diet probably due to higher mRNA levels of uncoupling proteins (UCP 1, 2, and 3). Therefore, it is possible that an increase in energy expenditure through the increased mRNA levels of PPARγ and subsequent upregulation of UCPs is involved in the antiobesity effect of soy protein (Takahashi and Ide 2008; Torre-Villalvazo et al. 2008).

4. Effect of Soy Protein in Reduction of Caloric Intake Protein, compared with carbohydrate and fat in isocaloric diets, has been shown to significantly increase satiety in lean and overweight men, reducing appetite hormones such as ghrelin and increasing satiety hormones including cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) (Bowen et al. 2008). Cope et al. (2008) reviewed the animal, epidemiologic, and clinical data on the effect of soy protein and caloric intake reduction. They concluded that, when included as part of the diet, soy protein caused an acute decrease in caloric intake; however, there are no reports to indicate that soy will reduce caloric intake chronically. Dunshea and Cos (2008) demonstrated that dietary protein intake reduced feed intake, fat, and weight gain, with the response being most pronounced in pigs consuming diets containing soy protein isolate rather than whey protein isolate. However, data from most clinical studies have demonstrated that soy protein is equivalent to other protein sources for reducing hunger and improving satiety (Cope et al. 2008).

5. Role of Soy Protein in Glycemic Control and Insulin Resistance Anderson and Pasupuleti (2008) published a comprehensive review on the effect of soy protein on

glucose metabolism; this relationship was also reviewed by Cope et al. (2008). A summary from these two sources shows that there is evidence for positive effects of soy protein on glucoregulatory function in in vitro and animal models. It is not clear whether soy protein is more beneficial than other protein sources for glucoregulatory function during weight loss, nor is there enough evidence to show that soy directly affects glucose metabolism independent of weight and fat loss in animal models. However, epidemiologic data in postmenopausal women indicate that soy protein consumption (>13 g/d) decreases the risk of glycosuria. Furthermore, clinical evidence demonstrated that a soybased meal replacement protocol can be used for weight reduction in diabetic patients, causing a positive effect on insulin secretion and glycemic control. Wagner et al. (2008) treated male monkeys for 8 weeks using diets containing casein/lactalbumin, soy protein with a high isoflavone concentration, or soy protein that was alcohol-washed to deplete isoflavones. It was observed in this study that consumption of isoflavone-containing soy proteins increased insulin responses to the glucose challenge and decreased plasma adiponectin. On the other hand, isoflavone-depleted soy protein decreased body weight. An epidemiological study conducted in a population-based prospective cohort of middle-aged Chinese women found an association between soymilk consumption and lower risk of type 2 diabetes mellitus (Villegas et al. 2008). A longitudinal randomized clinical trial was conducted among 41 type 2 diabetes patients (≥126 mg/dl) with nephropathy. The soy protein group consumed a diet containing 70% animal proteins and 30% vegetable proteins for 4 years. Soy protein consumption significantly reduced fasting plasma glucose (−18 vs. 11 mg/dl for control group) and significantly improved levels of serum total cholesterol, low-density lipoprotein (LDL) cholesterol and triglycerides, and proteinuria and urinary creatinine (Azadbakht et al. 2008). Although the exact mechanism behind the beneficial effect of soy proteins on glycemic control and IR is not well known, several of the proposed mechanisms of action are presented in Figure 6.4.

Chapter 6 Soy Protein for the Metabolic Syndrome

75

Figure 6.4. Proposed molecular mechanism of soy protein action on insulin resistance in the metabolic syndrome (adapted from Torres and Tovar 2007). CCK = Cholecystokinin; GLUT = Glucose transporter; MCP = Monocyte chemoattractant protein; PPAR = Peroxisome proliferator activated receptor; SREBP = Sterol regulatory element binding protein; TNF = Tumor necrosis factor.

Soy protein may improve insulin sensitivity by increasing insulin signaling in fat and liver since insulin receptor mRNA expression was reported to be increased in the liver and adipose tissues as reviewed by Tremblay et al. (2007). Further studies have shown that long-term consumption of soy protein diet reduces hyperinsulinemia, which in turn decreases the expression of SREBP-1C in liver, reducing hepatic steatosis (Asencio et al. 2004). Moreover, soy protein may have a preventive effect of type 2 diabetes by reducing the production of MCP-1 in adipose tissue, which causes macrophages infiltration leading to reduction in TNF-α expression responsible for the inhibition of insulin signaling and IR in peripheral tissues, such as adipose tissue and skeletal muscle and liver (Horio 2007). Noriega-Lopez et al. (2007)

investigated whether soy protein stimulates insulin secretion to a lower extent and/or reduces IR in pancreatic islets of rats with diet-induced obesity. Amino acid pattern seen after soy protein consumption stimulated insulin secretion to a lower extent decreasing PPARγ and glucose transporter isoform 2 (GLUT-2). GLUT-2 is the main transporter of glucose in the pancreas, and it is regulated by PPARγ (Noriega-Lopez et al. 2007). These results suggest that less glucose uptake by pancreatic islets results in lower insulin secretion. In obesity, hyperplasia of pancreatic β-cells as a consequence of elevated influx of serum FFA released from adipose tissue, and to a lesser extent from endogenous pancreatic fatty acid synthesis, leads to the production of insulin by the pancreas (Matsui et al. 2004). Moreover, soy protein consumption reduced

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SREBP-1c expression, inhibiting lipogenesis and downregulating insulin expression in the pancreas, therefore ameliorating hyperinsulinemia (NoriegaLopez et al. 2007). Dietary proteins are found to induce glucagon secretion (Calbert and Holst 2004). Soy protein consumption may lower the insulin/glucagon (I/G) ratio associated with decrease in serum glucose, FFA, and leptin (Noriega-Lopez et al. 2007). Generally, consumption of plant proteins gives low I/G ratios associated with low serum lipids, whereas animal protein produces the opposite effect (Torres et al. 2006). Nordentoft et al. (2008) demonstrated that cholecystokinin (CCK) and CCK receptor expression were downregulated when KKAy mice were fed soy protein that was rich in isoflavones. CCK stimulates the insulin secretion as well as the secretion of pancreatic amylase and activates phospholipase A2 in beta cells. Phospholipase A2 facilitates arachidonic acid (AA) production, which in turn is a stimulator of insulin secretion (Simonsson et al. 2000). Another possible mechanism of action of soy protein is via stimulation of adiponectin, a cytokine produced by fat cells that improves insulin sensitivity in animal models of IR (Funahashi and Matsuzawa 2006). Intravenous adiponectin infusion lowers hepatic glucose production by reducing the expression of enzymes involved in gluconeogenesis (Funahashi and Matsuzawa 2006). There is some evidence showing that soy protein intake is associated with increased plasma concentration of adiponectin in Wistar rats (Nagasawa et al. 2002; Koba et al. 2007). The molecular mechanisms of the soy protein diet that enhance expression of adiponectin remain unclear. One possible mechanism is that the soy protein diet promotes the conversion of large adipocytes to smaller ones exhibiting adipocytokines-producing activities (Funahashi and Matsuzawa 2006). Diabetic patients are suggested to lose their sensitivity toward incretins (e.g., glucagon-like peptide1 [GLP-1] and gastric inhibitory peptide [GIP]), signaling molecules from the gut that lead the pancreas to secrete insulin in response to elevated plasma glucose (Burcelin 2005). Proteins and protein hydrolysates or bioactive peptides may help

in diabetic patients promoting the release of incretins (Kloek et al. 2008). Besides stimulating the incretin response, inhibition of the breakdown of incretins is another way to improve glycemic control (Kloek et al. 2008). Inhibitors of the primary enzyme responsible for breakdown of incretins (DPP-IV) such as hydrolysates, as well as single or combined oligopeptides from food proteins, could be useful for glycemic control in diabetic patients (Kloek et al. 2008).

6. Effect of Soy Protein on Serum Lipids Evidence from animal studies indicates that soy protein will reduce serum cholesterol and the results appear to be independent of weight loss (Table 6.2). Soy protein consumption was associated with reduced LDL cholesterol among subjects that lost weight on soy-based meal replacements as well as in hypercholesterolemic subjects (Table 6.2). This observation agrees with results of several metaanalysis studies, although the magnitude of the effect is variable (Zhan and Ho 2005; Reynolds et al. 2006; Sacks et al. 2006). Sirtori et al. (2007) evaluated studies on soy proteins and serum cholesterol published after 1995 using a normogram. These authors concluded that there is apparently a threshold level that needs to be reached before a significant reduction in plasma cholesterol occurs, and the cholesterol response is far more dramatic in individuals with cholesterol levels below 240–250 mg/dl, most likely only a minimal (probably clinically insignificant) cholesterol reduction. Some reports indicate that soy protein increases high-density lipoprotein (HDL) cholesterol and reduces triglyceride levels in animal models and human clinical trials (Tables 6.2 and 6.3, respectively), but more experiments are needed to make definitive conclusions. However, a recent meta-analysis indicates that soy protein consumption can lead to a meaningful and significant reduction in blood cholesterol, thus protecting against heart disease (Samuel et al. 2008). This metaanalysis found reductions of 9.54 mg/dl in total cholesterol and 7.12 mg/dl in LDL cholesterol (about 4% and 5% reductions, respectively). The meta-analyses also looked at individuals who had

Table 6.3. Effect of soy protein diets on body weight loss, adiposity reduction, and metabolic parameters in recent human clinical studies. Subjects

N

Duration (Weeks)

Obese adults

90

Overweight and obese adults

Diet

Body Weight

Metabolic Parameters

References

18

Lifestyle education group vs. low-energy diet with soy-based meal replacement plus exercise vs. soy protein diet without exercise

All groups showed an improvement in metabolic parameters (lower serum HDL-c, LDL-c, insulin, glucose and leptin levels).

Deibert et al. 2004

52

12

5 soy-based shakes/day vs. 2 milk-based shakes/day as part of low-energy diet (1,200 Kcal/d)

Reductions in serum total cholesterol, LDL-c and TG vs. baseline values

Anderson and Hoie 2005

Overweight and obese adults

138

20

Soy candy (5 g β conglycinin/d) vs. placebo candy (5 g casein/d)

Casein and soy-fed groups did not affect serum lipids.

Kohno et al. 2006

Overweight adults

30

8

Soy-based low-calorie group vs. low-calorie group consuming 2/3 animal protein

Serum total cholesterol, LDL-c and TG levels decreased but no significant changes were observed in serum TG, HDL-c or fasting glucose in the soy group.

Liao et al. 2007

Obese women

43

16

Three servings casein or soy shakes/d and 5 servings of fruits and vegetables/d to have a total energy intake between 4.5 and 5 MJ/d

Soy diet groups lost more weight than lifestyle education group (8.9 Kg vs. 6.2 Kg), and there was a trend for subjects’ lifestyle education [8.8 and 9.4 (exercise) vs. 6.6 Kg]. Weight loss (9.0% of their initial body weight) and central adiposity reduction in the subjects on the soy-based diet but no significant differences were observed between groups. A significant reduction in body weight (−2.7 Kg) and visceral fat area occurred in the β-conglycinin group(−6.5 cm2). Body weight, body mass index, % body fat, and waist circumference significantly decreased in both groups. The decrease in % body fat in the soy group was greater vs. control (2.2% vs. 1.4%, respectively). Weight loss (13.4% of their initial body weight) in the subjects on the soy-based diet but no significant differences were observed between groups.

No changes in serum TG were observed. Lower total cholesterol, LDL-c, and HDL-c in soy group but no significant differences were observed between casein and soy.

Anderson et al. 2007

77

78

Table 6.3. Continued Subjects

N

Duration (Weeks)

Obese postmenopausal women

15

12

Overweight women

48

12

Premenopausal women

18

8

Skim milk-based low-calorie group vs. soymilk-based low-calorie group

Overweight and obese adults

90

6

Fat restricted low-calorie group vs. low-calorie high soy protein drink

Diet

Body Weight

Metabolic Parameters

References

Daily shake containing soy (20 g soy protein plus 160 mg isoflavones) vs. daily isocaloric casein placebo shake Caloric intake reduction by 500 Kcal/d and 15 g soy protein vs. dietary counseling

Significant reduction in total abdominal fat and subcutaneous abdominal fat with the soy supplement compared with casein placebo There was no difference between groups in change in percent fat mass (−5.31% for soy protein diet vs. −3.94% for control diet), % fat-free mass, and waist circumference. Body weight, % body fat, and waist circumference significantly decreased in both groups but no significant differences between groups were observed. Significantly higher reductions in body weight (−6.4 vs. −3.1 kg) and fat mass (−5.1 vs. 2.8 Kg) in the high soy protein group vs. control

Not determined

Sites et al. 2007

No significant effect of soy protein consumption in serum total, LDL-c, HDL-c, TG, glucose insulin levels

St-Onge et al. 2007

Not determined

Lukaszuk et al. 2007

No significant differences were observed between groups in total cholesterol, HDL-c, LDL-c, glucose, and insulin levels.

Konig et al. 2008

Chapter 6 Soy Protein for the Metabolic Syndrome

high and normal blood cholesterol and found that soy protein consumption resulted in a significant reduction in blood cholesterol for both groups. In regard to the mechanism underlying this hypolipidemic effect, studies have shown that soy protein, compared to casein, decreases cholesterol intestinal absorption and increases fecal bile acid excretion, thereby reducing hepatic cholesterol content and enhancing removal of LDL (Torres et al. 2006). In addition, soy protein or soy hydrolysates may increase LDL receptor expression (Tachibana et al. 2005; Torres et al. 2006; Cho et al. 2008), resulting in improved clearance of plasma LDL cholesterol. Duranti et al. (2004) demonstrated an increased binding of VLDL to liver membranes of hypercholesterolemic rats fed a diet containing α′ subunit from soy 7S globulin, suggesting altered hepatic metabolism with increased LDL and βVLDL removal by hepatocytes. Another study by Lovati et al. (1987) has shown that a soy protein diet consistently increased degradation of LDL by mononuclear cells from patients with hypercholesterolemia, even in the presence of an elevated cholesterol intake. The reduction in the insulin/glucagon ratio could be in part responsible for the hypolipidemic effect of soy protein (Torres et al. 2006). Increases in serum glucagon concentration by soy protein decrease insulin/glucagon ratio with subsequent reduction of SREBP-1 reducing gene expression of enzymes involved in fatty acid biosynthesis. In addition, SRBEP-1 regulates the expression of stearoyl-CoA desaturase (SCD-1). The reduction of SCD-1 mRNA in the liver of rats fed with soy protein appears to be an important mechanism to reduce hepatic triglycerides and circulating VLDL particles (Tovar et al. 2005). Some evidence also has shown that the orphan receptor LXRα is probably involved in the mechanism by which soy protein reduces fatty acid and cholesterol synthesis (Torres and Tovar 2007). Gudbrandsen et al. (2006) have shown that feeding obese Zucker rats with soy protein concentrate enriched with isoflavones (HDI) for 6 weeks reduced fatty liver and decreased the plasma levels of alanine transaminase and aspartate transaminase. These effects were accompanied by increased activities of mitochondrial and peroxi-

79

somal β-oxidation, acetyl-CoA carboxylase, fatty acid synthase, and glycerol-3-phosphate acyltransferase in liver, increased plasma triacylglycerol level, and decreased hepatic mRNA level of VLDL receptor. However, the decreased gene expression of VLDL receptor found in the liver was not observed in epididymal fat and skeletal muscle of rats fed HDI, indicating that the liver may be the primary organ responsible for the reduced clearance of triacylglycerol-rich lipoproteins from plasma after HDI feeding.

7. Effect of Soy Protein on Blood Pressure To date, there have been more than 25 reported cross-sectional population studies investigating the relationship between protein intake and blood pressure, with approximately two-thirds reporting an inverse relationship of protein intake and most of the remaining studies reporting no association (Lee et al. 2008). Some cross-sectional studies suggest stronger associations with plant protein rather than animal protein. There is only one large population study that has specifically investigated the relationship of soy intake and blood pressure, the Shanghai Women’s Health Study (Yang et al. 2005a). In that study, an increase in soy protein of approximately 25 g/day was associated with 2 mmHg lower systolic blood pressure in all women and of 5 mmHg in women older than 60 years. Several intervention trials have assessed the effect of soy protein on blood pressure. A reduction in blood pressure has been a consistent finding in these trials, with increases in protein and decreases in carbohydrate intakes ranging from 20 to 70 g/day (Washburn et al. 1999; Burke et al. 2001; He et al. 2005). In the first study, a randomized controlled crossover trial in perimenopausal women, 20 g/day of soy protein was compared with 20 g/day of carbohydrate and resulted in significant falls in clinical diastolic blood pressure (Washburn et al. 1999). The second study was a randomized controlled parallel trial in 36 hypertensive individuals; 66 g/day of soy protein was compared with 66 g/day of carbohydrate

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Part 2 Functions of Biologically Active Proteins and Peptides

maltodextrin (Burke et al. 2001). It resulted in a reduction in 24 hour ambulatory systolic and diastolic blood pressure by 5.9 and 2.6 mmHg, respectively. Finally, in a study of three Chinese communities, significantly lower systolic and diastolic blood pressure, by 4.3 and 2.8 mmHg, respectively, was found when consuming 26 g/day of soy protein in comparison to wheat carbohydrates (He et al. 2005). Several controlled trials have investigated whether the type of protein in the diet affects blood pressure by comparing soy protein with animal proteins (Hermansen et al. 2005; Kreijkamp-Kaspers et al. 2005; Sacks et al. 2006; Teede et al. 2006; Anderson et al. 2007; Matthan et al. 2007; Welty et al. 2007). The results of these studies have concluded that there was insufficient evidence for a comparative benefit of soy on blood pressure vs. other protein sources. In order to examine the association of dietary protein with blood pressure, Wang et al. (2008) analyzed data from PREMIER, an 18-month clinical trial (n = 810) that examined the effects of plant protein intake on blood pressure. Individuals met the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (1997) criteria. They concluded that increased intake of plant protein was significantly associated with a lower risk of hypertension at 6 but not at 18 months. Results of this study indicated that plant protein had a beneficial effect on blood pressure and was associated with a lower risk of hypertension at 6 months, although the exact mechanism linking plant or total protein intake to blood pressure is still unclear. A possible explanation is that an increase in protein intake may increase plasma amino acids that may directly influence proximal sodium reabsorption or alter cell permeability, subsequently increasing renal plasma flow, renal size, and glomerular filtration rate (Woods 1993; He et al. 1995). Second, the amino acid arginine may act as a vasodilator through nitric oxide and contribute to blood pressure lowering (Palloshi et al. 2004). Plant foods including soy products are good sources of arginine but so are animal foods such as animal meats, seafood, dairy products, and eggs. Third, soy proteins and soy

protein hydrolysates have been identified as angiotensin I-converting enzyme (ACE) inhibitors (Yang et al. 2005b). Numerous studies in spontaneously hypertensive rats as well as in hypertensive human volunteers have been performed to determine the antihypertensive effects of soy-derived ACE inhibitors as reviewed by Erdmann et al. (2008). These in vivo studies have demonstrated that several ACE inhibitory peptides significantly reduce blood pressure, either after intravenous or oral administration in hypertensive subjects. An important observation from these trials is that the peptides being studied have little or no effect on blood pressure of normotensive subjects, suggesting that they exert no acute hypotensive effect. A computational model has been developed with approximately 200 peptides to predict the catalytic inhibition of ACE, obtaining excellent correlation with experimental IC50 values (Figure 6.5) (de Mejia and Wang 2006). This method may facilitate the understanding of the effect of peptides produced by gastrointestinal digestion of proteins on the reduction of blood pressure through ACE inhibition. Also, the reduction in soluble vascular cell adhesion molecule-1 with soy nuts (25 g soy protein and 101 mg aglycone isoflavones) in women with hypertension, but not normotensive, suggests an improvement in endothelial function that may reflect an overall improvement in the inflammatory process underlying atherosclerosis (Nasca et al. 2008).

8. Conclusion Proteins play a role in promoting optimal human health. However, we need to understand their potential and elucidate their mechanisms of action in weight control, reduction of inflammation, insulin sensitivity, glucose homeostasis, satiety induction, and cardiovascular health. The evidence available to date suggests that protein consumption is important not only at the Recommended Daily Allowance but also at higher intakes (Millward et al. 2008). Diets with increased protein and reduced carbohydrates have shown to improve body composition and lipid and lipoprotein profiles, reducing blood pressure, metabolic syndrome, and glycemic regulations asso-

Chapter 6 Soy Protein for the Metabolic Syndrome

a.

81

b.

Figure 6.5. Docking of antihypertensive peptides into angiotensin-converting enzyme (ACE) C-terminal and validation of computational modeling with experimental inhibition of peptides (IC50). (a) Structure of testicular ACE showing the inhibition by Val-Try. (b) Docking scores showing high correlation with experimental data of ACE inhibition of the same peptides.

ciated with treatment of obesity and weight loss. Additional research is needed to determine specific levels of protein, carbohydrate, and fat for optimum health of individuals who differ in age, physical activity, and metabolic phenotypes (Layman et al. 2008). Soy protein is a promising dietary component that needs to be further evaluated in regard to its long-term effect on adipogenesis and potential reduction of blood pressure. This will allow determining the recommended intake of soy protein to reduce the MS risk in humans from different backgrounds and environments. Attention must also be paid to the technological processes in order to optimize the protein quality, its bioavailability, and the main protein components that will produce the most biologically active peptides. Research continues to show that consuming soy protein or other plant protein sources can be part of a healthy diet that may result in significant and meaningful reductions in total cholesterol and LDL cholesterol. Soy protein, for example, can easily be a part of a healthy, low-cholesterol, low-fat diet and can be incorporated into a variety of food forms, including bars, beverages, and cereals, to make heart-healthy eating convenient for consumers.

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Hermansen K, Hansen B, Jacobsen R, Clausen P, Dalgaard M, Dinesen B, Holst JJ, Pedersen E, Astrup A. 2005. Effects of soy supplementation on blood lipids and arterial function in hypercholesterolemic subjects. Eur J Clin Nutr 59:843–850. Heymsfield SB, van Mierlo CA, vander Knaap HC, Heo M, Frier JI. 2003. Weight management using a meal replacement strategy: Meta and pooling analysis from six studies. Intl J Obes Relat Metab Disord 27:537–549. Hoffman JR, Falvo MJ. 2004. Protein—Which is best? J Sports Sci Med 3:118–130. Hollander JM, Mechanick JI. 2008. Complementary and alternative medicine and the management of the metabolic syndrome. J Amer Diet Assoc 108:495–509. Horio F. 2007. Effect of dietary soy protein isolate on the production of adipocytokines such as adiponectin. Soy Protein Japan 10:63–66. Hu G, Qiao Q, Tuomilehto J, Balkau B, Borch-Johsen K, Pyorala K. 2004. Prevalence of the metabolic syndrome and its relation to all-cause and cardiovascular mortality in nondiabetic European men and women. Arch Intern Med 164:1066– 1076. International Diabetes Federation. 2004. The IDF consensus worldwide definition of the metabolic syndrome. Brussels, Belgium: International Diabetes Federation. Available at: http://www.idf.org/webdata/docs/metabolic_syndrome_ definition.pdf. Accessed October 1, 2008. Johnson LW, Weinstock RS. 2006. The metabolic syndrome: Concepts and controversy. Mayo Clin Proc 81:1516–1520. Johnston CS, Tjonn SL, Swan PD. 2004. High-protein, low-fat diets are effective for weight loss and favorably alter biomarkers in healthy adults. J Nutr 134:586–591. Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. 1997. The sixth report. Arch Intern Med 157:2413–2446. Kim F, Tysseling KA, Rice J. 2005. Free fatty acid impairment of nitric oxide production in endothelial cells is mediated by IKKbeta. Arterioscler Thromb Vasc Biol 25:989–994. Kloek J, Pasupuleti VK, van Loon LJC. 2008. Proteins, protein hydrolysates and bioactive peptides in the management of type 2 diabetes. In: VK Pasupuleti, JW Anderson, eds., Nutraceuticals, Glycemic Health and Type 2 Diabetes, 460– 489. Ames, IA: Wiley-Blackwell. Koba K, Akahoshi A, Tanaka K, Sugano M. 2007. Interaction of dietary β-conglycinin and conjugated linoleic acid on body fat mass and lipid metabolism in rats. Soy Protein Res Japan 10:67–71. Kohno M, Hirotsuka M, Kito M, Matsuzawa Y. 2006. Decreases in serum triacyglycerol and visceral fat mediated by dietary soybean β-conglycinin. J Atherosc Thromb 13:247–255. Konig D, Deibert P, Frey I, Landmann U, Berg A. 2008. Effect of meal replacement on metabolic risk factors in overweight and obese subjects. Ann Nutr Metab 52:74–78. Kreijkamp-Kaspers S, Kok L, Bots ML, Grobbee DE, Lampe JW, van der Schouw YT. 2005. Randomized controlled trial of the effects of soy protein containing isoflavones on vascular

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function in postmenopausal women. Amer J Clin Nutr 81:189–195. Laakso M, Kovanen PT. 2006. Metabolic syndrome: To be or not to be? Ann Med 38:32–33. Layman DK, Clifton P, Gannon MC, Krauss RM, Nuttall FQ. 2008. Protein in optimal health: Heart disease and type 2 diabetes. Amer J Clin Nutr 87(5):1571–1575S. Lee WY, Park JS, Noh SY, Rhee EJ, Kim SW, Zimmet PZ. 2004. Prevalence of the metabolic syndrome among 40,698 Korean metropolitan subjects. Diabetes Res Clin Pract 65:143–149. Lee YP, Puddey IB, Hodgson JM. 2008. Protein, fibre and blood pressure: Potential benefit of legumes. Clin Exp Pharmacol Physiol 35:473–476. Liao F-H, Shieh M-J, Yang S-C, Lin S-H, Chien Y-W. 2007. Effectiveness of a soy-based compared with a traditional lowcalorie diet on weight loss and lipid levels in overweight adults. Nutr 23:551–556. Lovati MR, Manzoni C, Canavesi A, Sirtori M, Vaccarino V, Marchi M, Gaddi G, Sirtori CR. 1987. Soybean protein diet increases low density lipoprotein receptor activity in mononuclear cells from hypercholesterolemic patients. J Clin Invest 80:1498–1502. Lukaszuk JM, Luebbers P, Gordon BA. 2007. Preliminary study: Soy milk as effective as skim milk in promoting weight loss. J Amer Diet Assoc 107:1811–1814. Magliano DJ, Shaw JE, Zimmet PZ. 2006. How to best define the metabolic syndrome? Ann Med 38:34–41. Martinez-Villaluenga C, Bringe NA, Berhow MA, Gonzalez de Mejia E. 2008. beta-Conglycinin embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytes in vitro. J Agric Food Chem 56:10533–10543. Maskarinec G, Arylward AG, Erber E, Takata Y, Kolonel LN. 2008. Soy intake is related to a lower body mass index in adult women. Eur J Nutr 47:138–144. Matsui J, Terauchi Y, Kubota N, Takamoto I, Eto K, Yamashita T, Komeda K, Yamauchi T, Kamon J, Kita S, Noda M, Kadowaki T. 2004. Pioglitazone reduces islet triglyceride content and restores impaired glucose-stimulated insulin secretion in heterozygous peroxisome proliferator-activated receptor-gamma-deficient mice on a high-fat diet. Diabetes 53:2844–2854. Matthan NR, Jalbert SM, Ausman LM, Kuvin JT, Kara RH, Lichtenstein AH. 2007. Effect of soy protein from differently processed products on cardiovascular disease risk factors and vascular endothelial function in hypercholesterolemic subjects. Amer J Clin Nutr 85:960–966. Millward DJ, Layman DK, Tomé D, Schaafsma G. 2008. Protein quality assessment: Impact of expanding understanding of protein and amino acid needs for optimal health. Amer J Clin Nutr 87:1576–1581S. Morifuji M, Sanbongi C, Sugiura K. 2006. Dietary soya protein intake and exercise training have an additive effect on skeletal muscle fatty acid oxidation enzyme activities and mRNA levels in rats. Br J Nutr 96:469–475.

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Moriyama T, Kishimoto K, Nagai K, Urade R, Ogawa T, Utsumi S, Maruyama N, Maebuchi M. 2004. Soybean betaconglycinin diet suppresses serum triglyceride levels in normal and genetically obese mice by induction of beta-oxidation, downregulation of fatty acid synthase, and inhibition of triglyceride absorption. Biosci Biotech Biochem 68:352–359. Nagasawa A, Fukui K, Funahashi T, Maeda N, Shimomura I, Kihara S, Waki M, Takamatsu K, Matsuzawa Y. 2002. Effects of soy protein diet on the expression of adipose genes and plasma adiponectin. Hormone Metabol Res 34:635–639. Nasca MM, Zhou JR, Welty FK. 2008. Effect of soy nuts on adhesion molecules and markers of inflammation in hypertensive and normotensive postmenopausal women. Amer J Cardiol 102:84–86. National Cholesterol Education Program (NCEP). 2001. Expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Panel Treatment III). Executive Summary of the Third Report. JAMA 285:2486–2497. Nordentoft I, Jeppesen PB, Hong J, Abudula R, Hermansen K. 2008. Increased insulin sensitivity and changes in the expression profile of key insulin regulatory genes and beta cell transcription factors in diabetic KKAy-mice after feeding with a soybean protein rich diet high in isoflavone content. J Agric Food Chem 56:4377–4385. Noriega-Lopez L, Tovar AR, Gonzalez-Granillo M, HernandezPando R, Escalante B, Santillan-Doherty P, Torres N. 2007. Pancreatic insulin secretion in rats fed a soy protein high fat diet depends on the interaction between the amino acid pattern and isoflavones. J Biol Chem 282:20657–20666. Palloshi A, Fragasso G, Piatt P, Monti LD, Setola E, Valsecchi G, Galluccio E, Chierchia SL, Margonato A. 2004. Effect of oral L-arginine on blood pressure and symptoms and endothelial function in patients with systemic hypertension, positive exercise tests and normal coronary arteries. Amer J Cardiol 93: 933–935. Pan A, Franco OH, Ye J, Demark-Wahnefried W, Ye X, Yu Z, Li H, Lin X. 2008. Soy protein intake has sex-specific effects on the risk of metabolic syndrome in middle-aged and elderly Chinese. J Nutr 138:2413–2421. Pella D, Singh RB, Tomlinson B, Kong CW. 2003. Coronary artery disease in developing and newly industrialized countries: A scientific statement of the International College of Cardiology. In: NS Dhalla, A Cholingham, HJ Berkowitz, PK Singal, eds., Frontiers of Cardiovascular Health, 473–483. Boston: Kluwer. Reaven GM. 1988. Role of insulin resistance in human disease. Diabetes 37:1595–1607. Reynolds K, Chin A, Lees KA, Nguyen A, Bujnowski D, He J. 2006. A meta-analysis of the effect of soy protein supplementation on serum lipids. Amer J Cardiol 98:633–640. Sacks FM, Lichtenstein A, Van Horn L, Harris W, Kris-Etherton P, Winston M. 2006. Soy protein isoflavones, and cardiovascular health. An American Heart Association Science Advisory for Professionals from the Nutrition Committee. Circulation 113:1034–1044.

Samuel P, Zakharkin S, Spitznagel E, Greaves K, Butteiger D, Krul E. 2008. Meta-analysis confirms soy protein’s cholesterol lowering efficacy. 8th International Symposium on the Role of Soy in Health Promotion and Chronic Disease Prevention and Treatment, November 11, 2008, Tokyo, Japan. Sattar N, Gaw A, Scherbakova O, Ford I, O’Reilly DS, Haffner SM, Isles C, Macfarlane PW, Packard CJ, Cobbe SM, Shepherd J. 2003. Metabolic syndrome with and without C-reactive protein as a predictor of coronary heart disease and diabetes in the West of Scotland Coronary Prevention Study. Circulation 108:414–419. Simonsson E, Karlsson S, Ahren B. 2000. Islet phospholipase A2 activation is potentiated in insulin resistant mice. Biochem Biophys Res Commun 272:539–543. Singh P, Kumar R, Sabapathy SN, Bawa AS. 2008. Functional and edible uses of soy protein products. Comprehen Rev Food Sci Food Safety 7:14–28. Sirtori CR, Eberini I, Arnoldi A. 2007. Hypocholesterolemic effects of soya proteins: Results of recent studies are predictable from the Anderson meta-analysis. Br J Nutr 97:816–822. Sirtori CR, Johnson SK. 2006. Soy proteins, cholesterolemia, and atherosclerosis. In: M Sugano, ed., Soy in Health and Disease Prevention, 17–41. Boca Raton, FL: CRC Press. Sites CK, Cooper MD, Toth MJ, Gastaldelli A, Arabshahi A, Barnes S. 2007. Effect of a daily supplement of soy protein on body composition and insulin secretion in postmenopausal women. Fertility Sterility 88:1609–1617. Stehno-Bittel L. 2008. Intricacies of fat. Phys Ther 88:1–14. St-Onge MP, Claps N, Wolper C, Heymsfield SB. 2007. Supplementation with soy-protein–rich foods does not enhance weight loss. J Amer Diet Assoc 107:500–505. Tachibana N, Matsumoto I, Fukui K, Arai S, Kato H, Abe K, Takamatsu K. 2005. Intake of soy protein isolate alters hepatic gene expression in rats. J Agric Food Chem 53:4253–4257. Takahashi Y, Ide T. 2008. Effects of soy protein and isoflavone on hepatic fatty acid synthesis and oxidation and mRNA expression of uncoupling protein and peroxisome proliferators-activated receptor γ in adipose tissues of rats. J Nutr Biochem 19:682–693. Teede HJ, Giannopoulos D, Dalais FS, Hodgson J, McGrath BP. 2006. Randomized, controlled, cross-over trial of soy protein with isoflavones on blood pressure and arterial function in hypertensive subjects. J Amer Coll Nutr 25:533–540. Torres N, Torre-Villalvazo I, Tovar AR. 2006. Regulation of lipid metabolism by soy protein and its implication in diseases mediated by lipid disorders. J Nutr Biochem 17:365–373. Torres N, Tovar A. 2007. The role of dietary protein on lipotoxicity. Nutr Rev 65:S64–68. Torre-Villalvazo I, Tovar A, Ramos-Barragan VE, CerbonCervantes MA, Torres N. 2008. Soy protein ameliorates metabolic abnormalities in liver and adipose tissue of rats fed a high fat diet. J Nutr 138:462–468. Torre-Villalvazo I, Tovar A, Torres N. 2006. Soy protein intake prevents adipocyte hypertrophy in rats fed a high fat diet. FASEB J 20:A595.

Chapter 6 Soy Protein for the Metabolic Syndrome

Tovar AR, Torre-Villalvazo I, Ochoa M, Elias AL, Ortiz V, Aguilar-Salinas CA, Torres N. 2005. Soy protein reduces hepatic lipotoxicity in hyperinsulinemic obese Zucher fa/fa rats. J Lipid Res 46:1823–1832. Trayhurn P, Wood IS. 2005. Signaling role of adipose tissue: Adipokines and inflammation in obesity. Biochem Soc Trans 33:1078–1081. Tremblay F, Lavigne C, Jacques H, Marette A. 2007. Role of dietary proteins and amino acids in the pathogenesis of insulin resistance. Ann Rev Nutr 27:293–310. Velasquez MT, Bhathena SJ. 2007. Role of dietary soy protein in obesity. Intl J Med Sci 4:72–82. Villegas R, Gao Y-T, Yang G, Li H-L, Elasy TA, Zheng W, Shu XO. 2008. Legume and soy food intake and the incidence of type 2 diabetes in the Shangai women’s study. Amer J Clin Nutr 87:162–167. Wagner JD, Zhang L, Shadoan MK, Kavanagh K, Chen H, Tresnasari K, Kaplan JR, Adams MR. 2008. Effects of soy protein and isoflavones on insulin resistance and adiponectin in male monkeys. Metab-Clin Exp 57:S24– 31. Wang YF, Yancy WS, Yu D Jr, Champagne C, Appel LJ, Lin P-H. 2008. The relationship between dietary intake and blood pressure: Results from the PREMIER study. J Human Hypertens 22:745–754. Washburn S, Burke GL, Morgan T, Anthony M. 1999. Effect of soy protein supplementation on serum lipoproteins, blood

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pressure, and menopausal symptoms in perimenopausal women. Menopause 6:7–13. Welty FK, Lee KS, Lew NS, Zhou JR. 2007. Effect of soy nuts on blood pressure and lipid levels in hypertensive, prehypertensive, and normotensive postmenopausal women. Arch Intern Med 167:1060–1067. Woods LL. 1993. Mechanisms of renal hemodynamic regulation in response to protein feeding. Kidney Intl 44:659–675. Xiao CW, Wood C, Huang W, L’Abbe MR, Gilani GS, Cooke GM, Curran I. 2006. Tissue-specific regulation of acetyl CoA carboxylase gene expression by dietary soya protein isolate in rats. Br J Nutr 95:1048–1054. Yang G, Shu XO, Jin F, Zhang X, Li HL, Li Q, Gao YT, Zheng W. 2005a. Longitudinal study of soy food intake and blood pressure among middle-aged and elderly Chinese women. Amer J Clin Nutr 81:1012–1017. Yang S-C, Liu S-M, Yang H-Y, Lin Y-H, Chen J-R. 2005b. Soybean protein hydrolysate improves plasma and liver lipid profiles in rats fed high-cholesterol diet. J Amer College Nutr 26:416–423. Ye X, Yu Z, Li H, Franco OH, Liu Y, Lin X. 2007. Distribution of C-reactive protein and its association with metabolic syndrome in middle-aged and older Chinese people. J Amer Coll Cardiol 49:1798–1805. Zhan S, Ho S. 2005. Meta-analysis of the effects of soy protein containing isoflavones on the lipid profile. Amer Soc Clin Nutr 81:397–408.

Chapter 7 Amyloidogenic Proteins and Peptides Soichiro Nakamura, Takanobu Owaki, Yuki Maeda, Shigeru Katayama, and Kosuke Nakamura

Contents 1. Introduction, 87 2. Definition of Amyloid and Amyloidosis, 89 3. Naturally Occurring Amyloidogenic Proteins/Peptides, 90 3.1. Prion, 90 3.2. β2-microglobulin, 90 3.3. Cystatin C, 90 3.4. Aβ, 91 3.5. Amylin, 91

1. Introduction To date, many different proteins and polypeptides have been recognized as naturally occurring amyloidogenic in humans (Table 7.1). These precursor proteins, clearly differing in their biochemical function and in their three-dimensional structure, may become prone to undergoing an irreversible transition from their native conformation into highly ordered amyloid fibrils. The extracellular deposition of amyloid fibrils in tissues is the basis of a wide range of human diseases, including neurodegenerative disorders of the central nervous system and various forms of systemic and cutaneous amyloidosis. Recently, it was revealed that the existence of

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

3.6. Others, 92 4. Amyloidogenicity of Small Peptides, 92 5. Strategies for Prevention of Amyloid Formation, 92 5.1. Phenolic Compounds, 92 5.2. Peptidic Compounds, 94 5.3. Others, 95 6. Conclusion, 95 7. References, 95

amyloid structure is associated with some specific sequence properties or secondary structural propensities (DePace et al. 1998; Makin et al. 2005; Perutz et al. 1994; Rochet and Lansbury 2000), in which phenylalanine pairs occur in Aβ and in the amyloidosis-related serum amyloid A; and a motif of three consecutive alanine residues and the motif AAXK are found throughout the repeat region of αsynuclein (Makin et al. 2005). In the meantime, accumulating evidence indicated that amyloid fibril can be formed by small peptides, based on experimental studies (Jaroniec et al. 2002; Lopez De La Paz et al. 2002; Petkova et al. 2002; Pastor et al. 2008; Inouye et al. 2006; Reches et al. 2002; Balbach et al. 2000; Gsponer et al. 2003; Nelson et al. 2005; Sawaya et al. 2007) and computer simulations (de Groot et al. 2007; Klimov and Thirumalai 2003; Ma and Nussinov 2002; Reches and Gazit 2003; Sambasivam et al. 2008; Seabloom et al. 2002; Straub et al. 2002; Thirumalai et al. 2003). Sawaya 87

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Table 7.1. Precursor and name of amyloid.1 Precursor

Name of Amyloid

Localization2

Syndrome or Involved Tissue

Reference

Immunoglobulin light chain Immunoglobulin heavy chain β2-microglobulin

AL

S, L

Glenner et al. 1971

AH

S, L

Aβ2M

S, L#

Transthyretin

ATTR

S, L#

(Apo)serum AA Apolipoprotein AI

AA AApoAI

S S, L

Apolipoprotein AII Apolipoprotein AIV Gelsolin Lysozyme Fibrinogen α-chain Cystatin C ABriPP ADanPP Aβ protein precursor (AβPP) Prion protein (Pro)calcitonin Islet amyloid polypeptide (Amylin) Atrial natriuretic factor Prolactin Insulin Lactadherin Keratoepithelin Lactoferrin Odontogenic ameloblastassociated protein Semenogelin I Tau

AApoAII AApoAIV AGel ALys AFib ACys ABri Adan Aβ

S S S S S S S L L

Primary (S), Myelomaassociated (L) Primary (S), Myelomaassociated (L) Hemodialysis-associated (S), Joints (L#) Familial and senile systemic (S), Tenosynovium (L#) Secondary and reactive Familial (S), Aorta (L), Meniscus (L) Familial Sporadic, associated with aging Familial, Finnish Familial Familial Familial Familial dementia, British Familial dementia, Danish Alzheimer ’s disease, aging

APrP ACal AIAPP

L L L

AANF APro AIns AMed AKer ALac AOaap

L L L L L L L

ASemI ATau

L L

Spongiform encephalopathies C-cell thyroid tumors Islets of langerhans, insulinomas Cardiac atria Aging pituitary, prolactinomas Iatrogenic Senile aortic, arterial media Cornea, familial Cornea Odontogenic tumors Vesicula seminalis Alzheimer ’s disease, frontotemporal dementia, aging, cerebral

Eulitz et al. 1990 Gejyo et al. 1985 Costa et al. 1978 Benditt et al. 1971 Nichols et al. 1988 Benson et al. 2001 Bergstrom et al. 2004 Maury and Baumann 1990 Pepys et al. 1993 Benson et al. 1993 Cohen et al. 1983 Vidal et al. 1999 Holton et al. 2002 Verbeek et al. 1997 Bolton et al. 1982 Sletten et al. 1976 Westermark et al. 1986 Johansson et al. 1987 Westermark et al. 1997 Clark and Nilsson 2004 Haggqvist et al. 1999 Korvatska et al. 2000 Ando et al. 2002 Murphy et al. 2008 Linke et al. 2005 Buee and Delacourte 1999

1

This table was mainly adapted according to the latest meeting of the Nomenclature Committee of the international Society of Amyloidosis (Westermark et al. 2007). 2 S, systemic amyloidosis; L, localized and organ restricted amyloidosis; L#, possibly localized amyloidosis.

et al. demonstrated that 30 segments from fibrilforming proteins formed amyloid-like fibrils and microcrystals (Sawaya et al. 2007). According to numerous studies on Aβ, suppression or prevention of transition from monomeric to oligomeric and polymeric forms has emerged as a goal in the development of a therapy for amyloidosis

(Diehl 1995; Selkoe 1995, 1997). Soto et al. (1996) postulated that fibrillogenesis of Aβ can be inhibited by short peptides’ partially homologous sequence acting as β-sheet breaker (blocker) peptides and demonstrated that a five-residue peptide inhibits amyloid fibril formation and prevents neuronal death induced by fibrils in cell culture (Soto et al.

Chapter 7 Amyloidogenic Proteins and Peptides

1998). Important clues to understanding the molecular basis of amyloidosis have emerged recently from the observation made in proteins unrelated to human diseases. It has been reported that numerous soluble proteins can be converted to insoluble amyloid-like fibrils. Myoglobin, the textbook example of αhelical and globular structure, shows the typical characteristics of pathological amyloid fibrils (Fandrich et al. 2001). Since numerous polypeptides that are not known to relate to any amyloidosis can adopt amyloid structure under appropriate conditions in vitro, it is suggested that nearly all proteins have the ability to form amyloid fibrils (Chiti et al. 1999; Dobson 1999, 2001; Stefani and Dobson 2003; Fandrich et al. 2001). As stated by Makin and Serpell (2005), some fibrils may in fact not be amyloid but simply have a sufficiently high β-sheet content to fall within an overbroad definition. Advances in investigations mentioned above are crucial to devising the most suitable strategies for expanding disorders such as Alzheimer ’s disease, hemodialysis-associated disease, bovine spongiform encephalopathies (prion disease), and so on. Recent collaborating works among medicine, biochemistry, molecular biology, and organic chemistry as well as computer science facilitate the discovery of small-molecule aggregation inhibitors and the development of more efficacious antiamyloid agents to treat and/or reverse the pathogenicities of amyloid diseases. Thus, there is a growing interest in gaining an improved understanding of amyloid and amyloidosis. This chapter will address the fundamental concept of amyloidogenic protein/ peptides and provide recent progress in curing and preventing strategies for amyloidosis.

2. Definition of Amyloid and Amyloidosis Amyloid is the general term for proteinaceous starch (amylo)-like (oid) substances, which was originally introduced in 1854 by a German physician, Rudolf Virchow, to denote specific macroscopic abnormalities that exhibited a positive iodine staining reaction for human tissues (Puchtler and Sweat 1966). Virchow found that waxlike deposits and degenera-

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tion of spleen, liver, and kidneys showed a starchlike reaction with iodine. A hundred years later, it was found that amyloid was formed in the protein deposits of a renal disease in man (Mellors and Ortega 1956). In 1956, Cohen and Calkins demonstrated the presence of specific fibrillar proteins with 80–100 Å width in the deposits based on the electron microscopic analysis (Cohen and Calkins 1959). According to Sunde and Blake (1998), all amyloids were assumed to share the following tinctorial, morphological, and structural characteristics: (1) stain with certain aromatic dyes like Congo Red and generate green birefringence under the polarization microscope observation; (2) can be observed as uniform, straight unbranched fibers with approximately 100 Å in diameter in the electron microscope; (3) show the ordered, repeating molecular structure of the fibrils consisting of polypeptide chains in an extended β-sheet-rich conformation, so-called the “cross-β” conformation (Glenner 1980a, 1980b). Today, it is widely believed that amyloid is a chemical that forms inside tissue in an amorphous way. Deposition of amyloid has been observed in a variety of human pathological disorders such as Alzheimer ’s, Parkinson’s, Huntington’s, and prion diseases. The disease associated with amyloid deposits is called “amyloidosis” (pl. amyloidoses). Although it is characterized by the abnormal deposition of amyloid fibrils in various tissues of the body, there is disagreement as to whether amyloid directly causes particular disease or is simply a sign of the disease downstream from the cause. Determination of the physico-chemical principles underlying amyloid fibril formation is fundamental to the identification of therapeutic strategies to prevent and/or cure amyloidosis. There is no reason to doubt that the main constituent of amyloid deposits is proteins or peptides; all amyloidoses are characterized by the transformation of soluble proteins/peptides into aggregated fibrils in different organs and tissues. Although all amyloidogenic proteins/peptides do not have any homology in their amino acid sequences, amyloid fibrils share similar physiochemical and ultrastructural features with a cross-β structure as determined by transmission electron microscopy x-ray fiber

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diffraction (Dobson 1999; Gazit 2002; Harper and Lansbury 1997; Sipe and Cohen 2000; Sunde et al. 1997; Wickner et al. 2000; Sunde and Blake 1998). Since the accurate mechanism that leads to the selfassembly of polypeptides into ordered fibrils is not fully understood, the spontaneous conversion of the soluble materials into aggregates or amyloid fibrils is a challenging problem to be solved urgently in biomedical sciences. As shown in Table 7.1, amyloidoses in humans comprise more than 20 diseases, including Alzheimer ’s disease, Parkinson’s disease, Huntington’s disease, Creutzfeldt-Jakob disease, hemodialysis-associated disease, bovine spongiform encephalopathy (prion disease), and so on. Amyloid protein deposited in a localized area is called localized amyloidosis, while deposition of amyloid throughout the body is referred to as systemic amyloidosis (Westermark et al. 2007). Systemic amyloidosis can cause serious changes in virtually any organ of the body. Since amyloid fibrils can be deposited in a localized area and may not be harmful or may only affect a single tissue of the body, there is disagreement as to whether amyloid directly causes the disease or is simply a sign of the disease downstream from the cause. Amyloidoses induced as a result of another illness, such as myeloma, tuberculosis, osteomyelitis, rheumatoid arthritis, or ankylosing spondylitis, are called “secondary.” Primary amyloidosis (AL) appears in a specialized cell in the bone marrow that contains fibrils derived from monoclonal immunoglobulin light chains. On the other hand, familial amyloidosis (ATTR) is defined as a rare form of inherited amyloidosis.

3. Naturally Occurring Amyloidogenic Proteins/Peptides 3.1. Prion Prion diseases are identified by the accumulation in the brain of an abnormal form of the host-encoded prion protein (PrP) (Prusiner and Scott 1997). Humans are subject to four prion diseases including Creutzfeldt-Jakob disease, Kuru disease, GermanStraussler-Scheinker syndrome, and fatal familial

insomnia. The normal isoform of the prion protein (PrPc) is a cell membrane–anchored glycoprotein expressed in the brain and in almost all peripheral tissues of mammals. The normal PrPc consists mainly of α-helices, but the converted PrPsc (from scrapie disease) is predominantly formed by βsheets. The PrPsc accumulates in lysosomes and eventually fills the organelle until it bursts, releasing the prions to attack other cells (Collinge 2001).

3.2. β2-microglobulin β2-microglobulin (β2-m) is the major protein constituent of dialysis-related amyloidosis (Koch 1992). β2-m is a 99 amino acid protein, which consists of seven β-strands organized in two β-sheets linked by one interchain disulphide bond (Bjorkman et al. 1987). It is the light chain component of class I major histocompatibility complex (MHC-1) and normally present in most biological fluids, including the serum. Patients with chronic renal failure on long-term hemodialysis end up with an accumulation of free β2-m in the blood. This condition facilitates the formation and deposition of β2-m amyloid fibrils in skeletal joints. Carpal tunnel syndrome and destructive arthropathy associated with cystic bone lesions are the major clinical features of dialysisrelated amyloidosis (Gejyo et al. 1986).

3.3. Cystatin C Cystatin C is a cysteine protease inhibitor found in the brain that accumulates in cortical vessels in hereditary cerebral hemorrhage with amyloidosis in Iceland (Gudmundsson et al. 1972). The active form of human cystatin C is a single nonglycosylated polypeptide chain consisting of 120 amino acid residues and containing 4 characteristic disulfide-paired cysteine residues. Human cystatin C is encoded by the CST3 gene, ubiquitously expressed at moderate levels. Cystatin C monomer is present in all human body fluids; it is preferentially abundant in cerebrospinal fluid and seminal plasma. Cystatin C L68Q variant is an amyloid fibril-forming protein with a high tendency to dimerize. It forms self-aggregates with massive amyloid deposits in the brain arteries

Chapter 7 Amyloidogenic Proteins and Peptides

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Figure 7.1. Amyloid fibril formation of an amyloidogenic protein, L68Q human cystatin C. Recombinant L68Q cystatin C expressed by Pichia pastris was incubated in pH 7.0, 100mM NaCl, at 37°C with continuous agitation. Process of amyloid fibrils formation of L68Q was monitored with Thioflavin T (ThT) fluorescence specifically binding amyloid fibrils. During lag period, dimer, trimer, and eventually oligomer nucleations ( ) are assumed to be formed. Elongation of seeded fibrils ( ) rapidly proceeds to mature fibrils at steady states.

of young adults, leading to lethal cerebral hemorrhage early in life and premature death. The process of amyloid formation of recombinant L68Q cystatin C was monitored by the thioflavin-T fluorescence assay for amyloid fibril detection, as shown in Figure 7.1. Amyloid fibrils have been demonstrated to assemble typically via nucleation and growth kinetics, characterized by an initial lag phase before fibril elongation. During lag period, dimmer, trimer, and eventually oligomer nucleation are formed. Seeded fibril elongation rapidly progresses to mature fibrils at steady states. The fibril formation was observed by transmission electron microscope in lag phase and elongated fibrils.

3.4. Aβ Alzheimer ’s disease (AD) is the most common form of dementia, a neurologic disease characterized by impairment of mental ability severe enough to interfere with normal activities of daily living (Hardy and Higgins 1992). AD is characterized by the progressive accumulation of amyloid β protein (Aβ) in areas of the brain serving cognitive functions such as memory and language. Aβ is a peptide consisting of 40–42 amino acids and is derived by β- and γ-

secretase cleavage from the Aβ protein precursor (APP), a type I transmembrane protein, which contains about 700 amino acid residues. Aβ42 is more hydrophobic than Aβ40 and is most closely linked with AD pathogenesis (Selkoe 1997). Increases in the concentration of Aβ in the course of the disease with subtle effects on synaptic efficacy will lead to gradual increase in the load of amyloid plaques and progression in cognitive impairment.

3.5. Amylin In 1987, Westermark and Cooper identified the major component of islet amyloid as a 37 amino acid peptide and named it islet amyloid polypeptide (IAPP) or amylin (Westermark et al. 1987, 1990; Clark et al. 1987). IAPP is a neuroendocine peptide hormone that is produced and co-secreted along with insulin from pancreatic β-cells. In type 2 diabetes, amylin aggregates to form fibrils that are thought to be toxic to β-cells. Islet IAPP has turned out to be a pathogenic factor, which is accompanied by death of β-cells and reduction of the insulinproducing capacity. Human IAPP exhibits a random coil structure with small components of α-helix and β-sheet conformations.

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3.6. Others α-synuclein is a natively unfolded 140 residue protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson’s disease (Polymeropoulos et al. 1997). Polyglutamine expansion in the huntingtin protein is responsible for the neurodegenerative disease Huntington’s disease (Martin and Gusella 1986). It is caused by a dominantly transmitted CAG (Cytosine-AdenineGuanine) repeat expansion mutation, called a triplet repeat disease.

4. Amyloidogenicity of Small Peptides Although the structure of amyloid fibrils remains limited in atomic detail, there is accumulating evidence from x-ray fiber diffraction studies that the core of the typical amyloid fibril is composed of β-sheets whose strands run perpendicular to the fibril axis (Sunde and Blake 1997). It has been shown that specific short segments in proteins are responsible for their fibril-forming behavior. The short peptides of amyloidogneic proteins are able to form a cross-β-structure, which is indistinguishable from those formed by the usual peptide chains (Lopez De La Paz et al. 2002; Jaroniec et al. 2002; Petkova et al. 2002). Amyloidogenic peptides known to date are summarized in Table 7.2. Although all mature amyloid fibrils show similar organized structure under x-ray fiber diffraction, electron microscopy, and specific chemical staining analyses, precursor proteins that form amyloid fibrils do not share sequence homology. The tripeptide VYK is a minimal core segment of fibril formation among the peptides identified from amyloid proteins (Inouye et al. 2006). It is well established that the aromatic residues play an important role in fibrillogenesis (Gazit 2002). The interaction between aromatic tyrosine residues is likely initiated to fibril formation. Besides, phenylalanine pairs occur in the Alzheimer ’s disease peptide Aβ because phenylalanine side chains are considered to stabilize the intersheet packing. Studies using de novo designed peptides revealed that Ile-Phe dipeptide analog is able to self-assemble as a network of fibrillar struc-

tures in aqueous solution. On the other hand, overexpression of hydrophobic homopolymeric amino acids, such as polyisoleucine, polyleucine, polyphenylalanine, and polyvaline, produced oligomerization and aggregation in cells (Oma et al. 2004). The hydrophobicity itself may trigger the oligomerization and aggregation of proteins. These small size peptides may serve as an excellent model system for investigating the role of amyloid formation and suppression.

5. Strategies for Prevention of Amyloid Formation 5.1. Phenolic Compounds Antioxidant molecules that inhibit amyloid formation and destabilized preformed amyloid may contribute to the prevention or control of amyloidosis (Ono and Yamada 2006). Several polyphenol molecules have been demonstrated to remarkably inhibit the formation of amyloid fibril assemblies (Porat et al. 2006). Nordihydroguaiaretic acid (NDGA), curcumin, rosmarinic acid, and ferulic acid inhibit amyloid fibril formation from Aβ and fibril extension dose-dependently in vitro (Naiki et al. 1998; Ono et al. 2003, 2004, 2005). Our observation using a mutant human cystatin C, L68Q, also revealed antiamyloid effects of polyphenols. It was demonstrated that dopamine, chlorogenic acid, ferulic acid, synephrine, and naringenin delay efficiently the transition period to the nucleation phase of the amyloidogenic L68Q protein, while quercetin, NDGA, curcumin, and genistein elongate the amyloid fibril of the protein. It is well known that cystatin C can form a stable dimmer via three-dimensional domain swapping in which there are two important domains in its feature, closed-interface and open-interface. As shown in Figure 7.2, the closed-interface is composed of α-helix and β-strand. Hydrophobic interaction between the position 34Y on the α-helix region and the position 96F on the β-strand may contribute to keeping the closed-interface in the domainswapped molecules. On the other hand, openinterface is composed of each β-strand that is newly generated when monomers become open structure.

Table 7.2. Amyloidogenic peptides. Origin Tau

Aβ

Prion human

Prion mouse Prion Syrian hamster Prion elk Cystatin C human Apolipoprotein A1 Calcitonin Yeast prion sup35

Yeast prion sup35 & huntingtin β2-microglobulin

Insulin A chain Insulin B chain Lysozyme human Lysozyme hen IAPP (Amylin)

RNase Myoglobin α-synuclein

Designed peptide

Region 590–595 274–279 308–310 307–310 305–310 16–21 16–22 37–42 35–40 178–183 244–249 245–250 138–144 170–175 138–144 138–144 170–175 98–103 13–18 15–19 15–18 7–13 8–13 8–11 — 83–88 83–89 62–67 64–69 13–18 12–16 56–61 55–60 22–27 28–33 14–20 15–20 108–113 51–56 66–74 86–92 — — — — — — — — — — —

AA Sequence

References

KVQIIN VQIINK VYK IVYK VQIVYK KLVFFA KLVFFAE GGVVIA MVGGVV DCVNIT ISFLIF SFLIFL IIHFGSD SNQNNF MIHFGND MMHFGND NNQNTF SFQIYA LAVLFL DFNKFH DFNKF GNNQQNY NNQQNY NNQQ QQQQQ NHVTLS NHVTLSQ FYLLYY LLYYTE LYQLE VEALY IFQINS ILQINS NFGAIL SSTNVG NFLVHSS SSTSAA SQAIIH GVATVA VGGAVVTGV GSIAAT KTVIIE STVIIE KTVIIT STVIIT KTVLIE KTVIVE KTVIYE STVIYE IIIII FF IF

Pastor et al. 2008 Inouye et al. 2006; von Bergen et al. 2001 Inouye et al. 2006 Inouye et al. 2006 Inouye et al. 2006; von Bergen et al. 2000 Pastor et al. 2008 Balbach et al. 2000 Sawaya et al. 2007 Sawaya et al. 2007 Pastor et al. 2008 Pastor et al. 2008 Pastor et al. 2008 Sawaya et al. 2007 Sawaya et al. 2007 Sawaya et al. 2007 Sawaya et al. 2007 Sawaya et al. 2007 Pastor et al. 2008 Pastor et al. 2008 Reches et al. 2002 Reches et al. 2002 Gsponer et al. 2003; Balbirnie et al. 2001 Nelson et al. 2005 Sawaya et al. 2007 Sawaya et al. 2007 Ivanova et al. 2004 Ivanova et al. 2004 Ivanova et al. 2004; Jones et al. 2003 Ivanova et al. 2004; Jones et al. 2003 Sawaya et al. 2007 Sawaya et al. 2007 Krebs et al. 2000 Krebs et al. 2000 Tenidis et al. 2000 Tenidis et al. 2000 Tenidis et al. 2000 Sawaya et al. 2007 Fandrich et al. 2003 Sawaya et al. 2007 Sawaya et al. 2007 Sawaya et al. 2007 Lopez De La Paz et al. 2002 Pastor et al. 2008; Lopez De La Paz et al. 2002 Lopez De La Paz et al. 2002 Lopez De La Paz et al. 2002 Lopez De La Paz et al. 2002 Lopez De La Paz et al. 2002 Lopez De La Paz et al. 2002 Lopez De La Paz et al. 2002 Sambasivam et al. 2008 Reches and Gazit 2003 de Groot et al. 2007

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Figure 7.2. Schematic model for the inhibitory mechanism of amyloid fibril formation by polyphenol compounds. Left figure shows a dimeric domain-swapped L68Q human cystatin C (http://www.rcsb.org/pdb, PDB ID = 1TIJ).

The open-interface is also presumed to be stable because of the interaction between the positions 102Y and 106W. The open-interface is thought of as playing an important role to make bigger oligomer or elongate their fibrils. Destabilization of closed-interface open-interface may be effective for preventing nucleation and for preventing elongatation of fibrils, respectively. It can be proposed that inhibitors having more compact structure, such as phenol carboxylic acid or phenol amine, may be more effective against nucleation formation but not so for amyloid fibril elongation. Bindings could be induced by hydrophobic interactions between polyphenol rings and the hydrophobic region of closedinterface, α-β interface in L68Q cystatin C, thus blocking three-dimensional domain swapping and stabilizing the amyloidogenic protein conformation. The inhibitory mechanism of dopamine is illustrated in Figure 7.2. Compared with compact molecules, large compounds with double rings such as flavonids or curcumin exerted an inhibitory effect on the amyloid fibril elongation phase. In this phase, oligomers stack with each other and elongate fibrils by β-interaction with long β-sheets termed openinterface. The cohesive new structural feature could be observed in the dimmer. Large molecules are hypothesized to be involved in inhibiting β-

interactions of open-interface with each oligomer. These interactions could be reinforced by H-bond between the hydroxyl group of phenolic rings and the donor/acceptor group of open-interface (Figure 7.2). These observations may be useful for treatment of amyloidosis.

5.2. Peptidic Compounds Highly amyloidogenic core sequences in amyloid proteins may play critical roles in the initiation and progression of amyloid fibril formation (Sawaya et al. 2007). Thus, it is postulated that fibrillogenesis of Aβ can be inhibited by short peptides’ partially homologous sequence acting as β-sheet breakers (Soto et al. 1996). A small peptide, LPFFD, which was designed from Aβ17-20 (LVFF), behaves as a β-sheet breaker and completely blocks the formation of amyloid fibrils in a rat brain model of amyloidosis (Soto et al. 1998). Findeis et al. successfully developed peptidic inhibitors with potent antipolymerization activity (1999). On the basis of optimization of the Aβ-derived peptides portion and size-reduction trials, they found that Cholyl-Leu-Val-Phe-Phe-AlaOH showed a potent inhibitory activity against amyloid formation. Murphy et al. (2008) proposed synthetic peptidic derivatives for inhibition of

Chapter 7 Amyloidogenic Proteins and Peptides

amyloid formation of Aβ, that is, proline-introduced, terminal-blocking, and N-methlylated homologous to Aβ15-25 (Ghanta et al. 1996; Lowe et al. 2001; Pallitto et al. 1999). Soto et al. demonstrated that a pentapeptide, LPFFD, can inhibit amyloid fibril formation from Aβ1-40 and Aβ1-42 (1998). Based on the evidence of the stability of dehydrophenylalanine (ΔPhe) residue to enzymatic degradation, a βsheet breaker, Boc-Phe-Ala-Ile-ΔPhe-Ala-Ome, was developed (Gupta et al. 2008). Since ΔPhe residue itself exhibited a potential ability to inhibit amyloid fibril formation, it can be used for designing peptidebased antifibrillization drugs that are resistant to enzymatic degradation in vivo. These data must provide a therapeutic potential of peptidic compounds in terms of preventing and retarding amyloid-related disorders.

5.3. Others Recently, Tanaka et al. reported that various disaccharides reduced the formation of polyglutamine aggregates through in vitro screening studies. Further in vivo experiments established that oral administration of treharose, the most effective disaccharide, inhibited the formation of huntingtin aggregates and extended lifespan in a transgenic mouse model of Huntington’s disease (Tanaka et al. 2004). The stabilization of aggregation-prone proteins with carbohydrate may provide a new therapeutic strategy for polyglutamine-mediated pathology because it can block the initial stage of the disease cascade.

6. Conclusion Under certain environments, soluble proteins and protein fragments (peptides) self-assemble into βsheet-rich structures (cross-β conformation) and form amyloid. Amyloid causes serious disorders known as amyloidosis, such as Alzheimer ’s disease, hemodialysis-associated disease, and bovine spongiform encephalopathies, which are characterized by the transformation of soluble proteins/peptides into aggregated fibrils in different organs and tissues. Recent reliable discovery of amyloid-forming sequences in amyloidogenic proteins should have a

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great impact on the development of antiamyloid agents. Indeed, peptidic modulators were developed based on the partially homologous sequence with the core sequences. Several observations demonstrated that polyphenols are capable of inhibiting amyloid fibril formation in vitro, where it is assumed that some of them act directly with the amyloidogenic core region. These findings must open new strategies for the development of amyloid-formation inhibitors.

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Chapter 8 Peptide-Based Immunotherapy for Food Allergy Marie Yang and Yoshinori Mine

Contents 1. Introduction, 101 2. Background Information, 102 2.1. Intestine Mucosal Barrier and Mechanisms of an Allergic Response, 102 2.1.1. Intestine Mucosal Barrier, 102 2.1.2. Allergic Response to Food Antigens: A Two-Phase Mechanism, 103 2.2. Roles of T Lymphocytes in Food Allergy, 103 2.3. Molecular Properties of Food Allergens, 105 2.4. Food Allergen B- and T-cell Epitopes, 105 2.4.1. Definitions, 105 2.4.2. Allergen Epitope Identification, 106 2.4.2.1. T-cell Epitope Mapping, 106 2.4.2.2. B-cell Epitope Mapping, 107 2.5. Current Management of Food Allergy, 107 2.6. Overview of Novel Immunotherapeutic Strategies, 108 3. Rationale, Strategies, and Potential Mechanisms of PIT, 108 3.1. Aim, Rationale, and Strategies, 108 3.1.1. Aim and Rationale, 108 3.1.2. Strategies, 108

3.1.2.1. Use of Overlapping Synthetic Peptides, 109 3.1.2.2. Food-Based Hydrolyzates, 109 3.1.2.3. B-cell Epitope-Based Immunotherapy (IgG-Binding), 110 3.1.2.4. T-cell Epitope-Based Immunotherapy, 110 3.2. Advantages of Peptide-Based Immunotherapy, 110 3.3. Suggested Mechanisms Underlying PIT, 111 4. Current Experimental Models of PIT in Food Allergy, 111 4.1. Cow’s Milk Allergy Model, 112 4.2. Peanut Allergy Model, 113 4.3. Egg Allergy Model, 113 4.4. Wheat and Beef Allergy Model, 114 4.5. PIT Investigations Carried Out with Inhalant Allergens, 114 5. Translation of PIT into Human Clinical Applications, 114 5.1. MHC Polymorphism, 115 5.2. Large-Scale Production of Peptides, 115 5.3. Other Practical Issues, 115 6. Conclusion, 115 7. References, 116

1. Introduction

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

Food allergy is classically defined as an adverse reaction to food components resulting from an overt response mediated by the immune system. Its prevalence has been estimated at 2–4% of adults and 6–8% of children in Western countries (Burks et al. 101

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2008a; Sicherer and Sampson 2008). These figures represent approximately twice those reported only a decade ago. The most common form of food allergy is mediated by the class of immunoglobulin E (IgE), a type I form of hypersensitivity according to the classification of Gell and Coombs (Rajan 2003). The clinical symptoms associated with an allergic response include skin rashes, wheezing, sneezing, swelling around the lips, bloating, vomiting, and diarrhea; in extreme cases a life-threatening reaction, known as anaphylaxis, can occur within minutes. Currently, the most medically advised approach for the management of food allergy is an avoidance diet; however, risks of inadvertent exposure to the offending food as well as possibilities for malnutrition render the approach unviable for allergic individuals. Thus the escalating incidence of food allergy and the current lack of efficient treatment call for the development of more active immunotherapeutic interventions. Of the innovative strategies currently investigated, peptidebased immunotherapy (PIT) represents an elegant approach to prevent or downregulate allergic responses and potentially induce long-term immune tolerance. While a number of investigations have already documented the efficacy of a PIT-based approach in respiratory allergies, recent studies have emerged reporting promising results on the therapeutic efficacy of the approach for food-induced allergy. This chapter focuses on the rationale and strategies behind peptide-based immunotherapy. Current experimental models of PIT for food allergy are described, and translation of the findings into human applications is discussed. Potential scenarios for the molecular and cellular mechanisms underlying PIT are also presented.

2. Background Information The gastrointestinal mucosa is constantly exposed to a multitude of innocuous food antigens that do not usually elicit an overt immune response. The reasons why certain individuals are more susceptible to developing an allergic response to food anti-

gens remain obscure; however, epidemiological studies suggest that both genetic and environmental influences are involved. While the causes underpinning food allergies remain to be fully elucidated, intensive investigations are focusing on characterizing the common properties of food allergens and on unraveling the molecular and cellular mechanisms underlying allergic responses. These aspects are briefly presented in the following section as background information prior to introducing the concept of PIT.

2.1. Intestine Mucosal Barrier and Mechanisms of an Allergic Response 2.1.1. Intestine Mucosal Barrier For many years, it was presumed that macromolecules did not enter the circulation via the gastrointestinal tract. The presence of mucosal secretions, binding by secretory IgA, and commensal bacteria were factors that were believed to ensure the barrier function of the intestinal tract. However, it has now been clearly shown that dietary antigens can cross the intestinal epithelial barrier, in both intact and partially digested forms, and can directly interact with the cells of the immune system (Chehade and Mayer 2005). Food antigens entering the gastrointestinal tract can thus be found in the vascular compartment in an intact form (Husby et al. 1985a, 1985b). The mechanisms by which food antigens traverse the gut into areas containing immunocompetent cells are multiple: they occur (1) through an invagination process, known as endocytosis, which is facilitated by the uptake of food antigens by immunoglobulin Fc receptors (mainly IgG isotypes); (2) through tight junction structures present between intestinal epithelial cells; (3) via an absorption mechanism mediated by a class of specialized cells, known as M cells—M cells differ from normal epithelial cells due the absence of surface microvilli, a poorly developed glycocalyx, and a lack of lysosomal organelles, suggesting little or no digestion of antigen; or (4) via an alternative pathway involving dendritic cells, which can extend their processes into

Chapter 8 Peptide-Based Immunotherapy for Food Allergy

the lumen to directly sample dietary antigens (Chehade and Mayer 2005). 2.1.2. Allergic Response to Food Antigens: A Two-Phase Mechanism The etiology of IgEmediated food allergy is classically described as a two-phase phenomenon: (1) the sensitization phase, without symptoms, during which the immune system is primed, resulting in the production of antibodies belonging to the immunoglobulin E class, and (2) the elicitation phase with specific clinical responses (i.e., allergic symptoms), which occurs after subsequent exposure to the offending compound. During the sensitization phase, it is believed that food antigens traverse the intestinal barrier and are captured by the underlying immune cells. They are processed and presented by a specialized class of immune cells known as antigen-presenting cells (APCs) for presentation to T lymphocytes. The cytokine environment encountered by a naive CD4+ T helper cell (Th0) plays a prominent role in determining whether Th0 will exhibit a Th1- or Th2-cell phenotype. Allergic individuals preferentially develop an interleukin (IL)-4-rich microenvironment that drives the immune response toward a Th2-biased response, which in turn promotes IgE production by B cells. Allergen specific-IgE then binds to highaffinity receptors (FcεRI) present on the surface of mast cells and basophils. The influences responsible for an IL-4 dominant environment are not completely understood, but innate immune cells such as natural killer (NK) and NK-T cells have been suggested as candidate sources of IL-4 (Yoshimoto et al. 1995). Upon subsequent ingestion of the food allergen (elicitation phase), allergenic fragments (epitopes) bind to receptor-bound IgE present on mast cells and basophils, triggering the aggregation of the receptors and the release of inflammatory and vasoactive mediators such as leukotrienes, prostaglandins, and histamine. Simultaneously, allergen presentation by APCs leads to the rapid stimulation of sensitized Th2 cells, as well as the local recruitment and activation of effector cells such as eosinophils and basophils (Figure 8.1). These events are responsible for the clinical reactions characteristics of an allergic response.

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2.2. Roles of T Lymphocytes in Food Allergy The contribution of T lymphocytes—and more particularly, CD4+ T cells—is pivotal in facilitating the production of allergen-specific IgE, and in their role of effector cells secreting autocrine and paracrine factors, of which cytokines are the main players. As mentioned earlier, the cytokine environment encountered by a naive T helper cell (Th0) plays a prominent role in determining whether Th0 will adopt a Th1 or a Th2 phenotype. While Th1 cells play a prominent role in cell-mediated immunity with the secretion of cytokines such as IFN-γ and TNF-β, which promote the development of cytotoxic T lymphocytes (CTLs) and macrophages (Chehade and Mayer 2005), Th2 cells primarily promote humoral immune responses, with the production of cytokines such as IL-4 and IL-13 (IgE synthesis), but also IL-5 (eosinophil proliferation) and IL-9 (mast cell activation). A key feature of the Th1/Th2 balance is that the two cell subsets are capable of counter-regulating one another (Mosmann and Coffman 1989; Mosmann et al. 1986). In recent years, although the paradigm of the Th1/Th2 balance is still recognized, more complex changes in lymphocyte responses were shown to arise in the course of an allergic response. The Th1/Th2 model has, therefore, evolved to encompass the heterogeneous and complex populations of regulatory T cells (Tregs). The revised model is schematically represented in Figure 8.2. This has spurred interest in the prospective use of Tregs for the treatment of allergic conditions (Elkord 2006). Indeed regulatory T cells, previously known as suppressor T cells, are believed to play a critical role in the maintenance of immunological tolerance (Verhagen et al. 2006) at mucosal surfaces. They have been defined as a class of immune cells able to “actively control or suppress the function of other immune and non-immune cells, generally in an inhibitory fashion” (Prioult and Nagler-Anderson 2005). Within this conceptual framework, it was suggested that a disruption of Tregs activity may promote the development of a Th2-skewed allergic response. Mechanistically, regulatory T cells have been reported to exert their inhibitory activity on T helper

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Figure 8.1. Schematic representation of the sequence of events leading to type I hypersensitivity. Adapted from Prioult and Nagler-Anderson 2005; Kaza et al. 2007. Note: A complex bidirectional interplay exists between mast cells/granulocytes and CD4+T cells (Ahern and Robinson 2005), but the phenomenon has not been illustrated in the figure.

cells (both Th1 and Th2) and their downstream effectors by either direct contact or by production of suppressive cytokines (for example, IL-10 and TGF-β) (Stock et al. 2006). Two major lineages of Tregs have been described: (1) natural thymusderived Tregs, which constitutively express CD4,

CD25, and FOXP3; and (2) inducible Tregs activated in the periphery after antigen stimulation. Inducible Tregs include T regulatory (Tr)-1 cells, characterized by production of both IL-10 and TGFβ; Th3 cells, characterized by their prominent production of TGF-β (Taylor et al. 2006); and also a

Chapter 8 Peptide-Based Immunotherapy for Food Allergy

Figure 8.2. Currently accepted view on T-cell regulation. An immune balance exists between the different subsets of CD4+ T cells involving Th1, Th2, and Treg cells. A disturbance in the balance can lead to a nonfavorable outcome for patients, such as an allergic response. Several populations of Treg have been reported, for example, IL-10 producing inducible Tr1 cells, TGF-β-producing Th3 cells, naturally occurring and inducible CD4+CD25+FOXP3 Treg cells. TGF-β and IL-10 could exert direct and/or indirect inhibitory effects on neighboring cells and are believed to play an important role in the induction of T-cell tolerance. Adapted from Jeurink and Savelkoul 2006.

class of peripheral FOXP3-expressing CD4+CD25+T cells. A major limitation in the study of Treg functions and cellular activities has been the lack of reliable and specific Treg molecular markers. This was further complicated by reports describing various types of Treg cells including innate immune cells such as natural killer T (NKT) cells, γδ-T cells, CD8+ T cells, and B-cell subsets (Verhagen et al. 2006; Nagler-Anderson et al. 2004), as well as a newly reported subset of CD4+ T cells known as Th17 cells (Oboki et al. 2008). There is undeniable evidence that T cells are essential in the pathogenesis and regulation of allergic reactions. However, it is important to note that allergic responses result from interactions involving many other players of the immune system (both cells and soluble mediators), contributing to the complexity of the allergic process and hindering the development of efficient curative strategies.

2.3. Molecular Properties of Food Allergens Foods are typically derived from both animal and vegetable sources. Examples of animal groups

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include birds (e.g., chicken), crustaceans (e.g., crab), and red meat (e.g., beef), while plant groups include the apple family, grass family (e.g., wheat), legume family (e.g., peanut), and walnut family (Burks 2004a). Proteins, lipids, and carbohydrates are the major constituents of foods; however, food allergens have been mainly characterized as glycoproteins having a molecular mass of 10–60 kDa (Bernstein et al. 2003) and soluble in water (i.e., albumins) or saline solutions (i.e., globulins) (Burks 2004b). They commonly own attributes that preserve them from the destructive effects of low pH, proteolysis, and thermal treatments (Bredehorst and David 2001; Huby et al. 2000; Breiteneder and Ebner 2000; Breiteneder and Mills 2005), but exceptions exist, especially for allergens found in fruits and vegetables (Bannon 2004). Other properties, such as the presence of disulfide bonds, N-glycosylation, structural aggregation, rheomorphic or repetitive structures, and lipid-binding capacity, are also believed to contribute to their inherent stability and, therefore, to their allergenicity (Breiteneder and Mills 2005). Structurally, comparisons of primary amino acid sequences failed to reveal any typical pattern (Ivanciuc et al. 2003a, 2003b). On the other hand, a recent study based on in silico approaches suggested that plant food allergens may be classified into four structural superfamilies based on their conserved native surface features (Jenkins et al. 2005) and supports the view that conserved spatial motifs of food proteins may be major determinants in their allergenicity (Sampson 2005). Features common to food allergens were gathered into a bioinfomatics databank and exploited in an attempt to create prediction tools for allergenicity (Kleter and Peijnenburg 2002; Ivanciuc et al. 2003a; Stadler and Stadler 2003; Soeria-Atmadja et al. 2006); however, to date unique structural motifs or determinants responsible for food allergenicity have yet to be identified.

2.4. Food Allergen B- and T-cell Epitopes 2.4.1. Definitions The immune system classically recognizes antigens through two classes of molecules: (1) immunoglobulins, in the form of soluble or membrane-bound molecules produced by

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B lymphocytes, or (2) T-cell receptors or TCRs, present at the surface of T lymphocytes in the context of major histocompatibility complex presentation by APCs. The molecular regions specifically recognized by immunoglobulins and TCRs are commonly known as B-cell and T-cell epitopes, respectively. B-cell epitopes can be either continuous (i.e., linear amino acid sequences) or discontinuous (i.e., residues forming the epitope arise from different positions brought together by folding). B-cell epitopes do not have a defined length and can vary from 5 to 20 amino acids in length. The importance of linear vs. conformational epitopes has been the object of several studies, and it was proposed that the recognition of linear epitopes by certain patients was predictive of a persistent form of food allergy in these individuals (Mine and Yang 2008). On the other hand, the length of T-cell epitope peptides associated with MHC class II molecules is much more restricted and varies from 10 to 20 amino acids (Zeiler and Virtanen 2008). T-cell epitopes are strictly linear sequences as a result of the degradation of antigens through the cytosol of APCs (Figure 8.3). In the past few years, a great deal of research has focused on the characterization of B- and T-cell

epitopes of food allergens to provide a better understanding of their recognition by immune cells. Knowledge of food allergen epitopes can provide important information on the pathogenesis of food allergy, and it can also be a guide for the development of pertinent experimental and therapeutic approaches for the prevention or the suppression of allergic disorders (Bohle 2006). 2.4.2. Allergen Epitope Identification A detailed description of the methods available for the identification of allergen epitopes (or epitope mapping) is beyond the scope of this chapter. A brief overview is presented in this section, but comprehensive information is available elsewhere (Morris 1996). 2.4.2.1. T-cell Epitope Mapping Activation and proliferation of CD4+ T lymphocytes is initiated upon contact with an APC presenting T-cell epitopes complexed with MHC class II molecules. This cognate recognition is critical to generation of the adequate immune response. Progress in the past decade allowed the identification of T-cell epitopes in any allergen with a known primary sequence, and such investigation has been initiated for major food allergens. Experimental approaches toward

Figure 8.3. Schematic representation of antigen B- and T-cell epitopes or determinants.

Chapter 8 Peptide-Based Immunotherapy for Food Allergy

identification of T-cell epitopes rely traditionally on the preparation of cell cultures. Cultures are prepared from peripheral blood mononuclear cells (PBMCs) in humans, and from lymph nodes or spleen when using murine models. Proliferation and cytokine assays are often conducted upon stimulation with libraries of overlapping sequences covering the entire primary sequence of the protein of interest to allow localization of the immunogenic regions. In humans, establishment of allergenspecific T-cell lines or clones is often preferred due to the low frequency of specific T cells and the weak responses obtained with PBMC cultures. The preparation of T-cell clones permits a detailed analysis of epitope specificity and cell phenotype (Zeiler and Virtanen 2008), but can be labor intensive and technically demanding. Alternative epitope mapping tools include the use of tetrameric MHC complexes and flow cytometry (Hoffmeister et al. 2003). Computer-based programs such as TEPITOPE (Bian et al. 2003) or COREX/BEST (Melton and Landry 2008) have also been designed to predict MHC class II binding regions and were shown to be applicable to allergenic proteins. While such algorithms may be useful for predicting epitopes prior to conducting the actual mapping experiments, complete agreement with data generated by in vitro methods is not always observed. 2.4.2.2. B-cell Epitope Mapping Food allergens are a group of antigens classically defined by their ability to induce the production of specific IgE. The crosslinking of allergen specific IgE at the surface of mast cells or basophils is the event responsible for the clinical symptoms characteristic of an allergic response. As such, characterization of IgEbinding sites (or B-cell epitopes) is considered an important step toward the characterization of allergens. Identification of antigenic determinants primarily requires (1) knowledge of the amino acid sequence of the protein and (2) the use of specific antibodies (monoclonal or polyclonal, obtained from animal or patient sera) for the antigen of interest. Indeed, conventional methods for identification of B-cell linear epitopes rely on the use of overlapping linear peptide fragments where antigenic deter-

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minants are assayed by ELISA or by methods in which the antibody has been labeled with a fluorochrome or other type of readily detectable reagent. The library of peptides can be provided on a solid support (e.g., SPOTS® membranes or PEPSCAN on paper discs) or in the form of peptide libraries displayed by bacteriophages (Zegers et al. 1995). On the other hand, methods to identify conformational B-cell epitopes are based on the use of x-ray crystallography often combined to sitedirected mutagenesis. This latter method, however, requires a high degree of purity of the antigen and remains complex and time consuming. Alternatively, recent technology based on the use of chemical “scaffolds” (i.e., Chemically Linked Peptides on Scaffolds, or CLIPS™), onto which one or more peptides are attached to closely resemble the native structure of protein, may allow identification of B-cell discontinuous epitopes yet to be tested for food allergens. Similar to T-cell epitopes, a multitude of predictive methods for identification of B-cell epitopes have been documented based on physicochemical and structural propensity scales (e.g., hydrophilicity, flexibility, exposure to the solvent, and occurrence of charged residues). Their limitations have been discussed in recent reviews (Ponomarenko and Bourne 2007; Roggen 2006).

2.5. Current Management of Food Allergy Current approaches toward the management of food-induced allergic conditions mainly consist of dietary avoidance and pharmacological interventions. Compliance with an avoidance regimen is often difficult because many prepackaged or prepared foods may contain minute amounts of milk, egg, or nut proteins, commonly referred to as “hidden allergens,” in a dose sufficient to provoke allergic symptoms (Arshad 2001; Zeiger 2003). These hidden allergens may result from allergen contamination during food processing, misformulation, or erroneous labeling (Puglisi and Frieri 2007). More importantly, the elimination of essential foods for an extended period of time may not represent a viable

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solution, particularly in young children, as it may lead to malnutrition (Sampson 1999). Additionally, recent studies have suggested that an elimination diet may not represent the safest alternative for allergic patients and may in fact have a deleterious impact by lowering the patient reactivity threshold (Morisset et al. 2007; Rolinck-Werninghaus et al. 2005). Current pharmacological treatments mainly induce symptomatic relief through the use of agents such as antihistamines, steroids, and, in severe cases, epinephrine (Thompson and Chandra 2002).

2.6. Overview of Novel Immunotherapeutic Strategies The current lack of efficient treatments for food allergy has prompted the development of novel preventive and therapeutic approaches that can provide safer alternatives for combatting food allergies. This chapter focuses on the key findings brought by peptide-based approaches. The interested reader is referred to a number of recent reviews focusing on the various immunotherapeutic strategies currently investigated for the treatment and prevention of IgEmediated food allergy (Sicherer and Sampson 2008; Nowak-Wegrzyn and Sicherer 2008), as are listed in Table 8.1. Immunotherapeutic approaches can be divided into specific and nonspecific approaches. Specific immunotherapy (SIT) targets the individual allergens responsible for the patient’s disease, whereas nonspecific immunotherapy aims at modulating the immune system in an allergen-independent manner (Mine and Yang 2008). Specific immunotherapies can be further divided into those using native forms of food allergens and those using recombinant or altered forms of allergens.

3. Rationale, Strategies, and Potential Mechanisms of PIT 3.1. Aim, Rationale, and Strategies 3.1.1. Aim and Rationale While conventional forms of IT are primarily based on injection or ingestion of intact food proteins, their use is not recommended due to unacceptably high rates of adverse—and sometimes life-threatening—side

effects. For this reason, new approaches are being investigated. In line with the scope of this book, the utilization of peptides will be the main topic described in this section. The concept behind peptide-based immunotherapy is not new and stems from the development of peptide-based vaccines designed to elicit a protective immune response toward pathogens such as viruses, bacteria, and parasites. Such vaccines are designed to incorporate immunoreactive regions derived from the protein native structure and give rise to a specific immune response toward the intact protein. In the context of food allergy, the terminology associated with a good therapeutic prognosis remains controversial and is often referred to as “desensitization” or “tolerance” induction (Niggemann et al. 2006). A recent monograph emphasized that discrimination should be made between true “tolerance,” which refers to situations where the food may be ingested without triggering any allergy symptoms even during elimination periods, and “desensitization,” which refers to cases where the allergen is ingested without symptoms during treatment but requires a maintenance regimen (Nowak-Wegrzyn and Sicherer 2008). Within this conceptual framework, a PIT-based approach aims at designing so-called “tolerogenic” peptides (Pecquet et al. 2000), that is, peptides capable of inducing a status of immune tolerance toward the allergen(s) of interest. Specific to food allergy, the rationale of PIT relies on the use of nonanaphylactic (synthetic or not) peptides, unable to crosslink IgE molecules (Ferreira et al. 2004). The reduced ability of peptides to react with IgE will prevent not only mediator release from mast cells and basophils (Figure 8.1) but also the uptake on IgE via Fc epsilon receptors of antigen presenting cells, which may contribute to an amplification of the allergic response. 3.1.2. Strategies Potential PIT-based strategies for food allergy are presented in the following paragraphs based on the source of peptides (i.e., synthetisized vs. enzymatically obtained peptides) and on the nature of the immunogenic sequences contained in the preparation (i.e., T-cell epitopes vs. B-cell epitopes).

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Table 8.1. Potential immunotherapeutic approaches for IgE-mediated food allergy. Adapted from Sicherer and Sampson 2008; Nowak-Wegrzyn and Sicherer 2008. Immunotherapeutic Approaches

Rationale

Current Status/Comments

Specific immunotherapy with native forms of food allergens Subcutaneous injection Antigen presentation in nonmucosal sites favors Th1-biased response Oral administration Antigen presentation at mucosal sites leads to desensitization and/or immune tolerance Sublingual administration

Induction of “tolerogenic” antigen presenting cells leads to immune tolerance.

Specific immunotherapy with altered forms of food allergens Recombinant mutant proteins Targeted mutations of IgE-binding sites Peptide-based immunotherapy Short peptides lack crosslinking activity but maintain T-cell activation potential. Conjugation with ISS sequences Enhance Th1 response by activating innate immune receptors Plasmid DNA-encoded allergens Chemically or physically modified allergens

Endogenous production of allergens may favor tolerance. Hypoallergenic preparation reduces risks of inducing severe side effects.

Nonspecific immunotherapy (immunomodulatory treatments) Anti-IgE antibodies Inactivate and bind IgE. Recommended use in combination with specific forms of immunotherapy Cytokine/anticytokine agents Blocks inflammatory signals

Chinese herbal medicine

Immunosuppressive mechanisms remain to be elucidated.

Probiotics

Stems from the conceptual framework of the “hygiene hypothesis”

No active development due to safety concerns; i.e., risks of severe side effects Efficiency documented by nonrandomized clinical trials and case reports only. Highly sensitive and anaphylactic patients remain excluded due to safety concerns. Preliminary data, including randomized DBPC trials, indicate promising results, but protocol optimization is still required. Efficiency documented in mouse models. Human studies envisaged Experimental studies using murine models expected to shed light on underlying mechanisms Isolated reports documented efficiency using murine models. No recent development reported in food allergy No active development in food allergy Applications likely to exist in the context of food industry Well investigated in the context of peanut allergy e.g., FDA-approved drug Omalizumab (trade name Xolair). Promising results obtained with murine studies. Anti-IL5 therapy being investigated for eosinophilic eosophagitis Promising results obtained. Safety studies in human under way. Identification of active component ongoing Immunomodulatory effects still under investigation. Elucidation of underlying mechanisms under way; e.g., innate receptors may be involved

DBPC, Double-Blind Placebo Controlled.

3.1.2.1. Use of Overlapping Synthetic Peptides Under experimental conditions, a library of overlapping synthetic peptides covering the entire sequence of the native protein is often used as a preliminary experiment to the identification of T-cell epitope regions. In a therapeutic context, the main advantage of using a series of overlapping peptides resides in the fact that the mapping or mutation of the epitope

regions is no longer required (Nowak-Wegrzyn and Sicherer 2008). This approach has been documented in murine models of peanut allergy, as will be described in section 4. 3.1.2.2. Food-Based Hydrolyzates Together with synthetic peptide libraries, the use of foodbased hydrolyzates represents a more random and

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probably less specific approach compared to interventions based on T- or B-cell epitopes. This approach has been particularly investigated in a prophylactic context and relies on the notion that peptides obtained from the enzymatic hydrolysis of native protein lack secondary structures—by virtue of their sizes—a feature that is necessary for the process of allergen sensitization and/or the crosslinking of IgE at the surface of mast cell and basophils. The nature of their immunomodulatory properties (e.g., peptide structures) remains, however, to be elucidated. The approach has been particularly researched in the context of cow’s milk allergy, one of the most prevalent forms of food allergy in infants and young children. The preventive use of milk-based infant hydrolyzed formula is discussed in section 4. 3.1.2.3. B-cell Epitope-Based Immunotherapy (IgG-Binding) The approach has been documented by a few reports using respiratory allergens represented by grass and birch pollen allergens in mice models (Vrtala et al. 2004; Focke et al. 2001, 2004). The authors tested the prophylactic effects of synthetic peptides (25–32 amino acids) rich in solvent-exposed amino acids but lacking both secondary structure and T-cell activating potential. The study hypothesized that such peptides might favor production of allergen-specific IgG to the detriment of specific IgE, in line with the “blocking IgG” theory. This theory is based on the belief that allergen-specific IgG can exert protective effects through mechanisms involving either (1) the direct inhibition of IgE-mediated release of inflammatory mediators from mast cells and basophils or (2) the inhibition of IgE-facilitated antigen presentation to T cells (Wachholz and Durham 2004; Larché and Wraith 2005). This latter mechanism relies on the presence of specific-IgE bound to high(FcεRI) or low-affinity IgE receptors (FcεRII, CD23) present at the surface of antigen-presenting cells, which can efficiently capture the allergen and promote its presentation to T cells (Wilcock et al. 2006; van Neerven et al. 2006). No such approach has yet been documented with models of food allergy.

3.1.2.4. T-cell Epitope-Based Immunotherapy The epitope mapping trend was initiated in the early 1980s with the identification of T-cell epitopes in parasitic and viral proteins and stands as a milestone in the development of vaccines (De Groot and Moise 2007). Within the same conceptual framework, T-cell epitope-containing peptides were put forward as an efficient approach toward the induction of tolerance in food allergy. By virtue of its central role in the pathoetiology of food allergy, the T-cell compartment represents a prime target for therapeutic interventions. For a preparation to be useful in immunotherapy, it is suggested that immunodominant T-cell epitopes be present because of the beneficial effects mediated by T lymphocytes (Zeiler and Virtanen 2008). The rationale behind this strategy relies on the use of peptides lacking the secondary or tertiary structure responsible for the crosslinking of IgE—similar to the use of foodbased hydrolyzates—but the main requirement is that peptides be capable of maintaining their T-cell activation potential. Studies carried out in animal models of respiratory allergies as well as in clinical settings have provided proof-of-principle demonstrating the induction of hyporesponsiveness to the allergen (Larché 2005). A clear association with the production of regulatory cytokine IL-10 was highlighted in these studies. We have investigated in our laboratory the feasibility of such an approach using a BALB/c mouse model of egg allergy, as presented in section 4.

3.2. Advantages of Peptide-Based Immunotherapy Peptide-based immunotherapy stands as an appealing approach based on the criterion that the manufacturing of nonsynthetic (e.g., food hydrolyzates) or synthetic peptides has today become a low-cost routine exercise (Thomas 2004). Standardization of synthesis methods, characterization of peptides, and analysis for purity are currently carried out using well-established analytical techniques, for example, liquid chromatography and mass spectrometry. This presents significant benefits compared to other approaches such as the diligent task

Chapter 8 Peptide-Based Immunotherapy for Food Allergy

of producing mutant recombinant proteins (Sampson 2001); but more importantly, it facilitates quality control and ultimately approval by regulatory authorities. In addition, the use of well-defined preparations alleviates the risks of generating new IgE specificity, as it has been reported with the use of native allergen extracts. Compared to whole allergens, peptides are expected to be better tolerated (Texier et al. 2000). Thus, high doses of peptides may be administered with low risk of inducing anaphylactic reactions; hence they are useful for highly sensitive individuals. The administration of peptides containing allergen epitope sequences bypass the problem of exogenous proteins not being processed in the appropriate manner (Zegers et al. 1995). Finally, a significant benefit of peptide-based approaches for food allergy is their versatility. For example, peptides may be coupled with various cytokines in order to modulate the immune response toward the desired effects, or they may be modified or mimicked (peptidomimetics) to improve their bioavailability, for example, solubility.

3.3. Suggested Mechanisms Underlying PIT Efforts targeted toward understanding the mechanisms of peptide-based immunotherapies have been hindered by the complexity of the immune system. Currently, the mechanisms by which peptide-based treatments exert their suppressive effect on allergic responses remain obscure. However, it has been hypothesized that, upon parenteral administration, some peptides may reach the peripheral circulation via a capillary network, may be captured by APCs as they migrate to lymphoid tissues, and may traffic to mucosal tissues, where they can induce activation and proliferation of regulatory T cells (Larché 2005). The question then arises, how are synthetic peptides presented to T cells? Vaccine developmental studies have investigated the phenotype of APC responsible for presentation of peptides. Although findings have been disputed, recent reports suggest that dendritic cells (rather than B cells or macrophages) are capable of directly binding peptides and presenting them to T cells without prior processing

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(Kern et al. 1998; Larché and Wraith 2005; Hochweller et al. 2006). In humans, it has been suggested that this phenomenon could involve the activity of chaperone molecules such as HLA-DM (Santambrogio et al. 1999). Once complexed with MHC molecules, how T-cell determinants induce regulatory mechanisms is poorly understood. It has been proposed that peptides may bind to MHC molecules without providing the co-stimulatory signals (e.g., CD80/CD86 family) required for activation and maturation of APCs (Hochweller et al. 2006), an activation state coined “tolerogenic.” As a result, presentation of peptide-MHC complexes by “tolerogenic” APC may yield to hyporesponsive allergenspecific T cells and to the generation of regulatory T cells. In the context of food allergy, PIT-based strategies aim at the modulation of CD4+ T cells and, more particularly, at the immune balance existing between Th1, Th2, and Treg cells (Figure 8.2). Of the different PIT-based strategies, T-cell epitopePIT represents a more targeted approach and receives, therefore, greater attention in current research. In clinical trials with cat allergic patients, T-cell epitope-based IT were documented to activate regulatory events involving regulatory cytokines IL-10, TGF-β, and FOXP3-expressing cells. Based on knowledge provided by current literature, we schematically represented the potential mechanisms underlying PIT based on the use of T-cell epitope peptides (Figure 8.4). Further investigation will be required to shed light on the intricacies of these complex events.

4. Current Experimental Models of PIT in Food Allergy More than 170 allergens have been identified from dietary sources (Hefle et al. 1996). Despite this diversity, only eight types of food accounted for more than 90% of all food-induced allergic reactions. These are referred to as the “big eight” and include cow’s milk, eggs, fish, crustaceans, peanuts, soybeans, tree nuts, and wheat (Breiteneder and Mills 2005). Some of them have been the object of PIT-based investigations. We thus describe in the

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Figure 8.4. Potential mechanisms underlying T-cell epitope-based immunotherapy. Upon peptide administration, exposure to multiple allergen T-cell epitopes may lead to activation of a population of FOXP3- and TGF-β-expressing cells, which are likely to be represented by CD4+CD25+ regulatory T cells and Th3 cells, respectively. TGF-β was documented to be capable of inducing FOXP3 expression in peripheral tissues and stimulating production of allergen-specific IgA at mucosal surfaces. As a result of the functional counter-inhibition between Th1- and Th2-biased responses, a Th1-cytokine profile may predominate, leading to the production of allergen-specific IgG in line with the “blocking IgG” theory. APC = antigen presenting cells; CD = cluster of differentiation; FOXP3 = Forkhead box protein 3; IFN = interferon; IL = interleukin; Ig = immunoglobulin; Th = T helper cells; TGF-β = transforming growth factor-β.

following section how milk allergy has been the ongoing focus of peptide-based approaches using enzymatic hydrolyzed formulas, while peanut and egg allergies have been the object of very recent PIT investigations using the mouse model of food allergy. Other allergen models, such as wheat and beef proteins, have been the focus of more scarce investigations.

4.1. Cow’s Milk Allergy Model Cow’s milk allergy is mainly reported as the most prevalent food allergy in infants and young children, and it affects 2–3% of the general population (Skripak et al. 2007). First, it is important that a distinction be made between the concept of “hypoallergenicity” and “tolerogenicity.” Hypoallergenicity

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refers to a situation where the food material is made into a preparation lacking IgE-binding activity and aiming at preventing an allergic response. On the other hand, the notion of “tolerogenicity” refers to the active induction of immune tolerance for the allergen prior to any exposure (prophylactic effect) or the capacity to downregulate allergic responses in already sensitized individuals (therapeutic effect). There are only a few examples of hypoallergenic foods that have been brought to the market, and enzymatically hydrolyzed milk proteins for infant formulations is probably the best-known example (Hays and Wood 2005). In spite of a number of randomized clinical trials, the prophylactic value of milk hydrolyzed formulas remains controversial (Osborn and Sinn 2006); and no therapeutic value has been attributed to any of them. A few years ago, interesting studies using murine models reported the isolation of so-designated “tolerogenic” peptides released from the tryptic digestion of β-lactoglobulin, a major milk allergen. Using a rat model, it was shown that a partially hydrolyzed formula administered before and during sensitization could induce a suppressive effect on the production of specific IgE, mast cell sensitization, and allergen-induced lymphocyte proliferation (Fritsché et al. 1997). Further studies by the same authors identified several tolerogenic peptides with sizes ranging from 8 to 23 amino acids, establishing a first step toward the understanding of the correlation between peptide structure and tolerogenicity (Pecquet et al. 2000). Whether such tolerogenic peptides may be used to induce immune tolerance in a therapeutic manner remains to be investigated, and translation of these findings into human applications demands further investigation.

4.2. Peanut Allergy Model Responsible for the most clinically severe form of food allergy, the peanut has been and continues to be the focus of intensive research. Various immunotherapeutic approaches were investigated using peanut allergy as a model, and multiple reviews have focused on the disease (Li 2005; de Leon et al. 2007). Of the reports investigating peptide-based approaches, one in vitro study showed that peptides

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derived from the peptic digestion of peanut allergens, which contained T-cell epitopes but no IgE-binding epitopes, could preferentially induce production of IFN-γ (Th1-cytokine) in PBMC cultures, in a concentration-dependent manner (Hong et al. 1999). Additionally, a recent review reported unpublished results demonstrating that the repeated subcutaneous administration of 30 overlapping peptides (3 doses/week for 4 weeks), covering the entire sequence of the major peanut allergen Ara h 2, led to a significant reduction of specific IgE and histamine levels in peanut-sensitized C3H/HeJ mice, compared to placebo-treated mice. These changes were accompanied by lower anaphylaxis scores and increased production of IFN-γ by splenocytes cultured in vitro with Ara h 2 (Sicherer and Sampson 2007). However, to the best of our knowledge, no further investigations were reported.

4.3. Egg Allergy Model Egg allergy was documented as the second-mostcommon food allergy in children in the United States (Eggesbø et al. 2001; Heine et al. 2006; Sampson 2004). In European and Asian industrialized countries, egg allergy has recently been reported as the most prevalent food hypersensitivity in the pediatric population, exceeding that of cow’s milk allergy (Crespo et al. 1995; Imai and Iikura 2003; Teuber et al. 2006; Han et al. 2004; Gustafsson et al. 2003; Chiang et al. 2007). In vivo studies using BALB/c mice were recently carried out in the laboratory and provided supportive evidence to the therapeutic potential of a peptidebased approach using epitope-containing peptides. Investigations of the immunomodulatory properties of a preparation of egg white hydrolyzate led to identification of immunodominant B- and T-cell epitope sequences of OVA. Using a BALB/c model of orally induced egg allergy, the oral administration of the egg hydrolyzate was shown to decrease the level of specific IgE and histamine titers. Spleen cell cultures revealed inhibitory effects on both allergen-induced Th1 and Th2 responses, while gene expression analyses indicated the induction of regulatory mechanisms involving FOXP3 and

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TGF-β (Yang et al. 2009). TGF-β is particularly well known as the “α-switch factor” (Coffman 1989), leading to the production and secretion of allergen-specific IgA at mucosal surfaces. A T-cell epitope based approach has also been investigated (Yang et al. 2010). BALB/c mice were administered with a cocktail of synthetic peptides containing a mixture of OVA immunodominant T-cell epitopes. The animals showed significant improvement in their clinical responses upon oral challenge, accompanied by a prominent decline in serum histamine and specific IgE titers. Mechanistically, a shift toward a Th1-biased cytokine response was not only seen in vitro at the protein level (spleen cell cultures), but its presence was also shown in vivo with an increase in the gene expression ratios of Th1-biased cytokines (IFN-γ, IL-18, and IL-12).

4.4. Wheat and Beef Allergy Model Better known for their deleterious effects in the infamous inflammatory immune disorder commonly known as celiac disease, wheat proteins belong to the “big eight” food allergens. A recent monograph reported the single clinical case of a 10-year-old patient who was subjected to ingestion of wheatflour hydrolyzed formula, containing T-cell reactive peptides for duration of 18 months, and who did not develop any severe side effects upon subsequent exposure with nonhydrolyzed wheat flour (Tanabe 2007). In vitro studies conducted with bovine serum albumin, the major beef allergen, reported that analogue peptides lacking IgE-binding ability could preserve T-cell reactivity and may have the capacity to induce immune tolerance. Analyses of PBMC cultures incubated with these peptide analogues indicated a shift toward a Th1-cytokine response, characterized by a marked increase in the production of IFN-γ (Tanabe et al. 2004).

4.5. PIT Investigations Carried Out with Inhalant Allergens Investigations carried out with inhalant allergens have provided the initial proof-of-principle that a peptide-based approach was a viable option in the

treatment of allergic disorders. It is thus interesting to present some of the key results obtained. Several inhalant allergens have, indeed, been the object of PIT investigations: the major cat allergen Fel d 1, the bee venom allergen Api m 1 (Fellrath et al. 2003; Tarzi et al. 2006), and more recently the ragweed allergen Amb a 1 (Larché 2007) and the dog allergen Can f 1 (Virtanen 2006). The most promising findings were obtained with cat allergens, as evidenced by studies completed in clinical settings (Larché 2007). Administration of peptides containing T-cell epitopes led to increased pulmonary functions in cat allergic patients and allowed exposure to cats with reduced symptoms (Maguire et al. 1999). In a double-blind placebo-controlled study, production of IL-10 was increased in PBMC obtained from patients with cat allergic asthma after treatment with Fel d 1 peptides (Oldfield et al. 2002). Reduction in allergen-specific proliferative response as well as in the production of Th1-biased and Th2-biased cytokines was also observed. Coculture experiments using PBMCs led to the isolation of CD4+ cells capable of actively suppressing allergen-specific proliferative responses of CD4− cells obtained prior to peptide treatment (Verhoef et al. 2005). In the context of food allergy, it is reasonable to expect that similar phenomena may occur. However, they may also differ on the basis of the differences between the sensitization mechanisms occurring at respiratory vs. intestinal mucosa.

5. Translation of PIT into Human Clinical Applications Translation of PIT experimental findings into a vaccine for human allergic disorders represents the ultimate achievement of current investigations. Since CD4+ T cells were described as central players in the induction and maintenance of immune tolerance (Hiroi and Takaiwa 2006), most PIT investigations were, to date, based on strategies using T-cell epitope peptides. We thus discuss the feasibility of translating PIT into clinical applications with regard to T-cell epitope-based immunotherapy. The design of peptide-based vaccines for food allergy faces several challenges encompassing the polymorphism

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of MHC molecules inherent to humans and the production of peptides at an industrial scale, as well as other practical issues such as route of administration and optimal dose of peptides. These different aspects are the objects of the following section.

5.1. MHC Polymorphism An epitope-based vaccine must be effective in a broad-range population, and must therefore take into account the MHC restriction of T-cell mediated immune response. Indeed, a highly desirable criterion is the design of peptides capable of inducing a tolerance response in individuals with different human leukocyte antigen (HLA) background. This hurdle may be overcome with the preparation of vaccines containing multiple peptides that would be effective in binding various MHC molecules, thus efficient in a larger MHC-heterogeneous population. A second alternative would be the selection of those epitope sequences restricted to the HLAs that are most frequent in the human population. For example, there has been a report of unpublished observations suggesting that short regions of allergen sequence could comprise multiple overlapping HLAbinding motifs (Larché and Wraith 2005). More interestingly, it was documented that the expression of seven alleles of HLA-DR—the most polymorphic HLA region in humans—was predominant in the Caucasian population: namely, DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501. Thus, peptides that would efficiently bind to these allele products would represent good PIT vaccine candidates (Texier et al. 2000).

5.2. Large-Scale Production of Peptides Peptide therapeutics is currently driven by the development of peptide-based vaccines for infectious diseases and tumors. In the United States, the peptide industry underwent a dramatic change in the past 5 years since the approval of the peptide drug Fuzeon (HIV-fusion inhibitor) by the U.S. Food and Drug Administration (FDA). A very recent review confirmed this renewed enthusiasm for peptide-based

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vaccines, illustrated by an impressive panel of ongoing preclinical and clinical trials (Purcell et al. 2007). Due to the recent progress in peptide chemistry, it is no longer unrealistic to envisage the production of synthetic peptides at metric ton scale (Hampton 2005). Besides automated peptide synthesis, plant-based expression systems have been suggested as novel methods for the large-scale production of peptide vaccines in the form of “edible vaccine.” Such an approach would address the stability issues associated with the administration of peptides by oral route. T-cell epitopes of Japanese cedar pollen allergens, Cry j 1 and Cry j 2, were expressed in the edible part of transgenic rice, a staple food in Asian cultures (Hiroi and Takaiwa 2006). Efficiency of the rice-based vaccine has shown promising results in mouse models, and whether such an approach is viable for the incorporation of food allergen T-cell epitopes would be interesting to explore.

5.3. Other Practical Issues Several aspects related to the development of peptide-based vaccines warrant further investigation. Route and frequency of administration ought to influence the outcome of the treatment. Parameters such as dose and volume, as well as the type of adjuvant (if any) classically determine the route and frequency of administration. With regard to doses, it has been proposed that doses of peptides fit in a model where too low a dose would be insufficient to induce any biological response, and too high a dose would lead to activation of adverse effects (Larché 2007). The same author also suggests that considerably higher doses of peptides are required to induce tolerance in murine systems and that the difference between mice and humans would stand around five order of magnitude (Larché 2001). And last, solubility, in vivo instability of synthetic peptides, and their short half-life are other issues that will need to be addressed.

6. Conclusion Peptide-based immunotherapy has been praised as a promising strategy for food allergy by a number of

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reviews (Li and Sampson 2004; Sicherer and Sampson 2007; Burks et al. 2008a, 2008b). While PIT investigations carried out with inhalant allergens have reached clinical settings with encouraging results, there is currently little information on the curative potential of PIT in food-induced allergies. However, recent PIT investigations using murine models of food allergy—in particular, those recently generated in our laboratory—point the way toward promising avenues. Of the various strategies proposed, an immunotherapy based on the use of T-cell epitope-containing peptides has been, to date, the object of most investigations. Elucidation of the mechanisms underlying PIT has guided research into further understanding of allergic responses, which may lead the way toward the design of more efficient immunotherapeutic approaches. Mechanistically, the induction of events mediated by the class of regulatory T cells and key molecules such as IL-10, TGF-β, and FOXP3 are actively researched. Clinical translation of PIT experimental data in the form of peptide-based “vaccine” for food allergy demands further investigation but is highly probable, and therapeutic trials in humans are eagerly awaited.

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ing and presentation by immature dendritic cells. Proc Natl Acad Sci USA 96(26):15056–15061. Sicherer SH, Sampson HA. 2008. Food allergy: Recent advances in pathophysiology and treatment. Annu Rev Med 60:261–277. ———. 2007. Peanut allergy: Emerging concepts and approaches for an apparent epidemic. J Allergy Clin Immunol 120(3): 491–503. Skripak J, Matsui EC, Mudd K, Wood RA. 2007. The natural history of IgE-mediated cow’s milk allergy. J Allergy Clin Immunol 120(5):1172–1177. Soeria-Atmadja D, Lundell T, Gustafsson MG, Hammerling U. 2006. Computational detection of allergenic proteins attains a new level of accuracy with in silico variable-length peptide extraction and machine learning. Nucleic Acids Res 34(13): 3779–3793. Stadler MB, Stadler BM. 2003. Allergenicity prediction by protein sequence. FASEB J 17(9):1141–1143. Stock P, DeKruyff RH, Umetsu DT. 2006. Inhibition of the allergic response by regulatory T cells. Curr Opin Allergy Clin Immunol 6(1):12–16. Tanabe S. 2007. Epitope peptides and immunotherapy. Curr Protein Pept Sci 8(1):109–118. Tanabe S, Shibata R, Nishimura T. 2004. Hypoallergenic and T cell reactive analogue peptides of bovine serum albumin, the major beef allergen. Mol Immunol 41(9):885–890. Tarzi M, Klunker S, Texier C, Verhoef A, Stapel SO, Akdis CA, Maillere B, Kay AB, Larché M. 2006. Induction of interleukin-10 and suppressor of cytokine signalling-3 gene expression following peptide immunotherapy. Clin Exp Allergy 36(4):465–474. Taylor A, Verhagen J, Blaser K, Akdis M, Akdis CA. 2006. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-beta: The role of T regulatory cells. Immunology 117(4):433–442. Teuber SS, Beyer K, Comstock S, Wallowitz M. 2006. The big eight foods: Clinical and epidemiological overview. In: SJ Maleki, AW Burks, RM Helm, eds., Food Allergy, 49–79. Washington DC: AMS Press. Texier C, Pouvelle S, Busson M, Hervé M, Charron D, Ménez A, Maillère B. 2000. HLA-DR restricted peptide candidates for bee venom immunotherapy. J Immunol 164(6):3177– 3184. Thomas WR. 2004. Allergen-derived peptides: From bench to bedside. Annual Meeting of the American College of Allergy, Asthma & Immunology, November 11–17, Boston. Thompson K, Chandra RK. 2002. The management and prevention of food prophylaxis. Nutr Res 22:89–110. Van Neerven RJ, Knol EF, Ejrnaes A, Würtzen PA. 2006. IgEmediated allergen presentation and blocking antibodies: Regulation of T-cell activation in allergy. Intl Arch Allergy Immunol 141(2):119–129. Verhagen J, Blaser K, Akdis CA, Akdis M. 2006. Mechanisms of allergen-specific immunotherapy: T-regulatory cells and more. Immunol Allergy Clin North Amer 26(2):207–213.

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Verhoef A, Alexander C, Kay AB, Larché M. 2005. T cell epitope immunotherapy induces a CD4+ T cell population with regulatory activity. PLoS Med 2(3e78):253–261. Virtanen T. 2006. Prospects for peptide-based immunotherapy for dog allergy. Curr Opin Allergy Clin Immunol 6(6):461– 465. Vrtala S, Focke-Tejkl M, Swoboda I, Kraft D, Valenta R. 2004. Strategies for converting allergens into hypoallergenic vaccine candidates. Methods 32(3):313–320. Wachholz P, Durham S. 2004. Mechanisms of immunotherapy: IgG revisited. Curr Opin Allergy Clin Immunol 4(4):313–318. Wilcock LK, Francis JN, Durham SR. 2006. IgE-facilitated antigen presentation: Role in allergy and the influence of allergen immunotherapy. Immunol Allergy Clin North Amer 26(2):333–347. Yang M, Yang C, Nau F, Pasco M, Juneja LR, Okubo T, Mine Y. 2009. Immunomodulatory effects of egg white enzymatic hydrolysates containing immunodominant epitopes in a

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BALB/c mouse model of egg allergy. J Agri Food Chem 57(6):2241–2248. Yang M, Yang C, Mine Y. 2010. Multiple T cell epitope peptides suppress allergic responses in an egg allergy mouse model by the elicitation of forkhead box transcription factor 3—and transforming growth factor beta-associated mechanisms. Clin Exp Allergy 40:668–678. Yoshimoto T, Bendelac A, Watson C, Hu-Li J, Paul WE. 1995. Role of NK1.1+ T cells in a TH2 response and in immunoglobulin E production. Science 270(5243):1845–1847. Zegers ND, Boersma WAJ, Claassen E. 1995. Immunological recognition of peptides in medicine and biology. Boca Raton, FL: CRC Press. Zeiger RS. 2003. Food allergen avoidance in the prevention of food allergy in infants and children. Pediatrics 111(6 Pt 3): 1662–1671. Zeiler T, Virtanen T. 2008. Mapping of human T-cell epitopes of allergens. Methods Mol Med 138:51–56.

Chapter 9 Gamma-Aminobutyric Acid Bo Jiang, Yuanxin Fu, and Tao Zhang

Contents 1. Introduction, 121 2. Physical Properties of GABA, 122 3. Sources and Properties of Glutamate Decarboxylase, 122 4. Functions of GABA, 122 4.1. Roles of GABA Synthesis in Plants, 122 4.1.1. pH Regulation, 123 4.1.2. Nitrogen Storage, 123 4.1.3. Plant Defense, 123 4.1.4. Plant Development, 123 4.1.5. Potential Modulator of Ion Transport, 124 4.2. Roles of GABA in Animals, 124 4.2.1. Regulating Mood, 124

1. Introduction Gamma-aminobutyric acid (GABA), a four-carbon nonprotein amino acid, has been well documented as a physiologically functional compound in nature. This chapter considers the current understanding of its physiological functions, biosynthesis, utilization in pathways, reaction mechanisms, and applications. GABA is a significant component of the free amino acid pool in most prokaryotic and eukaryotic cells. It was discovered first in potatoes more than

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

4.2.2. 4.2.3. 4.2.4. 4.2.5. 5. 5.1. 5.2. 5.3. 6. 7. 8. 9.

Curing Neurological Disease, 124 Preventing Brain Senescence, 124 Hypotensive Effects, 125 Other Roles of GABA in Animals, 125 Preparation of GABA, 125 GABA Tea, 125 Other GABA Foodstuffs, 125 Synthesis by Microbes, 126 Reaction Mechanism of GAD, 127 Metabolic Pathway of GABA, 127 Human Application of GABA, 129 References, 130

half a century ago and subsequently in rat brains (Steward 1949). Later, GABA was detected in vertebrates, nonvertebrates, and various plant components, and is well known as a neurotransmitter that regulates inhibitory neurotransmission in the mammalian central nervous system (CNS) (Davidson 1976). GABA has many biological functions such as neurotransmission and induction of hypotensive, diuretic, and tranquilizer effects (Shelp et al. 1999). Due to these physiological functions, developing functional foods that contain high concentrations of GABA has been actively pursued. GABA enrichment has been achieved in anaerobic-incubated tea and in rice germ soaked in water, and its production by various microorganisms has also been reported using bacteria, fungi, and yeast. 121

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Figure 9.1. Schematic representation of neutral (a) and zwitterionic (b) GABA structures.

2. Physical Properties of GABA GABA is similar in structure to an amino acid but has an amine group on the γ-carbon instead of the α-carbon, and it exists in an unbound form. Synonyms include 4-amino-n-butyric acid, piperidinic acid, and 4-aminobutyric acid. GABA is highly soluble in water; structurally, it is a flexible molecule that can assume several conformations in solution including a cyclic structure (Figure 9.1), and its solubility is linearly dependent on temperature over the range 25–55°C (Lin et al. 1994). It is zwitterionic (carries both a positive and negative charge) at physiological pH values with pK values of 4.03 and 10.56 (Shelp et al. 1999), and has an isoelectric point (pI) of 7.33 (Lin et al. 1994).

3. Sources and Properties of Glutamate Decarboxylase Glutamate decarboxylase (GAD; EC 4.1.1.15) is the pyridoxal-5′-L-phosphate (PLP)-dependent enzyme that synthesizes GABA (Sardana et al. 2006). In higher plants, GAD has been purified and characterized from cowpea (Johnson et al. 1997), squash (Matsumoto et al. 1986), potato (Satyanarayan and Nair 1985), and rice germ (Zhang et al. 2007). The optimum synthesis pH is between 5.5 and 6.5, and the optimum temperatures of most plants’ GAD activity is between 37 and 40°C (with the exception of squash GAD, which has an optimal temperature of 60°C). A unique feature of plant and yeast GAD is the ability to bind calmodulin (CaM), an acidic, bilobed protein (molecular mass of 16.7 kDa) that can activate around 40 distinct proteins in a calciumdependent manner. Most CaM-binding domain regions are basic, hydrophobic, and devoid of mul-

tiple negatively charged residues and have a propensity to form an α-helix (Yuan and Vogel 1998). In plants, transient elevation of cytosolic Ca2+ in response to different stresses may be responsible for GAD activation via CaM (Yang et al. 2004; Zik et al. 2006); for example, GAD activity was increased two- to eight-fold (depending on the tissue source) in the presence of Ca2+ and CaM at pH 7 (Snedden et al. 1995). Bacterial GAD has some features similar to those of the plant enzyme: it exhibits an acidic pH optimum (3.8–4.6), forms a hexamer (Sukhareva et al. 1994), and is expressed in response to environmental stresses (Blankenhorn et al. 1999). The E. coli enzyme is probably the best-characterized enzyme among the investigated GADs, mainly due to its availability. However, it is the causative agent of a serious food-borne pathogen and cannot be used to produce GABA in food. GAD purified from commercial lactic acid bacteria (Nomura et al. 1999) has also been studied. Purified enzymes showed a single protein band, and the subunit molecular mass ranged from 54 to 65 kDa (Table 9.1) (Huang et al. 2007; Park and Oh 2007; Xu et al. 2004; Ueno et al. 1997). The activity was stable at acidic pH values, with maximum activity at pH 4.4–4.7, and no activity in neutral pH range.

4. Functions of GABA 4.1. Roles of GABA Synthesis in Plants GABA is widely distributed in plant tissues, such as tea (Millin and Rustidge 1967), soybean (Oh and Choi 2001), radish leaves (Streeter and Thompson 1972), rice germ, germinated brown rice, and so on. The average levels of GABA in plants range from

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Table 9.1. Properties of GAD from lactic acid bacteria. Species Lactococcus lactis subsp. Lactobacillus brevis CGMCC 1306 Lactobacillus brevis IFO 2005 Lactococcus lactis 1.009 Lactobacillus brevis OPK-3

Subunit MW (kDa)

Optimum pH

Optimum Temperature

Km (mM)

Reference

54 62 60 65 53.4

4.7 4.4 4.2 4.7 —

— 37 30 50 —

0.51 8.22 9.3 1.9 —

Nomura et al. 1999 Huang et al. 2007 Ueno et al. 1997 Xu et al. 2004 Park and Oh 2007

0.03 to 2.00 μmol/g fresh weight (Steward 1949). GABA can be accumulated rapidly under several environmental stress conditions, such as mechanical stimulation, damage, cold shock (Wallace et al. 1984), heat shock, hypoxia (Shelp et al. 1995), cytosolic acidification, darkness, water stress (Handa et al. 1983), phytohormones, and drought stress (Serraj et al. 1998). In plants, rapid, stress-induced GABA accumulation is well documented, with levels increasing 20- to 40-fold within 5 minutes. However, the role of GABA accumulation is contentious. 4.1.1. pH Regulation In vitro GAD from a variety of tissues has a sharp pH optimum around 5.8, with 10–40% of maximal activity at pH 7.0 (Bown and Shelp 1997). Because GAD activity increases as pH decreases and glutamate decarboxylation is a proton consuming reaction, it follows that GABA production could act as a sink for excess protons in the cytoplasm and, thus, regulate pH (Carroll et al. 1994; Crawford et al. 1994; Snedden et al. 1992). In vivo 15N and 31P NMR spectroscopy also shows that GABA synthesis is stimulated by a decrease in cytoplasmic pH. 4.1.2. Nitrogen Storage The conversion of glutamate to GABA increases under conditions that inhibit glutamine synthesis, reduce protein synthesis, or enhance protein degradation (Scott-Taggart et al. 1999). This prompts the hypothesis that GABA is a temporary nitrogen store. In addition, glutamate flux through the GABA shunt is comparable to the direct incorporation of glutamate into protein (Tuin and Shelp 1996). Evidence from Micallef and Shelp

(1989) also indicates that glutamate and GABA are produced during protein storage and mobilization as a means of recycling arginine-derived nitrogen and carbon. 4.1.3. Plant Defense Phytophagous activity by insects and other invertebrates destroys vacuolar compartmentalization, increases H+ levels in the cytosol, and stimulates GABA synthesis. Ramputh and Bowm (1996) investigated the hypothesis that mechanical stimulation or damage resulting from phytophagous insects stimulated GABA accumulation, which in turn deterred insect growth and development. Results showed that increasing GABA levels in a synthetic diet from 1.6 to 2.6 μmol ·g−1 fresh weight reduced the growth, developmental, and survival rates of cultured Harris larvae. 4.1.4. Plant Development GAD is a soluble protein with a Ca2+/calmodulin binding domain, and its expression level is regulated during development by transcription and posttranscriptional processes. Genetic studies show that changing GABA concentration had severe consequences on plant development (Bouché et al. 2003). Transgenic plants that ectopically expressed a constitutively active GAD enzyme have a higher concentration of GABA and a lower concentration of glutamate, resulting in abnormal growth and development. Plant GADs carry a C-terminal extension that binds to Ca2+/ calmodulin to modulate enzyme activity. Kazuhito and Fumio (2007) investigated rice, which possesses two distinct GADs, OsGAD1 and OsGAD2. Both have a C-terminal extension, but the former peptide

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contains an authentic CaM-binding domain, while the latter does not. The results suggested that the C-terminal extension of OsGAD2 is a strong autoinhibitory domain and that truncation of this domain causes the enzyme to act constitutively with higher activity both in vitro and in vivo. 4.1.5. Potential Modulator of Ion Transport Kinnersley and Lin (2000) studied duckweed to investigate whether plants also possessed GABA receptors. Results supported the hypothesis that GABA activity in plants involved interaction with a receptor, an effect on mineral transport, and suggested a way that GABA could amplify stress perception in plants. The association between growth and mineral content of GABA-treated plants supported the hypothesis that one of the functions of GABA in plants involved regulating ion transport.

4.2. Roles of GABA in Animals In 1950, GABA was discovered in mammalian brain tissue (Roberts and Frankel 1950). The concentration of GABA in these tissues is 1,000 times greater than amine. GABA is now recognized as the most important inhibitory neurotransmitter in the mammalian CNS. From 30–50% of all central synapses are GABA-ergic (Paredes and Agmo 1992). GABA has several physiological functions, such as neurotransmission, tranquilization, and hypotensive and diuretic effects. 4.2.1. Regulating Mood Several lines of preclinical and clinical evidence suggest that the amino acid neurotransmitter GABA has a role in mood disorders, particularly depression (Emrich et al. 1980; Shiahs and Yatham 1998). GABA-A receptors are heteropentameric ligand-gated chloride channels that mediate the majority of rapid inhibitory synaptic transmission (Sieghart 1992). GABA-B receptors appear to be of major importance in synaptic processing within the brain and are present at both post- and presynaptic sites. Their activation can hyperpolarize neurons and diminish neurotransmitter release from presynaptic terminals (Bowery 1993). The effects of antidepressant drugs on GABA

receptors have been studied. A single injection of low doses of different antidepressants does not seem to affect brain GABA-A receptor binding in rat brains. Repeated or prolonged administration of antidepressants is either without effect or downregulates GABA-A receptor complexes. There are conflicting reports with regard to effects of antidepressants on GABA receptor binding sites. For example, several different classes of antidepressant agents including tricyclic antidepressants, monoamine oxidase inhibitors, and selective serotonin reuptake inhibitors induce an increase in GABA receptor binding sites in the rat frontal cortex and hippocampus following chronic administration. 4.2.2. Curing Neurological Disease Stiff-person syndrome (SPS) is a rare neurologic disease characterized by muscle rigidity and episodic painful spasms (Yamamoto et al. 2007). Although the etiology is not well understood, it is postulated that the pathophysiology of SPS is created by antibodies against GAD (Moersch and Woltman 1956). The loss of GABA-ergic input from inhibitory spinal interneurons and impaired supraspinal GABA-ergic neurons leads to the hyperexcitability of motor neurons and subsequent progressive muscle rigidity (Sandbrink et al. 2000). In Parkinson’s, GABA is reduced in an area of the brain called the subthalamic nucleus. This region is working in “overdrive” in the disease process, and it is important to calm this hyper-reactive circuit because GABA is an inhibitory transmitter. There is normally an increase in extracellular K+ during a seizure. Along with the increase in intracellular Na+ and depolarization, carrier-mediated GABA release would be greater during seizures and would reduce excitability. Indeed, the anticonvulsant gabapentin can enhance nonvesicular GABA release induced via heteroexchange release by the GABA analog nipecotic acid. Along with the recently described effect of gabapentin on calcium currents, enhancing nonvesicular GABA release could inhibit seizures. 4.2.3. Preventing Brain Senescence Human cerebral cortical function degrades during aging.

Chapter 9 Gamma-Aminobutyric Acid

Much of this change may result from a degradation of intracortical inhibition during senescence (Hwang et al. 2005). Leventhal and co-workers (2003) used multibarrel microelectrodes to study the effects of electrophoretic application of GABA, the GABA-A receptor agonist muscimol, and the GABA-B receptor antagonist bicuculline on the properties of V1 cells in old monkeys. Bicuculline exerted a much weaker effect on neuronal responses in old compared to young animals, confirming degradation of GABA-mediated inhibition. On the other hand, administering GABA and muscimol resulted in improved visual function. Many treated cells in the V1 area of old animals displayed responses typical of young cells. 4.2.4. Hypotensive Effects GABA regulates cardiovascular functions, such as blood pressure and heart rate. GABA can reduce blood pressure in experimental animals and spontaneously hypertensive rats (Hayakawa et al. 2005). Inoue and collaborators (2003) studied 39 mildly hypertensive patients who consumed fermented milk containing GABA or a placebo daily (weeks 1–12), followed by 2 weeks of no intake (weeks 13 and 14). They found that there was a significant decrease of blood pressure within 2–4 weeks, and it remained decreased throughout the 12-week intake (Inoue et al. 2003). In the CNS, nitric oxide (NO) enhances the spontaneous as well as the evoked release of several neurotransmitters, such as glutamate and GABA (Lonart et al. 1992). Studying the blood pressure responses to intrathecally injected sodium nitroprusside in anesthetized rats concluded that NO generated from sodium nitroprusside in the spinal cord exerts inhibitory and excitatory effects on blood pressure, probably through the release of GABA and glutamate. The inhibitory action on blood pressure involves the stimulation of spinal GABA-A and GABA-B receptors, whereas the excitatory response to glutamate appears to be mediated through the activation of spinal AMPA/kainate receptors (García et al. 1998). 4.2.5. Other Roles of GABA in Animals Inhibit acute hyperammonemia: GABA has the potential to prevent acute hyperammonemia by increasing

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detoxification of ammonia to glutamine and decreasing diffusion of ammonia from the blood into the brain (Paul 2003). Weight loss (Van den Pol 2003): Body weight is maintained via a homeostatic system involving the CNS. Recently, a study found that GABA and glutamate were present in key neurons that regulate body weight and, consequently, may represent important orexigenic/anorexigenic mediators that convey information to other neurons within the hypothalamus as well as from the hypothalamus to other brain regions that participate in energy regulation (Meister 2007). Regulate sleep: Luppi et al. (2007) found that the tonic inhibition of locus coeruleus noradrenergic and dorsal raphe serotonergic neurons during sleep was due to a tonic GABA-ergic inhibition by neurons localized in the dorsal paragigantocellular reticular nucleus and the ventrolateral periaqueductal gray.

5. Preparation of GABA 5.1. GABA Tea In 1987, Tsushida and Murai accidentally found that a large amount of GABA accumulated in green tea under anaerobic conditions. They further examined the GABA content of green, oolong, and black tea made under anaerobic conditions and found that GABA accumulated in all teas. Methods to increase GABA concentrations in food have been studied. Sawai et al. (2001) found that the amount of GABA increased more by repeating anaerobic and aerobic incubations than by treating only with anaerobic incubation. Wang et al. (2006) compared the bioactive components in GABA tea and green tea produced in Taiwan. The results suggests that GABA tea was high in GABA, valine, isoleucine, and leucine, which may improve liver function, and showed that GABA tea contained more essential amino acids than other teas.

5.2. Other GABA Foodstuffs Since the physiological functions of GABA have been published, many researchers have devoted

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themselves to developing GABA-rich foods. Examples include Monascus-fermented rice (Lin et al. 2008; Wang et al. 2003), soybean seedlings (Oh and Choi 2001), germinated soybean extract (Park and Oh 2007), pregerminated brown rice (Ohtsubo et al. 2005), and germinated brown rice (GBR) (Komatsuzaki et al. 2007). GBR was produced by soaking brown rice grains in water to promote germination, and GABA accumulated in the grains during this process. GBR contains vitamins, minerals, fiber, and effective components such as phytic acid (Hunt et al. 2002; Graf et al. 1987) and ferulic acid (Tian et al. 2004). Although normal brown rice has high nutritional value, its popularity is low because it cannot be cooked in conventional rice cookers. However, GBR is easily cooked, and the texture is softer than that of regular brown rice. Therefore, GBR could become a popular health food later. Ohtsubo et al. (2005) processed grains of cultivars with a large germ by soaking and gaseous treatment. After soaking for 3 hours and treating with nitrogen gas for 21 hours at 35°C, the content of GABA in GBR (24.9 mg/100 g) was higher than that by the conventional soaking method (10.1 mg/100 g).

5.3. Synthesis by Microbes Although GABA and GAD are widely distributed in bacterial, plant, insect, and mammalian (Ueno 2000) tissues, GABA is relatively hard to extract from plants and animals because the concentration is very low and the extraction technique is complicated. Many researchers biosynthesize GABA using microorganisms, such as Escherichia coli (Tramonti et al. 2003), Monascus (Kono and Himeno 2000), Lactobacillus (Komatsuzaki et al. 2005; Xu et al. 2004), Streptococcus (Yang et al. 2007), and yeast (Takahashi et al. 2004; Coleman et al. 2001). GABA synthesized by Monascus and Lactobacillus is considered to be safe and is commercially obtained. These strains could be used as starters in the production of GABA-containing yogurt, cheese, and other fermented functional foods. Kono and Himeno (2000) examined the change of GABA content during beni-koji making. They

found that koji would increase GABA production by increasing the proportion of tomo and that GABA production peaked on the fifth day and declined thereafter. The peak content was 1.2 μmol/g of dry koji. Su et al. (2003) discovered Monascus purpureus CCRC 31615 for high levels of GABA production. Using sodium nitrate during solid-state fermentation, the GABA content was 1,267.6 mg/ kg. Furthermore, GABA productivity increased to 1,493.6 mg/kg when dipotassium hydrophosphate was added to the medium. Komatsuzaki et al. (2005) screened a strain of Lactobacillus paracasei NFRI 7415 from a Japanese traditional fermented fish (funa-sushi), which showed high GABA-producing ability. The GABA production was further improved by the addition of PLP to the culture medium and maintaining the culture medium at pH 5.0. Under optimal cultivation conditions, strain NFRI 7415 produced GABA at a concentration of 302 mM when the glutamate concentration in the culture medium was 500 mM. Yogurt is a nutrient-rich fermented food made of milk, and it is very popular in Asian countries. When starter is added to soymilk for yogurt, GABA concentration is increased (Park and Oh 2007). The conventional methods yield GABA levels less than 1.5 g/g dry weight, but production with starter and substrate lead to a GABA concentration of 424.67 g/ gDW. Soymilk was fermented with five lactic acid bacteria (Lactobacillus casei, Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium longum) for up to 30 hours at 42°C, and GABA content increased from 93.9 to 361.6 mg/100 g. During cheese ripening, casein is degraded into peptides and amino acids by proteolytic enzymes. Some amino acids may subsequently undergo degradation reactions such as decarboxylation. Nomura et al. (1998) found that GABA levels increased linearly in cheese as the pH decreased, and the content could reach 1,797 μg/g cheese. Lactobacillus brevis hjxj-01 was treated with UV and 60Co γ-rays by Xia et al. (2006). Based on high positive mutation rates, the final mutagenesis conditions were 30 W UV light, an irradiation distance of 45 cm, an irradiation time of 50 seconds, and 60Co

Chapter 9 Gamma-Aminobutyric Acid

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Table 9.2. Biosynthesis of GABA by microorganisms. Microorganisms

GABA Contents

Researchers

Monascus pilosus IFO 4520 Lactobacillus paracasei + Lb. plantarum Lactobacillus paracasei Lactobacillus brevis OPY-1 (KFCC 11337) Lactobacillus casei, Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium longum Lactobacillus brevis OPK-3 Lactococcus lactis ssp. Lactobacillus brevis IFO-1200 Lactococcus lactis 1.009 Lactococcus lactis SK005 Lactobacillus brevis hjxj-08119

120 μmol/g 89.1 mg/100g (DW) 302 mM 424.67 g/g (DW)

Kono and Himeno 2000 Irigoyen et al. 2007 Komatsuzaki et al. 2005 Park and Oh 2007

361.6 mg/100 g 84.292 mg/L/h 21% 10.18 mM 2.5 g/L 3.63 g/L 17 g/L

Tsai et al. 2006 Park and Oh 2007 Nomura et al. 1998 Yokoyama et al. 2002 Xu 2004 Zhong et al. 2004 Xia et al. 2006

γ-rays irradiation at 500 Gy. The mutant strain had a high GABA production ability. The fermented GABA content was 17 g/L, which was 142.9% of the origin strain. The biosynthesis information of GABA by microorganisms is presented in Table 9.2.

reprotonated on Cα to give the corresponding amine as the product (Figures 9.2 and 9.3). The proposed transformation of L-Glu to GABA by GAD is shown in Figure 9.4.

7. Metabolic Pathway of GABA 6. Reaction Mechanism of GAD GABA is a ubiquitous nonprotein amino acid that is produced primarily by α-decarboxylation of glutamate catalyzed by GAD, which is a PLP-dependent enzyme. The first and common step for all PLP-dependent enzyme catalyzed reactions is a Schiff-base exchange reaction (transamination). PLP enzymes exist in their resting state as a Schiff-base (internal aldimine) with an active site lysine residue. The incoming amine-containing substrate displaces the lysine ε-amino group from the internal aldimine in the process, forming a new aldimine with the substrate (external aldimine). This is a multistep process that includes several facile steps and is frequently very rapid compared to the central steps in the reaction mechanism (e.g., deprotonation of Cα). From the external aldimine intermediate, carbanions formed by heterolytic cleavage of any one of the bonds to Cα (except for the C-N bond) can be stabilized. Loss of CO2 gives a carbanion that is commonly

In plants, the pathway that converts glutamate to succinate via GABA is called the GABA shunt. The first step of this shunt is the direct and irreversible α-decarboxylation of glutamate by glutamate decarboxylase. The second enzyme involved in the GABA shunt, GABA transaminase (GABA-T; EC 2.6.1.19), catalyzes the reversible conversion of GABA to succinic semialdehyde using either pyruvate or α-ketoglutarate as amino acceptors. The last step of the GABA shunt is catalyzed by succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.16), which irreversibly oxidizes succinic semialdehyde to succinate (Bown and Shelp 1997). The metabolic pathway in mammals is the same as in plants. GABA is formed in neurons by the decarboxylation of the amino acid L-glutamic acid (Figure 9.5). The rate-determining enzyme that catalyzes this step is GAD. The essential cofactor of the enzyme is PLP. The enzyme responsible for eliminating GABA is GABA-T. The resulting product, succinic semialdehyde, is converted to succinic

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Figure 9.2. Formation of a Schiff-base by substrate and PLP (Toney 2005).

Figure 9.3. Conversion of intermediate to GABA (Toney 2005).

acid, which has negative-feedback inhibition on the enzyme GAD, thereby decreasing the conversion of L-glutamic acid to GABA. Therefore, if an insufficient quantity of GABA is present, GAD is activated due to a lack of end-product inhibition caused by the end product, succinic acid (http://www.uic.edu/ classes/phar/phar402/). Alternate sources of GABA include putrescine, spermine, spermidine, and ornithine, which produce GABA via deamination and decarboxylation reactions, while L-glutamine is an additional source of glutamic acid via deamination. GAD from mammalian brain occurs in two molecular forms, GAD65 and GAD67 (referring to the subunit relative molecular weight in kilodaltons). These different forms of GAD are products of dif-

ferent genes, which differ in nucleotide sequence, immunoreactivity, and subcellular localization. The presence and characteristics of GAD have been investigated in a wide variety of nonneural tissues including liver, kidney, pancreas, testis, ova, oviduct, adrenal, sympathetic ganglia, gastrointestinal tract, and circulating erythrocytes. In some tissues, one form (GAD65 or GAD67) predominates. GABA-T has been located in most of the same tissues, primarily through histochemical and/or immunochemical methods; GABA-T is also present in a variety of circulating cells, including platelets and lymphocytes. SSADH, the final enzyme in GABA catabolism, has been detected in some of the tissues in which GAD and GABA-T have been identified,

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Figure 9.4. Proposed transformation of L-Glu to GABA by GAD (Xu et al. 2004).

although the presence of this enzyme has not been found in mammalian pancreas, ova, oviduct, testis, or sympathetic ganglia (Tillakaratne et al. 1995). In humans, disruption of SSADH or GABA-T causes severe clinical manifestations associated with the accumulation of neurotransmitters in physiological fluids (http://www.uic.edu/classes/phar/phar402/).

8. Human Application of GABA GABA is a nonessential amino acid and has been generally recognized as safe by the Food and Drug Administration. GABA was first introduced into the

market in foodstuffs by Japan. In addition to GABArich foods, such as GABA tea, Monascus-fermented rice, soybean seedlings, germinated soybean extract, pregerminated brown rice, and germinated brown rice, GABA has been used more in a variety of foods, including cola, chocolate, bread, instant noodles, and other foods. Several petitions for GABA use in foods are currently in process in other countries. As a dietary supplement, no recommended daily allowance exists for GABA. In children, doses of as little as 10–100 mg are sufficient when administering GABA to promote healthy neurological and nervous system function. Adults

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Figure 9.5. Biosynthetic pathway and GABA metabolism (Tillakaratne et al. 1995).

are advised to take between 250 and 1,000 mg to provide positive support for low energy levels and mental fatigue caused by occasional anxiety and stress.

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Inoue K, Shirai T, Ochiai H, Kasao M, Hayakawa K, Kimura M, Sansawa H. 2003. Blood-pressure–lowering effect of a novel fermented milk containing gamma-aminobutyric acid (GABA) in mild hypertensives. Eur J Clinical Nutrition 57:490– 495. Irigoyen A, Ortigosa M, Juansaras I, Oneca M, Torre P. 2007. Influence of an adjunct culture of Lactobacillus on the free amino acids and volatile compounds in a Roncal-type ewe’smilk cheese. Food Chemistry 100:71–80. Johnson BS, Singh NK, Cherry JH, Locy RD. 1997. Puridication and characterization of glutamate decarboxylase from cowpea. Phytochemistry 46:39–44. Kazuhito A, Fumio T. 2007. C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and over expression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells. J Experimental Botany 58:2699–2707. Kinnersley AM, Lin F. 2000. Receptor modifiers indicate that 4-aminobutyric acid (GABA) is a potential modulator of ion transport in plants. Plant Growth Regulation 32:65–76. Komatsuzaki N, Shima J, Kawamoto S, Momose H, Kimura T. 2005. Production of γ-aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented food. Food Microbiology 22:497–504. Komatsuzaki N, Tsukahara K, Toyoshima H, Suzuki T, Shimizu N, Kimura T. 2007. Effect of soaking and gaseous treatment on GABA content in germinated brown rice. J Food Engineering 78:556–560. Kono I, Himeno K. 2000. Changes in γ-aminobutyric acid content during beni-koji making. Bioscience, Biotechnology, Biochemistry 64(3):617–619. Leventhal AG, Wang Y, Pu M, Zhou Y, Ma Y. 2003. GABA and its agonists improved visual cortical function in senescent monkey. Science 300:812–815. Lin CH, Gabas N, Canselier JP, Tanori J, Pezron I, Clausse D, Pèpe G. 1994. Surfactant effects in crystallization: Nucleation and crystal habit of γ-aminobutyric acid. Progress Colloid Polymer Science 97:174–178. Lin YL, Wang TH, Lee MH, Su NW. 2008. Biologically active components and nutraceuticals in the Monascus-fermented rice: A review. Applied Microbiology Biotechnology 77:965– 973. Lonart G, Wang J, Johnson KM. 1992. Nitric oxide induces neurotransmitter release from hippocampal slices. Eur J Pharmacology 220:271–272. Luppi PH, Gervasoni D, Verret L, Goutagny R, Peyron C, Salvert D, Leger L, Fort P. 2007. Paradoxical (REM) sleep genesis: The switch from an aminergic–cholinergic to a GABAergic– glutamatergic hypothesis. J Physiology Paris 100:271–283. Matsumoto T, Yamaura I, Funatsu M. 1986. Purification and properties of glutamate decarboxylase from squash. Agricultural Biological Chemistry 50:1413–1417. Meister B. 2007. Neurotransmitters in key neurons of the hypothalamus that regulate feeding behavior and body weight. Physiology & Behavior 92:263–271.

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Chapter 10 Food Proteins or Their Hydrolysates as Regulators of Satiety Martin Foltz, Mylene Portier, and Daniel Tomé

Contents 1. Introduction, 135 2. Protein-Induced Satiety and Food Intake Inhibition—Evidence from Animal and Human Studies, 136 2.1. High-Protein Preload and High-Protein Meal-Induced Satiety and Food Intake Inhibition, 136 2.2. Satiety and Food Intake Inhibition in High-Protein Diets, 137 2.3. Roles of Protein Sources and Types in Protein-Induced Satiety, 138

1. Introduction Food intake in man is determined by both physiological and psychological factors. All factors are centrally received, organized, and integrated balancing both short-term and long-term energy intake with energy expenditure. The regulation of energy intake is determined by both the energy content and the energy source (fat, carbohydrate, and protein) of the meal, each of them having unique effects on satiation and satiety regulatory mechanisms. Although when strictly used the term “satiety” refers to postprandial events that affect the interval and the size of the next meal, we use this term as a

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

3. Mechanisms in Protein-Induced Satiety, 139 3.1. Peripheral, Gut-Derived Satiety Hormones, 139 3.2. Energy Expenditure and Glucose as Metabolic Signals in Protein-Induced Satiety, 142 3.3. Plasma Amino Acids as Central Satiety Signals, 142 3.4. Protein-Induced Satiety and Central Neuronal Pathways, 143 4. Conclusion, 144 5. References, 145

combined term for both “satiety” as well as “satiation” events, that is, including processes that promote meal termination. In this chapter, we center on protein-specific satiety; in particular, we focus on mechanisms and regulation of protein-induced satiety. The evidence derived from studies with different experimental concepts with animals and humans is given; our emphasis is on the underlying biochemical and neural mechanisms. A variety of studies comparing isoenergetic loads of fat, protein, and carbohydrates with respect to their food intake inhibitory and satiety stimulatory effects established that under most conditions protein is more satiating than the isoenergetic ingestion of carbohydrate or fat (Marmonier et al. 2000; Bensaid et al. 2002; Bertenshaw et al. 2008). Furthermore, various studies in humans and animals have shown that high-protein–induced suppression 135

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in food intake is greater than explained by its energy content alone, suggesting that there is a direct effect of the protein or its constituents in regulating satiety (Anderson et al. 1984). In addition, several studies demonstrated that a sustained high-protein diet frequently improves weight loss and body composition (Arciero et al. 2008; Pichon et al. 2008). Up to now there is good evidence from animal and human studies that protein has greater food intake inhibitory effects compared to carbohydrate or fat. Data from human studies comparing effects of different protein sources on postingestive behavior show, however, so far no conclusive results as to whether a specific protein source or fraction is superior in food intake inhibition. In vitro studies suggest that protein hydrolysates, that is, proteins enzymatically cleaved into peptides varying in size prior to ingestion, have a greater effect on triggering satiety hormone secretion than intact proteins (CordierBussat et al. 1997). Protein hydrolysates have also been suggested to reduce in animals food intake more efficiently when compared with intact proteins (Covasa and Ritter 2001; Pupovac and Anderson 2002; Nishi et al. 2003; Reidelberger et al. 2003). This effect has so far not conclusively been confirmed in randomized, placebo-controlled human intervention trials. However, the totality of research in animals and humans indicates that in addition to total ingested energy, protein-specific mechanisms are involved in the induction of satiety and in the control of food intake. Several mechanisms such as secretion of gut satiety hormones, increase in energy expenditure, or increase in plasma amino acid concentrations are candidate signals for protein and peptide-induced satiety.

2. Protein-Induced Satiety and Food Intake Inhibition—Evidence from Animal and Human Studies 2.1. High-Protein Preload and High-Protein Meal-Induced Satiety and Food Intake Inhibition Relatively high-protein diets (meals and diets with on average >15% of energy from protein are repre-

sentative of high-protein meals and diets) have been given attention as they showed the potential to regulate body weight and provide metabolic conditions needed for weight maintenance and weight loss. Protein-induced satiety has been investigated using different study designs. A design that has been used very often is that of the short-term preload paradigm. A preload is given to subjects and animals followed by a test-meal after a specified length of time. Different preload approaches are used that vary in form of the preload (i.e., total energy content, amount and type of protein, solid or liquid form) and in the delay between the preload and the test-meal (i.e., spontaneous or fixed delay, duration of the fixed delay). Furthermore, the meal offered subsequent to the preload can differ in nature and composition. In both animals and humans, the presence or absence of previous adaptation to the preload paradigm may influence the results. Comparisons between studies differing in one or more of these parameters should therefore be handled with care. Studies with a spontaneous delay showed that a high-protein preload increases the delay between the preload and the test-meal compared to a high-fat or a high-carbohydrate preload (Marmonier et al. 2000, 2002). Rolls et al. (1988) tested the effect of a preload on ad libitum energy intake of a subsequent meal with a fixed delay of 120 minutes. They showed a reduction in mean energy intake after the ingestion of both a high-carbohydrate (starch) and a high-protein preload compared to an isocaloric but not isovolumetric preload of fat and sucrose. Similarly, Bertenshaw et al. (2008) demonstrated that a high-protein preload lowers the ad libitum intake of the test-meal in comparison with a highcarbohydrate preload, independent of the fixed delay between the preload and the test-meal (30 or 120 minutes). However, not all human studies show differences in satiety and food intake when comparing highprotein with carbohydrate and fat preloads. In recent work by Blom et al. (2006), a high-protein breakfast did not modify food intake at a test-meal served 3 hours later compared to a high-carbohydrate breakfast. A similar trend was obtained in a study by Smeets et al. (2008), where postprandial satiety and

Chapter 10 Food Proteins or Their Hydrolysates as Regulators of Satiety

hunger perceptions were compared after a highprotein lunch and an isocaloric high-carbohydrate lunch. Although increased satiety was observed 30 and 180 minutes postprandially after the highprotein meal, the differences were minor (< 10 mm on a 100 mm visual analogue scale score) and disappeared 210 minutes after lunch (Smeets et al. 2008). The absence of a satiety effect of proteins in these studies was perhaps due to a too-long delay between preload (which is the breakfast in this case) and the ad libitum test-meal. Indeed, several authors have shown that the consumption of a preload more than 2 hours before a test-meal does not reduce subsequent food intake at the test-meal (Porrini et al. 1997; Anderson et al. 2004). A possible explanation is that the preload is already metabolized and its effect on the food intake control system has dissipated during the long delay between preload and test-meal. This would suggest that the protein preloads influence primarily satiation and short-term satiety.

2.2. Satiety and Food Intake Inhibition in High-Protein Diets A high-protein diet is generally defined as a diet in which high-protein meals are offered at each meal lasting over 24 hours. The difference between a high-protein meal and a high-protein diet is that in the latter situation, the subject gets into a condition where his or her respiratory quotient gets close to the foods quotient. In addition, on a high-protein diet metabolic reactions have been fully established (Lejeune et al. 2006). A large body of evidence that high-protein diets affect body weight and energy metabolism is coming from animal studies showing reduction in weight gain and increase in lean body mass in response to high-protein feeding. For example, in a rat study, Pichon et al. (2006) demonstrated that the consumption of a high-protein diet (55% of energy from proteins) over several days leads to a physiological relevant reduction in cumulative food intake, thereby lowering weight gain and adiposity compared to a normal-protein diet (14% of energy from proteins). However, the protein loads used in animal studies are often very high (>50% energy from protein) and

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in most cases exceed feasible and physiologically more sensible amounts for human consumption. Nevertheless, in humans, several randomized, placebo-controlled intervention studies have shown most frequently comparable, sometimes even superior, effects of high-protein diets (20–30% energy from protein) compared with lower-protein diets on weight loss, preservation of lean body mass, and satiety in humans (Westerterp-Plantenga et al. 1999; Lejeune et al. 2006; Johnstone et al. 2008; Leidy et al. 2007; Veldhorst et al. 2008). For example, Westerterp-Plantenga et al. (1999) compared the effect of a high-protein, high-carbohydrate diet (30/60/10% energy from protein, carbohydrate, fat) and a normal-protein diet (10/30/60% energy from protein, carbohydrate, fat) on satiety and metabolic rate over 24 hours. When in energy balance, the high-protein diet led to higher satiety and lower hunger and appetite over 24 hours compared to the high-carbohydrate, low-fat diet. In a similar experimental setup, the same normal-protein diet was compared to a high-protein, high-fat diet (30/40/30% energy from protein, carbohydrate, fat) (Lejeune et al. 2006). The high-protein, high-fat diet showed increased satiety and decreased appetite and hunger over a 24-hour period similar to the high-protein, low-fat diet (Westerterp-Plantenga et al. 1999). In the latter experiment, the content of fat was kept constant, whereas in the earlier experiment protein was exchanged against both carbohydrate and fat, suggesting that the effect on satiety is indeed dependent on the high-protein load and not on the exchange with the type of other macronutrients. A study by Johnstone et al. (2008) investigated further whether the amount of carbohydrate modulates satiety and ad libitum food intake in high-protein diets. Giving either a high-protein diet (30% energy from protein) with very low or medium levels of carbohydrate (4 or 35% energy from carbohydrate, respectively) over 4 weeks, they showed that the high-protein, very low-carbohydrate diet was superior in both reducing ad libitum food intake and increasing satiety. Overall, high-protein diets have repeatedly been shown to induce higher satiety throughout the day with the effect persisting over longer intervention

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periods up to 4 weeks. The safety of high-protein diets has yet to be determined in longer-term studies.

2.3. Roles of Protein Sources and Types in Protein-Induced Satiety Different types of proteins may differ in their satiety-inducing and food intake-inhibiting properties. Indeed, several groups have suggested that different protein sources may differently affect food intake or satiety ratings in high-protein diets or high-protein preloads. Particularly, more rapid gastric emptying and postprandial increase in plasma amino acid concentration is suggested to increase satiety because of a greater stimulatory effect on gastrointestinal hormones such as cholecystokinin (CCK) and GLP-1. Despite the fact that several studies reported source-dependent and hydrolysis-dependent effects on satiety hormone release in vitro (Cordier-Bussat et al. 1997; Foltz et al. 2008), so far only a few human studies have examined the effect of protein source and the degree of protein hydrolysis on satiety and food intake (Lang et al. 1998, 1999; Hall et al. 2003; Anderson et al. 2004; Bowen et al. 2006a, 2006b; Faipoux et al. 2008; Diepvens et al. 2008). Most evidence that the protein source may play an important role for protein-induced satiety is coming from studies comparing the food intake inhibitory properties of whey and casein. For example, Hall et al. (2003) reported a significantly lower energy intake after a whey protein preload compared with a casein preload. Other studies, however, show less clear results when comparing the satiating properties of different protein sources. Comparing the effect of casein, gelatine, and soy protein as part of mixed meals on appetite perception and subsequent meal intake, Lang et al. (1999) obtained only weak effects on satiety and failed to show an effect on food intake. The effect of the protein source in high-protein diets on body weight reduction has rarely been compared. Anderson et al. (2007) evaluated the effect of high-soy and highcasein meal replacement shakes (4 shakes daily, 20 g protein per shake) on body weight reduction and body composition in a 16-week intervention study. Although both study groups lost significant amounts

of weight, differences in weight loss and body composition changes between casein and soy treatments were not significant. In general, in most of the human studies, protein sources can differ with respect to their satiety hormone-releasing properties, but this is seldom related to differences in energy intake or satiety feelings. For example, 50 g of liquid whey, soy, and gluten protein preload evoked different responses in postprandial plasma concentrations of GLP-1, CCK, and ghrelin; but no differences in energy intake of a subsequent meal was observed for the different protein sources (Bowen et al. 2006a, 2006b). Satiety is triggered in part by luminal events within the gastrointestinal tract, including activation of nutrient sensing systems for sugar, fat, and proteins. As protein is digested into peptides and amino acids upon ingestion, it has been hypothesised that predigested proteins, that is, protein hydrolysates, are more efficient in stimulating satiety than intact proteins. Indeed, a few in vitro studies testing the effect of protein hydrolysates on satiety hormone secretion illustrate that hydrolysates induce increased concentrations of CCK and GLP-1 (Cordier-Bussat et al. 1997; Foltz et al. 2008). This effect has been partly confirmed in animal studies, but with fewer differences observed than one could have expected based on the in vitro data. For example, intragastric infusion of pea protein hydrolysate reduced significantly short-term food intake compared to pea protein but had no effect on 24-hour energy intake (Häberer et al. submitted). In the same study, a similar trend was observed for soy and casein hydrolysates compared to their corresponding proteins. Additional evidence that hydrolysates stimulate satiety more than intact proteins is given by Aziz and Anderson (2003). They investigated the interaction between a GLP-1 receptor agonist (Exendin-4) and proteins (intact proteins, hydrolysates, and a mixture of amino acids) in rats. The ingestion of a preload of hydrolyzed proteins (whey and casein) or of an amino acid mixture led to reduced energy intake 3 hours after administration compared to ingestion of the intact protein. In this study, products of protein digestion seem to be more efficient in reducing food intake than the corresponding intact

Chapter 10 Food Proteins or Their Hydrolysates as Regulators of Satiety

proteins. However, such an effect could not be reproduced so far in a human intervention study. Diepvens and co-workers (2008) obtained only subtle effects of pea protein hydrolysate on satiety and hunger perceptions compared to the nonhydrolyzed protein. Taken together, these studies demonstrated that proteins from different sources and proteins vs. protein hydrolysates differ with respect to their satiety hormone-releasing properties. However, considering the total scientific evidence, it is not conclusive so far whether the protein source as well as the integrity in humans has a clinical relevant effect on a behavioral response, that is, reduction in food intake and/or perception of satiety.

3. Mechanisms in Protein-Induced Satiety 3.1. Peripheral, Gut-Derived Satiety Hormones It has been hypothesized that protein-induced satiety could be due to alterations of the secretion of gut neuropeptides by either an increased secretion of the anorexigenic gut hormones CCK and GLP-1 or a decreased secretion of the orexigenic gut hormone ghrelin as candidate mechanisms. This hypothesis has been strengthened by showing that intraluminal application of proteins induces an increase in plasma concentrations of gut-derived satiety hormones. In some studies the increase in plasma concentration of CCK was greater after high-protein meals compared to high-carbohydrate meals (Blom et al. 2006). The role of CCK as a major endocrine determinant in food intake regulation is well documented (Figure 10.1). CCK acts on a multitude of physiological processes such as gallbladder contraction, pancreatic secretion, intestinal mobility, and gastric emptying. Moreover, the role of peripheral CCK in satiety has been demonstrated in works of Reidelberger et al. (2003, 2004) with CCK receptor antagonists and vagotomized rats, resulting in abolished anorexia normally produced by gastric or duodenal nutrients.

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Figure 10.1. Schematic diagram of putative CCK satiety mechanisms based primarily on studies of the effects of exogenous CCK on food intake. Solid lines represent direction of nutrient flux. Broken lines represent putative regulatory feedback mechanisms to suppress or increase feeding. CCK acts indirectly through control of gastric emptying. Chyme entering the small intestine during and after ingestion of a meal stimulates secretion of CCK from endocrine cells in the upper intestine. Circulating CCK slows gastric emptying, which produces an increased rate of gastric distention, activation of vagal afferent neurons, and inhibition of the brain feeding system. Recent evidence suggests that CCK may also directly activate the vagal afferent neurons that mediate the satiety effects of gastric distention. Moreover, food intake causes the release of brain CCK, which inhibits the brain feeding system (adapted from Reidelberger 1994).

Secretion of CCK occurs in response to food intake by enteric endocrine I-cells that are scattered throughout the mucosa of the small intestine (Cummings and Overduin 2007; Little et al. 2005). Basal plasma CCK concentrations are generally in the low picomolar range in most species and increase approximately 5- to 10-fold following meal ingestion, with dietary fat and protein shown as the most potent stimulators of CCK release (Douglas et al. 1988). It is believed that CCK release by dietary protein is regulated by endogenous, trypsinsensitive CCK-releasing peptides (Liddle 1995), but some studies, both in vivo and in vitro, have found that dietary protein and its peptide react on CCKproducing cells directly to stimulate CCK release (Beucher et al. 1994; Cordier-Bussat et al. 1997; Nemoz-Gaillard et al. 1998). The presence of protein hydrolysate in the duodenum effectively stimulates the release of CCK from endocrine cells, resulting

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in the activation of CCK1 receptor (CCK1R) on vagal afferent nerve terminals (Raybould 1998). Studies in animals and humans using different protein sources suggest that in order to stimulate CCK secretion efficiently, the protein needs to be hydrolyzed to short-chain peptides and amino acids (Darcel et al. 2005). In addition, selected proteins and hydrolysates have been suggested to increase CCK secretion from proteolysis due to competitive peptidase inhibition (Liddle 1995). Recently a receptor, GPR93, was identified in the apical membrane of enterocytes and described to be activated by protein hydrolysates (Choi et al. 2007a). When overexpressed in the enteroendocrine cell line STC-1, GPR93 activation by protein hydrolysates induced CCK secretion from STC-1 cells (Choi et al. 2007b). Several works have shown that inhibition of gastric motility in response to protein hydrolysates is mediated by a CCK1R and vagal afferent reflex pathway (White et al. 2000; Raybould and Lloyd 1994). Indeed, stimulation of vagal afferent activity in response to protein digests is blocked by specific antagonists of CCK1R; and activation of the marker of neuronal activation, fos, in the brainstem is increased by intestinal perfusion of protein hydrolysates via a CCK1R mechanism (Zittel et al. 1994; Glatzle et al. 2001). The precise mechanism and the cascade of events by which a protein hydrolysate is detected by intestinal epithelial cells and causes the release of CCK from endocrine cells remain unclear. A main process for the uptake of protein hydrolysates in the intestine is the proton-coupled oligopeptide transporter PepT1, a member of the family of peptide transporters found in all species from bacteria to humans. This transporter, localized to the apical membrane of enterocytes, is the exclusive oligopeptide transporter of the intestinal mucosa and has a requirement for the di- and tripeptide structure (Rubio-Aliaga and Daniel 2002). Darcel et al. (2005) have demonstrated that the ability of luminal perfusion of protein hydrolysates to stimulate CCKresponsive vagal afferent fiber discharge and inhibit gastric motility was blocked in the presence of a competitive inhibitor of PepT1. Moreover, Cefaclor, a specific substrate for PepT1, increased CCKresponsive vagal afferent fiber activity. Taken

together, these findings support the hypothesis that entry of di- and tripeptides into cells in the intestinal mucosa via PepT1 is involved in the sensory transduction process. The protein hydrolysate content of the intestinal lumen is signalled to vagal afferent nerve terminals to induce activation of a neural pathway and a vagal reflex inhibition of gastric motility. Recently Foltz et al. (2008) have demonstrated that the source of protein hydrolysates could impact stimulation of CCK1R in a dose-dependent manner. Indeed, during protein hydrolysis, shortchain peptides are formed, which possibly act as bioactive peptides at CCK1R. In addition, one should not exclude a direct action of CCK on the central nervous system (at the level of the area postrema or hypothalamus). Indeed, some works of Bowen et al. (2006a, 2006b) showed that in normal and overweight subjects, a preload of 1.1 MJ containing 50 g of whey, soy, and gluten proteins increases the duration of the postprandial blood elevation of CCK compared to a 1.1 MJ glucose preload. The same result was obtained with 1 MJ preloads containing 55 g of whey and casein with a correlation between a concentration of postprandial CCK and appetite ratings, but not with energy intake that remains unchanged after a highprotein preload or a high-carbohydrate (glucose) preload. Finally, the involvement of CCK in acute protein-induced satiety seems to be dose-dependent. Indeed, the replacement of 25 g of whey proteins by 25 g of fructose leads to a lower elevation of CCK concentration compared to a 50 g whey protein preload in obese people (Bowen et al. 2007). However, as pointed out earlier, despite the fact that high loads of proteins induced changes in plasma levels of satiety hormones, this is not accompanied in all studies by changes in subsequent food intake (Bowen et al. 2006a). GLP-1 is a gastrointestinal hormone that is released in response to food intake from the distal small intestine. Kieffer and Habener (1999) have demonstrated that it is derived from proglucagon and produced by cleavage of glucagon in the pancreas, small intestine, and central nervous system. Its peripheral release during meal intake reduces food intake as shown by Chelikani et al. (2005) in

Chapter 10 Food Proteins or Their Hydrolysates as Regulators of Satiety

Cumulative Food Intake (g)

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GLP-1 pmol·kg–1·min–1 0 170

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Figure 10.2. Effect of 3-hour intravenous infusions of GLP-1 on cumulative food intake in rats. Rats that were normally fed received 3-hour jugular vein infusions of GLP-1 at 0 and 170 pmol kg-1 min-1 beginning 15 minutes before dark onset. Food intake was determined from continuous computer recordings of changes in food bowl weight. Values are means ± SE; † p < 0.01; ‡ p < 0.001 (from Chelikani et al. 2005).

rats (Figure 10.2) and in humans as shown by Verdich et al. (2001). Turton et al. (1996) have also shown that central injection of GLP-1 leads to a reduction of food intake. The anorexigenic actions of GLP-1 include a glucose-dependent insulinotropic effect on the pancreatic B cells as shown by Kreymann et al. (1987) and Mojsov et al. (1987); and an inhibition of gastric emptying (Nauck et al. 1997). Recently, studies of Bowen et al. (2006a) have showed in humans that the ingestion of a highprotein preload leads to an increase in plasma concentration of GLP-1 relative to a high-carbohydrate preload. GLP-1 plasma concentrations peak later after the high-protein preload, which results in maintaining a high concentration. The long-term action of GLP-1 has been studied by Lejeune et al. (2006). Lean women were fed in energy balance with a normal or high-protein diet that contained 10/30/60% or 30/40/30% of energy from protein/ carbohydrate/fat during 4 days (4-day total consumption of about 60 and 180 g of protein in normal and high-protein diet). On the fourth day, GLP-1 concentration was measured throughout the day.

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GLP-1 was found to be higher with the high-protein diet than with the normal-protein diet after dinner, which confirms similar results of Johnson and Vickers (1993) in men. However, in acute conditions, Smeets et al. (2008) did not obtain differences in concentrations of satiety-related hormones including GLP-1 and PYY after a single normal or highprotein diet (10% or 25% of energy brought by proteins, respectively). As suggested before, the source of proteins could also influence the postprandial profile of GLP-1. Hall et al. (2003) have shown that a large load of whey proteins leads to a greater increase in plasma GLP-1 concentrations than casein. Moreover, the degree of protein fractionation seems to affect GLP-1 secretion. In a study by Aziz and Anderson (2003), the GLP-1 receptor agonist, Exendin-4, was given intraperitoneally to rats. Food intake was measured when Exendin-4 was given alone or with preloads of intact whey and casein proteins, their hydrolysates, and amino acid mixtures. Both Exendin-4 and the preloads suppressed food intake. Since the effect of Exendin-4 was reduced by the protein hydrolysates and by the amino acid preloads, the results support a role for the end products of protein digestion and GLP-1 release in the suppression of food intake in response to protein ingestion. However, some contradiction exists. In a human study, Calbet and Holst (2004) observed similar GLP-1 and PYY plasma responses after intragastric administration of complete cow milk protein and three different hydrolysates thereof varying in degree of hydrolysis. Thus, in this study plasma GLP-1 responses seem to be independent of the degree of protein fractionation. Ghrelin is produced by the stomach and is, thus far, the only orexigenic hormone identified. Peripheral administration of ghrelin increases food intake in rodents and humans. Ghrelin mediates its orexigenic effect via stimulation of NPY/AgRP arcuate neurons. Bowen et al. (2006a) have recently demonstrated that the postprandial suppression of ghrelin seems to be prolonged after a high-protein liquid preload containing 50 g of whey, soy, or gluten compared to a high-carbohydrate preload in lean men. The same result appears in overweight men after consumption of 55 g of either whey or

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casein (Bowen et al. 2006b). However, Lejeune et al. (2006) did not show a difference in ghrelin concentration after 4 days of high-protein vs. normal-protein diet, which supports findings from Moran et al. (2005) showing that the satiating effect of protein is not related to postprandial ghrelin secretion.

3.2. Energy Expenditure and Glucose as Metabolic Signals in Protein-Induced Satiety Metabolic signals, including an increase in energy expenditure and the production of glucose through gluconeogenesis, have also been hypothesized as signals in protein-induced satiety. It is currently accepted that protein stimulates diet-induced thermogenesis to a greater extent than other macronutrients (Raben et al. 2003). The Atwater factor for protein (17 kJ/g) gives a reflection of the net metabolizable energy value; the factor accounts for digestive and urinary losses. Diet-induced thermogenic losses induced by protein are above this value and are higher than those for either carbohydrate or fat. Lejeune et al. (2006) reported that the higher satiety effect of a protein-rich diet was related to an increase in total and diet-induced thermogenesis in women (diet-induced thermogenesis of 0.91 ± 0.25 and 0.69 ± 0.24 MJ/day for a high and a normal-protein diet, respectively). These differences of about 2% change in daily energy intake are not huge but could be important in long-term high-protein diets. Several hypotheses were proposed to explain these observations, including an increase in liver amino acid oxidation and metabolism. The main gluconeogenic organ is the liver and de novo synthesis of glucose in the liver from gluconeogenic precursors, including amino acids, is stimulated by a high-protein diet in the fed state. This process could be involved in the satiating effect of protein through a modulation of glucose homeostasis and glucose signalling to the brain. Azzout-Marniche et al. (2007) have demonstrated that when the protein content of the diet is increased, phosphoenolpyruvate carboxylase kinase (PEPCK), which controls the initial conversion of oxaloacetate to phosphoenolpyruvate, is upregulated in the fasted and in the fed state. In contrast, glucose 6-phosphatase

(G6Pase), which controls the last step of gluconeogenesis, is upregulated in the fasted state and downregulated in the fed state. The existence of intestinal gluconeogenesis and glucose portal sensing through portal vagus afferent fibers has also been hypothesized as an alternative mechanism for the elevated satiety related to a high-protein diet (Mithieux et al. 2005). Supporting evidence that protein-induced satiety is partly mediated via nonvagal pathways, of which portal glucose sensing could be one possibility, is coming from vagal deafferiation studies. L’Heureux-Bouron et al. (2003) have shown that the depressing effect of a high-protein diet on food intake is not abolished after vagotomy in the rat. However, the opposite, that is, significant reduction of the protein-induced food intake inhibitory effect, was observed in a similar study by Froetschel et al. (2001). Taken together, these observations do not support intestinal gluconeogenesis and its role through glucose portal sensing in the effect of highprotein feeding on food intake. The relevance and physiological significance of intestinal gluconeogenesis stays thus as a subject of debate and needs to be confirmed (Azzout-Marniche et al. 2007; Habold et al. 2005; Martin et al. 2007).

3.3. Plasma Amino Acids as Central Satiety Signals A rise in plasma amino acid concentration in response to either ingestion of protein, protein hydrolysates and amino acids or by infusion of an amino acid mixture, appears to be accompanied by a waning of appetite. Indeed, it has been suggested that an elevated concentration of plasma amino acids, which cannot be channeled into protein synthesis, may serve as a satiety signal for a food intake-regulating mechanism and thereby result in depressed food intake. The variations in plasma hormones and free amino acid concentrations can be directly recorded by the central nervous system mainly through the concomitant variations in their intracerebral level. The role of histidine and tyrosine as precursors of histamine and dopamine has been questioned. Indeed, histidine has been reported to decrease food intake possibly through the activation

Chapter 10 Food Proteins or Their Hydrolysates as Regulators of Satiety

of histamine neurons. In rats, diet supplementation with histidine has been reported to be negatively correlated with food intake, weight gain, and weight of fat pads by Kasaoka et al. (2004, 2005). Moreover, meal pattern has been reported to be affected by central factors related to the interaction between dopamine and serotonin within the lateral hypothalamic area (LHA) and ventromedial nucleus (VMN) (Meguid et al. 2000), which is known to have an appetite-suppressing effect. Indeed, Blundell and Latham (1978) have demonstrated a serotonininduced dose-related reduction in both meal size and feeding rate, whereas analysis of dopaminergic mechanisms show that dopamine D2 receptor blockade rate is associated with an enhancement of meal size and a decrease of meal frequency. However, Bassil et al. (2007) failed to show an effect of a 5%-supplemented diet of either histidine or tyrosine on food intake in Sprague-Dawley rats. In contrast, Cota et al. (2006) have shown that an intracerebroventricular (ICV) administration of leucine reduces food intake and body weight and improves glucose and cholesterol metabolism in rats and mice (Figure 10.3) to the same extent as an increase in dietary leucine (Zhang et al. 2007). Moreover, Morrison et al. (2007) have demonstrated that this effect is specific to leucine, since

Figure 10.3. Intracerebroventicular administration of Lleucine in fasted rats decreases 4 hours and 24 hours food intake. The mean ± SE of 6–7 rats used for each treatment group is shown. *p < 0.05 versus PBS-treated rats; #p < 0.05 versus rats treated with 0.2 μg of L-leucine in 2 μl of PBS (leu 0.2) (from Cota et al. 2006).

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ICV-applied leucine alone has the same effect on food intake as a mixture of ICV-applied amino acids. However, it remains questionable whether under physiological conditions cerebal leucine levels can be sufficiently increased after oral application of a leucine-rich protein source. Ropelle et al. (2008) showed also that the AMPActivated Protein Kinase (AMPK) and the mammalian Target of Rapamycin (mTOR) are involved in the satiety induced by high-protein diets. AMPK is the downstream component of a kinase cascade that acts as a sensor of cellular energy charge and is activated by an increase in the AMP-to-ATP ratio. Once activated, AMPK phosphorylates acetyl-CoA carboxylase (ACC) and switches on energy-producing pathways at the expense of energy-depleting processes (Ropelle et al. 2008). A high-protein diet and ICV leucine administration suppress AMPK and ACC phosphorylation in the hypothalamus of rats, which is accompanied by a decreased AMP-to-ATP ratio. In this respect, mTOR has been discussed to function as a molecular sensor and switch in dependence of the metabolic status of the cell. It has been demonstrated that a high-protein diet as well as ICV administration of free amino acids or leucine alone led to an activation of mTOR in the hypothalamus (Morrison et al. 2007; Ropelle et al. 2008). Moreover, high-protein diets modulate AMPK and mTOR in the same specific neuronal subset, the arcuate and paraventricular nuclei of the hypothalamus (ARC and PVN, respectively). AMPK and mTOR may have overlapping and reciprocal functions (Cota et al. 2006; Kimura et al. 2003) (Figure 10.4). Finally, the activation of mTOR and the suppression of AMPK phosphorylation activity seem to modulate hypothalamic neuropeptides: decrease of concentration of Neuropeptide Y (NPY) and Agouti related peptide (Agrp), both orexigenic neuropeptides, and increased Pro-opiomelanocortin (POMC) expression, which is anorexigenic.

3.4. Protein-Induced Satiety and Central Neuronal Pathways At the brain level, two afferent pathways are involved in protein and amino acid monitoring: the

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Figure 10.4. Proposed model for the role of mTOR signaling in the hypothalamic regulation of energy balance. Anorectic signals, such as amino acids (L-leucine), leptine, and refeeding, increase hypothalamic mTOR signaling. Increased mTOR activity leads to a decrease in food intake. Rapamycin inhibits hypothalamic mTOR, causing an increase in food intake. Opposite to their effect on mTOR, leptin and rerefeeding decrease hypothalamic AMPK. mTOR is inhibited by AMPK-dependent mechanisms in vitro. Thus, reciprocal interaction might exist between hypothalamic mTOR and AMPK (from Cota et al. 2006).

indirect neuro-mediated (mainly vagus-mediated) and the direct blood pathways. The neuro-mediated pathways transfer preabsorptive and visceral information through the vagus nerve that innervates a part of the orosensory zone (stomach, duodenum, and liver). Localized in the brain stem, the nucleus of the solitary tract (NTS) is the main projection of the vagus nerve and integrates sensory information of oropharyngeal, intestinal, and visceral origin. The involvement of vagal afferent pathways in protein sensing and signalling to the brain is supported by results of Jean et al. (2001) showing that intraduodenal protein activates vagal afferent fibers and that high-protein feeding induces the transcription factor c-Fos expression in neurons within the NTS. Faipoux et al. (2008) have shown that the decrease of food intake after a high-protein load (compared to a normal-protein load) results from the activation of the noradrenergic neurons that are related to CCKinduced anorexia. Moreover, it has been shown that neurons expressing GLP-1 were not activated, which is consistent with the fact that protein-induced satiety is not associated with aversive behavior or conditioned taste aversion (CTA) as evidenced by Bensaid et al. (2003). However, Phifer and Berthoud

(1998) have shown that, when infusing glucose (4.5 kcal), linoleic acid, or an amino acid mixture (both 1.5 kcal) into the duodenum of unanaesthetized rats, all compounds suppress sham feeding, but the amino acid mixture triggers relatively little c-fos in the NTS and area postrema compared to glucose and linoleic acid. This suggests that amino acids, although potent inducers of satiety, affect ingestion by processes different from those subserving lipids and carbohydrates. This result is in agreement with the work of Zittel et al. (1994) showing that expression of c-fos-like immunoreactivity was seen in the NTS after infusions of lipid emulsion (2.7 kcal), D-glucose (2.9 kcal), but not after infusions of glucose at 1.1 kcal, mannitol, hydrochloric acid, and casein hydrolysate (1.2 kcal). Peripheral information is further centralized in the hypothalamus, which is critical in the regulation of food intake and energy homeostasis. Energy homeostasis-regulating circuits are found within and connecting these nuclei. In the ARC, the activation of POMC neurons induces a reduction in food intake. These neurons are responsible for the anorectic response induced by circulating leptin (Cowley et al. 2001) through the activation of neurons in the PVN (Balthasar et al. 2005). Activation of POMC neurons is also inseparable from the behavior of another population in the ARC, NPY/Agrp neurons whose activation is potent in increasing food intake and inhibiting POMC neuron activation. Faipoux et al. (2008) showed that, in the case of ingestion of a high-protein meal, the number of double-labeled Fos (marker of the activation of POMC neurons) and α-MSH (characterizing POMC neurons) increased. There is also a decrease in the activation of non-POMC neurons. This result is less pronounced when a high-protein diet is consumed for several days (21 days) rather than briefly. Moreover, because arcuate neurons are mainly POMC or NPY, it could be hypothesized that NPY neurons are less activated after high-protein meals.

4. Conclusion Proteins and their digestion products such as hydrolysates and amino acids are strong determinants of

Chapter 10 Food Proteins or Their Hydrolysates as Regulators of Satiety

short-term satiety and volume of food eaten. Several human studies have demonstrated that high-protein meals and diets show comparable and sometimes superior effects on satiety, food intake, and even weight loss. So far, there is no scientific consensus whether the protein source and integrity has a significant effect on behavioral endpoints in humans, although a variety of in vitro and animal studies have suggested this. Complex and complementary pathways mediate satiety and food intake inhibition in response to protein ingestion. Mechanisms that have been proposed to explain the increased satiety response are mainly nutrient-specific. Peripheral hormones, in particular CCK and GLP-1, are involved in the mechanism of short-term protein-induced satiety, and some works suggest that these mechanisms are more efficient when protein is hydrolyzed compared to intact proteins. Although the protein source determines the response in satiety hormone secretion in vitro and in some studies in vivo, further research has to clarify the potency needed to achieve relevant changes in satiety and food intake. In addition, at the peripheral level, both the increase in energy expenditure and the production of glucose through gluconeogenesis have been hypothesized as metabolic signals in protein-induced satiety. The influence of specific amino acids such as leucine or other precursors of neuropeptides and neurotransmitters may impact protein-induced satiety via central mechanisms. Several regions of the brain are involved in the central regulation of food intake and it has been shown that protein modulates neuronal pathways involved in satiety, in specific areas of the brain stem and the hypothalamus. Other areas that are involved in detecting high-protein meals include the area postrema, anterior piriform cortex, or lateral hypothalamus with orexin-containing neurons. Next studies should aim to identify the potency of the protein-induced gut-derived satiety signals and determine the effect of longer persistence of these short-term effects. Finally, further research should shed more light on the interplay between the various protein-induced mechanisms involved in regulation of energy homoeostasis.

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Diepvens K, Häberer D, Westerterp-Plantenga M. 2008. Different proteins and biopeptides differently affect satiety and anorexigenic/orexigenic hormones in healthy humans. Intl J Obesity 32:510–518. Douglas BR, Woutersen RA, Jansen JB, de Jong AJ, Lamers CB. 1988. The influence of different nutrients on plasma cholecystokinin levels in the rat. Experientia 44:21–23. Faipoux R, Tomé D, Gougis S, Darcel N, Fromentin G. 2008. Proteins activate satiety-related neuronal pathways in the brainstem and hypothalamus of rats. J Nutrition 138:1172– 1178. Foltz M, Ansems P, Schwarz J, Tasker MC, Lourbakos A, Gerhardt CC. 2008. Protein hydrolysates induce CCK release from enteroendocrine cells and act as partial agonist of the CCK1 receptor. J Agricultural and Food Chemistry 56:837– 843. Froetschel MA, Azain MJ, Edwards GL, Barb CR, Amos HE. 2001. Opioid and cholecystokinin antagonists alleviate gastric inhibition of food intake by premeal loads of casein in mealfed rats. J Nutrition 131(12):3270–3276. Glatzle J, Kreis ME, Kawano K, Raybould HE, Zittel TT. 2001. Postprandial neuronal activation in the nucleus of the solitary tract is partly mediated by CCK-A receptors. Amer J Physiology 281:R222–229. Häberer D, Tasker MC, Foltz M, Diepvens K, Geary N, Langhans W. Intragastric infusion of pea protein hydrolysate reduces test meal size more than pea protein in rats. Submitted for publication in Physiology and Behavior. Habold C, Foltzer-Jourdainne C, Le Maho Y, Lignot JH, Oudart H. 2005. Intestinal gluconeogenesis and glucose transport according to body fuel availability in rats. J Physiology 566:575–586. Hall WL, Millward DJ, Long SJ, Morgan LM. 2003. Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite. Br J Nutrition 89:239–248. Jean C, Rome S, Mathe V, Huneau JF, Aattouri N, Fromentin G, Achagiotis CL, Tomé D. 2001. Metabolic evidence for adaptation to a high protein diet in rats. J Nutrition 131:91–98. Johnson J, Vickers Z. 1993. Effects of flavor and macronutrient composition of food servings on liking, hunger and subsequent intake. Appetite 21:25–39. Johnstone AM, Horgan GW, Murison SD, Bremner DM, Lobley GE. 2008. Effects of a high-protein ketogenic diet on hunger, appetite, and weight loss in obese men feeding ad libitum. Amer J Clinical Nutrition 87:44–55. Kasaoka S, Kawahara Y, Inoue S, Tsuji M, Kato H, Tsuchiya T, Okuda H, Nakajima S. 2005. Gender effects in dietary histidine-induced anorexia. Nutrition 21(7–8):855–858. Kasaoka S, Tsuboyama-Kasaoka N, Kawahara Y, Inoue S, Tsuji M, Ezaki O, Kato H, Tsuchiya T, Okuda H, Nakajima S. 2004. Histidine supplementation suppresses food intake and fat accumulation in rats. Nutrition 20(11–12):991–966. Kieffer TJ, Habener JF. 1999. The glucagon-like peptides. Endocrinology Review 20(6):876–913.

Chapter 10 Food Proteins or Their Hydrolysates as Regulators of Satiety

Kimura N, Tokunaga C, Dalal S, Richardson C, Yoshino K, Hara K, Kemp BE, Witters LA, Mimura O, Yonezawa K. 2003. A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells 8:65–79. Kreymann B, Williams G, Ghatei MA, Bloom SR. 1987. Glucagon-like peptide-1 7-36: A physiological incretin in man. Lancet 2(8571):1300–1304. Lang V, Bellisle F, Alamowitch C, Craplet C, Bornet FR, Slama G, Guy-Grand B. 1999. Varying the protein source in mixed meal modifies glucose, insulin and glucagon kinetics in healthy men, has weak effects on subjective satiety and fails to affect food intake. Eur J Clinical Nutrition 53(12):959–965. Lang V, Bellisle F, Oppert JM, Craplet C, Bornet FR, Slama G, Guy-Grand B. 1998. Satiating effect of proteins in healthy subjects: A comparison of egg albumin, casein, gelatin, soy protein, pea protein, and wheat gluten. Amer J Clinical Nutrition 67:1197–1204. Leidy HJ, Carnell NS, Mattes RD, Campbell WW. 2007. Higher protein intake preserves lean mass and satiety with weight loss in pre-obese and obese women. Obesity (Silver Spring) 15:421–429. Lejeune MP, Westerterp KR, Adam TC, Luscombe-Marsh ND, Westerterp-Plantenga MS. 2006. Ghrelin and glucagon-like peptide 1 concentrations, 24-h satiety, and energy and substrate metabolism during a high-protein diet and measured in a respiration chamber. Amer J Clinical Nutrition 83:89–94. L’Heureux-Bouron D, Tomé D, Rampin O, Even PC, LarueAchagiotis C, Fromentin G. 2003. Total subdiaphragmatic vagotomy does not suppress high protein diet-induced food intake depression in rats. J Nutrition 133:2639–2642. Liddle RA. 1995. Regulation of cholecystokinin secretion by intraluminal releasing factors. Amer J Physiology 269:G319– 327. Little T, Horowitz M, Feinle-Bisset C. 2005. Role of cholecystokinin in appetite control and body weight regulation. Obesity Review 6:297–306. Marmonier C, Chapelot D, Fantino M, Louis-Sylvestre J. 2002. Snacks consumed in a nonhungry state have poor satiating efficiency: Influence of snack composition on substrate utilization and hunger. Amer J Clinical Nutrition 76(3):518–528. Marmonier C, Chapelot D, Louis-Sylvestre J. 2000. Effects of macronutrient content and energy density of snacks consumed in a satiety state on the onset of the next meal. Appetite 34:161–168. Martin G, Ferrier B, Conjard A, Martin M, Nazaret R, Boghossian M, Saadé F, Mancuso C, Durozard D, Beverel G. 2007. Glutamine gluconeogenesis in the small intestine of 72 h-fasted adult rats is undetectable. Biochemical J 401:465–473. Meguid MM, Fetissov SO, Varma M, Sato T, Zhang L, Laviano A, Rossi-Fanelli F. 2000. Hypothalamic dopamine and serotonin in the regulation of food intake. Nutrition 16(10): 843–857. Mithieux G, Misery P, Magnan C, Pillot B, Gautier-Stein A, Bernard C, Rajas F, Zitoun C. 2005. Portal sensing of intestinal

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Raybould HE, Lloyd KC. 1994. Integration of postprandial function in the proximal gastrointestinal tract. Role of CCK and sensory pathways. Annals of the NY Academy of Sciences 713:143–156. Reidelberger RD. 1994. Cholecystokinin and control of food intake. J Nutrition 124(8 Suppl):1327–1333S. Reidelberger RD, Heimann D, Kelsey L, Hulce M. 2003. Effects of peripheral CCK receptor blockade on feeding responses to duodenal nutrient infusions in rats. Amer J Physiology— Regulatory, Integrative and Comparative Physiology 284: R389–398. Reidelberger RD, Hernandez J, Fritzsch B, Hulce M. 2004. Abdominal vagal mediation of the satiety effects of CCK in rats. Amer J Physiology—Regulatory, Integrative and Comparative Physiology 286:R1005–1012. Rolls RJ, Hetherington M, Burley VJ. 1988. The specificity of satiety: The influence of foods of different macronutrient content on the development of satiety. Physiology and Behavior 43:145–153. Ropelle ER, Pauli JR, Fernandes MF, Rocco SA, Marin RM, Morari J, Souza KK, Dias MM, Gomes-Marcondes MC, Gontijo JA, Franchini KG, Velloso LA, Saad MJ, Carvalheira JB. 2008. A central role for neuronal AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) in high-protein diet induced weight loss. Diabetes 57:594– 605. Rubio-Aliaga I, Daniel H. 2002. Mammalian peptide transporters as targets for drug delivery. Trends in Pharmacological Sciences 23(9):434–440. Smeets AJ, Soenen S, Luscombe-Marsh ND, Ueland O, Westerterp-Plantenga MS. 2008. Energy expenditure, satiety, and plasma ghrelin, glucagon-like peptide 1, and peptide tyro-

sine–tyrosine concentrations following a single high-protein lunch. J Nutrition 138:698–702. Turton MD, O’Shea D, Gunn I, Beak SA, Edwards CM, Meeran K, Choi SJ, Taylor GM, Heath MM, Lambert PD, Wilding JP, Smith DM, Ghatei MA, Herbert J, Bloom SR. 1996. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 379(6560):69–72. Veldhorst M, Smeets A, Soenen S, Hochstenbach-Waelen A, Hursel R, Diepvens K, Lejeune M, Luscombe-Marsh N, Westerterp-Plantenga M. 2008. Protein-induced satiety: Effects and mechanisms of different proteins. Physiology and Behavior 94:300–307. Verdich C, Flint A, Gutzwiller JP, Näslund E, Beglinger C, Hellstrom PM, Long SJ, Morgan LM, Holst JJ, Astrup A. 2001. A meta-analysis of the effect of glucagonlike peptide-1 (7-36) amide on ad libitum energy intake in humans. J Clinical Endocrinology and Metabolism 86(9):4382–4389. Westerterp-Plantenga MS, Rolland V, Wilson SA, Westerterp KR. 1999. Satiety related to 24 h diet-induced thermogenesis during high protein/carbohydrate vs high fat diets measured in a respiration chamber. Eur J Clinical Nutrition 53(6):495–502. White W, Schwartz GJ, Moran TH. 2000. Role of endogenous CCK in the inhibition of gastric emptying by peptone and Intralipid in rats. Regulatory Peptides 88:47–53. Zhang Y, Guo K, LeBlanc RE, Loh D, Schwartz GJ, Yu YH. 2007. Increasing dietary leucine intake reduces diet induced obesity and improves glucose and cholesterol metabolism in mice via multimechanisms. Diabetes 56:1647–1654. Zittel TT, de Giorgio R, Sternini C, Raybould HE. 1994. Fos protein expression in the nucleus of the solitary tract in response to intestinal nutrients in awake rats. Brain Research 663:266–270.

Part 3 Examples of Food Proteins and Peptides with Biological Activity

Chapter 11 Health-Promoting Proteins and Peptides in Colostrum and Whey Hannu J. Korhonen

Contents 1. Introduction, 151 2. Occurrence and Isolation of Bioactive Whey Proteins, 152 3. Production of Bioactive Peptides, 154 4. Biological Functions and Applications of Whey Proteins, 155 4.1. Immunoglobulins, 156 4.2. α-lactalbumin, 156

1. Introduction The high nutritional value of bovine milk proteins is widely recognized. Also, the multiple functional properties of major milk proteins are well characterized and exploited by various industries. Over the last 2 decades, milk proteins have attracted growing scientific and commercial interest as sources of biologically active molecules having distinct characteristics. Such proteins and their derivatives are found both in casein and whey protein fractions. Many of these specific compounds can be enriched and purified industrially on a large scale (Korhonen and Pihlanto 2007a). In particular, the proteins occurring naturally in the whey fraction have attracted considerable scientific and commercial interest owing to their diverse physico-chemical and

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

4.3. 4.4. 4.5. 4.6. 5. 6. 7. 8.

β-lactoglobulin, 157 Lactoferrin, 157 Lactoperoxidase, 159 Glycomacropeptide, 160 Growth Factors, 160 Novel Applications of Bioactive Peptides, 161 Conclusion, 162 References, 163

biological functionalities (Mulvihill and Ennis 2003). The best characterized bioactive bovine whey proteins include immunoglobulins (Igs), lactoferrin (Lf), lactoperoxidase (LP), and growth factors. They occur in colostrum in much higher concentrations than in milk, reflecting their importance to the health of the neonate. These native proteins are known to exert a wide range of physiological activities, for example, enhancement of nutrient absorption, stimulation of cell growth, enzymatic activity, inhibition of enzyme activity, modulation of the immune system, and defense against microbial infections (Pakkanen and Aalto 1997; Pihlanto and Korhonen 2003; Marshall 2004; Tripathi and Vashishtha 2006; Yalcin 2006). Furthermore, recent research has brought up new information about the putative biological functions of alpha-lactalbumin (α-la) and beta-lactoglobulin (β-lg), the two most abundant proteins in whey. Accordingly, the scope of potential health benefits attributable to whey proteins has expanded greatly in recent years. For example, whey protein–based formulations have proven beneficial 151

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in weight management and regulation of events related to the metabolic syndrome (Walzem et al. 2002; Schaafsma 2006a, 2006b; Luhovyy et al. 2007; Madureira et al. 2007). As a result, commercial applications of these proteins have rapidly increased. In addition to bioactivities exerted by native protein molecules, it is now well established that milk proteins possess further physiological functions resulting from the numerous bioactive peptides encrypted within the intact protein molecules. Such peptides can be liberated in gastrointestinal digestion of milk proteins and fermentation of milk by proteolytic starter cultures, as is the case, for example, in the manufacture of yogurt, sour milk, and cheese (Korhonen and Pihlanto 2006; Gobbetti et al. 2007). The discovery of natural bioactive peptides is now opening plenty of new opportunities for their exploitation in promotion of human health through a regular diet and food supplements or even in the form of biopharmaceuticals. This chapter focuses on the health-promoting biological properties of major native colostral and whey proteins and peptides derived from these proteins.

2. Occurrence and Isolation of Bioactive Whey Proteins Whey proteins represent about 60–80% of the total protein in bovine colostrum, whereas in normal, mature milk the caseins are the predominant protein fraction, accounting for about 80% of the total protein. Also, the concentrations of whey proteins in colostrum differ greatly from those present in mature milk. The foremost protein fractions in colostrum are the immunoglobulins (IgG, IgM, IgA). A majority of Igs in mammary secretions is derived from blood serum. The transport of Igs from serum to milk is a selective process favoring in most species homologous IgG. Specific receptors are involved in the process, enabling the characteristic concentration of Ig isotypes in milk and colostrum. In many species, the absorption of Igs from intestine is selective and receptor mediated. However, in ruminants (for example, in the cow), the absorption of Igs takes

place nonselectively during the first 12–36 hours after birth of the offspring. Ruminant neonates are born virtually without Igs and the colostral Igs are essential for survival. Thus, in ruminants the colostrum contains remarkably higher amounts of Igs than the mature milk (Butler 1998; Levieux and Ollier 1999). In the first milking, the total Ig amount may vary between 20 and 200 g/L, and the IgG concentration postpartum ranges from 15 to 180 g/L, the mean being approximately 60 g/L. Thereafter, the IgG concentration falls sharply to the level below 1 g/L in mature milk. Both the concentrations of α-la and β-lg are highest (on average 3.0 and 8.0 g/L, respectively) in the first milkings of colostrum and decrease sharply thereafter to the average levels of 1.2 g/L for α-la and 3.3 g/L for β-lg. In parallel, the Lf concentration in colostrum varies from 1 to 2 g/L, decreasing soon after parturition to the level of about 0.1 g/L in mature milk of healthy cows. Lactoperoxidase is the major enzyme in bovine mammary secretions. Colostrum contains about 30 mg/L of LP, and about the same amount is found in the mature milk. In contrast to a relatively high concentration (0.36 g/L) in human colostrum, the concentration of lysozyme (LZM) enzyme is about a thousand times less in cow’s colostrum, ranging from 0.3 to 0.8 mg/L and declining to 0.1 mg/L in mature milk (Korhonen et al. 2000a; Hurley 2003; Pihlanto and Korhonen 2003). Colostrum and milk contain many factors that influence cell growth and differentiation. At present, the following growth factors have been identified in bovine mammary secretions: BTC (beta cellulin), EGF (epidermal growth factor), FGF1 and FGF2 (fibroblast growth factor), IGF-I and IGF-II (insulinlike growth factor), TGF-β1 and TGF-β2 (transforming growth factor), and PDGF (platelet-derived growth factor). The concentrations of all known growth factors are highest in colostrum during the first hours after calving and decrease substantially thereafter (Pakkanen and Aalto 1997; Pouliot and Gauthier 2006). In cow’s colostrum, the EGF concentration varies from 4 to 320 μg/L and that of mature milk from 2 to 155 μg/L. Beta cellulin is present in colostrum (2.30 μg/L) and at the same level in cheese whey (2.59 μg/L). The insulin-like

Chapter 11 Health-Promoting Proteins and Peptides in Colostrum and Whey

growth factors 1 and 2 (IGF-I and IGF-II) are single chain polypeptides structurally resembling insulin. Bovine colostrum contains much higher concentrations of IGF-I than does human colostrum (32–800 μg/L as compared with 17–52 μg/L) with lower concentrations in mature bovine milk (4– 27 μg/L). IGF-II is present in bovine colostrum and milk at concentrations of 150–600 μg/L and 2–100 μg/L, respectively. TGF-β1 is detected in bovine colostrum and milk (12–43 μg/L and 0.8– 4 μg/L, respectively), but TGF-β2 is the predominant isoform present at high concentrations in bovine colostrum and milk (150–1,150 μg/L and 13–71 μg/L, respectively). Table 11.1 summarizes the major bioactive whey proteins found in bovine colostrum and milk and provides information about their concentration, molecular weight, and documented or suggested biological functions.

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Fractionation, enrichment, and purification of bioactive, health-enhancing milk proteins has in recent years emerged as a new lucrative sector for dairy industries and specialized bio-industries. To this end, chromatographic and membrane separation techniques have been developed to fractionate and isolate many of these components from colostrum, milk, and cheese whey on a pilot, semi-industrial, or industrial scale (see reviews by Smithers et al. 1996; Korhonen 2002; Kulozik et al. 2003; Pouliot et al. 2006; Korhonen and Pihlanto 2007a). Advances in these techniques and their applications have also been described in two recent seminar proceedings (IDF 2004, 2007) and a special supplement of International Dairy Journal (Korhonen 2006). Membrane separation processes, such as ultrafiltration (UF), reverse osmosis (RO), and diafiltration (DF), are now industrially applied in the manufacture of ordinary whey powder and whey protein

Table 11.1. Major bioactive whey proteins of bovine colostrum and milk: Concentration, molecular weight, and potential biological functions.* Concentration (g/L) Protein Immunoglobulins

Colostrum 20–150

Milk 0.5–1.0

Molecular Weight Daltons 150.000–1000.000

β-lactoglobulin

8.0

3.3

18.400

α-lactalbumin

3.0

1.2

14.200

Lactoferrin

1.5

0.1

80.000

Lactoperoxidase

0.02

0.03

78.000

Lysozyme

0.0004

0.0004

14.000

Milk basic protein

N.A.

N.A.

10.000–17.000

Growth factors

50 μg–40 mg/L

<1 μg-2mg/L

6.400–30.000

Established or Suggested Biological Activity Immune protection through specific antibodies and complement system, synergistic effects with lactoferrin, lactoperoxidase, and lysozyme Potential carrier for vitamins, lipids, and peptides, potential antioxidant, precursor for bioactive peptides Effector of lactose synthesis in mammary gland, calcium carrier, immunomodulatory, precursor for bioactive peptides, potentially anticarcinogenic Antimicrobial, antioxidative, anticarcinogenic, anti-inflammatory, iron transport, cell growth regulation, precursor for bioactive peptides, immunomodulatory, stimulation of osteoblast proliferation Antimicrobial, synergistic effects with immunoglobulins, lactoferrin, and lysozyme Antimicrobial, synergistic effects with immunoglobulins, lactoferrin, and lactoperoxidase Stimulation of osteoblast protein proliferation and suppression of bone resorption Stimulation of cell growth, intestinal cell protection and repair, regulation of immune system

* Modified from Pihlanto and Korhonen 2003 and Korhonen and Pihlanto 2007a; N.A. = not announced.

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concentrates (WPCs) with a protein content of 30– 80%. Gel filtration and ion-exchange chromatography techniques can be employed in the manufacture of whey protein isolates (WPI) with a protein content of 90–95% (Kelly and McDonagh 2000; Etzel 2004; Strohmaier 2004). The chemical composition and functionality of whey protein preparations are largely affected by the method used in the process. The biological properties of such preparations may also be affected and are difficult to standardize due to the complex nature of bioactivities exerted by different proteins (Korhonen et al. 1998; Korhonen 2002; Foegeding et al. 2002). Therefore, growing interest has been focused on development of specific techniques for isolation of pure whey protein components. Such large-scale techniques are already available for purification of α-la, β-lg, Igs, Lf, LP, some growth factors, and glycomacropeptide (GMP) from bovine colostral or cheese whey. Current and potential techniques have been reviewed in recent articles (IDF 2004; Chatterton et al. 2006; Korhonen 2004; Korhonen and Pihlanto 2007a; Konrad and Kleinschmidt 2008; Lozano et al. 2008). The progress of proteomics has facilitated the use of proteomic techniques, for example, two-dimensional gel electrophoresis, in identification and characterization of novel whey protein molecules (O’Donnell et al. 2004; Lindmark-Månsson et al. 2005; Fong et al. 2008).

3. Production of Bioactive Peptides Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions and may ultimately influence health (Kitts and Weiler 2003). The activity of peptides is based on their inherent amino acid composition and sequence. The size of active peptide sequences may vary from 2 to 20 amino acid residues, and many peptides may exert several bioactivities. At present, milk proteins are considered the most important sources of bioactive peptides. Over the last decade, a great number of peptide sequences with different bioactivities have been identified in various milk proteins. The best-characterized milk peptides exhibit antihypertensive, antithrombotic, antimicrobial, antioxidative, immunomodulatory, and opioid

activities (Korhonen and Pihlanto 2006). Bioactive peptides have been found in enzymatic protein hydrolysates and fermented dairy products, but they can also be released during gastrointestinal digestion of proteins, as reviewed in many articles (Clare and Swaisgood 2000; Meisel and FitzGerald 2003; Meisel 2005; Korhonen and Pihlanto 2003, 2006, 2007b; Gobbetti et al. 2007; Hartmann and Meisel 2007). Bioactive peptides are inactive within the sequence of the parent protein molecule and can be released from precursor proteins in the following ways: (1) enzymatic hydrolysis by digestive enzymes, (2) fermentation of milk with proteolytic starter cultures, and (3) proteolysis by enzymes derived from microorganisms or plants. A successive combination of (1) and (2) or (1) and (3), respectively, has proven effective in generating many active peptides both from caseins and whey proteins (Korhonen and Pihlanto 2007a). Digestive enzymes, such as pepsin, trypsin, and chymotrypsin, have been shown to liberate a great number of antihypertensive, antibacterial, immunomodulatory, and opioid peptides from different whey protein molecules, such as α-la, β-lg, and Lf (Yamamoto et al. 2003; FitzGerald et al. 2004; Gobbetti et al. 2004; Bennett et al. 2005; Gauthier et al. 2006b; Ferreira et al. 2007). Blood pressure–reducing peptides that inhibit the angiotensin-converting enzyme I (ACE) are the most studied peptides (see reviews by López-Fandiño et al. 2006; Murray and FitzGerald 2007; Saito 2008). Casein hydrolysates have in some studies (Otte et al. 2007) produced higher ACE-inhibitory activity than whey protein hydrolysates, but also whey peptides (for example, AlaLeu-Pro-Met-His-Ile-Arg [ALPMHIR] or lactokinin from tryptic digest of β-lg) have been identified with strong antihypertensive activity (Mullally et al. 1997; Maes et al. 2004; Ferreira et al. 2007). Also other proteolytic enzymes, such as alcalase, thermolysin, subtilisin, and successive treatment with pepsin and trypsin in order to simulate gastrointestinal digestion have been employed to release from individual whey proteins or whey protein products various bioactive peptides, including antihypertensive or ACE inhibitory (Pihlanto-Leppälä et al. 1998; Murakami et al. 2004; da Costa et al. 2007;

Chapter 11 Health-Promoting Proteins and Peptides in Colostrum and Whey

Roufik et al. 2006), antibacterial (López-Expósito and Recio 2006; López-Expósito et al. 2007), antioxidative (Pihlanto 2006), immunomodulatory (Gauthier et al. 2006a; Saint-Sauveur et al. 2008), and opioid-like (Meisel 2005). Many industrially employed lactic acid bacteria (LAB)–based starter cultures are highly proteolytic and can release different bioactive peptides from milk proteins through microbial proteolysis (Matar et al. 2003; Fitzgerald and Murray 2006; Gobbetti et al. 2007). In particular, Lactobacillus helveticus strains have been shown capable of releasing antihypertensive peptides, the best known of which are casein-derived ACE-inhibitory tripeptides Val-ProPro and Ile-Pro-Pro. The hypotensive capacity of these peptides has been demonstrated in many in vitro, rat model and human studies (Nakamura et al. 1995; Hata et al. 1996; Sipola et al. 2002; Seppo et al. 2003; Jauhiainen et al. 2005). Also yogurt bacteria, cheese starter bacteria, and commercial probiotic bacteria have been demonstrated to produce different bioactive peptides in milk during fermentation (Fuglsang et al. 2003; Gobbetti et al. 2004; Virtanen et al. 2006; Donkor et al. 2007). Recently, Chen et al. (2007) showed that fermentation of milk with a commercial starter culture mixture of five LAB strains followed by hydrolysis with a microbial protease increased ACE inhibitory activity of the hydrolysate, and two strong ACEinhibitory tripeptides (Gly-Thr-Trp and Gly-ValTrp) were identified. The hypotensive effect of the hydrolysate containing these peptides was demonstrated in an animal model study using spontaneously hypertensive rats (SHR). It can be speculated that events, as described in the above study, may happen also under in vivo conditions in the gastrointestinal tract, resulting in the release of peptides with different bioactivities. Over the last decade, large-scale technologies, based on membrane separation techniques, have been developed for the purpose of enrichment and isolation of peptides with a specific molecular weight range (Kitts and Weiler 2003; Pouliot et al. 2006; Korhonen and Pihlanto 2007a). In particular, nanofiltration and ultrafiltration techniques are often employed to fractionate and enrich specific

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bioactive peptides from casein or whey protein hydrolysates.

4. Biological Functions and Applications of Whey Proteins It is well documented that intact bovine whey proteins exert distinct physiological functions in vivo (Walzem et al. 2002; Clare et al. 2003; Floris et al. 2003; Pihlanto and Korhonen 2003; Madureira et al. 2007; Zimecki and Kruzel 2007; Möller et al. 2008). Increasing evidence from animal model and human clinical studies suggests that milk whey proteins provide many health benefits and attract, therefore, increasing commercial interest (Smithers 2008). In fact, several whey proteins or their combinations are already commercially available (Playne et al. 2003; Rowan et al. 2005; Mehra et al. 2006; Krissansen 2007). These formulated products are targeted to boost, for example, the immune system, reduce elevated blood pressure, combat gastrointestinal infections, and help control body weight (FitzGerald et al. 2004; Korhonen and Marnila 2006; Hartmann and Meisel 2007; Luhovyy et al. 2007). Also, there is increasing evidence that many milk-derived components are effective in reducing the risk of metabolic syndrome, which may lead to various chronic diseases, such as cardiovascular disease and diabetes (Mensink 2006; Pfeuffer and Schrezenmeir 2006; Scholz-Ahrens and Schrezenmeir 2006). The potential beneficial health effects attributed to the total whey protein complex include the following: 1. Improvement of physical performance, recovery after exercise, and prevention of muscular atrophy (Ha and Zemel 2003; Krissansen 2007) 2. Satiety and weight management (Schaafsma 2006a, 2006b; Luhovyy et al. 2007) 3. Cardiovascular health (Murray and FitzGerald 2007; Möller et al. 2008) 4. Anticancer effects (Parodi 1998; Bounous 2000; Gill and Cross 2000) 5. Wound care and repair (Smithers 2004, 2008) 6. Management of microbial infections and mucosal inflammation (Korhonen et al. 2000b; Korhonen and Marnila 2006)

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7. Hypoallergenic infant nutrition (Crittenden and Bennett 2005) 8. Healthy aging (Smilowitz et al. 2005). In the following sections, the health-promoting properties of major bovine whey proteins and examples of their commercial applications will be described briefly.

4.1. Immunoglobulins The biological function of colostral Igs is to give the offspring an immunological protection against microbial pathogens and toxins and to protect the mammary gland against infections. To this end, the major mechanisms provided by colostral and milk Igs are augmenting phagocytosis and cell-mediated cytotoxicity reactions by leukocytes, agglutination of bacteria, neutralization of microbes and toxins, and activation of the complement system in milk (Korhonen and Marnila 2000a). The importance of colostral Igs to the newborn calf in protection against microbial infections is well documented (Butler 1998; Hurley 2003). Colostral Ig preparations designed for farm animals are commercially available, and colostrum-based products have found a growing worldwide market as dietary supplements for humans (Scammel 2001; Tripathi and Vashishtha 2006; Struff and Sprotte 2007; Wheeler et al. 2007). Bovine colostrum contains natural antibodies against a wide variety of nonpathogenic and potentially pathogenic microorganisms found in the cow’s environment and feeds. Specific antibodies can be raised in colostrum and milk by immunizing cows with vaccines made of different microorganisms or their antigenic components (Korhonen et al. 2000b). Recent advances in bioseparation techniques have made it possible to fractionate and enrich these antibodies and formulate so-called immune milk preparations to treat humans against microbial infections (Korhonen 2004; Mehra et al. 2006). The concept of “immune milk” dates back to the 1950s, when Petersen and Campbell first suggested that orally administered bovine colostrum could provide passive immune protection for humans (Campbell

and Petersen 1963). Since the late 1980s, a great number of clinical studies have demonstrated that such immune milk preparations can be effective in prevention of human and animal diseases caused by different pathogenic microbes, for example, rotavirus, Escherichia coli, Candida albicans, Clostridium difficile, Shigella flexneri, Streptococcus mutans, Cryptosporidium parvum, and Helicobacter pylori. The therapeutic efficacy of these preparations appears, however, to be quite limited (for reviews see Weiner et al. 1999; Korhonen et al. 2000b; Korhonen and Marnila 2006; Hammarström and Krüger-Weiner 2008). A few commercial immune milk products are on the market in some countries, but the unclear regulatory status of these products in many countries has emerged as a constraint for global commercialization (Hoerr and Bostwick 2002; Mehra et al. 2006). During the last decade, antibiotic-resistant strains causing endemic hospital infections have emerged as global problems. The development of appropriate immune milk products to combat these infections appears to be a highly interesting challenge for future research. To this end, encouraging results have been obtained from preliminary intervention trials with immune milk preparations against Clostridium difficile enterotoxins (Numan et al. 2007; Young et al. 2007).

4.2. α-lactalbumin α-la is the major whey protein in human milk and accounts for about 20% of the proteins in bovine whey. α-la is fully synthesized in the mammary gland, where it acts as coenzyme for biosynthesis of lactose. The biological functions of α-la have long been obscured, but recent research suggests that this protein can provide beneficial health effects through (1) the intact whole molecule, (2) peptides of the partly hydrolyzed protein, and (3) amino acids of the fully digested protein (Chatterton et al. 2006). α-la is a good source of the essential amino acids tryptophan and cystein, which are precursors of serotonin and glutathion, respectively. It has been speculated that the oral administration of α-la could improve the ability to cope with stress. A human clinical study with a group of stress-vulnerable sub-

Chapter 11 Health-Promoting Proteins and Peptides in Colostrum and Whey

jects showed that an α-la enriched diet favorably affected biomarkers related to stress relief and reduced depressive mood (Markus et al. 2000). In a later study, the same researchers (Markus et al. 2002) observed that α-la improved cognitive functions in stress-vulnerable subjects by increased brain tryptophan and serotonin activity. In another recent clinical study, Scrutton et al. (2007) demonstrated that daily administration of 40 g of α-la to healthy women increased plasma tryptophan levels and its ratio to neutral amino acids, but no changes in emotional processing were observed. Moreover, many animal model studies suggest that α-la can provide a protective effect against induced gastric mucosal injury (Matsumoto et al. 2001; Ushida et al. 2003). Bovine α-la exhibits a high degree of amino acid homology to human α-la. Therefore, α-la enriched or purified from cow’s milk and hydrolysates of this protein are well suited as an ingredient for infant formulas. A few bovine α-la enriched formulas are already commercially available.

4.3. β-lactoglobulin β-lg is the predominant whey protein in bovine milk, accounting for about 50% of the proteins in whey, but it is not found in human milk. β-lg exerts a variety of functional and nutritional characteristics that have made this protein a multifunctional ingredient material for many food and biochemical applications (Smithers 2008). β-lg has excellent heat-set gelation properties that make it suitable for a wide range of applications in products where water binding and texturization are required. The putative biological activities of β-lg have been reviewed recently by Chatterton et al. (2006) and include, for example, the following: antiviral, prevention of pathogen adhesion, anticarcinogenic and hypocholesterolemic, and ability to bind hydrophobic components, including retinol and long-chain fatty acids. It has been speculated that this protein may also play a role in the absorption and subsequent metabolism of fatty acids. Furthermore, β-lg has proven an excellent source of peptides with a wide range of bioactivities (see chapter 3).

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4.4. Lactoferrin Lactoferrin is an iron-binding glycoprotein found in different biological fluids of mammals and in neutrophils. Lf is considered to be an important host defense molecule and exhibits a diverse range of physiological functions, such as antimicrobial, antiinflammatory, antioxidant, and immunomodulatory activity. Experimental and clinical research carried out over the last 30 years has accumulated increasing evidence about the potential beneficial health effects of Lf and its derivatives. These benefits may include, for example, anti-infective, anticancer, and anti-inflammatory effects, as reviewed in many articles (Lönnerdal 2003; Wakabayashi et al. 2006; Pan et al. 2007; Weinberg 2007; Zimecki and Kruzel 2007). Furthermore, several antimicrobial peptides, such as lactoferricin B f(18-36) and lactoferrampin f(268-284) can be cleaved from Lf by the action of digestive enzyme pepsin. Lf is considered to play an important role in the body’s innate defense system against microbial infections and degenerative processes induced, for example, by free oxygen radicals. The biological properties of Lf have been the subject of scientific research since its discovery in the early 1960s. Initially, the role was confined largely to antimicrobial activity alone, but now the multifunctionality of Lf has been well recognized. The major known or speculated in vivo activities of Lf in lacteal secretions are 1. Defense against infections of the mammary gland and the gastrointestinal tract (antimicrobial activity, regulation of the immune system) 2. Nutritional effects (bioavailability of iron, source of amino acids) 3. Mitogenic and trophic activities on the intestinal mucosa and gastrointestinal tract associated lymphoid tissue and on bone tissue 4. Antineoplastic activity in the gastrointestinal tract. The antimicrobial activity of Lf and its derivatives has been attributed mainly to three mechanisms: (1) iron binding from the medium leading to inhibition of bacterial growth, (2) direct binding of Lf to the microbial membrane, especially to lipopolysaccharide in Gram-negative bacteria, causing

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fatal structural damage to outer membranes and inhibition of viral replication, and (3) prevention of microbial attachment to epithelial cells or entrocytes. As reviewed by Pan et al. (2007), the bactericidal effect of Lf can be augmented by the action of lysozyme or antibodies. Lf can also increase susceptibility of bacteria to certain antibiotics, such as vancomycin, penicillin, and cephalosporins. The in vitro antimicrobial activity of Lf and the derivatives has been demonstrated against a wide range of pathogenic microbes, including enteropathogenic E. coli, Clostridium perfringens, Candida albicans, Haemophilus influenzae, Helicobacter pylori, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, S. enteriditis, Staphylococcus aureus, Streptoccccus mutans, Vibrio cholerae, and hepatitis C, G, and B virus, HIV-1, cytomegalovirus, poliovirus, rotavirus, and herpes simplex virus (Farnaud and Evans 2003; Pan et al. 2007). One possible in vivo effect of Lf is blocking biofilm development of bacteria by chelating iron from the environment. Iron deprivation stimulates bacterial surface motility, causing the bacteria to wander across the surface instead of forming cell clusters and biofilms. The bacterial transition from a free-living form into biofilms can be devastating for the host, because biofilms protect bacteria effectively from the host defense mechanisms and antibiotics. Lf has been shown to block biofilm development of Pseudomonas aeruginosa at concentrations below those that kill or prevent growth. The antitumor activity of Lf has been studied intensively over the last decade and many mechanisms have been suggested, for example, iron chelation related antioxidative property, immunoregulatory, and anti-inflammatory functions (Wakabayashi et al. 2006). In in vitro experiments, Lf has been shown to regulate both cellular and humoral immune systems by (1) stimulation of proliferation of lymphocytes, (2) activation of macrophages, monocytes, natural killer cells, and neutrophils, (3) induction of cytokine and nitric oxide production, as well as (4) stimulation of intestinal and peripheral antibody response (Pan et al. 2007).

Over the last 4 decades, a great number of animal and human studies have shown that oral administration of Lf can exert many beneficial health effects. These studies have been compiled and reviewed in several excellent articles (Teraguchi et al. 2004; Wakabayashi et al. 2006; Pan et al. 2007; Weinberg 2007; Zimecki and Kruzel 2007). Animal studies with mice or rats have demonstrated that orally administered Lf and related compounds can suppress the overgrowth and translocation of certain intestinal bacteria, such as E. coli, Streptococcus, and Clostridium strains, but does not affect the growth of intestinal bifidobacteria. Also, oral administration of Lf and lactoferricin reduces the infection rate of H. pylori, Toxoplasma gondii, candidiasis, and tinea pedis, as well as prevents clinical symptoms of influenza virus infection. Further animal studies have demonstrated that orally ingested Lf and related compounds can reduce iron-deficient anemia and drug-induced intestinal inflammation, colitis, and arthritis and decrease mortality caused by endotoxin shock. Also, Lf stimulates weight gain in preweaning calves. A recent animal study by Cornish et al. (2004) has shown that oral Lf administration to mice regulates the bone cell activity and increases bone formation. In addition, animal studies have shown beneficial effects of Lf ingestion on inhibition of carcinogen-induced tumors in colon, esophagus, lung, tongue, bladder, and liver. Clinical studies in infants have demonstrated that oral administration of bovine Lf preparations increases the number of bifidobacteria in fecal flora and the serum ferritin level, while the ratios of Enterobacteriaceae, Streptococcus, and Clostridium tend to decrease. A recent clinical study (King et al. 2007) has shown that Lf supplementation to healthy infants for 12 months was associated with fewer lower respiratory tract illnesses and higher hematocrits as compared to the control group, which received regular infant formula. Other human studies have shown that orally administered Lf increases eradication rate of H. pylori gastritis when given in connection with triple therapy. Also, in further human studies, Lf ingestion has been demonstrated to decrease the incidence of bacteremia and severity of infection in neutropenic patients, alleviate symptoms of

Chapter 11 Health-Promoting Proteins and Peptides in Colostrum and Whey

hepatitis C virus infection, and reduce small intestine permeability in drug-induced intestinal injury. Lf ingestion has also been shown to promote the cure of tinea pedis (Tomita et al. 2008). Recent human studies reviewed by Zimecki and Kruzel (2007) suggest that oral bovine Lf administration could be beneficial in stress-related neurodegenerative disorders and treatment of certain cancer types. Lf has attracted increasing commercial interest and many products containing added Lf have already been launched on the market in Asian countries, in particular. Lf is produced industrially by many companies worldwide, and it is expected that its use as an ingredient in functional foods and pharmaceutical preparations will increase drastically in the near future (Tamura 2004; Tomita et al. 2008). Current commercial applications of Lf include, for example, yogurt products marketed in Japan and Taiwan, and baby foods and infant formulas marketed in South Korea, Japan, and China. In addition, Lf has been applied in different dietary supplements that combine, for example, Lf and bovine colostrum and/ or probiotic bacteria. Due to potential synergistic actions, Lf has been incorporated together with LZM and LP into human oral health care products, such as toothpastes, mouth rinses, moisturizing gels, and chewing gums (Wakabayashi et al. 2006). The U.S. Food and Drug Administration (FDA) has approved the use of bLf (at not more than 2% by weight) as a spray to reduce microbial contamination on the surface of raw beef carcasses. FDA has granted a “Generally Recognized as Safe” (GRAS, GRN 67) status to bLf, and this determination accounts for uses at defined levels in beef carcasses, subprimals, and finished cuts (Taylor et al. 2004).

4.5. Lactoperoxidase LP is a glycoprotein that occurs naturally in colostrum, milk, and many other human and animal secretions. LP is the most abundant enzyme in milk and can be recovered in substantial quantities from whey using chromatographic techniques (Kussendrager and van Hooijdonk 2000). The biological function of LP has been associated mainly with its ability to catalyze an unspecific antimicro-

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bial system. In this system, which was originally suggested in the 1960s by Reiter and Oram (1967), LP catalyzes peroxidation of thiocyanate anion or some halides in the presence of a hydrogen peroxide source to generate short-lived oxidation products of SCN-, primarily hypothiocyanate (OSCN-), which kill or inhibit the growth of many species of microorganisms, including bacteria, viruses, fungi, molds, and protozoa (Seifu et al. 2005). The hypothiocyanate anion causes oxidation of sulphydryl (SH) groups of microbial enzymes and other membrane proteins, leading to intermediary inhibition of growth or killing of susceptible microorganisms. Nowadays, the LP/SCN/H2O2 antimicrobial system is considered to be an important part of the natural host defense system of mammals (Boots and Floris 2006). One possible in vivo function of the LP system is related to the presence of LP in human airway epithelia, where it is likely to actively combat respiratory infections caused by microbial invaders (Gerson et al. 2000). The LP system is known to be bactericidal against Gram-negative pathogenic and spoilage bacteria, such as E. coli, Salmonella spp., Pseudomonas spp., and Campylobacter spp. On the other hand, the system is bacteriostatic against many Gram-positive bacteria, such as Listeria spp., Staphylococcus spp., and Streptococcus spp. Also, this mechanism is inhibitory to Candida spp. and the protozoan Plasmodium falciparium and it has been shown to inactivate in vitro the HIV type 1 and polio virus (Seifu et al. 2005). The LP system is considered to provide a natural method for preserving raw milk, as milk contains naturally all necessary components to make the system functional. The natural concentrations of thiocyanate and H2O2 can, however, be critical in milk, and the system usually requires activation by addition of a source of these components. The effectiveness of the activated LP system in raw, uncooled milk has been demonstrated in many pilot studies and field trials worldwide (Anonymous 2005). Since 1991, the LP system has been approved by the Codex Alimentarius Committee for preservation of raw milk under conditions where facilities for milk cooling are insufficient. The method is now being utilized in practice in a number of developing

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countries. The LP system has also found other applications; for example, in dental health care products and animal feeds. A potential new target is gastritis caused by H. pylori, as this pathogen is killed in vitro by the LP system (Shin et al. 2002). More novel applications have been envisaged for temporary shelf-life extension of different items, for example, meat, fish, vegetables, fruits, and flowers (Boots and Floris 2006).

4.6. Glycomacropeptide Glycomacropeptide is a C-terminal glycopeptide f(106-169) released from the κ-casein molecule at 105Phe-106Met by the action of chymosin. GMP is hydrophilic and remains in the whey fraction in the cheese manufacturing process. GMP has a molecular weight of about 8,000 Daltons and contains a significant (50–60% of total GMP) carbohydrate (glycoside) fraction, which is composed of galactose, N-acetyl-galactosamine, and N-neuraminic acid. The nonglycosylated form of GMP is termed caseinomacropeptide or CMP. GMP is the most abundant protein/peptide in renneted cheese whey, amounting to 20–25% of the proteins (ThomäWorringer et al. 2006). The biological activities of GMP have received considerable attention in recent years. It has been shown in many in vitro studies that GMP inactivates microbial toxins of E. coli and V. cholerae, inhibits adhesion of cariogenic Str. mutans and Str. sobrinus bacteria and influenza virus, modulates immune system responses, promotes growth of bifidobacteria, suppresses gastric hormone activities, and regulates blood circulation through antihypertensive and antithrombotic activity (Brody 2000; Manso and López-Fandiño 2004). Rhoades et al. (2005) have demonstrated that GMP can inhibit effectively in vitro adhesion of pathogenic (VTEC and EPEC) E. coli strains to human HT29 colon carcinoma cells. In another study (Brück et al. 2003), oral GMP administration was shown to reduce E. coli–induced diarrhea in infant rhesus monkeys. The glycocide moiety of GMP seems to endow this glycoprotein with many interesting nutritional and physico-chemical properties. GMP is rich in

branched-chain amino acids and low in methionin, which makes it a useful ingredient in diets for patients suffering from hepatic diseases. GMP contains no phenylalanine, making it suitable for patients suffering from phenylketonuria. On the other hand, animal model studies have suggested that the high sialic acid content of GMP may deliver beneficial effects for brain development and improvement of learning ability (Wang et al. 2001). The potential role of GMP in regulation of intestinal functions has been investigated in a number of studies (Manso and López-Fandiño 2004). GMP has been shown to inhibit gastric secretions and slow down stomach motility by means of stimulating the release of cholecystokinin (CKK). This satiety-inducing hormone is involved in controlling food intake and digestion in the duodenum of animals and humans (Yvon et al. 1994). Commercial products containing GMP or CMP have been launched on the market in recent years for the purpose of appetite control and weight management. The clinical efficacy of such products remains, however, to be established. It has been suggested that GMP may have a beneficial role in modulation of gut microflora, as this protein has been shown to promote the growth of bifidobacteria (Manso and Lopéz-Fandiño 2004). Recently, Burton-Freeman (2008) suggested, concluding from a small clinical trial, that GMP alone is not critical in premeal whey-induced satiety: however, it may have a unique role in compensatory intake regulation managing daily energy intake. Obviously, more research is needed to elucidate better the potential biological functions of this versatile peptide molecule that could find in the future many food and nonfood applications.

5. Growth Factors The presence of factors with growth-promoting or growth-inhibitory activity for different cell types was first demonstrated in human colostrum and milk during the 1980s and later in bovine colostrum, milk, and whey (Pakkanen and Aalto 1997; Gauthier et al. 2006a; Pouliot and Gauthier 2006). At present the following growth factors have been identified in bovine mammary secretions: BTC (beta cellulin),

Chapter 11 Health-Promoting Proteins and Peptides in Colostrum and Whey

EGF (epidermal growth factor), FGF1 and FGF2 (fibroblast growth factor), IGF-I and IGF-II (insulinlike growth factor), TGF-β1 and TGF-β2 (transforming growth factor), and PDGF (platelet-derived growth factor). As described in section 2, “Occurrence and Isolation of Bioactive Whey Proteins,” the concentrations of all known growth factors are highest in colostrum during the first hours after calving and decrease substantially thereafter. Chemically, growth factors are polypeptides and their molecular masses range between 6,000 and 30,000 Daltons with amino acid residues varying from 53 (EFG) to about 425 (TGF-β2), respectively. An important characteristic of the growth factors present in colostrum and milk is that they seem to withstand pasteurization and even UHT (ultra high temperature) heat treatment of milk (Gauthier et al. 2006a) relatively well. The main biological functions of milk growth factors have been reviewed recently (Gauthier et al. 2006a; Pouliot and Gauthier 2006; Tripathi and Vashishtha 2006). In essence, EGF and BTC stimulate the proliferation of epidermal, epithelial, and embryonic cells. Furthermore, they inhibit the secretion of gastric acid and promote wound healing and bone resorption. The TDF-β family plays an important role in the development of embryo, tissue repair, formation of bone and cartilage, and in regulation of the immune system. Both forms of TGF-β are known to stimulate proliferation of connective tissue cells and inhibit proliferation of lymphocytes and epithelial cells. Both forms of IGF stimulate proliferation of many cell types and regulate some metabolic functions, for example, glucose uptake and synthesis of glycogen (Pouliot and Gauthier 2006). Contradictory results are reported from studies concerning the stability of growth factors in the gastrointestinal tract. Many animal model studies have, however, shown that EGF, IGF-I, and both TGF forms can provoke various local effects on the gastrointestinal tract and can be absorbed intact or partially from intestine into blood circulation. At present, there are very few human studies performed about the potential physiological effects of oral administration of bovine growth factors. Mero et al. (2002) have demonstrated that oral administration

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of a dietary colostrum-based food supplement increases serum IGF-I in male athletes during a short-term endurance and speed training. According to Gauthier et al. (2006a), increasing evidence supports the view that orally administered growth factors would retain their biological activity and exhibit in the body a variety of local and systemic functions. The main health targets of suggested product concepts have been skin disorders, gut health, and bone health (Donnet-Hughes et al. 2000). Playford et al. (2000) suggested that colostrumbased products containing active growth factors could be applied to prevent the side effects of nonsteroidal anti-inflammatory drugs (NSAIDs) and symptoms of arthritis. To this end, specific methods for extraction of certain growth factors from acid casein or cheese whey have been developed. An acid casein extract rich in TGF-β2 has been tested successfully in children suffering from Crohn’s disease. Another growth factor extract from cheese whey has shown promising results in animal models and humans in treatment of oral mucositis and wound healing, of leg ulcers, for example (Pouliot and Gauthier 2006; Smithers 2008). Other proposed applications could be treatment of psoriasis, induction of oral tolerance in newborn children against allergies, and cytoprotection against intestinal damages caused by chemotherapy. Another interesting protein complex isolated from whey is milk basic protein (MBP), which consists of several low-molecular compounds, such as kininogen and cystatin and HMG-like protein (Kawakami 2005). In cell culture and animal model studies, MBP has been shown to stimulate bone formation and simultaneously suppress bone resorption. The bone-strengthening effects of MBP have been confirmed in human trials and the product has been evaluated for safety and commercialized in Japan.

6. Novel Applications of Bioactive Peptides In many recent studies, traditional, fermented dairy products, such as yogurt, sour milk, dahi, quark, and different types of cheese, have proven to be rich

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sources of bioactive peptides formed during the fermentation process (Chobert et al. 2005; FitzGerald and Murray 2006; Gobbetti et al. 2007). The occurrence, specific activity, and amount of such peptides in fermented dairy products depend on many factors, including type of starters used, type of product, time of fermentation, and storage conditions (Korhonen and Pihlanto 2006; Ardö et al. 2007; Ong et al. 2007). The formation of peptides can be regulated to some extent by starter cultures used, but the stability of desired peptides during storage seems difficult to control (Ryhänen et al.2001; Bütikofer et al. 2007). The potential health benefits attributable to bioactive peptides formed in traditional dairy products warrant extensive research. A study by Ashar and Chand (2004) demonstrated that administration of an Indian fermented milk, “dahi,” reduced blood pressure in middle-aged hypertensive male subjects. The technological optimization of release of bioactive peptides in traditional dairy products and the potential health benefits of these peptides deserve further research. Recently, commercial proteolytic enzymes and starter cultures have been employed successfully for production of specific bioactive peptides from various milk protein fractions. Such preparations are now commercially available and are being used as ingredients in different consumer products, such as dairy and fruit-based drinks, confectionery, chewing gum, pastilles, and capsules. Bioactive peptides added into these products may possess antihypertensive, anticariogenic, mineral-binding, or stressrelieving properties (Korhonen and Pihlanto 2006). So far, the best-studied peptide products are the fermented milk products Calpis® and Evolus®, which are targeted to subjects having mild hypertension. The blood pressure–reducing effects of these two products have been established in many human studies (Hata et al. 1996; Seppo et al. 2003; Mizuno et al. 2004; Aihara et al. 2005; Jauhiainen et al. 2005). Both products contain hypotensive tripeptides Val-Pro-Pro and Ile-Pro-Pro derived from both β-casein and κ-casein. Casein hydrolysates have been demonstrated to produce many peptides with strong bioactivities (Silva and Malcata 2005; LópezExpósito et al. 2007; Saito 2008), but also bovine

α-la and β-lg have proven good precursor proteins of bioactive peptides with a wide range of bioactivities, such as antihypertensive, antimicrobial, antioxidative, anticarcinogenic, immunomodulatory, opioid, hypocholesterolemic, and other metabolic effects (Chatterton et al. 2006). Of particular interest are the antihypertensive peptide β-lactosin B (AlaLeu-Pro-Met; f(142-145)), which has shown significant antihypertensive activity when administered orally to SHR rats (Murakami et al. 2004), and a tryptic peptide of β-lg (Ile-Ile-Ala-Glu-Lys; f(7175)), which has exerted hypocholesterolemic activity in rat model studies (Nagaoka et al. 2001). Also an opioid peptide, β-lactorphin (Tyr-Leu-Leu-Phe; f(102-105)), has been shown to improve arterial functions in SHR rats (Sipola et al. 2002). β-lgderived peptides carry much biological potential, but further in vivo studies are essential to validate the physiological effects. This view is supported by a study of Roufik et al. (2006), who showed that a β-lg-derived ACE-inhibitory peptide (ALPMHIR; f[142-148]) was rapidly degraded upon in vitro incubation with human serum and after oral ingestion could not be detected in sera of human subjects. An increasing number of ingredients containing specific bioactive peptides derived from milk protein hydrolysates have been launched on the market within the past few years or are currently under development. Examples of these commercial ingredients and their applications are listed in Table 11.2.

7. Conclusion The global interest in developing heath-promoting functional foods provides a timely opportunity to tap the many innate bioactive milk proteins for inclusion in such formulations. Moving from science to commercial applications now seems appropriate as the fundamental biological properties and mechanism of action of milk proteins, bioactive peptides, and growth factors are reasonably well established. Also, industrial or semi-industrial scale processing techniques are available for fractionation and isolation of such components from colostrum and milk. Fractionation and marketing of bioactive milk

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Table 11.2. Examples of commercial dairy products and ingredients based on bioactive peptides.* Brand Names

Types of Products

Functional Bioactive Peptides

Health/Function Claims

Manufacturers

Calpis/ Ameal S

Sour milk

Blood pressure reduction

Calpis Co., Japan

Evolus

Fermented milk

Blood pressure reduction

Valio Oy, Finland

BioZate

Hydrolysed whey protein isolate Whey protein isolate

Val-Pro-Pro, Ile-Pro-Pro, derived from β-casein and κ-casein Val-Pro-Pro, Ile-Pro-Pro, derived from β-casein and κ-casein β-lactoglobulin fragments

Blood pressure reduction

Davisco, USA

κ-casein f(106-169) (Glycomacropeptide)

Anticariogenic, antimicrobial, Antithrombotic, Stress symptoms relief

Davisco, USA

BioPURE-GMP

PeptoPro

Flavored milk drink, confectionery, capsules Flavored drink

αs1-casein f(91–100) (Tyr-Leu-Gly-Tyr-Leu-GluGln-Leu-Leu-Arg) Casein derived di- and tripeptides

Vivinal Alpha

Ingredient

α-lactalbumin rich whey protein hydrolysate

Praventin

Capsule

Lactoferrin enriched whey protein hydrolysate

ProDiet F200/ Lactium

Improves athletic performance and muscle recovery after exercise Aids relaxation and sleep

Helps reduce acne

Ingredia, France

DSM Food Specialties, the Netherlands Borculo Domo Ingredients (BDI), the Netherlands DMV International, the Netherlands

* Modified from Korhonen and Pihlanto 2006.

proteins and peptides is emerging as a new lucrative sector for the dairy industries and specialized bioindustries. Use of proteomic tools for discovery of active peptides and metabolomics for assessment of their effects in the body are likely to contribute tremendously to the development in this field in coming years. The dairy industry has already commercialized a few milk protein and peptide-based products, but it can be envisaged that in the near future more similar products will be launched on worldwide markets. They could be targeted to infants, elderly, and immune-compromised people, as well as to maintenance of good health status and prevention of dietrelated chronic diseases. In view of the current global trend of increasing prevalence of obesity and related diseases, type 2 diabetes in particular, more experimental research should be focused on bioactive milk peptides that can regulate appetite and manage blood glucose balance. Other new areas where more

research with bioactive milk proteins and peptides is warranted are impairment of cognitive functions, memory-related diseases, and mood control. In this context, antioxidative and opioid properties of many milk peptides may be worth further investigation. Also, potential to reduce oxidative stress in the body via oral administration of antioxidative peptides could be of considerable interest in view of the inflammatory events caused by production of reactive oxygen species (ROS) in the living cells.

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Chapter 11 Health-Promoting Proteins and Peptides in Colostrum and Whey

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Chapter 12 Functional Food Products with Antihypertensive Effects Naoyuki Yamamoto

Contents 1. Introduction, 169 2. Functional Food Products in the Market, 170 3. Preparation of ACE Inhibitory Peptides, 171 3.1. Enzymatic Hydrolysis, 171 3.2. Fermentation Products, 171 4. In Vivo Effect, 173 4.1. Effects on an Animal Model, 173

1. Introduction This chapter reviews antihypertensive peptides originating from fermented milk products and enzymatic hydrolysis of food proteins such as milk casein whey proteins and fish meat. Most antihypertensive peptides with proven effects on spontaneously hypertensive rats have angiotensin I-converting enzyme inhibitory activities. Clinical experiences for these antihypertensive peptides were also reviewed to discuss the potential of antihypertensive peptides for high blood pressure. Studies reporting an in vivo mechanism, based mainly on animal studies, were also included. The potential of antihypertensive peptides on people with high blood pressure is discussed throughout this review, based on our experiences in Japan. Various physiologically functional peptides, such as opioid peptides (Meisel 1996; Teschemacher

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

4.2. 5. 5.1. 5.2. 6. 7.

Clinical Trials, 173 Mode of Action, 174 Peptide Absorption, 174 ACE Inhibitory Effects, 174 Conclusion, 174 References, 175

et al. 1997), immunostimulating peptides (MiglioreSamour et al. 1989), mineral soluble peptides (FitzGerald 1998), antihypertensive peptides (Meisel and Bockelmann 1999; Yamamoto 1997; Yamamoto and Takano 1999; Takano 2002), and antimicrobial peptides (Bellamy et al. 1992), have been isolated from enzymatic hydrolyzes of raw materials. Among various physiologically functional peptides, antihypertensive peptides have been extensively studied. Hypertension is a major risk factor in cardiovascular disease, such as heart disease and stroke. In order to prevent disease incidence, pharmacological substances can be used to decrease high blood pressure to within the normal range. There are five categories of antihypertensive pharmaceutical products: (1) angiotensin I-converting enzyme (kininase II; EC 3.4.15.1) (ACE) inhibitors, (2) calcium channel antagonists, (3) beta- and alpha-blockers, (4) diuretic agents, and (5) endothelin antagonist. Recently, a peptide was shown to have moderate antihypertensive effects when taken daily for a few weeks (Meisel and Bockelmann 1999; Yamamoto 1997; Yamamoto and Takano 1999; Takano 2002). To study 169

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antihypertensive effects, peptides with ACE inhibitory (ACEI) activities, which play an important role in blood pressure regulation, are used as selection markers to quantitate the in vivo effect of peptides. A dipeptidyl carboxypeptidase ACE catalyzes the production of a vasoconstrictor, angiotensin II from angiotensin I, and inactivates a vasodilator, bradykinin (Skeggs et al. 1956; Yang et al. 1970). ACE is an unusual zinc-metallopeptidase in that it is activated by chloride and has a wide in vitro substrate specifity (Erdös and Skidgel 1987). ACE is predominantly expressed as a membrane-bound ectoenzyme in vascular endothelial cells and several other cell types, including absorptive epithelial, neuroepithelial, and male germinal cells (Caldwell et al. 1976; El-Dorry et al. 1982; Defendini et al. 1983). The first competitive inhibitors to ACE were reported as naturally occurring peptides isolated from snake venom (Ferreira et al. 1970; Ondetti et al. 1971). Ferreira et al. (1970) isolated nine biologically active peptides from Bothrops jararaca, and the peptides contained 5–13 amino acid residues per molecule. Among them, a peptide with the sequence pyrrolodonecarboxyl-Lys-Trp-Ala-Pro showed the highest ACEI activity. Ondetti et al. (1971) reported other strong ACEI peptides from the venom of B. jararaca. In the first part of this chapter, a brief overview is given of products launched in the market with the potential to control blood pressure. The findings of in vitro testing of ACEI peptides and in vivo studies of antihypertensive peptides to spontaneously hypertensive rats (SHR) obtained from various food proteins will then be presented. Finally, based on our studies, the mode of action of the antihypertensive peptides will be discussed.

2. Functional Food Products in the Market Functional food regulation in terms of “foods for specified health use” (FOSHU) was established by the Ministry of Health and Welfare of Japan in 1991. FOSHU products contain dietary ingredients that have beneficial effects on the physiological functions of the human body, maintain and promote

health, and improve health-related conditions. The labeling of health claims on foods should always be based on scientific evidence. For the FOSHU approval, publications and internal reports on safety and effectiveness, using animal and human studies, are required by the Japanese government, with a product-by-product-based concept. Evidence supporting the mechanism of the health benefit and the major active components, along with an explanation for a final product, which has a significant effect in clinical trials on Japanese people, are also required. More than 794 items were approved as FOSHU products in Japan, as of July 2008. These include foods to improve gastrointestinal conditions and foods for people concerned about their blood glucose, cholesterol or triglyceride levels, or mild hypertension. Most of the FOSHU products are foods that maintain a healthy gastrointestinal condition; however, the number of foods with lifestylerelated health benefits is increasing. The total market of FOSHU is still growing and was estimated at over $6 billion in 2007. Among the FOSHU products, the major foods with health claims for blood pressure–lowering effects in humans are summarized in Table 12.1. Most of the products contain ACE inhibitory peptides as the active component. These active peptides are relatively short, rich in Pro at the C-terminus, and contain branched chain amino acids. Ameal S® was initially prepared from Lactobacillus helveticus fermented milk; however, the active peptides, Val-Pro-Pro and Ile-Pro-Pro, are now routinely prepared by enzymatic hydrolysis of casein by an Aspergillus oryzae protease. The casein hydrolyzate was used recently in a new vegetable juice and approved as a FOSHU product. In the United States, the casein hydrolyzate containing Val-Pro-Pro and Ile-Pro-Pro has been used in a supplement product, Ameal bp®. Valio, a Finnish company, launched a fermented milk product containing Val-Pro-Pro and Ile-Pro-Pro in Finland in 2002. Blood pressure–lowering effects in Finnish people drinking the fermented milk product have been proven (Seppo et al. 2002). Since then, similar products with different brand names have been launched in various European countries, MS LH

Chapter 12 Functional Food Products with Antihypertensive Effects

171

Table 12.1. FOSHU products including antihypertensive peptides. Products

Ameal S

Peptide soup

Marine peptide

Sesamine tea

Active components Preparation Protein source Mechanism Launch Company

Val-Pro-Pro, Ile-Pro-Pro LAB fermentation, proteolysis Milk casein ACE inhibition 1997 Calpis

Leu-Lys-Pro-Asn-Met Proteolysis Bonito ACE inhibition 1997 Japan supplement

Val-Tyr Proteolysis Sardine ACE inhibition 2000 SenmiEkisu

Leu-Val-Tyr Proteolysis Sesamine ACE inhibition 2007 Suntory

FOSHU: Foods for Specified Health Use first established as Functional Food Products in Japan (1991).

(L. helveticus) in Iceland (2004), Kaiku Vita in Spain (2005), and Emmi Evolus in Portugal and Switzerland (both 2005). As described in Table 12.1, other peptides derived from food proteins and showing significant health benefits in Japanese people were approved as FOSHU products in Japan. Val-Tyr with ACE inhibitory activity was prepared from sardine proteins by the proteolytic action of a Bacillus alkaline protease (Matsui et al. 1993). Leu-lys-Pro-Asn-Met is prepared by hydrolysis of bonito with thermolisin (Yokoyama et al. 1992). Leu-Val-Tyr was prepared from sesame proteins by food-grade enzymes. In the manufacture of biogenic peptides, commercially available food-grade enzymes to hydrolyze food proteins are advantageous because they are cheap and safe.

3. Preparation of ACE Inhibitory Peptides 3.1. Enzymatic Hydrolysis Generally, commercially available proteases such as trypsin, subtilisin, chymotrypsin, thermolysin, pepsin, proteinase K, papain, plasmin, and so forth are used for the preparation of peptides from food proteins. Bovine caseins and whey proteins are excellent protein sources of functional peptides. ACEI peptides have been identified in the enzymatic hydrolysates of bovine casein (Maruyama and Suzuki 1982; Maruyama et al. 1985, 1987a, 1987b; Karaki et al. 1990), human casein (Kohmura et al. 1989), zein (Maruyama et al. 1989; Miyoshi et al. 1991a, 1991b), gelatin (Oshima et al. 1979), soy

sauce (Kinoshita et al. 1993), soybean, corn, wheat, and other food proteins. Japan is one of the largest consumers of fish meat in the world, and consumption is higher in Japan than the whole of the European Union. In Japan, attempts have been made to isolate bioactive peptides from fish products like sardine muscle (Matsui et al. 1993, 1999), tuna muscle (Kohama et al. 1988, 1991; Yokoyama et al. 1992; Fujita et al. 2000), and bonito (Matsumura et al. 1993a, 1993b) as described above in Japan. Part of the reported peptides with ACEI activity or antihypertensive effects on spontaneously hypertensive rats isolated from milk proteins, fish meat, maize, and sake proteins are summarized in Table 12.2. The source of the functional peptides and their amino acid sequence are also described in Table 12.2. Peptides with high ACEI activity were relatively short, with a proline residue at the C-terminus. Interestingly, prodrug type peptides that can be activated to potent peptides by digestive enzyme action in vivo have been reported. An ACEI peptide with the sequence Leu-Lys-Pro-Asn-Met (IC50 = 2.4 μM), isolated from a thermolysin digest of dried bonito (Yokoyama et al. 1992), may be converted to Leu-Lys-Pro, which is a more potent ACEI peptide (IC50 = 0.32 μM) (Fujita et al. 2000). These results suggest that the breakdown of ingested peptides by gastrointestinal enzymes could happen in vivo.

3.2. Fermentation Products Fermented food products prepared using various microbes have a long history in their uses because of their many beneficial effects. Since the beginning

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Table 12.2. Antihypertensive peptides derived from caseins by proteolytic action. Peptide

Source

Preparation

Enzymatic hydrolysate FFVAPFPEVFGK αs1-casein AVPYPQR β-casein TTMPLW αs1-casein LKPNM Aldolase LKP Aldolase IPA β-lactogloblin VYPFPG β-casein GKP β-microglobulin FP β-casein, albumin YKVPQL αs1-casein

Trypsin Trypsin Trypsin Thermolysin Chicken muscle Proteinase K Proteinase K Proteinase K Proteinase K Proteinase

Fermented products RF Sake lees VW Sake lees YW Sake lees VY Sake IYPRY Sake VPP β-casein

Brewing Brewing Brewing Brewing Brewing Fermentation

IC50(μM)

Dose (mg/kg)

SBP(mmHg)

77 15 16 2.4 0.32 141 221 352 315 22

100 100 100 60 60 8 8 8 8 1

−13.0 −10.0 −13.6 −23 −18 −31 −22 −26 −27 −12.5

Maruyama et al. 1987b Matsumura et al. 1993b Matsumura et al. 1993b Fujita et al. 2000 Fujita et al. 2000 Matsumura et al. 1993b Matsumura et al. 1993b Matsumura et al. 1993b Matsumura et al. 1993b Yamamoto et al. 1994a

— 1.4 10.5 7.1 4.1 9

100 100 100 100 100 1.6

−17 −10 −28 −31 −19 −20

Saito et al. 1994 Saito et al. 1994 Saito et al. 1994 Saito et al. 1994 Saito et al. 1994 Nakamura et al. 1995a, 1995b Nakamura et al. 1995a, 1995b Yamamoto and Takano 1999

IPP

β- and κ-casein

Fermentation

5

1

−15.1

YP

αs1, β- and κ-casein

Fermentation

720

1

−27.4

of this century, many studies have suggested beneficial effects of lactic acid bacteria and cultured dairy products on the nutrition and health status of animals and humans. Microbe fermentation involves the proteolytic processing of proteins to release peptides for use as a nitrogen source. Some peptides accumulated in the fermented media during the fermentation process are also a good source of bioactive peptides. Lactic acid bacteria are suitable microbes for milk fermentation because they have a proteolytic system, which decomposes casein and contains lactose digestive enzymes. Peptides released from casein by an extracellular proteinase of lactic acid bacteria have been reported (Thomas and Mills 1981; Smid et al. 1991; Pritchard and Coolbear 1993; Tan et al. 1993; Yamamoto et al. 1993, 1994a). In our study, ACE inhibitory peptides were isolated from sour milk produced by a starter containing L. helveticus and Saccharomyces cerevisiae (Nakamura et al. 1995a, 1995b). Two kinds of ACE inhibitory peptides were purified from the sour milk, which had

Reference

been fermented until the pH reached approximately 3.3; these two substances were identified as ValPro-Pro and Ile-Pro-Pro. The concentrations of peptides producing 50% inhibition of ACE (IC50) were 9 and 5 μM, respectively. The amino acid sequences of Val-Pro-Pro and Ile-Pro-Pro were found in the primary structure of bovine β-casein (84-86), and (74-76) and κ-casein (108-110), respectively. These peptides may have been processed from the casein molecule by an extracellular proteinase, followed by peptidase action during fermentation. Moreover, bioactive peptides have been reported from other fermented milk products. A recent study showed production of ACEI peptides in fermented milk started by Lactococcus lactis subsp. cremoris FT4 (Gobbetti et al. 2000) and from cheese whey (Amhar et al. 1996; Saito et al. 1997). Eleven different kinds of Japanese fermented foods were analyzed for ACEI peptides (Okamoto et al. 1995). As a result, potent inhibitory activities were detected in soy sauce, fish sauce, natto, fermented tofu, and

Chapter 12 Functional Food Products with Antihypertensive Effects

cheese, but not in mirin, sake, or vinegar (Okamoto et al. 1995). Moreover, Saito et al. (1992, 1994) reported ACEI peptides in sake and sake lee (rice wine and its by-product, respectively). Recently, a potent ACEI peptide, with the sequence His-HisLeu, was reported from fermented soybean paste, a popular food in the Korean diet (Shin et al. 2001).

4. In Vivo Effect 4.1. Effects on an Animal Model Spontaneously hypertensive rat (SHR) is a useful animal model to evaluate the antihypertensive activity of selected ACEI peptides. Some of the reported ACEI peptides have demonstrated strong antihypertensive effects by oral administration in SHR. In our study, oral administration of sour milk containing Val-Pro-Pro and Ile-Pro-Pro to SHR, at a single dose of 5 ml/kg body weight, significantly decreased systolic blood pressure between 6 and 8 hours after administration (Okamoto et al. 1995). The antihypertensive effect of these two chemically synthesized peptides was also observed between 2 and 8 hours after administration, and the effects were dose-dependent. L. helveticus has a higher proteolytic activity than other lactic acid bacteria species, and the antihypertensive effect in SHR was specific to milk fermented by the L. helveticus strain (Yamamoto et al. 1994b). There is discordance between the ACEI activity and antihypertensive activity of this peptide in SHR. Several factors, including stability, processing, intestinal absorption, and mechanism of action, may affect antihypertensive activity. A strong blood pressure–lowering activity was reported for the peptide Leu-Arg-Pro, isolated from α-zein hydrolysate prepared with thermolysin, in a study using 30 mg/kg in SHR (Miyoshi et al. 1991a). Previously described was a prodrugtype antihypertensive peptide that was activated to mature form after an oral administration. Antihypertensive effects of Leu-Lys-Pro-Asn-Met, the proform, and Leu-Lys-Pro, the mature form, were compared to that of captopril in SHR by oral administration (Fujita and Yoshikawa 1999). The minimum effective doses of Leu-Lys-Pro-Asn-Met,

173

Leu-Lys-Pro, and captopril were 8, 2.25, and 1.25 mg/kg, respectively. In addition, the peptide Val-Pro, from the peptide Arg-Val-Pro isolated from an alkaline protease hydrolysate from sardine muscle, also showed strong blood pressure–decreasing activity (Matsufuji et al. 1994). Interestingly, there was a larger decrease in diastolic blood pressure than systolic blood pressure after oral administration of the Val-Tyr peptide (Matsufuji et al. 1995). For a better understanding of the mechanism of the antihypertensive effect in SHR, in vivo mode of action should be studied for each active peptide.

4.2. Clinical Trials Antihypertensive effects in humans have not been proven for most of the peptides obtained by processing food proteins. Recently, the antihypertensive effect of milk, which contains Val-Pro-Pro and IlePro-Pro, was tested in hypertensive patients in Japan (Hata et al. 1996) and Finland (Loffler 2000). Patients were randomly assigned to two groups; the first group ingested 95 ml of milk, which contained 3.4 mg of Val-Pro-Pro and Ile-Pro-Pro, daily for 8 weeks, the second group ingested the same amount of artificially acidified milk as a placebo, for 8 weeks. In the fermented milk group, systolic blood pressure was significantly decreased between 4 and 8 weeks after the beginning of ingestion. No change in systolic blood pressure was observed in the placebo group. The antihypertensive effect of ValTyr on mild hypertensive subjects was investigated using a randomized double-blind placebo-controlled study (Hata et al. 1996). A 100 ml drink containing 3 mg of Val-Tyr and a 100 ml placebo drink were prepared and administered twice a day for 4 weeks. In the Val-Tyr group, systolic and diastolic blood pressure was reduced from 9.7 to 5.3 mmHg (P < 0. 001) at 1 week, and from 9.3 to 5.2 mmHg (P < 0.001) at 4 weeks. Many clinical trials with ACE inhibitory peptides have shown significant blood pressure–lowering effects on humans. Recently, meta-analysis for 12 clinical trials of ValPro-Pro and Ile-Pro-Pro performed in Finland and Japan was reported (Xu et al. 2008). Many of the clinical studies described above strongly suggest

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antihypertensive peptides have the potential to treat people with mild hypertension or prehypertension.

5. Mode of Action 5.1. Peptide Absorption To achieve the antihypertensive in vivo effect, the active peptide has to be absorbed from the intestine in an active form. It is known that small peptides, such as di- and tripeptides, are easily adsorbed in the intestine (Adibi 1971; Hara et al. 1984). However, reports on the adsorption of biologically functional peptides and digestive enzyme-resistant peptides in the intestine are limited. In SHR fed with Val-Pro-Pro and Ile-Pro-Pro containing fermented milk, ACE activities in the aorta, heart, liver, testes, kidney, lung, and brain were measured (Nakamura et al. 1996). Of the various organs measured, ACEI activity in the aorta was significantly lower in the animals fed with the sour milk group than the control group. Ikemoto et al. (1986) reported that ACE activity in the aorta in stroke-prone SHR was significantly higher than that in normotensive rats. The importance of the decrease in ACE activity in the aorta for the expression of antihypertensive activity of the sour milk in SHR was suggested (Masuda et al. 1996). Moreover, the major antihypertensive peptides in the sour milk, Val-Pro-Pro and Ile-ProPro, were detected in a heat-treated solubilized fraction from the abdominal aorta of rats fed with the sour milk, but not in rats fed with unfermented milk (Masuda et al. 1996). Masuda et al. (1996) suggested that these peptides were absorbed directly and transported to the abdominal aorta where they inhibited ACE, producing an antihypertensive effect in SHR. However, recent reports showed a low level of bioavailability of Val-Pro-Pro in circulating blood in a human study (Foltz et al. 2007). Matsui et al. also reported a limited amount of absorption of Val-Tyr for mild hypertension subjects and normotensive subjects (Fujimura et al. 1986; Matsui et al. 2002). However, accumulation of Val-Tyr in some organs of SHR was suggested by Matsui et al. (Matsui et al. 2004). The antihypertensive effect of captopril was observed a few hours after administra-

tion, and the general antihypertensive effects of this peptide continued for more than 10 hours. These results may link to the half-lives of these components; captopril in blood was estimated to be 60 minutes (Fujimura et al. 1986) and approximately 3.5 hours for Val-Tyr (Matsui et al. 2004). These results suggest the need for a better understanding of pharmacokinetic study for target organs.

5.2. ACE Inhibitory Effects As previously described, ACE may modulate blood pressure by catalyzing the conversion of propeptide angiotensin I to vasoconstrictor peptide angiotensin II. Potent ACEI peptides, selected by an in vitro test, had antihypertensive effects in SHR; however, the ACEI activity was still lower than previously reported pharmacological agents such as captopril (Meyer et al. 1981). There appeared to be no reason why these selected peptides had stronger antihypertensive effects in SHR, and even the ACEI activity was lower than for ACEI drugs. Taking these findings into account there may be differences in mode of action and metabolism of peptides. Previous results showed that ingestion of Val-Tyr had no effect on angiotensin I and angiotensin II levels in circulating blood (Fujimura et al. 1986; Matsui et al. 2004). However, angiotensin II levels in the abdominal aorta and lung were significantly decreased by a single oral administration of Val-Tyr in SHR. These results strongly suggest that Val-Tyr may only have beneficial health effects in target organs such as the aorta and lung. Recently, DNA microarray analysis of the aorta of SHR after repeated feeding of Val-Pro-Pro and Ile-Pro-Pro correlated with results obtained with ACE inhibitory drugs (Yamaguchi et al. 2009) such as captopril and ramipril (Jin et al. 2001; Kohlstedt et al. 2005).

6. Conclusion In this report, the benefits of antihypertensive peptides with ACE inhibitory activities generated from food proteins were introduced. Some of the peptides

Chapter 12 Functional Food Products with Antihypertensive Effects

with antihypertensive effects in SHR demonstrated significant efficacies in humans. In Japan, some ACE inhibitory peptides with proven significant antihypertensive effects in humans have been approved as FOSHU products with health claims concerning high blood pressure. Taking into account the mild effects of ACE inhibitory peptides, which have no adverse effects, we consider them to be ideal food-derived natural functional ingredients to keep blood pressure within the normal range.

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related to the tandem repeated sequence of a maize endosperm protein. Agricul Biol Chem 53:1077. Maruyama S, Nakagome K, Tomizuka N, Suzuki H. 1985. Angiotensin I-converting enzyme inhibitor derived from an enzymatic hydrolysate of casein. II. Isolation and bradykininpotentiating activity on the uterus and the Ileum of rats. Agric Biol Chem 49:1405. Maruyama S, Suzuki H. 1982. A peptide inhibitor of angiotensin I converting enzyme in the tryptic hydrolysate of casein. Agric Biol Chem 46:1393. Masuda O, Nakamura Y, Takano T. 1996. Antihypertensive peptides are present in aorta after oral administration of sour milk containing these peptides to spontaneously hypertensive rats. J Nutr 126:3063. Matsufuji H, Matusi T, Ohshige S, Kawasaki T, Osajima K, Osajima Y. 1995. Antihypertensive effects of angiotensin fragments in SHR. Biosci Biotech Biochem 59:1398. Matsufuji H, Matusi T, Seki E, Osajima K, Nakashima M, Osajima Y. 1994. Angiotensin I-converting enzyme inhibitory peptides in an alkaline protease hydrolyzate derived from sardine muscle. Biosci Biotech Biochem 58:2244. Matsui T, Imamura M, Oka H, Osajima K, Kimoto K, Kawasaki T, Matsumoto K. 2004. Tissue distribution of antihypertensive dipeptide, Val-Tyr, after its single oral administration to spontaneously hypertensive rats. J Pept Sci 10:535. Matsui T, Li CH, Osajima Y. 1999. Preparation and characterization of novel bioactive peptides responsible for angiotensin I-converting enzyme inhibition from wheat germ. J Pept Sci 5:289–297. Matsui T, Matsufuji H, Seki E, Osajima K, Nakashima M, Osajima Y. 1993. Inhibition of angiotensin I-converting enzyme by Bacillus licheniformis alkaline protease hydrolyzates derived from sardine muscle. Biosci Biotech Biochem 57:922. Matsui T, Tamaya K, Seki E, Osajima K, Matsumo K, Kawasaki T. 2002. Absorption of Val-Tyr with in vitro angiotensin I-converting enzyme inhibitory activity into the circulating blood system of mild hypertensive subjects. Biol Pharm Bull 25:1228. Matsumura N, Fujii M, Takeda Y, Shimizu T. 1993b. Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from bonito bowels. Biosci Biotech Biochem 57:1743. Matsumura N, Fujii M, Takeda Y, Sugita K, Shimizu, T. 1993a. Angiotensin I-converting enzyme inhibitory peptides derived from bonito bowels autolysate. Biosci Biotech Biochem 57:695. Meisel H. 1996. Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein. FEBS Lett 196–223. Meisel H, Bockelmann, W. 1999. Bioactive peptides encrypted in milk proteins: Proteolytic activation and thropho-functional properties. Antonie van Leewenhoek 76:207. Meyer RF, Nicolaides ED, Tinney FJ, Lunney EA, Holmes A, Hoefle, ML, Smith RD, Essenburg AD, Kaplan HR, Almquist RG. 1981. Novel synthesis of (S)-1-[5-(benzoylamino)-1,4-

dioxo-6-phenylhexyl]-L-proline and analogues: Potent angiotensin converting enzyme inhibitors. J Med Chem 24:964. Migliore-Samour D, Floch F, Jollès P. 1989. Biologically active casein peptides implicated in immunomodulation. J Dairy Res 56:357. Miyoshi S, Ishikawa H, Kaneko T, Fukui F, Tanaka H, Maruyama S. 1991a. Structures and activity of angiotensin-converting enzyme inhibitors in an alpha-zein hydrolysate. Agricul Biol Chem 55:1313. Miyoshi S, Kaneko T, Yoshizawa Y, Fukui F, Tanaka H, Maruyama S. 1991b. Hypotensive activity of enzymatic α-zein hydrolyzate. Agricul Biol Chem 55:1407–1408. Nakamura Y, Masuda O, Takano T. 1996. Decrease of tissue angiotensin I-converting enzyme activity upon feeding sour milk in spontaneously hypertensive rats. Biosci Biotech Biochem 60:488. Nakamura Y, Yamamoto N, Sakai K, Okubo A, Yamazaki S, Takano T. 1995a. J Dairy Sci 78:777. Nakamura Y, Yamamoto N, Sakai K, Takano T. 1995b. Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. J Dairy Sci 78:1253. Okamoto A, Hanagata H, Matsumoto E, Kawamura Y, Koizumi Y, Yanagida F. 1995. Angiotensin I converting enzyme inhibitory activities of various fermented foods. Biosci Biotechnol Biochem 59:1147. Ondetti MA, Williams NJ, Sabo EF, Pluscec J, Weaver ER, Kocy O. 1971. Angiotensin-converting enzyme inhibitors from the venom of Bothrops jararaca. Isolation, elucidation of structure, and synthesis. Biochemistry 10:4033. Oshima G, Shimabukuro H, Nagasawa K. 1979. Peptide inhibitors of angiotensin I-converting enzyme in digests of gelatin by bacterial collagenase. Biochim Biophys Acta 566:128. Pritchard GG, Coolbear T. 1993. The physiology and biochemistry of the proteolytic system in lactic acid bacteria. FEMS Microbiol Rev 12:179. Saito T, Abubakar A, Itoh T, Arai I, Aimar MV. 1997. Development of a new type of fermented cheese whey beverage with inhibitory effects against angiotensin converting enzyme. J Agric Res 48:15. Saito Y, Nakamura K, Kawato A, Imayasu, S. 1992. [No available Japanese title.] Nippon Nôgeikagaku Kaishi (in Japanese) 66:1081. Saito Y, Wanezaki K, Kawato A, Imayasu S. 1994. Structure and activity of angiotensin I converting enzyme inhibitory peptides from sake and sake lees. Biosci Biotech Biochem 58:1767. Seppo L, Kerojoki O, Suomalainen T, Korpela R. 2002. The effect of a Lactobacillus helveticus LBK-16 H fermented milk on hypertension—a pilot study on humans. Milchwissenschaft 57:124. Shin ZI, Yu R, Park SA, Cheng DK, Ahn CW, Nam HS, Kim KS, Lee HJ. 2001. His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo. J Agric Food Chem 49:3004.

Chapter 12 Functional Food Products with Antihypertensive Effects

Skeggs LT, Kahn JR, Shumway NP. 1956. The preparation and function of the hypertensin-converting enzyme. J Exp Med 103:295. Smid EJ, Poolman B, Konings WN. 1991. Casein utilization by lactococci. Appl Environ Microbiol 57:2447. Takano T. 2002. Anti-hypertensive activity of fermented dairy products containing biogenic peptides. Antonie van Leewenhoek 82:333. Tan PST, Poolman B, Konings WN. 1993. Proteolytic enzymes of Lactococcus lactis. J Dairy Res 60:269. Teschemacher H, Kock G, Brantl V. 1997. Milk protein-derived opioid receptor ligands. Biopoly 43:99. Thomas TD, Mills, OE. 1981. Proteolytic enzymes of starter bacteria. Neth Milk Dairy J 35:255. Xu JY, Qin LQ, Wang PY, Li W, Chang C. 2008. Effect of milk tripeptides on blood pressure: A meta-analysis of randomized controlled trials. Nutrition 24:933. Yamaguchi N, Kawaguchi K, Yamamoto N. 2009. Study of the mechanism of antihypertensive peptides VPP and IPP in spontaneously hypertensive rats by DNA microarray analysis. Eur J Pharmacol. 620:71.

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Yamamoto N. 1997. Antihypertensive peptides derived from food proteins. Biopoly 43:129. Yamamoto N, Akino A, Takano T. 1994a. Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from Lactobacillus helveticus CP790. J Dairy Sci 77:917. Yamamoto N, Akino A, Takano T. 1994b. Antihypertensive effects of different kinds of fermented milk in spontaneously hypertensive rats. Biosci Biotech Biochem 58:776. Yamamoto N, Akino A, Takano T. 1993. Purification and specificity of a cell-wall-associated proteinase from Lactobacillus helveticus CP790. J Biochem 114:740. Yamamoto N, Takano T. 1999. Antihypertensive peptides derived from milk proteins. Nahrung 43:159. Yang HYT, Erdös EG, Levin Y. 1970. A dipeptidyl carboxypeptidase that converts angiotensin I and inactivates bradykinin. Biochem Biopys Acta 214:374. Yokoyama K, Chiba H, Yoshikawa M. 1992. Peptide inhibitors for angiotensin I-converting enzyme from thermolysin digest of dried bonito. Biosci Biotech Biochem 56:1541.

Chapter 13 Secreted Lactoferrin and Lactoferrin-Related Peptides: Insight into Structure and Biological Functions Dominique Legrand, Annick Pierce, and Joël Mazurier

Contents 1. Introduction, 179 2. Origin of Secreted Lf, 180 3. Structure of Lf, 180 3.1. Lf Gene Structure and Amino Acid Sequence, 180 3.2. 3D Structure of Lf, 181 3.3. Glycosylation of Lf, 181 4. Iron-Binding Properties and Dynamics of Lf, 181 5. Structure of the Biologically Active Peptides of Lf, 182 5.1. The Biologically Active Peptide Sequences of Lf, 182 5.2. Structure of Lfcin, 184 5.3. Structure of Lfampin, 185

1. Introduction Lactoferrin (Lf) is an iron-binding glycoprotein of the transferrin family that is expressed in most biological fluids and is a major component of the innate immune system of mammals. Its protective effect ranges from direct antimicrobial activities against a large panel of microorganisms including bacteria, viruses, fungi, and parasites, to anti-inflammatory

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

6. 6.1. 6.2. 6.3. 7.

Biological Roles of Lf in Host Defense, 185 Antimicrobial Activities of Lf, 185 Anti-inflammatory Properties of Lf, 187 Pro-inflammatory Properties of Lf, 188 Biological Roles of Lf in Cancer and Cell Proliferation, 189 8. Biological Properties of the Lf-Related Peptides, 191 8.1. Biological Properties and Mechanisms of Action of Lfcin, 191 8.2. Biological Properties and Mechanisms of Action of Lfampin, 193 9. Applications of Lf and Related Peptides, 193 10. Conclusion, 194 11. References, 194

and anticancer activities. This plethora of activities is made possible by mechanisms of action implementing not only the capacity of Lf to bind iron but also interactions of Lf with molecular and cellular components of both host and pathogens. Interestingly, most interactions involve specific cationic peptide sequences of the N-terminal lobe of the molecule that can mimic, sometimes with a higher potency than the whole protein, the biological properties of lactoferrin. Those peptides, either purified from biological fluids or chemically synthesized, were proven useful tools to study the mechanisms of action of lactoferrin, as well as new therapeutical compounds. This review will summarize our 179

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knowledge on the structure and biological functions of Lf and of the Lf-derived peptides. Lactoferrrin has been the subject of intensive structural and functional studies since it was first purified from human and bovine milks, in 1960 (Montreuil et al. 1960). Its most striking characteristic, an intense red color when incubated in the presence of Fe3+ ions, suggested that Lf was an analogue of the serum transferrin (Tf), and led to its naming as “lactoferrin” or “lactotransferrin.” Intriguingly, Lf was found not only in milk but also in other external secretions, such as saliva, tears, semen, and mucosal secretions (Masson et al. 1966), and as an important constituent of the neutrophilic granules of leukocytes (Baggiolini et al. 1970). From the beginning of the research on Lf biological functions, it appeared certain that the molecule owns crucial antimicrobial properties. Although these properties were formerly attributed to its tremendous metal-binding ability, which deprives microbes of iron, it soon became clear that Lf has far more than one string to its bow. Since then, big steps forward have indeed been taken in the understanding of its roles in host defense and the innate immunity system, not only against bacteria but also against viruses, parasites, and fungi. Actually, it is now well established that the main antimicrobial activities of the protein lie in particular domains or peptides that have been isolated and characterized. One of them is lactoferricin (Lfcin), a major antimicrobial basic peptide located within the 50 first N-terminal amino acids of Lf and isolated more than a decade ago. A novel important peptide is lactoferrampin (Lfampin), which is in close proximity to Lfcin and exhibits strong anti-Candida activities. This review will describe our current knowledge on the structure-functions relationships of Lf. It will also focus on the Lf-derived peptides and their potential applications for health.

2. Origin of Secreted Lf Lf is synthesized either continuously in exocrine fluids, under hormonal control in genital tract and mammary gland (Teng et al. 2002), or at welldefined stages of cell differentiation in neutrophils

(PMNs) (Masson et al. 1969). Lf is secreted in the apo-form (iron-free form) from epithelial cells in most exocrine fluids including milk (Montreuil et al. 1960). In human milk, Lf concentration may vary from 1 g/l (mature milk) to 7 g/l (colostrum). In mature bovine milk, its mean concentration is 30 mg/l. Furthermore, Lf is synthesized during the transition from promyelocytes to myelocytes, and it is thus a major component of the secondary granules of PMNs (Masson et al. 1969). During inflammation and in pathologies, Lf levels of biological fluids, such as plasma and milk, may increase greatly (from 0.4 to 2 mg/l to up to 200 mg/l in plasma) and constitute a marker for inflammatory diseases.

3. Structure of Lf 3.1. Lf Gene Structure and Amino Acid Sequence The Lf gene is highly conserved (Teng 2002). Lf gene polymorphisms are widely distributed in the human population and may lead to modulation of Lf activity. Transcription of the Lf gene, mapped to human chromosome 3 at 3p21.3, leads to two products, Lf and delta-Lf (ΔLf) mRNAs, which result from the use of alternative promoters (Siebert and Huang 1997; Liu et al. 2003). The main product, Lf mRNA, codes for the secreted Lf protein found in biological fluids, while the ΔLf mRNA codes for a cytoplasmic N-terminally truncated Lf that can locate in the nucleus and play antiproliferative activities (Mariller et al. 2007). The sequences of human, mouse, cow, horse, pig, goat, sheep, buffalo, and camel Lfs are now known, but the major advances in understanding Lf function came with the determination of the amino acid sequence of human Lf (hLf) (Metz-Boutigue et al. 1984). All Lfs are 80 kDa proteins that comprise about 690 residues and share identities of at least 65%. All Lfs, like any member of the Tf family, possess a two-fold internal sequence repeat with about 40% identity, indicating a gene duplication event in evolution (Metz-Boutigue et al. 1984). However, unlike Tfs, Lfs are very basic proteins (pI ≥ 8.5).

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3.2. 3D Structure of Lf The structure of Lf has been described in detail for hLf (Anderson et al. 1989; Haridas et al. 1995), and its structure-functions relationships have been reviewed (Baker and Baker 2004, 2005, 2009). Crystallographic studies on the Lfs from bovine, mare, buffalo, and camel have shown the same basic structures (Moore et al. 1997; Sharma et al. 1998; Karthikeyan et al. 1999; Khan et al. 2001; Baldridge et al. 2005). According to the sequence homologies between the two halves of Lfs, the peptide chain is folded into two globular lobes formed by residues 1–333 and 345–692 in hLf (Figure 13.1A). Unlike in Tfs, the two lobes are connected by a peptide of 10–15 residues (residues 334–344 in hLf), which forms a three-turn α-helix, and stabilized by noncovalent hydrophobic interactions. Both lobes have the same fold, consistent with their sequence homology. In each lobe, two domains, referred to as N1 and N2, or C1 and C2, enclose a deep cleft within which is the iron-binding site (Figure 13.1A). The fold of each lobe corresponds to a classic two-domain “binding protein fold” that is shared by a large family of bacterial periplasmic proteins that transport ions and small molecules (Bruns et al. 1997), and suggests a common evolutionary origin.

3.3. Glycosylation of Lf All Lfs contain biantennary N-acetyllactosamine type glycans, α,1-6-fucosylated on the Nacetylglucosamine residue linked to the polypeptide chain (Spik et al. 1988). HLf may also possess additional poly-N-acetyllactosamine antennae that may be α,1-3-fucosylated on N-acetylglucosamine residues, whereas Lf of other species contain additional high-mannose type glycans (Coddeville et al. 1992). Both the number and location of the glycosylation sites vary among species. Furthermore, heterogeneity in the number of glycosylated sites is observed in individuals. The role of the glycan moiety seems to be restricted to a decrease in the immunogenicity of the protein and its protection from proteolysis (Spik et al. 1988; van Veen et al. 2004).

Figure 13.1. Overall structure of Lf. The structure of diferric hLf from the pdb x-ray diffraction data (reference 2BJJ) of a recombinant hlf produced in the milk of transgenic cows is shown (Thomassen et al. 2005). The structure is quite similar to the structure published formerly by Haridas et al. (1995) but shows the N-terminal 1-5 residues stretch (GRRRR). (A) The structure shows the organization of Lf in four domains: N1 and N2, and C1 and C2 for the N-t and C-t lobes, respectively. The iron-binding sites are located at the interfaces between each pair of domains. The positions of the two ferric ions, the two glycans, and the α-helix hinge region are indicated. (B) The figure shows the solvent-accessible surface of the molecule with the distribution of the positively charged amino acids on the molecule (black). Molecular modeling was performed using the Chimera software (http:// www.cgl.ucsf.edu/chimera/).

4. Iron-Binding Properties and Dynamics of Lf Like any other member of the Tf family, Lf binds ferric iron (Fe3+) together with carbonate CO32− (Aisen and Leibman 1972; Baker and Baker 2004). It may also bind other metal ions such as Ga3+, Al3+,

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VO2+, Mn3+, Co3+, Cu2+, Zn2+, and lanthanides, though with lesser affinity than Fe3+ (Baker and Lindley 1992). Iron binding by Lf is very tight but reversible (KD ∼ 10−22 M). Although Lfs strongly differ from Tfs in terms of iron-binding pH stability (typically pH 3–4, compared with pH 5–6 for Tf) (Mazurier and Spik 1980), all Lfs and Tfs so far characterized have identical metal and anion-binding sites. Each site comprises four protein ligands (2 Tyr, 1 Asp, 1 His) that provide three negative charges to balance the ionic charge of Fe3+, together with an Arg side chain whose positive charge balances the negative charge on the CO32− anion. The dynamics of iron binding and release by Lfs are now well known (reviewed in Baker and Baker 2009). They provide sound explanations to the observation that (1) the C-terminal lobe possesses a higher iron-binding pH stability than that of the C-terminal lobe and (2) Lfs have much higher pH stabilities than Tfs (Mazurier and Spik 1980). Iron binding indeed induces a “closed” structure in which the iron ligands of the two domains of each lobe enclose both Fe3+ and CO32− ions (Anderson et al. 1989). When no Fe3+ is present within the site, the N2 domain of the N-terminal lobe swings 54° away from the other to open up the binding cleft. This movement is made possible by a hinge in two polypeptide strands that run behind each iron-binding site (Anderson et al. 1990; Gerstein et al. 1993; Jameson et al. 1998). This event leads to the formerly reported “open” Lf structure (Anderson et al. 1990). Further crystallographic studies have shown that similar conformational change may occur for the C-terminal lobe, but in a lesser extent, and also that an iron-free N-terminal lobe may also exist in a closed structure (Baker et al. 2002). In the absence of a bound metal ion to lock the two domains of each lobe together, the apo form is flexible. These results lead to important conclusions on Lf conformational dynamics and the iron-release properties of Lf.

5. Structure of the Biologically Active Peptides of Lf It was originally thought that the antimicrobial activity of Lf was only dependent on its ability to

scavenge iron away from invading bacteria. It is now known that this is not its only mechanism of action. When the protein is digested at acidic pH by proteases such as pepsin that are found in the stomach, the products yield highly active antimicrobial peptides (Tomita et al. 1991; Bellamy et al. 1992). In biological fluids, Lf exists in the iron-free form that is very susceptible to proteolysis. It cannot be excluded that a posttranslational process of maturation by proteolysis leads to the release of Lfderived active peptides in biological fluids (Goldman et al. 1990). Two of these biologically active Lfderived peptides, Lfcin (Bellamy et al. 1992) and Lfampin (van der Kraan et al. 2004), have been characterized in Lf and their structural and functional properties thoroughly investigated.

5.1. The Biologically Active Peptide Sequences of Lf Most of Lf functions depend on its ability to bind to other macromolecules such as proteins, DNA, and polysaccharides. These activities, in turn, must depend on surface features of Lf. All known Lfs have high isoelectric points (pI ≥ 8.5); and this feature, which differentiates Lfs from Tfs, is a major factor in the ability of Lf to bind to many anionic molecules. The distribution of surface charge is highly uneven, with three notable concentrations of positive charges at the N-terminus (residues 1–7), along the outside of the first helix (residues 13–30), and in the interlobe region (Figure 13.1B). The N-terminus is, without contest, the part of Lf that interacts with most molecular partners (Legrand et al. 2008). Residues 1–5 of hLf, like residues 417– 432, were indeed reported as nuclear localization sequences (Penco et al. 2001; Mariller et al. 2007); residues 8–25 as a glycation site (Li et al. 1995); residues 1–47 as a bacterial porin-binding site (Sallmann et al. 1999); residues 1–6 and 28–31 as a proteoglycan-binding site (Mann et al. 1994) and an sCD14/lipopolysaccharide (LPS)-binding site (Elass-Rochard et al. 1995); residues 19–29 as an LPS-binding site (Japelj et al. 2005); residues 25–30 as Lipoprotein Receptor-related Protein (LRP) (Meilinger et al. 1995) and lymphocyte

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Figure 13.2. Localization of the Lfcin domain and Lfampin sequence on Lf. The figure shows the front (A and C) and left side (B and D) views of the Lf molecule (pdb reference 2BJJ) with the positions of LfcinH (black and light grey), LfcinB (light grey), and LfampinB or LfampinH (dark grey) (Bellamy et al. 1992; van der Kraan et al. 2004). Ribbon foldings of the peptide domains are shown in A and B, and the solvent-accessible surface of the molecule is shown in C and D. Molecular modeling was performed using the Chimera software (http://www.cgl.ucsf.edu/chimera/).

receptor–binding sites (Legrand et al. 1992); and residues 1–31 as a DNA-binding site (van Berkel et al. 1997). The first helix (residues 12–31 in hLf) forms the major part of the bactericidal domain identified by Bellamy et al. (Bellamy et al. 1992), described as the Lfcin domain (Gifford et al. 2005) (Figure 13.2). Recent results show that this bactericidal domain, which is a highly exposed feature of the Lf surface, can also serve as a macromolecular binding surface. A recent crystallographic analysis shows that the N-lobe of hLf binds specifically through this region to a bacterial cell surface protein, the pneumococcal surface protein A (PspA) from the important human pathogen Streptococcus pyogenes (Senkovich et al.

2007). Another example of complex formation by Lf, this time with ceruloplasmin, has been localized to the N-lobe and may involve this region (Sabatucci et al. 2007). Recently, another region of the N1 region of Lf encompassing residues 265–284 of the bovine protein (and its equivalent in other Lfs), called Lfampin, was shown to be essential in the antimicrobial activities of the molecule (van der Kraan et al. 2004, 2006). As shown in Table 13.1, the Lfampin sequence is close to the Lfcin domain. The cationic charges at its C-terminal end contribute to the antimicrobial activity of Lf, while its N-terminal end forms an amphipathic helix (van der Kraan, Nazmi et al. 2005; van der Kraan, van der Made et al. 2005). Lastly, it should be mentioned

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Table 13.1. Sequences of the Lfcin and Lfampin peptides from hLf and bLf. Peptide

Sequence

LfcinH LfcinB LfampinH LfampinB

GRRRRSVQWCAVSQPEATKC-FQWQRNMRKVRGPPVSCIKR-DSPIQCIQA FKCRRWQWRMKKLGAPSITCVRRAE WNLLRQAQEKFGKDKSP WKLLSKAQEKFGKNKSR

No. of aa

Net charge

49 25 17 17

+9 +8 +2 +5

that a serine protease activity has been detected in the N-terminal lobe of Lf that could play a role in its antibacterial activity against Hemophilus spp. K73 and S259 of hLf are essential residues for the protease activity (Hendrixson et al. 2003).

5.2. Structure of Lfcin Antimicrobial peptides encompass a wide variety of structures, of which many have α-helical motifs. The majority of these peptides are cationic and amphipathic, but there are also hydrophobic αhelical peptides that possess antimicrobial activity, and there are antimicrobial peptides that are rich in a certain specific amino acid such as Trp or His (Chan et al. 2006). In spite of the structural diversity, a common feature of the cationic antimicrobial peptides is that they all have an amphipathic structure that allows them to bind to the membrane interface. Limited proteolysis of Lf by trypsin leads to the release of the N-t and C-t lobes and the N-2 domain (Legrand et al. 1984), while hydrolysis of Lf by pepsin yields the Lfcin peptide (Bellamy et al. 1992). Bovine Lfcin (LfcinB) has been investigated most extensively since it is the most active Lfcin found across various species and also the most readily available (reviewed in Gifford et al. 2005). The Lfcin peptides from different species display drastic sequence differences but still share some similarities. LfcinB encompasses 25 residues, including 2 Trp residues at positions 6 and 8 and a disulfide bond between its 2 cysteine residues (Table 13.1; Figure 13.3). The structure of LfcinB in aqueous solution was solved by homonuclear NMR and shows that the peptide adopts a short, twisted, antiparallel β-sheet conformation (Figure 13.3) (Hwang et al. 1998).

Figure 13.3. Structure of LfcinB. The figure shows the ribbon foldings of (A) the LfcinB domain in bLf (residues 17–41) as determined by x-ray diffraction (pdb reference 1BLF) (Moore et al. 1997), and (B) the LfcinB 25-amino-acid peptide in aqueous solution as determined by NMR spectroscopy (pdb reference 1LFC) (Hwang et al. 1998). The positions of the positively charged amino acid residues are indicated, as well the positions of the Trp residues 19 and 21 in bLf. Molecular modeling was performed using the Chimera software (http:// www.cgl.ucsf.edu/chimera/).

The β-sheet gives the peptide an amphipathic structure, with most of the hydrophobic residues residing on one side of the β-sheet plane, while the majority of the hydrophilic residues rest on the other side. The LfcinB structure in solution at low salt concentration contrasts the conformation of the peptide in the Lf (Figure 13.3). In the crystal structure, an

Chapter 13 Secreted Lactoferrin and Lactoferrin-Related Peptides

α-helix stretches over half the peptide, whereas a β-sheet is present in solution (Hwang et al. 1998) (Figure 13.3). The peptide therefore undergoes a major conformational change when it is cleaved from Lf. The transition from helix to sheet has been modeled by molecular dynamics simulations (Zhou et al. 2004). Human Lfcin (LfcinH) is significantly larger than the bovine peptide, consisting of 49 amino acids and possessing a similar loop to LfcinB that is constrained by a disulfide bond (Table 13.1). Its peptide sequence was determined by NMR, Edman degradation, and mass spectrometry analysis, following pepsin digestion of hLf from milk (Hunter et al. 2005). Originally, another preparation yielded a slightly different product that contained two separate fragments held together by an additional disulfide bond (Bellamy et al. 1992). The structure of LfcinH exhibits a partially folded structure in water and is well defined in a more hydrophobic solvent, where it adopts an amphipathic, partially helical structure (Hunter et al. 2005). This, unlike LfcinB, is similar to the conformation seen in the crystal structure of hLf. Short peptides (15, 11, and 5 amino acids) derived from LfcinB and corresponding to sequences 17–31, 20–30, and 20–25, respectively, were shown to possess antimicrobial properties similar to LfcinB (Rekdal et al. 1999; Schibli et al. 1999; Hunter et al. 2005; Nguyen et al. 2005).

5.3. Structure of Lfampin An antimicrobial 17 amino acid peptide sequence named LfampinB was identified in the N-terminal lobe of bLf at residues 268–284 (van der Kraan et al. 2004) (Table 13.1; Figure 13.2). Following the initial discovery of LfampinB, a slightly longer sequence was published that included an additional N-terminal helix cap region, comprised of Asp-LeuIle. It was found that the longer peptide (cap-LfampinB) had a higher activity than the regular LfampinB peptide (van der Kraan, Nazmi et al. 2005). This increased activity was attributed to an increased stability of the α-helix at the N-terminus of the peptide (van der Kraan et al. 2006). Unlike Lfcin, Lfampin

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was never evidenced as a free peptide in secretions, although its release from the whole protein was demonstrated using a one-step proteolysis protocol with AsnP protease from Pseudomonas fragi (Bolscher et al. 2006) . Recently, human Lfampin (LfampinH) peptides corresponding to residues 269–285 in hLf were synthesized and characterized using NMR spectroscopy, fluorescence, and differential scanning calorimetry to determine which residues were important for the antimicrobial activity against Candida albicans (Haney et al. 2009). The authors concluded that the amphipathic N-terminal α-helix appears to be responsible for mediating the interaction between the peptide and the phospholipid bilayer, but the antimicrobial activity of the LfampinH peptides is dictated by the net cationic charge of the flexible C-terminal region.

6. Biological Roles of Lf in Host Defense 6.1. Antimicrobial Activities of Lf Lf plays a crucial role in the innate immunity against a broad range of pathogens: bacteria, viruses, fungi, and parasites (reviewed in Valenti and Antonini 2005; Jenssen and Hancock 2009). In the case of bacteria, Lf affects many Gram+ and Gram− pathogens including E. coli spp., Haemophilus influenzae, Salmonella typhimurium, Shigella dysenteriae, Listeria monocytogenes, Streptococcus spp., Vibrio cholerae, Legionella pneumophila, Enterococcus spp., Staphylococcus spp., Bacillus stearothermophilus, and Bacillus subtilis. However, it may promote the growth of beneficial bacteria like Lactobacillus and Bifidobacteria (Sherman et al. 2004). Concerning viruses, Lf has effects against the Friend virus complex, cytomegalovirus (CMV), polyomavirus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), hepatitis B and C (HBV and HCV) viruses, rotaviruses, and adenovirus. Finally, Lf exhibits antifungal activity against many Candida spp., more particularly C. albicans, and antiparasitic activities against Entamoeba histolytica, Tritrichomonas foetus, Trypanosoma cruzi, T. brucei,

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Plasmodium falciparum, T. gondii, and Eimeria stiedai. It is now clear that all these antimicrobial activities are supported by different mechanisms. Because Lf is a strong and stable iron chelator (Mazurier and Spik 1980), it may compete with the iron acquisition systems of pathogens. Indeed, Lf competes efficiently with most of the bacterial siderophores and can limit the growth of a broad range of bacteria. However, this iron limitation effect was not observed with fungi such as Candida spp. A role for the iron-binding ability of Lf on the adhesive properties of bacteria and the formation of biofilms during respiratory and oral infections was also proposed (Singh 2004). It is, however, interesting to note that Neisseriaceae, possibly Helicobacter pylori, and parasites like T. foetus may use Lf as a source of iron for their growth (reviewed in Ling and Schryvers 2006). Finally, Lf could destabilize the cell wall of Gram− bacteria by chelating Ca2+ and Mg2+ (Ellison et al. 1990). Although iron chelation was initially identified in vitro as the major mechanism for Lf bactericidal activity, it is now well established that this activity is mainly due to a direct binding of Lf and Lfderived peptides to microbes, thus leading to the destabilization of the membranes of a broad spectrum of Gram− and Gram+ bacteria (reviewed in Ling and Schryvers 2006) and fungi (Candida) (Xu et al. 1999). In Gram− bacteria, these negative charges may consist of the LPS themselves (see later), and it was shown that the negatively charged external loops of porins, such as E. coli OmpC and PhoE, may bind Lf (Sallmann et al. 1999). Another important effect of Lf binding to microbes is the modification of the interactions of microbes between themselves, with the host cells or with the extracellular matrix. This results in modified motility and aggregation of microbes and decreased endocytosis into host cells. As regards bacteria, experiments on Shigella flexneri (Gomez et al. 2003) and H. felis (Dial and Lichtenberger 2002) have shown that the glycans of Lf may bind to bacterial adhesins. It is now admitted that Lf may significantly limit the formation of biofilms and thus ensure the protection of mucosa, as shown with several E. coli strains (Kawasaki et al. 2000).

Concerning viruses, direct binding by Lf to HSV (Marchetti et al. 1996), HIV (Swart et al. 1996), hepatitis C virus (Yi et al. 1997), adenovirus (Pietrantoni et al. 2003), and rotavirus (Superti et al. 1997) particles was evidenced and connected, at least in part, with decreased infection of host cells by Lf. Recently, a serine-type endopeptidase activity, although much lower than that of trypsin, was evidenced for Lf. This proteolytic activity could be used to degrade virulence factors from H. influenzae (Qiu et al. 1998), S. flexneri (Gomez et al. 2003), enteropathogenic E. coli, and Actinobacillus actinomycetemcomitans (Ochoa et al. 2003). Besides its direct binding to microbes, Lf binds to most mammal cells and thus protects the host against infections, particularly against viral infections. Viruses indeed use common co-receptors at the surface of host cells, namely the glycomaninoglycans (GAGs) and integrins. Because Lf can bind to GAGs (Damiens, El Yazidi et al. 1998), it would be able to interfere with virus and other microbes and to prevent their internalization into cells. Such interference could account for the decreased infectivity of HBV on cells preincubated with Lf (Hara et al. 2002), a result that designates Lf as a possible candidate for the treatment of chronic hepatitis. Furthermore, it was demonstrated that Lf interferes with the binding of HSV glycoprotein C to heparan sulphate (HS) and/or chondroitin sulphate (CS) (Hasegawa et al. 1994). Lf may also compete with adenovirus that binds to HS-GAGs (Di Biase et al. 2003). Although the cationic N-terminus of Lf was reported as the major GAG-binding region of Lf (Mann et al. 1994; Legrand et al. 1997) and is important for its antiadenovirus activity, both lobes seem important for the anti-HSV activity (Siciliano et al. 1999), suggesting other mechanisms than a simple competition between Lf and the particles for GAG binding. Interestingly, Lf also binds to three of the many co-receptors of HIV, namely GAGs, surface nucleolin (Legrand et al. 2004), and the DCSIGN receptor (Groot et al. 2005). The interaction of Lf with surface nucleolin was shown to block the attachment and entry of HIV particles into HeLa P4 cells (Legrand et al. 2004). This interaction, together

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with Lf binding to proteoglycans and to the V3-loop of gp120 on viral particles (Swart et al. 1996), probably contributes to the anti-HIV activity of Lf. Concerning bacterial infections, Lf bound to the surface of epithelial cells would be able to inhibit the adherence of bacteria and the formation of biofilms. While many experiments showed no beneficial effect of Lf incubated with host cells prior to infection, some demonstrated the inhibition of enteroinvasive E. coli HB101 entry into cultured cells by bLf (Longhi et al. 1993). In the case of fungi, the effect of Lf on Candida spp. has been thoroughly studied and often related to Lf adsorption onto fungi and cell wall destabilization (Xu et al. 1999). Concerning parasites, Lf adsorption on host cells has been related to the enhancement of T. cruzi phagocytosis and lysis by macrophages (Lima and Kierszenbaum 1987) and also to the inhibition of the intracellular growth of P. falciparum in erythrocytes (Fritsch et al. 1987). It was also demonstrated that Lf might limit the infection of fibroblasts by Plasmodium, probably through its binding to HS-GAGs and to the LRP, which is recognized by the CS protein of Plasmodium (Shakibaei and Frevert 1996). Finally, adsorption of Lf on host cells might involve many more active processes than a simple competition for binding to microbial receptors. Thus, Lf may be able to activate immune cells through nuclear targeting and/or activation pathways. For example, in the case of cytomegalovirus infection, an increased activity of NK cells has been reported (Shimizu et al. 1996). Furthermore, a recent study suggests that the antimycotic activity of Lf is related to an activation of the immune system (Yamaguchi et al. 2004).

6.2. Anti-inflammatory Properties of Lf Lf is a potent modulator of inflammatory and immune responses, revealing host-protective effects, not only against microbial infections but also in inflammatory disorders including allergy, arthritis, and cancer (Legrand et al. 2005, 2008; Ward et al. 2005; Yamauchi et al. 2006). The up- or downregulating effects of Lf are related to its ability to interact

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with pro-inflammatory bacterial components, mainly LPS (Appelmelk et al. 1994; Elass-Rochard et al. 1995), and specific cellular receptors on a wide range of epithelial and immune cells (reviewed in Suzuki et al. 2005). This results in the modulation of the production of various cytokines and of the recruitment of immune cells at the infected sites. Lf may limit the inflammation associated with various bacterial infections, thus preventing septic shock. In particular, orally administred bLf has a beneficial effect on infections and protects animals against a lethal dose of LPS (Zagulski et al. 1989; Dial et al. 2005). Additionally, Lf plays anti-inflammatory roles in noninfectious pathologies such as rheumatoid arthritis, inflammatory bowel disorders, neurodegenerative diseases, and skin allergy. It has been shown that administration of Lf protects against chemically induced cutaneous inflammation (Cumberbatch et al. 2003) and nonsteroidal anti-inflammatory drug-induced intestinal injury (Dial et al. 2005). In collagen-induced and septic arthritis mouse models, peri-articular injection of hLf reduced inflammation (Guillen et al. 2000). The protection against bacterial infections is well understood and is explained by the ability of Lf to enhance the secretion of anti-inflammatory cytokines such as IL-10 and to inhibit the production of several pro-inflammatory cytokines, mainly tumor necrosis factor TNF-α, IL-1β, IL-6, and IL-8 (reviewed in Legrand et al. 2005, 2008). It is now admitted that these effects are mostly mediated by the neutralization by Lf of pro-inflammatory microbial molecules such as LPS and bacterial unmethylated CpG-containing oligonucleotides (Britigan et al. 2001). Lf, through its Lfcin domain, binds to the lipid A of LPS (Appelmelk et al. 1994). The sequestration of LPS by Lf prevents the interaction of endotoxin with the LPS-binding protein (LBP), therefore preventing LPS binding to membrane CD14 (mCD14) (Elass-Rochard et al. 1998) and its further presentation to the cell-signaling Toll-like receptor 4 (TLR4). Furthermore, other mechanisms may account for the inhibition of LPS-induced cytokine release by Lf. High-affinity interactions were indeed evidenced between the cationic N-terminal

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lobe of Lf and soluble CD14 (sCD14), either in a free form or complexed with LPS (Baveye, Elass, Fernig et al. 2000). Through this mechanism, Lf inhibits the secretion of IL-8 by endothelial cells (Elass et al. 2002). Interestingly, it should be observed that following pretreatment with the Lf-LPS complex, cells are rendered tolerant to LPS challenge (Na et al. 2004). The presence of specific receptors on immune cells suggests that the modulation of host responses by Lf may be related to a direct effect via receptor-mediated signaling pathways. Indeed, Lf downregulates IL-6 secretion in monocytes through a mechanism involving Lf translocation to the nucleus and inhibition of NF-κB activation (Haversen et al. 2002). The protective roles of Lf in noninfectious pathologies are not clearly elucidated. However, as demonstrated in skin allergy (Cumberbatch et al. 2003) and in adjuvant-stimulated arthritis in rats (Hayashida et al. 2004), a decrease in proinflammatory cytokines, particularly TNF-α and IL1-β and an increase in anti-inflammatory cytokines, including IL-10, were observed. In skin and lung allergies, Lf could be internalized into mast cells and interact with tryptase, chymase, and cathepsin G, three potent inflammatory proteases (He et al. 2003). Moreover, anti-IgE induced histamine release from colon mast cells can be inhibited by Lf. During inflammation, reactive oxygen species (ROS) are overproduced by activated granulocytes and their synthesis can be catalyzed in the presence of iron. Thus, the chelation of iron by apoLf may prevent the formation of hydroxyl radicals and subsequent lipid peroxidation, particularly in patients with chronic hepatitis (Konishi et al. 2006). Lastly, during neurodegenerative diseases, transcytosis of plasma Lf through the blood-brain barrier may contribute to limiting iron deposits and oxidative stress in the brain (Fillebeen et al. 1999). Cell migration is critical for a variety of biological processes. Recently, during influenza virus infection (pneumonia), bLf was shown to reduce the number of infiltrating leukocytes in bronchoalveolar lavage fluid, thus suppressing the hyperreaction of the host (Yamauchi et al. 2006). Although the exact mechanism of action of Lf in pneumonia is not

defined, it has been demonstrated in vitro that Lf may modulate the activation of human umbilical endothelial cells by LPS and the expression of the adhesion molecules E-selectin and ICAM-1 (Baveye, Elass, Mazurier et al. 2000). Lf also decreases the recruitment of eosinophils, reduces pollen antigeninduced allergic airway inflammation in a murine model of asthma (Kruzel et al. 2006), and reduces migration of Langherans cells in cutaneous inflammation (Griffiths et al. 2001). Finally, Lf can modulate fibroblast motility by regulating MMP-1 gene expression, a matrix metalloproteinase involved in the extracellular matrix turnover and the promotion of cell migration (Oh et al. 2001).

6.3. Pro-inflammatory Properties of Lf Lf displays immunological properties influencing both innate and acquired immunities. In particular, oral administration of bLf seems to influence mucosal and systemic immune responses in mice (Sfeir et al. 2004). It was also recently demonstrated that a complex of Lf with monophoshoryl lipid A is an efficient adjuvant of the humoral and cellular immune responses (Chodaczek et al. 2006). Its stimulating effect on the immune system concerns mainly the maturation and differentiation of T-lymphocytes, the Th1/Th2 cytokine balance (Fischer et al. 2006), and the activation of phagocytes. Very recently, it has been shown that Lf acts as an alarmin to promote the recruitment and activation of antigen-presenting cells and antigen-specific immune responses (de la Rosa et al. 2008). It was also reported as a novel maturation factor for dendritic cells (Spadaro et al. 2008). Under nonpathogenic conditions, Lf is able to stimulate the differentiation of T cells from their immature precursors through the induction of CD4 antigen (Dhennin-Duthille et al. 2000). A similar effect was described on isolated thymocytes incubated overnight with Lf. Furthermore, oral delivery of Lf significantly increased the number of CD4positive cells in lymphoid tissus (Kuhara et al. 2000). Lf induces a Th1 polarization in diseases in which the ability to control infection or tumor relies on a strong response, but may also reduce Th1 cyto-

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kines to limit excessive inflammatory responses, as previously described. Thus, Lf enhances both the ability of Th cells to assist the fungicidal actions of macrophages (Wakabayashi et al. 2003) and BCG vaccine efficacity against challenge with mycobacterium tuberculosis (Hwang et al. 2005, 2009). An upregulation of the Th1 response was associated with S. aureus clearance in Lf transgenic mice (Guillen et al. 2002). Oral administration of Lf increased the splenocyte production of IFN-γ and Il-12 in response to herpes simplex virus type 1 infection (Wakabayashi et al. 2004). Furthermore, the eradication of chronic hepatitis C virus by IFN therapy is enhanced by administration of bLf that induces a Th-1 cytokine dominant environment in peripheral blood (Ishii et al. 2003). The proposed mechanism is that oral Lf induces IL-18 production in the small intestine, therefore leading to an increase in the level of Th1 cells. Furthermore, it has been shown that bLf may promote systemic host immunity by activating the transcription in the small intestine of important genes such as IL-12p40, IFN-β, and NOD2 (Yamauchi et al. 2006). During infection, Lf secreted from neutrophil granules can bind to PMNs and monocytes/ macrophages (Gahr et al. 1991) and promotes the secretion of inflammatory molecules such as TNF-α (Sorimachi et al. 1997). A recent study has shown that Lf may activate macrophages via TLR4dependent and independent signaling pathways (Curran et al. 2006). This activation induces CD40 expression and IL-6 secretion. The binding of Lf to macrophages also enhances the phagocytosis of pathogens, as demonstrated during the infection of bovine mammary gland by S. aureus (Kai et al. 2002). Lf may also influence myelopoiesis by modulating granulocyte-macrophage colony stimulating factor (GM-CSF) production (Sawatzki and Rich 1989).

7. Biological Roles of Lf in Cancer and Cell Proliferation Although the current views on the physiological role of Lf are dominated by its activity as an antimicrobial agent and immune modulator, Lf also directly

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affects cell growth and acts as a regulatory element in defense against tumorigenesis and metastasis. Its antitumoral activities have long been suggested since numerous in vivo studies showed that bLf significantly reduces chemically induced tumorigenesis when administered orally to rodents (reviewed in Tsuda et al. 2002, 2004). Both bLf and recombinant hLf (rhLf) also possess antimetastatic effects, and injection or ingestion of bLf inhibits the growth of transplanted tumors and prevents experimental metastasis in rodents (Bezault et al. 1994; Iigo et al. 1999; Varadhachary et al. 2004). The spliced isoform of Lf (ΔLf) also possesses antitumor activities (Breton et al. 2004). Concomitant administration of bLf and carcinogens to rodents inhibits the induction of activating enzymes for carcinogenic heterocyclic amines, modulates lipid peroxidation, and activates antioxidant and carcinogen detoxification enzyme activities, blocking cancer development (Tsuda et al. 2002; Chandra Mohan, Kumaraguruparan et al. 2006). Lf promotes NK cell cytotoxicity, as shown by in vitro and in vivo studies (Bezault et al. 1994; Sekine et al. 1997; Damiens, Mazurier et al. 1998). In animal models of carcinogenesis, a marked increase in the number of NK and CD8+, CD4+, and IFNγ positive cells was observed in the mucosal layer of the small intestine, as well as in the peripheral cell population of bLf-treated rodents (Iigo et al. 1999). Oral administration of rhLf to tumorbearing mice also led to an enhancement of both local mucosal and systemic immune responses (Varadhachary et al. 2004). Recently, a mechanism involving the enhancement by Lf of caspase-1 activity followed by a transient increase in the active form of IL-18 has been reported to elevate both mucosal and systemic immune responses via the regulation of cytokine production, the mechanism of which remains to be elucidated (Iigo et al. 2004; Varadhachary et al. 2004; Kuhara et al. 2006). Moreover, IL-18, as an antiangiogenic compound, might also be partially responsible for bLf inhibition of angiogenesis (Shimamura et al. 2004). Curiously, bLf and hLf exert opposite effects on angiogenesis. Whereas

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orally administrated bLf inhibits angiogenesis in rats (Norrby et al. 2001) and tumor-induced angiogenesis in mice (Shimamura et al. 2004), hLf exerts a specific pro-angiogenic effect (Norrby 2004). Recently, Lf was shown to stimulate in vivo angiogenesis via the upregulation of the VEGF receptor KDR/Flk-1 subsequently promoting VEGF-induced proliferation and migration of endothelial cells (Kim, Son et al. 2006). Since tumor growth is angiogenesis-dependent, the extensive therapeutic potential warrants further studies to elucidate the contradictory effects of Lf on angiogenesis. In contrast to IL-18, some cytokines such as IL-8, IL-6, and IL-1β are potent angiogenic stimulators and Lf action in vivo might therefore partially involve suppression of the production of angiogenic cytokines (Haversen et al. 2002). Lf is able to promote apoptosis in cancerous cells. Bovine Lf (bLf) changed apoptosis-related gene expression in colon mucosa (Fujita et al. 2004a) and in chemically induced lung proliferative lesions (Matsuda et al. 2007). Thus, activation of caspases (3 and 8), death inducing receptor (Fas), and pro-apoptotic Bcl-2 members (Bid, Bax) has been reported (Fujita et al. 2004b; Chandra Mohan, Devaraj et al. 2006). Caspase-3 plays a central role in various apoptosis cascades and, recently, Lf has been shown to enhance procaspase-3 maturation in vitro. The Y679–K695 domain of Lf, which binds procaspase-3, is able as a synthetic peptide to compete with Lf and block procaspase-3 processing (Katunuma et al. 2006). This result is further strengthened by in vivo data since in D-galactosamineinduced apoptotic rat liver, Lf was shown to translocate from lysosomes into the cytoplasm of hepatocytes by an unknown mechanism and to lead to procaspase-3 activation (Katunuma et al. 2006). Other reports indicate that Lf could either promote or inhibit apoptosis in a dose-dependent manner. In vitro studies on PC12 cells showed that high doses of Lf lead to activation of caspases-3 and -8 and to a decrease in the protein expression of phosphorylated ERK1/2 and Bcl-2, whereas low doses upregulate phosphorylated ERK1/2 and Bcl-2 expression and protect cells from FasL-induced apoptosis (Lin et al. 2005; Lee et al. 2009). Lf may also indirectly

interfere with apoptosis by inhibiting retinoid signaling pathways since translocation of exogeneous Lf to the nucleus in the presence of retinoids leads to the alteration of retinoid-induced gene transactivation in mammary cells in vitro (Baumrucker et al. 2006). Relationships between Lf and retinoids are complex, since the Lf gene promoter contains a retinoid response element (Teng 2006), and require further investigation. Lf also limits the growth of tumor cells by inducing cell cycle arrest at the G1/S transition. In the MDA-MB-231 breast cancer cell line, the molecular mechanism underlying this G1 arrest associates both inhibition of cdk2 and cdk4 activities and an increase in cdk inhibitor (CKI) p21 expression, involving the MAPK pathway (Damiens et al. 1999). In head and neck cancer cells, Lf downregulation of cell growth is mediated through the p27/cyclin E-dependent pathway involving changes in the phosphorylation status of AKT (Xiao et al. 2004). The NF-κB signaling pathway has been implicated in HeLa cervical carcinoma cells, and Lf was shown to upregulate the tumor suppressor protein p53 and its target genes mdm2 and p21 (Oh et al. 2004). Recently, retinoblastoma protein (Rb)-mediated growth arrest has been demonstrated. Thus, Lf induces overexpression of Rb in tumor cell lines. A majority of Rb then remains as a hypophosphorylated isoform, and this form binds to E2F1, downregulating its transcriptional activity and leading therefore to cell cycle arrest (Son et al. 2006). In addition, Lf-induced overexpression of the CKI protein p21 was also observed and appears to occur independently of p53, whereas the expression of p27, another member of the Kip/Cip group of CKI, was not altered by Lf (Son et al. 2006). Expression of cytoplasmic ΔLf also leads to cell cycle arrest (Breton et al. 2004) and upregulation of both Rb and Skp1 (S-phase kinase associated protein) genes (Mariller et al. 2007). Since Skp1 belongs to the SCF (Skp1/Cullin-1/F-box ubiquitin ligase) complex responsible for the ubiquitinylation of proteins leading to their degradation by the proteasome at the G1/S transition, ΔLf may therefore indirectly modulate the half-life of cell cycle actors. δLf acts as a transactivating factor via a direct in situ

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interaction with two Lf response elements detected in both Skp1 and Rb promoters (Mariller et al. 2007). The GGCACTTGC sequence was also found to be functional in the IL-1β promoter and shown to be responsible for IL-1β transactivation by Lf in vitro (Son et al. 2002). Very recently, the gene of DcpS, an mRNA scavenger decapping enzyme, was identified as a ΔLf target gene (Mariller et al. 2009). Further studies will be necessary to confirm whether Lf induces in situ transcription of genes and, if so, to determine whether both Lf isoforms target the same genes. Since Lf isoforms exert cancer suppressive effects, they may act as tumor supressor proteins, the expression of which would be suppressed upon carcinogenesis, and their gene downregulated in cancerous cells. Lf expression has been found to be decreased or absent in numerous cancers (reviewed in Ward et al. 2005). Downregulation is due to structural alterations such as mutations, allelic loss of part of chromosome 3, and modification of the degree as well as the pattern of methylation (Teng et al. 2004; Iijima et al. 2006). This genetic and epigenetic inactivation of the Lf gene in cancer may therefore provide the tumor cells with a selective growth advantage. ΔLf expression was observed in normal tissues but was downregulated in their malignant counterparts (Siebert and Huang 1997; Liu et al. 2003; Benaïssa et al. 2005). Interestingly, it was shown that the expression level of either Lf or ΔLf mRNAs was of good prognosis value in human breast cancer with high concentrations being associated with longer relapse-free and overall survival, suggesting the usefulness of the detection of either Lf or ΔLf transcripts as markers for the follow-up of breast cancer patients (Benaïssa et al. 2005). Lf, which was also identified as a cancer-specific marker of endocervical adenocarcinomas, may also be useful for the early detection of disease and for prognosis (Farley et al. 1997). Recently, Lf has been shown to stimulate osteoblast proliferation in vitro and to exert an anabolic effect promoting bone formation in vivo (Cornish et al. 2006). It also limits bone resorption by inhibiting osteoclastogenesis (Lorget et al. 2002; Cornish

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et al. 2006). Taken together these data suggest that Lf has a physiological role in bone growth and might therefore act as a potential therapeutic agent in osteoporosis.

8. Biological Properties of the Lf-Related Peptides The evidence that the surface properties of Lf account for many of its biological activities led to isolate, synthesize, and characterize the peptides responsible for these activities. Most studies were performed on bLf, which represents a cheap potential source of biologically active peptides. They focused on two particular domains: the Lfcin and Lfampin domains.

8.1. Biological Properties and Mechanisms of Action of Lfcin The Lfcin peptide, when released by proteolysis from the intact protein, is a potent bactericidal agent, probably because it is able to form amphipathic structures that disrupt cell membranes. It is also a potent antifungal, antiparasitidal, antiviral, antitumor, and immunomodulatory agent (reviewed in Gifford et al. 2005; Chan et al. 2006). Both LfcinB and LfcinH have a broad range of action against many bacteria, either Gram+ (Bacillus, Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Listeria, Streptococcus, and Staphylococcus spp.) or Gram− (E. coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas fluorescens, Yersinia enterocolitica, and Salmonella spp.) (reviewed in Chan et al. 2006). The cationic character of the peptides and the negatively charged membranes of bacteria (peptidoglycan, LPS, teichoic acid) provide an ideal interaction to attract the peptides to these cells. Although electrostatic forces are important for selectivity and the interactions of peptides and membranes, Lfcins probably don’t disrupt the membranes. However, studies have shown that negatively charged liposomes display subtle changes upon LfcinB binding (Vogel et al. 2002). Both LfcinH

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and bLfcinB are capable of binding LPS, indicating that they do interact with the outer membrane at least in Gram− bacteria. In fact, an 11-residue peptide of LfcinH has been shown to form ordered aggregates when LPS is present, which could create a large amphipathic structure that has an increased ability to disrupt the outer membrane and through this membrane damaging effect, permit individual peptides to move across (Chapple et al. 2004). Therefore, Lfcins could act on intracellular targets, which interferes with the normal functioning of microorganisms. LfcinB transmission electron microscopy data shows that the peptide indeed moves into the cytoplasm of both S. aureus and E. coli (Shin et al. 1998; Haukland et al. 2001). Furthermore, the observation that β-sheet peptides translocate across the cytoplasmic membrane better than α-helical or extended peptides (Vorland et al. 1998) possibly explains the increased activity of LfcinB compared with LfcinH. The two Trp residues (Figure 13.3), which anchor the peptide in the membrane, are particularly important in LfcinB. Mutation of either of these residues indeed results in a complete loss of antimicrobial activity (Strom et al. 2000). This pattern is also seen in a number of other peptides such as buforin II and penetratin that have short regions responsible for their spontaneous transfer across the plasma membrane (Joliot and Prochiantz 2004). These peptides are known as cellpermeable peptides and interact with the membrane via electrostatic and/or hydrophobic interactions after which they may form pores or inverted micelles to shuttle inside the cell (Joliot and Prochiantz 2004). Similar to antimicrobial peptides, Arg residues and cation-π interactions play a crucial role in the interactions of cell-permeable peptides with membranes (Lensink 2008). Once inside the cell, LfcinB would be capable of binding and affecting DNA and RNA synthesis in both Gram-negative and Gram-positive bacteria (Ulvatne et al. 2004). The observation that Lfcins act intracellulary explains how these peptides can have such a broad range of target organisms. As long as an initial favorable electrostatic interaction takes place between the cationic peptide and anionic cells of bacteria, fungi, or even tumor cells, the peptide will be able to traverse

the membrane and move inside, where it performs its activity (Chan et al. 2006). In addition to bacteria, Lfcin is effective at inhibiting the growth of a number of yeasts, dermatophytes, and filamentous fungi, including the pathogen C. albicans (Bellamy et al. 1993; Wakabayashi et al. 1998; Lupetti et al. 2000). The results suggest that Lfcin has two antifungal mechanisms, including a fungicidal activity. Both Lfcins appear to bind fungal cells and to affect the cytoplasm. Aggregation of cytoplasmic material and secretion of ATP from mitochondria have been observed with Lfcin-exposed cells. Interestingly, this ATP is released extracellularly, where it can interact with extracellular ATP binding sites on the plasma membrane, giving rise to pore formation and cell death (Lupetti et al. 2000; Ueta et al. 2001). The second antifungal action of Lfcin appears to involve upregulating host defense through the Candida cellkilling activity of neutrophils. It was indeed shown to induce the generation of reactive oxygen species and the generation of Lf and defensins from neutrophils (Lupetti et al. 2000; Tanida et al. 2001; Ueta et al. 2001). Although Lfcin is parasiticidal against protozoans such as Eimeria stieda, Giardia lamblia, and Toxoplasma gondii, not much is known about how Lfcin exerts this effect (Tanaka et al. 1995; Turchany et al. 1995; Omata et al. 2001). It is possible that the interaction of Lfcin with parasites releases structural components that subsequently activate host defense systems. Lfcin also plays antiviral activity against adenovirus, feline calcivirus, HSV, cytomegalovirus, and HIV, although with a much lower efficiency, suggesting that either the size of the molecule is important or that other regions of Lf contribute to the antiviral activity (Siciliano et al. 1999; Andersen et al. 2001; Berkhout et al. 2002; Di Biase et al. 2003; McCann et al. 2003; Jenssen et al. 2008; Marr et al. 2009). Lfcin, like Lf, has been shown to have antitumor effects against a number of cell lines including leukemic (Yoo, Watanabe, Koike et al. 1997; Yoo, Watanabe, Watanabe et al. 1997; Onishi et al. 2008), fibrosarcomas, melanomas, and colon carcinomas

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(Eliassen et al. 2002), at a concentration that does not affect normal fibroblasts and erythrocytes. Lfcin binds to the tumor cell and is thought to disrupt the cell membrane and to trigger apoptosis through an oxidant-dependent pathway (Eliassen et al. 2002). Interestingly, the structural parameters that describe the antitumor effects of Lfcin are very similar to those that describe the antibacterial activity, that is, a high net positive charge (Yang et al. 2004) and an amphipathic structure (Yang et al. 2003).

8.2. Biological Properties and Mechanisms of Action of Lfampin A recent study has shown that Lfampin can be released from Lf using a one-step proteolysis protocol with the AsnP protease from Pseudomonas fragi (Bolscher et al. 2006). However, unlike Lfcin which can be released by proteolysis of Lf under physiological conditions in the digestive tract, Lfampin is not a “natural” peptide and its presence in secretion fluids has never been demonstrated. Interestingly, the synthetic LfampinB peptide exhibited activity against pathogenic yeast C. albicans, which was substantially higher than the activity of Lf. Furthermore, LfampinB was active against the Gram+ and Gram− bacteria B. subtilis, E. coli, and P. aeruginosa, but not against the fermenting bacteria Actinomyces naeslundii, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus sanguis (van der Kraan et al. 2004; van der Kraan, Nazmi et al. 2005). As described above, studies performed either on LfampinB or LfampinH demonstrated the importance of an increased stability of the α-helix at the N-terminus of the peptide and also the importance of the net cationic charge of the flexible C-terminal region (van der Kraan et al. 2006; Haney et al. 2009). Recently, the solution structure of LfampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles, used as model membranes, was determined using 2D NMR spectroscopy (Haney et al. 2007). LfampinB adopts an amphipathic α-

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helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11 and is seen in both micelle-bound structures. Both residues seem to serve as anchors for the lipid bilayer. These results support a two-step model of antimicrobial activity, where the initial attraction of LfampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix, which binds to the surface of the microbial lipid bilayer (Haney et al. 2007).

9. Applications of Lf and Related Peptides The large-scale preparation of bLf from cheese whey or skim milk (up to 100 metric tons per year) and of recombinant hLf produced in microorganisms and plants makes Lf available for human and animal (fish farming) health purposes and commercial applications. The first major application of bLf was the supplementation of infant formulas, but it is now added to cosmetics, pet care supplements, and immune-enhancing nutraceuticals, including drinks, fermented milks, and chewing gums (reviewed in Tomita et al. 2009). In all these media, Lf is expected to exert its natural antimicrobial, antioxidative, anti-inflammatory, anticancerous, and immunomodulatory properties. Furthermore, clinical trials demonstrated the efficiency of Lf against infections and in inflammatory diseases. For example, a recent clinical study concluded that the combination of Lf and fluconazole at the threshold minimal inhibitory concentrations elicited potent synergism, leading to total fungistasis of C. albicans and C. glabrata vaginal pathogens (Naidu et al. 2004). Lf was also reported as a potent molecule in the treatment of common inflammatory diseases (reviewed in Legrand et al. 2005) and iron deficiency in pregnant women (Paesano et al. 2009). As regards the anticancerous activity of Lf, extensive clinical trials are underway in Japan to further explore its preventive potential against colon carcinogenesis (Tsuda et al. 2002).

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Lf also offers applications in food preservation and safety, either by retarding lipid oxidation (Medina et al. 2002) or by limiting the growth of microbes. For example, incorporation of Lf into edible films has a great potential to enhance the safety of foods since the film can function as a physical barrier, as well as an antimicrobial agent. Lf can also be directly used as a spray applied to beef carcasses (Taylor et al. 2004). Lastly, Lf can be used as a clinical marker of inflammatory diseases since Lf levels in blood and biological fluids may greatly increase in septicemia or during Severe Acute Respiratory Syndrome (Reghunathan et al. 2005). In the same way, fecal Lf levels quickly increase with the influx of leukocytes into the intestinal lumen during inflammation. Fecal Lf is thus used as a noninvasive diagnostic tool to evaluate the severity of intestinal inflammation in patients presenting abdominal pain and diarrhea (Greenberg et al. 2002). This biomarker has been shown to be a sensitive and specific marker of disease activity in chronic inflammatory bowel disease (Kane et al. 2003) and in Crohn’s disease (Buderus et al. 2004). The powerful antimicrobial activities of the Lfderived peptides, more particularly Lfcin and related peptides, make these molecules very promising tools for health applications or food applications (Enrique et al. 2008). Several studies report the possibility of producing these peptides on a large scale, either by chemical synthesis (Haug et al. 2007), by isolation from food grade bLf (Chan and Li-Chan 2007), or by recombinant technologies (Kim, Chun et al. 2006; Wang et al. 2007). Other studies aim to direct peptide expression in mammary gland to prevent mastitis (Zhang et al. 2007) or, in Streptococcus thermophilus, to enhance the nutritional value of dairy food products (Renye and Somkuti 2008). Very recently, Lfcin and Lfampin chimeras with higher bacterical activities than their constituent peptides have been designed (Bolscher et al. 2009). The recent application of these peptide chimera to piglet development (Tang et al. 2009) or against halophilic Vibrio parahaemolyticus (LeonSicairos et al. 2009) highlight them as very interesting molecules for health applications.

10. Conclusion When Lf was discovered in milk in the early sixties, it would have been difficult to imagine that it could exert so many biological activities. Its protective roles against pathogens, inflammation, and cancer make the molecule a centerpiece of the nonspecific immune system. The time has come for the development and application of promising health-enhancing nutraceuticals for food and pharmaceutical applications of this intriguing molecule and its related peptides.

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Legrand D, Pierce A, Elass E, Carpentier M, Mariller C, Mazurier J. 2008. Lactoferrin structure and functions. Adv Exp Med Biol 606(1):163–194. Legrand D, van Berkel PH, Salmon V, van Veen HA, Slomianny MC, Nuijens JH, Spik G. 1997. The N-terminal Arg2, Arg3 and Arg4 of human lactoferrin interact with sulphated molecules but not with the receptor present on Jurkat human lymphoblastic T-cells. Biochem J 327(Pt 3):841–846. Legrand D, Vigie K, Said EA, Elass E, Masson M, Slomianny MC, Carpentier M, Briand JP, Mazurier J, Hovanessian AG. 2004. Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells. Eur J Biochem 271(2):303–317. Lensink MF. 2008. Membrane-associated proteins and peptides. Methods Mol Biol 443:161–179. Leon-Sicairos N, Canizalez-Roman A, de la Garza M, ReyesLopez M, Zazueta-Beltran J, Nazmi K, Gomez-Gil B, Bolscher JG. 2009. Bactericidal effect of lactoferrin and lactoferrin chimera against halophilic Vibrio parahaemolyticus. Biochimie 91(1):133–140. Li YM, Tan AX, Vlassara H. 1995. Antibacterial activity of lysozyme and lactoferrin is inhibited by binding of advanced glycation-modified proteins to a conserved motif. Nat Med 1(10):1057–1061. Lima MF, Kierszenbaum F. 1987. Lactoferrin effects on the interaction of blood forms of Trypanosoma cruzi with mononuclear phagocytes. Intl J Parasitol 17(6):1205–1208. Lin TY, Chiou SH, Chen M, Kuo CD. 2005. Human lactoferrin exerts bi-directional actions on PC12 cell survival via ERK1/2 pathway. Biochem Biophys Res Commun 337(1):330–336. Ling JM, Schryvers AB. 2006. Perspectives on interactions between lactoferrin and bacteria. Biochem Cell Biol 84(3): 275–281. Liu D, Wang X, Zhang Z, Teng CT. 2003. An intronic alternative promoter of the human lactoferrin gene is activated by Ets. Biochem Biophys Res Commun 301(2):472–479. Longhi C, Conte MP, Seganti L, Polidoro M, Alfsen A, Valenti P. 1993. Influence of lactoferrin on the entry process of Escherichia coli HB101 (pRI203) in HeLa cells. Med Microbiol Immunol 182(1):25–35. Lorget F, Clough J, Oliveira M, Daury MC, Sabokbar A, Offord E. 2002. Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity. Biochem Biophys Res Commun 296(2):261–266. Lupetti A, Paulusma-Annema A, Welling MM, Senesi S, van Dissel JT, Nibbering PH. 2000. Candidacidal activities of human lactoferrin peptides derived from the N terminus. Antimicrob Agents Chemother 44(12):3257–3263. Mann DM, Romm E, Migliorini M. 1994. Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin. J Biol Chem 269(38):23661– 23667. Marchetti M, Longhi C, Conte MP, Pisani S, Valenti P, Seganti L. 1996. Lactoferrin inhibits herpes simplex virus type 1 adsorption to Vero cells. Antiviral Res 29(2–3):221–231.

Chapter 13 Secreted Lactoferrin and Lactoferrin-Related Peptides

Mariller C, Benaïssa M, Hardiville S, Breton M, Pradelle G, Mazurier J, Pierce A. 2007. Human delta-lactoferrin is a transcription factor that enhances Skp1 (S-phase kinase-associated protein) gene expression. FEBS J 274(8):2038–2053. Mariller C, Hardiville S, Hoedt E, Benaïssa M, Mazurier J, Pierce A. 2009. Proteomic approach to the identification of novel deltalactoferrin target genes: Characterization of DcpS, an mRNA scavenger decapping enzyme. Biochimie 91(1):109–122. Marr AK, Jenssen H, Moniri MR, Hancock RE, Pante N. 2009. Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1. Biochimie 91(1):160– 164. Masson PL, Heremans JF, Dive C. 1966. Studies of the proteins of secretions from two villous tumours of the rectum. Gastroenterologia 105(5):270–282. Masson PL, Heremans JF, Schonne E. 1969. Lactoferrin, an ironbinding protein in neutrophilic leukocytes. J Exp Med 130(3):643–658. Matsuda Y, Saoo K, Hosokawa K, Yamakawa K, Yokohira M, Zeng Y, Takeuchi H, Imaida K. 2007. Post-initiation chemopreventive effects of dietary bovine lactoferrin on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in female A/J mice. Cancer Lett 246(1–2):41– 46. Mazurier J, Spik G. 1980. Comparative study of the iron-binding properties of human transferrins. I. Complete and sequential iron saturation and desaturation of the lactotransferrin. Biochim Biophys Acta 629(2):399–408. McCann KB, Lee A, Wan J, Roginski H, Coventry MJ. 2003. The effect of bovine lactoferrin and lactoferricin B on the ability of feline calicivirus (a norovirus surrogate) and poliovirus to infect cell cultures. J Appl Microbiol 95(5):1026–1033. Medina I, Tombo I, Satue-Gracia MT, German JB, Frankel EN. 2002. Effects of natural phenolic compounds on the antioxidant activity of lactoferrin in liposomes and oil-in-water emulsions. J Agric Food Chem 50(8):2392–2399. Meilinger M, Haumer M, Szakmary KA, Steinbock F, Scheiber B, Goldenberg H, Huettinger M. 1995. Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor and transport to endosomes. FEBS Lett 360(1):70–74. Metz-Boutigue MH, Jolles J, Mazurier J, Schoentgen F, Legrand D, Spik G, Montreuil J, Jolles P. 1984. Human lactotransferrin: Amino acid sequence and structural comparisons with other transferrins. Eur J Biochem 145(3):659–676. Montreuil J, Tonnelat J, Mullet S. 1960. Preparation and properties of lactosiderophilin (lactotransferrin) of human milk. Biochim Biophys Acta 45(1):413–421. Moore SA, Anderson BF, Groom CR, Haridas M, Baker EN. 1997. Three-dimensional structure of diferric bovine lactoferrin at 2.8 A resolution. J Mol Biol 274(2):222–236. Na YJ, Han SB, Kang JS, Yoon YD, Park SK, Kim HM, Yang KH, Joe CO. 2004. Lactoferrin works as a new LPS-binding protein in inflammatory activation of macrophages. Intl Immunopharmacol 4(9):1187–1199.

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Naidu AS, Fowler RS, Martinez C, Chen J, Tulpinski J. 2004. Activated lactoferrin and fluconazole synergism against Candida albicans and Candida glabrata vaginal isolates. J Reprod Med 49(10):800–807. Nguyen LT, Schibli DJ, Vogel HJ. 2005. Structural studies and model membrane interactions of two peptides derived from bovine lactoferricin. J Pept Sci 11(7):379–389. Norrby K. 2004. Human apo-lactoferrin enhances angiogenesis mediated by vascular endothelial growth factor A in vivo. J Vasc Res 41(4):293–304. Norrby K, Mattsby-Baltzer I, Innocenti M, Tuneberg S. 2001. Orally administered bovine lactoferrin systemically inhibits VEGF(165)-mediated angiogenesis in the rat. Intl J Cancer 91(2):236–240. Ochoa TJ, Noguera-Obenza M, Ebel F, Guzman CA, Gomez HF, Cleary TG. 2003. Lactoferrin impairs type III secretory system function in enteropathogenic Escherichia coli. Infect Immun 71(9):5149–5155. Oh SM, Hahm DH, Kim IH, Choi SY. 2001. Human neutrophil lactoferrin trans-activates the matrix metalloproteinase 1 gene through stress-activated MAPK signaling modules. J Biol Chem 276(45):42575–42579. Oh SM, Pyo CW, Kim Y, Choi SY. 2004. Neutrophil lactoferrin upregulates the human p53 gene through induction of NFkappaB activation cascade. Oncogene 23(50):8282–8291. Omata Y, Satake M, Maeda R, Saito A, Shimazaki K, Yamauchi K, Uzuka Y, Tanabe S, Sarashina T, Mikami T. 2001. Reduction of the infectivity of Toxoplasma gondii and Eimeria stiedai sporozoites by treatment with bovine lactoferricin. J Vet Med Sci 63(2):187–190. Onishi J, Roy MK, Juneja LR, Watanabe Y, Tamai Y. 2008. A lactoferrin-derived peptide with cationic residues concentrated in a region of its helical structure induces necrotic cell death in a leukemic cell line (HL-60). J Pept Sci 14(9):1032–1038. Paesano R, Pietropaoli M, Gessani S, Valenti P. 2009. The influence of lactoferrin, orally administered, on systemic iron homeostasis in pregnant women suffering of iron deficiency and iron deficiency anaemia. Biochimie 91(1):44–51. Penco S, Scarfi S, Giovine M, Damonte G, Millo E, Villaggio B, Passalacqua M, Pozzolini M, Garre C, Benatti U. 2001. Identification of an import signal for, and the nuclear localization of, human lactoferrin. Biotechnol Appl Biochem 34 (Pt 3):151–159. Pietrantoni A, Di Biase AM, Tinari A, Marchetti M, Valenti P, Seganti L, Superti F. 2003. Bovine lactoferrin inhibits adenovirus infection by interacting with viral structural polypeptides. Antimicrob Agents Chemother 47(8):2688–2691. Qiu J, Hendrixson DR, Baker EN, Murphy TF, St Geme JW 3rd, Plaut AG. 1998. Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae. Proc Natl Acad Sci USA 95(21):12641–12646. Reghunathan R, Jayapal M, Hsu LY, Chng HH, Tai D, Leung BP, Melendez AJ. 2005. Expression profile of immune response genes in patients with Severe Acute Respiratory Syndrome. BMC Immunol 6(1):2.

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Rekdal O, Andersen J, Vorland LH, Svendsen JS. 1999. Construction and synthesis of lactoferricin derivatives with enhanced antibacterial activity. J Pept Sci 5(1):32–45. Renye JA Jr., Somkuti GA. 2008. Cloning of milk-derived bioactive peptides in Streptococcus thermophilus. Biotechnol Lett 30(4):723–730. Sabatucci A, Vachette P, Vasilyev VB, Beltramini M, Sokolov A, Pulina M, Salvato B, Angelucci CB, Maccarrone M, Cozzani I, Dainese E. 2007. Structural characterization of the ceruloplasmin: Lactoferrin complex in solution. J Mol Biol 371(4):1038–1046. Sallmann FR, Baveye-Descamps S, Pattus F, Salmon V, Branza N, Spik G, Legrand D. 1999. Porins OmpC and PhoE of Escherichia coli as specific cell-surface targets of human lactoferrin. Binding characteristics and biological effects. J Biol Chem 274(23):16107–16114. Sawatzki G, Rich IN. 1989. Lactoferrin stimulates colony stimulating factor production in vitro and in vivo. Blood Cells 15(2):371–385. Schibli DJ, Hwang PM, Vogel HJ. 1999. The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles. FEBS Lett 446(2–3):213–217. Sekine K, Ushida Y, Kuhara T, Iigo M, Baba-Toriyama H, Moore MA, Murakoshi M, Satomi Y, Nishino H, Kakizoe T, Tsuda H. 1997. Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane. Cancer Lett 121(2):211–216. Senkovich O, Cook WJ, Mirza S, Hollingshead SK, Protasevich, II, Briles DE, Chattopadhyay D. 2007. Structure of a complex of human lactoferrin N-lobe with pneumococcal surface protein A provides insight into microbial defense mechanism. J Mol Biol 370(4):701–713. Sfeir RM, Dubarry M, Boyaka PN, Rautureau M, Tome D. 2004. The mode of oral bovine lactoferrin administration influences mucosal and systemic immune responses in mice. J Nutr 134(2):403–409. Shakibaei M, Frevert U. 1996. Dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (LRP) and heparan sulfate proteoglycans. J Exp Med 184(5):1699–1711. Sharma AK, Karthikeyan S, Sharma S, Yadav S, Srinivasan A, Singh TP. 1998. Structures of buffalo and mare lactoferrins. Similarities, differences, and flexibility. Adv Exp Med Biol 443(1):15–21. Sherman MP, Bennett SH, Hwang FF, Yu C. 2004. Neonatal small bowel epithelia: Enhancing anti-bacterial defense with lactoferrin and Lactobacillus GG. Biometals 17(3):285– 289. Shimamura M, Yamamoto Y, Ashino H, Oikawa T, Hazato T, Tsuda H, Iigo M. 2004. Bovine lactoferrin inhibits tumorinduced angiogenesis. Intl J Cancer 111(1):111–116. Shimizu K, Matsuzawa H, Okada K, Tazume S, Dosako S, Kawasaki Y, Hashimoto K, Koga Y. 1996. Lactoferrinmediated protection of the host from murine cytomegalovirus

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Chapter 13 Secreted Lactoferrin and Lactoferrin-Related Peptides

Tang Z, Yin Y, Zhang Y, Huang R, Sun Z, Li T, Chu W, Kong X, Li L, Geng M, Tu Q. 2009. Effects of dietary supplementation with an expressed fusion peptide bovine lactoferricinlactoferrampin on performance, immune function and intestinal mucosal morphology in piglets weaned at age 21 d. Br J Nutr 101:998–1005. Tanida T, Rao F, Hamada T, Ueta E, Osaki T. 2001. Lactoferrin peptide increases the survival of Candida albicans-inoculated mice by upregulating neutrophil and macrophage functions, especially in combination with amphotericin B and granulocyte-macrophage colony-stimulating factor. Infect Immun 69(6):3883–3890. Taylor S, Brock J, Kruger C, Berner T, Murphy M. 2004. Safety determination for the use of bovine milk-derived lactoferrin as a component of an antimicrobial beef carcass spray. Regul Toxicol Pharmacol 39(1):12–24. Teng C, Gladwell W, Raphiou I, Liu E. 2004. Methylation and expression of the lactoferrin gene in human tissues and cancer cells. Biometals 17(3):317–323. Teng CT. 2006. Factors regulating lactoferrin gene expression. Biochem Cell Biol 84(3):263–267. Teng CT. 2002. Lactoferrin gene expression and regulation: An overview. Biochem Cell Biol 80(1):7–16. Teng CT, Beard C, Gladwell W. 2002. Differential expression and estrogen response of lactoferrin gene in the female reproductive tract of mouse, rat, and hamster. Biol Reprod 67(5):1439–1449. Thomassen EA, van Veen HA, van Berkel PH, Nuijens JH, Abrahams JP. 2005. The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin. Transgenic Res 14(4):397–405. Tomita M, Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K. 1991. Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. J Dairy Sci 74(12): 4137–4142. Tomita M, Wakabayashi H, Shin K, Yamauchi K, Yaeshima T, Iwatsuki K. 2009. Twenty-five years of research on bovine lactoferrin applications. Biochimie 91(1):52–57. Tsuda H, Ohshima Y, Nomoto H, Fujita K, Matsuda E, Iigo M, Takasuka N, Moore MA. 2004. Cancer prevention by natural compounds. Drug Metab Pharmacokinet 19(4):245– 263. Tsuda H, Sekine K, Fujita K, Ligo M. 2002. Cancer prevention by bovine lactoferrin and underlying mechanisms—A review of experimental and clinical studies. Biochem Cell Biol 80(1):131–136. Turchany JM, Aley SB, Gillin FD. 1995. Giardicidal activity of lactoferrin and N-terminal peptides. Infect Immun 63(11): 4550–4552. Ueta E, Tanida T, Osaki T. 2001. A novel bovine lactoferrin peptide, FKCRRWQWRM, suppresses Candida cell growth and activates neutrophils. J Pept Res 57(3):240–249. Ulvatne H, Samuelsen O, Haukland HH, Kramer M, Vorland LH. 2004. Lactoferricin B inhibits bacterial macromolecular

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Wakabayashi H, Takakura N, Teraguchi S, Tamura Y. 2003. Lactoferrin feeding augments peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans. Microbiol Immunol 47(1):37–43. Wang H, Zhao X, Lu F. 2007. Heterologous expression of bovine lactoferricin in Pichia methanolica. Biochemistry (Mosc) 72(6):640–643. Ward PP, Paz E, Conneely OM. 2005. Multifunctional roles of lactoferrin: A critical overview. Cell Mol Life Sci 62(22): 2540–2548. Xiao Y, Monitto CL, Minhas KM, Sidransky D. 2004. Lactoferrin down-regulates G1 cyclin-dependent kinases during growth arrest of head and neck cancer cells. Clin Cancer Res 10(24):8683–8686. Xu YY, Samaranayake YH, Samaranayake LP, Nikawa H. 1999. In vitro susceptibility of Candida species to lactoferrin. Med Mycol 37(1):35–41. Yamaguchi H, Abe S, Takakura N. 2004. Potential usefulness of bovine lactoferrrin for adjunctive immunotherapy for mucosal Candida infections. Biometals 17(3):245–248. Yamauchi K, Wakabayashi H, Shin K, Takase M. 2006. Bovine lactoferrin: Benefits and mechanism of action against infections. Biochem Cell Biol 84(3):291–296. Yang N, Lejon T, Rekdal O. 2003. Antitumour activity and specificity as a function of substitutions in the lipophilic sector of helical lactoferrin-derived peptide. J Pept Sci 9(5):300–311.

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Chapter 14 Bioactive Peptides and Proteins from Fish Muscle and Collagen Nazlin K. Howell and Chitundu Kasase

Contents 1. Introduction, 203 2. Fish Proteins, 204 3. Fish Gelatin, 204 4. ACE Inhibitory Peptides Derived from Fish Proteins, 207 4.1. ACE and Blood Pressure, 207 4.2. ACE Inhibitors, 207 4.3. ACE Inhibitory Peptides Isolated from Fish Proteins, 208 4.4. In Vitro ACE Inhibition by Peptides Derived from Fish Proteins, 208 4.5. ACE Inhibition Mechanism, 211 4.6. In Vivo ACE Inhibition by Peptides Derived from Fish Proteins, 212

1. Introduction Fish comprises proteins, lipids including polyunsaturated fatty acids, vitamins, and minerals that provide a number of health benefits including about 20% of the average animal protein requirements to 2.6 billion people worldwide (Kelleher 2005). As marine fish supplies are threatened due to excessive fishing and poor management, the need to obtain fish protein from sustainable sources like aquaculture and improved processing has increased. Postharvest

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

5. 5.1. 5.2. 5.3. 6. 6.1. 6.2. 6.3. 7. 8.

Antioxidant Peptides Derived from Fish Proteins, 214 Introduction, 214 Mechanism of Lipid Oxidation, 214 Enzymatic Oxidation, 214 Food Antioxidants, 215 Natural Antioxidants, 215 Synthetic Antioxidants, 215 Antioxidant Fish Peptides, 215 Conclusion, 218 References, 219

losses, such as discards at sea, deterioration during storage, and wastage during processing, are substantial (Shahidi 1994; Kelleher 2005). Therefore utilizing and upgrading waste from fish processing into valuable food products is a major goal. Like mammalian muscle proteins, fish myofibrillar proteins display excellent gelation and emulsifying properties. In the last decade, fish collagen from skin and bones has been used to produce gelatin (Badii and Howell 2006). The nutritional quality of a protein depends on its amino acid content and on the physiological utilization of specific amino acids after digestion and absorption (Friedman 1996). Recent scientific evidence has shown that proteins and in particular peptides may exert specific biological activities besides the established nutritional 203

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function (Korhonen and Pihlanto 2003); this has increased interest in the production of biologically active peptides from food proteins (Friedman 1996; Meisel 1997). Hydrolytic proteolysis by gastrointestinal enzymes or during processing through enzymemediated proteolysis of the parent protein releases bioactive peptides which, when absorbed, exert a biological function in the human body and impact health (Korhonen and Pihlanto 2003; Yoshikawa et al. 2000). Bioactive peptides are relatively resistant to hydrolysis and may be transported across the intestinal mucosa due to their small size to exert biological activity; alternatively, the peptides can exert local effects in the gastrointestinal tract (Vermeirssen et al. 2004). In vivo, depending on the nature and sequence of amino acids, bioactive peptides exhibit biological activities including angiotensin I-converting enzyme (ACE) inhibition and mineral binding as well as antioxidant, antimicrobial, opioid, and immunomodulatory activities. This chapter reviews hydrolysates and peptides from fish muscle and collagen and their ACE inhibitory and antioxidant activity that may lead to their applications as bioactive ingredients in functional foods, conventional foods, and neutraceuticals (Meisel 1997; Clare and Swaisgood 2000; Korhonen and Pihlanto 2003; Mine and Shahidi 2006; Gobbetti et al. 2004).

2. Fish Proteins Fish proteins comprise myofibrillar, sarcoplasmic, and connective tissue proteins. The myofibrillar proteins constitute 70–80% of the total proteins and include myosin, actin, tropomyosin, and troponin, which are responsible for muscle contraction and also for gelation and emulsification properties in comminuted products. The amino acid composition of fish myofibrillar proteins is similar to mammalian muscle proteins. Sarcoplasmic proteins are small water-soluble globular proteins and include globulins and the majority of enzymes. In contrast, the connective tissue proteins are not soluble in water or salt solution and need to be hydrolyzed with acid, alkali, or enzymes. Connective tissue proteins consist predominantly of collagen, which comprises

only 3–10% of fish muscle proteins, compared with 17% in most mammals. The chemical and physical properties of collagen proteins differ depending on the tissues such as skin, swim bladder, and the myocommata in muscle. Fish collagen is heat sensitive due to labile crosslinks; and compared to mammals, the hydroxyproline content is lower, varying from 4 to 10% (Sato et al. 1989). However, different fish species contain varying amounts of collagen in the body tissues that reflect the swimming behavior and influence the textural characteristics of fish muscle (Yoshinaka et al. 1988; Montero and Borderias 1989). Fish proteins are a source of energy and essential amino acids, required for growth and maintenance. Fish proteins also contribute to the physicochemical and sensory properties of various protein-rich foods. In addition, fish peptides, when liberated from proteins by processing or digestion, are reported to exert specific biological activities that affect numerous physiological functions. The properties of fish collagen hydrolysates like gelatin and bioactive peptides are discussed below.

3. Fish Gelatin Collagen contains large quantities of hydroxyproline, with the primary structure repeating sequence of glycine-proline-hydroxyproline-glycine amino acids; in vertebrates, at least 12 different forms have been identified, each with a characteristic sequence of amino acids that allows the formation of a lefthanded helical secondary structure. Three helices combine to form tropocollagen, a right-handed super helix that assembles and aggregates to form fibrils and consequently fibers (Lawrie 1991). Collagen is hydrolyzed into gelatin that is used as a gelling agent in desserts and meat products as well as for making glue and in photography. About 200,000 metric tons per year of gelatin, derived from beef and pork skin and bones, is used globally. The physicochemical properties, gel formation, film formation, and foaming and emulsification properties and interactions with other biopolymers of mammalian gelatin have been researched extensively because of the popularity of gelatin and its

Chapter 14 Bioactive Peptides and Proteins from Fish Muscle and Collagen

characteristic mouth feel properties (Choi and Regenstein 2000; Badii and Howell 2006). In recent years the demand for nonbovine and nonporcine gelatin has increased due to the bovine spongiform encephalopathy (BSE) crisis and for religious and social reasons. Fish gelatin is acceptable for Islam, and there are minimum restrictions for Judaism. In addition, fish skin is a major byproduct of the fish-processing industry that contributes to wastage and pollution and could provide a valuable source of gelatin. Research on gelatins obtained from different fish species and their behavior in processed foods is therefore gaining interest (Choi and Regenstein 2000; Gilsenan and RossMurphy 2000; Badii and Howell 2006; Cheow et al. 2007). The physical and chemical properties of gelatin are influenced by intrinsic properties of collagen and processing parameters. Methods for preparing gelatin commercially include prolonged pretreatment of raw materials with cold alkaline solution and extraction at neutral pH. However, the main method for preparing pig skin gelatin involves soaking raw materials in acid solution for a few hours prior to extraction of gelatin with hot water at pH 4.0 (Hinterwaldner 1977). There are several other methods that involve modifications and combinations of these acid and alkaline processes (Sarabia et al. 2000). We have used a combination of sodium hydrogen carbonate (0.125%), sodium hydroxide (0.2%), sulphuric acid (0.2%), and citric acid (0.7%) followed by filtration and deionization to obtain gelatin from North Sea horse mackerel (Trachurus trachurus) (Badii and Howell 2006). The proximate composition of the extracted fish skin gelatin indicated that the ash, moisture, color, weight average molecular weight (195.8 kDa), Bloom (230), and pH (6.0) compared well with commercial fish gelatin. In addition, collagenolytic enzymes such as collagenase, a group of matrix metalloproteinase in a family of zinc-dependent enzymes, can also catalyse the hydrolysis of collagen and gelatin (Sikorski et al. 1990; Sovik and Rustard 2006; Intarasirisawat et al. 2007). Collagenases cleave the triple helices at a single site to a quarter or three-quarters of

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the original length. Metallo-collagenase MMP-13 (collagenase-3, EC 3.4.24) has been isolated from rainbow trout fibroblasts (Saito et al. 2000). Additionally, serine collagenases have been obtained from Atlantic cod (Kristjánsson et al. 1995) and shrimp (Aoki et al. 2003). The effect of autolysis of bigeye snapper skin by indigenous serine proteinases resulted in a higher yield of gelatin but increased degradation; autolysis was inhibited in the presence of soyabean trypsin inhibitor (Intarasirisawat et al. 2007). In contrast, transglutaminase has been added to enhance the gelation of gelatin obtained from herring processing wastes (Norziah et al. 2009). During acid or alkaline hydrolysis, collagen fibers are denatured irreversibly due to the rupture of covalent bonds. When heated in hot water (40°C), the breakdown of hydrogen and electrostatic bonds in the triple helical structure of collagen produces one, two, or three random-chain gelatin molecules that increase the solution viscosity (Flory and Weaver 1960). On cooling, partial reordering of triple helices like the parent collagen result in crosslinks or junction zones as well as disordered regions. In our study, all samples contained more than 30% glycine, which is the most dominant amino acid in gelatin (Balian and Bowes 1977; Rother 1995; Sarabia et al. 2000; Arnesen and Gildberg 2002). The small glycine molecules are required to occupy every third position for close packing of the triple helix (te Nijenhuis 1997). It has been reported that the substitution of glycine in a tripeptide GLY-X-Y by alanine alters the conformation with a local untwisting of the triple helix because close packing of the chains near the central axis is not possible (Bella et al. 1994). The chemical composition of gelatin is similar to that of the parent collagen (Eastoe and Leach 1977), but the molecular weight, number of each kind of amino acid residue, and number of polypeptide chains depend on the position of the breaks. These parameters influence important properties like gel strength and viscosity (Wainewright 1977). Generally, a high gel strength or Bloom value (220– 300) is desirable and more expensive than a medium (150–220) or low (<150) Bloom gelatin (JohnstonBank 1983).

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Gelatin properties and production processes are affected by species, breed, age, environment, feed, and storage conditions of raw materials (Hinterwaldner 1977). It has been noted that collagen and gelatin prepared from low-temperature fish species contain lower amounts of proline and hydroxyproline, contain a lower number of hydrogen bonds, and have a lower melting point than species from a higher temperature environment (Arnesen and Gildberg 2002; te Nijenhuis 1997; Badii and Howell 2006). The high imino acid content of mammalian and warm water fish gelatins results in a lower critical concentration and a higher melting point (Badii and Howell 2006; Abbey et al. 2008). These authors indicated that the imino acids content was significantly lower in nongelling cod gelatin compared with tilapia, flying gurnard (Dactylopterus volitans), and horse mackerel gelatin. Similarly, the melting point by differential scanning calorimetry of tropical fish shortfin scad and sin croaker were higher than cod skin gelatin (Cheow et al. 2007). Effective formation and stabilization of the collagen triple helix structure require the presence of the repeating sequence (GLY-X-Y), where X and Y can be any amino acid with an average of at least one proline or hydroxyproline in every other triplet (Piez and Sherman 1970). Therefore, GLYPRO-Y, GLY-X-HYP, and GLY-PRO-HYP are important for the stabilization of the collagen structure (Privalov 1982) and the transition temperature of (GLY-X-HYP)n is higher than that of (GLYPRO-Y)n (Thakur et al. 1986). Gelatins with a lower amount of imino acid content have a higher critical concentration and lower melting point, compared with those that contain a high amount of imino acids (Gilsenan and Ross-Murphy 2000). The amount of serine, which has a free hydroxyl group, is higher in nongelling gelatin. Conversely, thr and tyr were higher in a gelling horse mackerel and commercial tilapia gelatin compared with nongelling cod gelatin (Badii and Howell 2006). Hydrogen bonds between water molecules and free hydroxyl groups of amino acids also influence gelatin gel strength (Arnesen and Gildberg 2002). The regular water structure near the macromolecules stabilizes the collagen structure (Privalov and

Tiktopulo 1970; Privalov et al. 1971). Thus, the change from random coil into a triple helix conformation results in negative entropy due to the decreased entropy of the polypeptide chain itself, and more importantly because of the immobilization and orientation of the water molecules in the triple helix (te Nijenhuis 1997). Hydrogen bonding stabilizes the triple helix (Bella et al. 1994) and interchain hydrogen bonding also occurs between polypeptide chains (Privalov 1982). It has been reported that negative entropy leads to negative transition enthalpy not only due to the hydrogen bonding between the three helices but also due to electrostatic interactions between positively charged groups (lysine and arginine) primarily in the Y position with negatively charged glutamic and aspartic acid residues mainly in the X position of the GLY-X-Y triplets (Privalov 1982; Venugopal et al. 1994). In our study, however, the amounts of positively and negatively charged residues were similar for the three types of gelatin samples. Hydrophobic amino acids are also considered to contribute to the high Bloom value that was confirmed by Badii and Howell (2006); a lower value of hydrophobic amino acids (ala, val, leu, iso leu, pro, phe, and met) was found in the nongelling cod gelatin, compared to tilapia and horse mackerel gelatin. FT-Raman spectroscopy confirmed that hydrophobic groups (tyrosine doublet ratio 850/830 cm−1 and CH stretch region), as well as alpha-helix and random coil, for horse mackerel gelatin were in between those of tilapia and cod gelatin (Badii and Howell 2006). It has been suggested that extraction conditions may affect the hydrophobic amino acid composition and distribution that influence the physical properties of gelatin, even more than imino acid content (Montero and Gomez-Guillen 2000). A high Bloom value may also result from a higher molecular weight MW (300 kDa) compared with a low MW gelatin. In addition, the number and distribution of polypeptides, which are influenced by the manufacturing method and pH, also affect gelation properties. The weight average molecular weight of horse mackerel gelatin (196 kDa) was similar to the commercial tilapia gelatin and was

Chapter 14 Bioactive Peptides and Proteins from Fish Muscle and Collagen

higher than the limed ossein standard gelatin (147 kDa) used for calibration (Badii and Howell 2006). In general, a high MW gelatin gives a high Bloom value, which was reflected in the horse mackerel sample. Collagen hydrolysates and gelatin have an important functional role in many food products. In addition, they can be used to manufacture bioactive peptides that have antihypertensive and antioxidant properties that are discussed below.

4. ACE Inhibitory Peptides Derived from Fish Proteins 4.1. ACE and Blood Pressure High blood pressure is a major risk factor associated with cardiovascular disease, the biggest cause of death. As many synthetic drugs like ACE inhibitors have side effects, peptides from food sources provide an attractive alternative. A brief background on the regulation of blood pressure is included to help understand the role of ACE inhibitors. Within the body, the major physiological pathways involved in the regulation of blood pressure (BP) are the rennin-angiotensin system (RAS), the kinin-nitric oxide system (KNOS), the renninchymase system (RCS), and neutral endopeptidase system (NEPS). The RAS is perhaps the most important of various humoral vasoconstrictor and vasodilator mechanisms implicated in blood pressure regulation. It regulates BP, electrolyte balance, and renal, neuronal, and endocrine functions associated with cardiovascular control in the body. In the RAS, through the action of kallikrein, renin is released from the precursor compound prorenin (Beldent et al. 1993; Ondetti and Cushman 1982). Renin cleaves angiotensinogen to release angiotensin I (Ang-I), a decapeptide (Asp-Arg-ValTyr-Ile-His-Pro-Phe-His-Leu). The ACE hydrolyzes the Ang-I by removing the C-terminal dipeptide His-Leu to give an octapeptide, angiotensin II (Ang-II) (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), a potent vasoconstrictor. ACE is a specific component of vascular endothelium in most mammalian organs (Caldwell et al. 1976); however, the lung is thought to be the physiologically important site for

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generating angiotensin II destined for the systemic arterial circulation (Ng and Vane 1968). Ang-II regulates blood pressure, body fluid volume, and the activity of gonadotrophic-hormone-releasing hormone (GHRH) and pituitary hormones during the reproductive cycle and pregnancy, in addition to controlling neurotransmitter interactions (Philips 1987). In the kinin-kallikrein system, bradykinin is generated from kininogen by kallikrein, either directly or via the intermediate kallidin. In vivo, bradykinin is rapidly degraded by kininases, the most important of which are the metallopeptidases ACE and NEP, aminopeptidase P, and the carboxypeptidases M and N (Dendorfer et al. 1997; Kokkonen et al. 2000). Therefore, the two compounds angiotensin II and bradykinin are important peptides involved in the regulation of blood pressure. Ang II is a vasoconstrictor and growthpromoting substance, whereas bradykinin is a potent vasodilator and growth inhibitor. ACE is a key enzyme in the regulation of blood pressure and has been targeted in the control of hypertension. ACE inhibition prevents the formation of the vasoconstrictory (hypertensive) agent angiotensin II and potentiates the vasodilatory (hypotensive) properties of bradykinin, leading to lowering of blood pressure. ACE inhibitors, which are now widely used for the treatment of hypertension and heart failure, not only block the generation of Ang II from Ang I but also prevent the degradation of bradykinin.

4.2. ACE Inhibitors The first naturally occurring angiotensin Iconverting enzyme inhibitors (ACEI) of a peptide nature were isolated from snake venom (Ferreira et al. 1970; Ondetti et al. 1971). Cushman and Ondetti developed ACE inhibitors by constructing the active site of ACE, using computer-assisted studies (Ondetti 1977). Many prototypes of ACE inhibitors were developed, including succinyl-1proline and 2-D-methylsuccinyl-1-proline. The inhibitory potency was increased by replacing the carbonyl group with a thiol group, which eventually led to the development of the first synthetic ACE

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inhibitor, Captropil (Laurence et al. 1997). However, due to many reported zinc binding and resultant zinc deficiency effects of captopril, subsequent ACE inhibitors like enalapril, lisinopril, and ramipril were designed to replace the thiol group with moieties that do not bind intracellular zinc. Nevertheless, there are other side effects such as skin rash, headache, dry cough, and loss of taste perception that are associated with synthetic ACE inhibitors. Therefore, current research is now focused on ACE inhibitory (ACEI) peptides from natural sources such as milk, eggs, meat, fish, and plants (Meisel et al. 2006; Vercruysse et al. 2005; Gobbetti et al. 2004). The potency of an ACE inhibitory peptide is usually expressed as an IC50 value, which expresses the concentration of peptide mediating a 50% inhibition of activity. The intrinsic bioactivities of the peptides encrypted in food proteins are latent until they are released and activated by enzymatic hydrolysis; for example, during gastrointestinal digestion and food processing.

4.3. ACE Inhibitory Peptides Isolated from Fish Proteins ACE inhibitory peptides have been isolated and characterized from a number of fish sources, either from the muscle proteins or from waste and discards from fish processing. To date, ACE inhibitory peptides have been isolated and characterized from salmon, sardine, bonito, tuna, Alaska pollack, sea bream, pelagic thresher, fish sauce, and yellowfin sole. Table 14.1 shows the amino acid sequence of ACE inhibitory peptides derived from fish proteins, their origin, the enzymes used for hydrolysis, and the IC50 values.

4.4. In Vitro ACE Inhibition by Peptides Derived from Fish Proteins Suetsuna and Osajima (1986) first reported ACE inhibitory peptides derived from fish muscle proteins. Sardine hydrolyzed by a protease from Aspergillus oryzae (denazyme AP) produced ACE inhibitory peptides with IC50 value of 3.79 mg/L; the amino acid sequence for the peptide was, however,

not identified. In another study that involved sardine muscle protein, 11 new ACE inhibitory peptides, comprising 2–4 amino acid residues, were isolated from the hydrolyzate. The IC50 value of most of these peptides was below 100 μM except for three peptides that were higher. The highest inhibitory activity was observed for a dipeptide KY, IC50 = 1.63 μM (Matsufuji et al. 1994). Ariyoshi (1993) isolated five peptides from sardine with IC50 values ranging from 11 μM for an octapeptide to 188 μM for a tripeptide. In dried bonito (Katsuobusi, a Japanese traditional food processed from bonito muscle and intestines, hydrolyzed by various proteases), a thermolysin digest showed the most potent inhibitory activity. Eight inhibitory peptides with amino acid sequences IKPLNY, IVGRPRHQG, IWHHT, ALPHA, FQP, LKPNM, IY, and DYGLYP were isolated from the digest using high-pressure liquid chromatography (HPLC). By searching for the sequence homology in many proteins, four of them were found to originate from the primary structure of actin. Fragments of these peptides were prepared by further enzymatic digestion or chemical synthesis and their ACE-inhibitory activities were measured. Among them, ILP, IW, LKP, and LWP had higher inhibitory activity than their parental peptides (Yokoyama et al. 1992). Of these isolated peptides, the parent peptide LKPNM (IC50 = 2.4 mM) hydrolyzed by ACE produced LKP (IC50 = 0.32 mM) with eightfold higher ACE-inhibitory activity relative to LKPNM, suggesting that LKPNM functions as a prodrug-type ACE-inhibitory peptide (Fujita and Yoshikawa 1999). From the bonito intestinal autolysate, six ACE inhibitory peptides were isolated with amino acid sequences YRPY, GHF, VRP, IKP, LRP, and IRP and IC50 values 320; 1,100; 2.2; 2.5; 1.0; and 1.8 μM, respectively (Matsumura et al. 1993). Defatted muscle proteins of chum salmon hydrolyzed with thermolysin showed high inhibitory activity against angiotensin I-converting enzyme (IC50 = 27.9 protein g/mL) in vitro. Six active, tryptophan residue-containing ACE inhibitory dipeptides were isolated from the hydrolyzate by chromatography; these included: WA, VW, WM,

Table 14.1. ACE inhibitory peptides derived from fish proteins. Source

Parent Protein

Enzyme

Amino Acid Sequence

IC50 (μM)

Reference

Sardine Tuna

Meat Muscle Muscle Muscle Muscle Muscle Muscle Actin

A. oryzae protease Acid Acid Acid Acid Acid Thermolysin Thermolysin

— PTHIKWGD IF VWIG LTF IFG LKP IY

3.97 mg/l N/A 70.0 110.0 330.0 >1,000 0.32 2.31

Actin Muscle

Thermolysin Thermolysin

IVGRPRHQG LKPNM

2.4 2.4

Actin Actin

Thermolysin Thermolysin

IWH IWHHT

3.5 5.8

Muscle Actin Actin Muscle Muscle Muscle

Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin B.licheniformis alkaline protease Autolysate Autolysate Autolysate Autolysate Autolysate Autolysate Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease Alkaline protease — — — — — Trypsin, chymotrypsin, pronase E + pepsin Trypsin, chymotrypsin, pronase E + pepsin

IKP ALPHA FQP IKPLNY DYGLYP —

6.9 10 12 43 62 0.015 mg/ml

Suetsuna and Osajima 1986 Kohama et al. 1988 Kohama et al. 1988 Kohama et al. 1988 Kohama et al. 1988 Kohama et al. 1988 Fujita and Yoshikawa 1999 Yokoyama et al. 1992, Fujita and Yoshikawa 1999 Yokoyama et al. 1992 Yokoyama et al. 1992 Fujita and Yoshikawa 1999 Fujita and Yoshikawa 1999 Yokoyama et al. 1992 Fujita and Yoshikawa 1999 Fujita and Yoshikawa 1999 Yokoyama et al. 1992 Yokoyama et al. 1992 Yokoyama et al. 1992 Yokoyama et al. 1992 Matsui et al. 1993

LRP IRP VRP IKP YRPY GHF KY AKK GWAP VY IY GRP LY VF MF RY YL MY RVY RFH LKVGVKQY KVLAGM HQAAGW VKAGF LKL CWLPVY

1.0 1.8 2.2 2.5 320 1,100 1.63 3.13 3.86 10 10.5 20 38.5 43.7 44.7 51 82 193 205.6 330 11.0 30.0 60.0 83.0 188.0 22.20

Matsumura et al. 1993 Matsumura et al. 1993 Matsumura et al. 1993 Matsumura et al. 1993 Matsumura et al. 1993 Matsumura et al. 1993 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Matsufuji et al. 1994 Ariyoshi 1993 Ariyoshi 1993 Ariyoshi 1993 Ariyoshi 1993 Ariyoshi 1993 Astawan et al. 1995

VAWKL

31.97

Astawan et al. 1995

Bonito

Sardine Bonito bowels

Sardine

Sardine

Tuna (skipjack)

Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle — — — — — Flesh Flesh

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Table 14.1. Continued Source

Parent Protein

Enzyme

Tuna (skipjack)

Flesh

Trypsin, chymotrypsin, pronase E + pepsin Trypsin, chymotrypsin, pronase E + pepsin Fermentation Fermentation Fermentation Fermentation Autolysate Autolysate Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Alcalase, pronase + collagenase Alcalase, pronase + collagenase Pepsin Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Alkaline protease Alkaline protease Alkaline protease Alkaline protease Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Chymotrypsin Papain Pancreatic digest Pancreatic digest Pancreatic digest Pancreatic digest Thermolysin Thermolysin

Flesh Salmon Sardine Sardine Dried bonito Squid

Sauce Sauce Sauce Sauce

Bonito

Alaska pollack

Skin Skin

Pelagic thresher

Salmon

Sea breams

Salmon

Yellowfin sole Salmon Atlantic salmon Coho salmon Alaska pollack Blue whiting Atlantic salmon Coho salmon

210

Frame protein Muscle + viscera extracts

Muscle Muscle Muscle Muscle Muscle Muscle Scales (gelatin) Scales (gelatin) Scales (gelatin) Scales (gelatin) Muscle Muscle Muscle Muscle Muscle Frame protein Muscle Muscle Muscle Muscle Muscle Muscle Muscle

Amino Acid Sequence

IC50 (μM)

Reference

SKVPP

74.22

Astawan et al. 1995

YSKVL

156.3

Astawan et al. 1995

— — — — YALPHA GYALPHA IKP IY LKPNM IWHHT IVGRPRHQG GPL

1.69 mg/ml 1.43 mg/ml 3.15 mg/ml 0.24 mg/ml 9.8 27.3 1.6 2.1 2.4 5.8 2.4 mg/ml 2.60

Okamoto et al. 1995 Okamoto et al. 1995 Okamoto et al. 1995 Wako et al. 1996 Wako et al. 1996 Fujita et al. 2000 Fujita et al. 2000 Fujita et al. 2000 Fujita et al. 2000 Fujita et al. 2000 Byun and Kim 2001, 2002

GPM

17.13

Byun and Kim 2001, 2002

FGASTRGA IKW

14.7 0.54

Je et al. 2004 Nomura et al. 2002

VW MW FRVFTPN LWA VSW VTR VW IW MW LW WM WA VIY VY GY GF AW FL WL LF WV MIFPGAGGPEL IW Hydrolysate Hydrolysate Hydrolysate Hydrolysate Hydrolysate Hydrolysate

1.68 3.76 9.59 12.7 23.2 135.9 2.5 4.7 9.9 17.4 96.6 277.3 7.5 16 265 708 6.4 13.6 34.1 383.2 500.5 28.7 μg/ml 1.2 5.0 mg/ml 3.7 mg/ml 2.9 mg/ml 3.69 mg/ml 0.078 mg/ml 0.138 mg/ml

Nomura et al. 2002 Nomura et al. 2002 Nomura et al. 2002 Nomura et al. 2002 Nomura et al. 2002 Nomura et al. 2002 Ono et al. 2003 Ono et al. 2003 Ono et al. 2003 Ono et al. 2003 Ono et al. 2003 Ono et al. 2003 Fahmi et al. 2004 Fahmi et al. 2004 Fahmi et al. 2004 Fahmi et al. 2004 Ono et al. 2006 Ono et al. 2006 Ono et al. 2006 Ono et al. 2006 Ono et al. 2006 Jung et al. 2006 Enari et al. 2008 Nakajima et al. 2009 Nakajima et al. 2009 Nakajima et al. 2009 Nakajima et al. 2009 Nakajima et al. 2009 Nakajima et al. 2009

Chapter 14 Bioactive Peptides and Proteins from Fish Muscle and Collagen

MW, IW, and LW with IC50 values ranging from 2.5 to 277.3 μM. In a subsequent study, also involving chum salmon and thermolysin hydrolysis, five ACE inhibitory peptides, FL, LF, AW, WV, and WL, were characterized. The IC50 value of peptide FL was 13.6 μM. However, in the later study, the reverse sequence of the dipeptide LF showed ACE inhibitory activity with an IC50 value of 383.2 μM. Thus LF has a lower inhibitory potency than FL despite the fact that both dipeptides comprise the same type of amino acids (Ono et al. 2003, 2006). The products of a proteolytic digestion of gelatin extracts from Alaska pollack (Theragra chalcogramma) skin hydrolyzed by alcalase, pronase E, and collagenase using a three-step recycling membrane reactor demonstrated a high ACE inhibitory activity. Using gel filtration, ion-exchange chromatography, and reverse-phase HPLC, two peptides with an amino acid sequence of GPL and GPM IC50 values of 2.6 and 17.13 μM, respectively, were isolated (Byun and Kim 2001). Alaska pollack frame protein hydrolyzed with pepsin produced a fraction with ACE inhibitory activity. Further separation and purification by ultrafiltration, and consecutive gel filtration, ion-exchange, and HPLC, resulted in a novel ACE inhibitory peptide with sequence FGASTRGA and IC50 value of 14.7 μM (Je et al. 2004). Yellowfin sole (Limanda aspera) frame protein, hydrolyzed by α-chymotrypsin and ultrafiltered, indicated ACE inhibitory activity in the <5 kDa fraction. After purification by chromatography, the fraction with a molecular mass of 1.3 kDa yielded an ACE inhibitory peptide (YFP) comprising 11 amino acids with a sequence MIFPGAGGPEL and IC50 value of 28.7 μg/ml (Jung et al. 2006). Fahmi et al. (2004) reported an IC50 value of 0.57 mg/ml for collagen peptides from sea bream scales hydrolyzed with an alkaline protease. Four peptides were isolated from the scale hydrolysate with ACE inhibitory activities 5–20 times higher than that of the unpurified hydrolysate. The amino acid sequences of inhibitory peptides and IC50 values were GY (265 μM), VY (16 μM), GF (708 μM), and VIY (7.5 μM), respectively.

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Indonesian dried-salted skipjack tuna, when hydrolyzed with trypsin, chymotrypsin, pronase E, and pepsin, exhibited ACE inhibitory activity (Astawan et al. 1995). The pepsin digest showed the highest inhibitory activity (IC50 0.63 mg protein/ml) that decreased after 6 months of storage. After purification, four inhibitor peptides with amino acid sequences VAWKL, WSKVVL, SKVPP, and CWLPVY and IC50 values of 31.97, 156.28, 74.22, and 22.20 μM, respectively, were identified. Table 14.1 shows that fish peptides with inhibitory potency range from 2 to 10 amino acid residues, and IC50 values range from 0.32 μM for a tripeptide and 28.7 μM for a decapeptide. The di- and tripeptides showed high inhibitory activity with IC50 values ranging from 1.63 to 708 μM and 0.32 to 1,100 μM, respectively. The most potent inhibitory fish peptide is the tripeptide LKP (IC50 = 0.32 μM) isolated from bonito with 15 times less inhibitory potency compared to the synthetic ACE inhibitor, captopril (Fujita and Yoshikawa 1999). The same peptide was isolated from chicken muscle (Fujita et al. 2000) and pea vicilin (Vermeirssen 2003) with IC50 value of 0.30 μM and 0.32 μM, respectively.

4.5. ACE Inhibition Mechanism Competitive or noncompetitive inhibition activity of ACEI peptides have been determined using Lineweaver-Burk plots. The proper sequence and composition of amino acid residues are essential for the interaction of peptides with ACE to effect inhibition. In particular, dipeptides and tripeptides are recognized to be useful inhibitors. Captopril, the first synthetic ACEI to treat hypertension, was synthesized on the basis of a dipeptide configuration (Ondetti et al. 1977). Three different forms of ACE have been identified including somatic and testicular ACE and ACE homologue (ACEH). The somatic and testicular forms of ACE consist of two homologous N- and C-domains (Inagami 1992) that contain active sites for the catalytic hydrolysis of angiotensin I (Wei et al. 1992). ACE inhibitors may act on either ACE domain; however, the C-domain appears to be responsible for controlling blood pressure,

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signifying that the C-domain is the principal angiotensin-converting site (Natesh et al. 2003). Structure-activity correlations among different peptide inhibitors of ACE indicate that binding to the enzyme is strongly influenced by the C-terminal tripeptide sequence of the substrate. ACE appears to have a preference for substrates or competitive inhibitors that have hydrophobic (aromatic or branched side chains) amino acid residues at each of the three C-terminal positions (Meisel et al. 2006). Through computer-assisted molecular modeling, the mechanism of ACE inhibition is also considered to involve inhibitor interaction with an anionic binding site that is distinct from the catalytic site (Meisel 1993). Some inhibitory peptides possess a C-terminal glutamate residue that inhibits activity possibly by chelating the zinc ion, an important catalytic component bound at the active site of ACE (Wei et al. 1992). Several naturally occurring peptidic inhibitors include the highly active short-chain peptides that contain proline at the C-terminus. The majority of di- and tripeptide ACE inhibitors have a tyrosine, phenylalanine, tryptophan, or proline residue at the C-terminal end with tryptophan reported to be most effective in increasing ACE inhibition (Meisel et al. 2006). Table 14.1 confirms the above finding as fish di- and tripeptides with tyrosine, phenylalanine, tryptophan, or proline at the C-terminal showed higher IC50 values. In ACE-inhibitory peptides with lysine or arginine as the C-terminal residue, structure-activity data showed that the positive charge on the chain contributed significantly to the inhibitory potency. The replacement of arginine at the C-terminal end resulted in inactive analogues (Ondetti et al. 1977; Ariyoshi 1993; Cheung et al. 1980). Tripeptide substrates HHL, HFR, and HAP, which have the same COOH-terminal dipeptides as angiotensin I, showed similar binding affinities for ACE. The same order of enzyme-binding affinities was also found with competitive dipeptide inhibitors HL, PR, and AP (Cheung et al. 1980). This suggests that selective binding of the COOH-terminal dipeptide residue is an important determinant of the substrate specificity of ACE. ACE is highly specific

with regard to the terminal dipeptide residues of peptide substrates or competitive inhibitors. When two dipetides, VW and PG, were investigated for ACE inhibitory potency, VW (IC50 = 0.3 μM) was 10,000 times more inhibitory than the weakest inhibitor, PG (Cheung et al. 1980). Among the N-terminal amino acids of di- and tripeptides, the branched-chain aliphatic amino acids isoleucine and valine are predominantly highly active inhibitors. On the other hand, for long-chain peptides, the peptide conformation, that is, the structure adopted in a specific environment of the binding site, is responsible for ACE inhibitor potency (Meisel et al. 2006). Cheung et al. (1980) have further shown that NH2-terminal glycyl dipeptides vary 300-fold in inhibitory potency, those with tryptophan, tyrosine, or proline being the most effective inhibitors. COOH-terminal glycyl dipeptides vary only 16-fold in inhibitory activity, with valine, isoleucine, and arginine as the most effective NH2-terminal residues. Ono et al. (2006) isolated a peptide FL (IC50 = 13.6 μM), an ACE inhibitor from hydrolysate of chum salmon muscle with noncompetitive behavior. When the sequence of the peptide was reversed, the resulting peptide LF exhibited less inhibitory activity (IC50 = 383.2 μM) than FL, and competitive inhibition. Tryptophan-containing dipeptides with tryptophan as the C-terminal residue (AW, VW, MW, IW, LW), also isolated from salmon muscle hydrolysate, showed noncompetitive inhibition, while the reversed sequence with W at the N-terminal showed competitive inhibition, except WL (Ono et al. 2006). These results indicate that the sequence of ACE dipeptides influences both inhibitory power and the inhibition mechanisms.

4.6. In Vivo ACE Inhibition by Peptides Derived from Fish Proteins There are limited human studies (Kawasaki et al. 2000) on ACE inhibition by fish peptides. Spontaneous hypertensive rat (SHR), a strain of Rattus norvegicus with elevated blood pressure, is extensively used as a model for studying hypertension in animals. SHR has been used to investigate the efficacy of ACEI peptides to regu-

Chapter 14 Bioactive Peptides and Proteins from Fish Muscle and Collagen

late blood pressure. When orally or intravenously administrated to SHR, various animal and plant peptides have demonstrated antihypertensive effects through the reduction in blood pressure (Meisel et al. 2006). ACE-inhibitory fish peptides gave systolic

213

BP (SBP) responses ranging from 0.00 to 80 mmHg (Table 14.2). The wide variation in blood pressure responses may be due to variation in sample type, the dosage and duration of administration, the mechanism of delivery, that is, oral or intravenous

Table 14.2. Hypotensive effects of fish-derived peptides in spontaneously hypertensive rats. Origins of Peptides Main Peptides

Doses

Study Designs

Results

References

20 mg/kg BW

SHR, 12 wk old, Japan, I.V. SHR, 12 wk old, Japan, I.V. SHR, 16–25 wk old, Japan, SOD

SBP—7.2 mmHg and DBP—28.8 mmHg SBP—18 mmHg and BP—35 mmHg SBP—23 mmHg at 4 h

Matsufuji et al. 1995

SHR, 16–25 wk old, Japan, SOD SHR, 16–25 wk old, Japan, I.V. SHR, 16–25 wk old, Japan, I.V. SHR, Japan, I.V. and SOD

SBP—18 mmHg at 2 h

SBP—45.0 mmHg (I.V.), SBP—19.0 mmHg (SOD +2 h)

SHR 10 wk SOD

SBP—55.0 mmHg (I.V.), SBP—22.0 mmHg (SOD +2 h) SBP—70.0 mmHg (I.V.), SBP—20.0 mmHg (SOD +6 h) SBP—70.0 mmHg (I.V.), SBP—30.0 mmHg (SOD +4 h) SBP—25.0 mmHg (I.V.), SBP—17.0 mmHg (SOD +6 h) SBP—80.0 mmHg (I.V.), SBP—23.0 mmHg (SOD +6 h) SBP—60.0 mmHg (I.V.), SBP—26.0 mmHg (SOD +6 h) SBP—0.00 mmHg (I.V.), SBP—14.0 mmHg (SOD +8 h) SBP—22 mmHg +3 h

SHR SOD

SBP decreased +2–4 h

—

SHR SOD

SBP decreased

300 mg/kg BW

SHR SOD

SBP—20 mmHg +3 h

Sardine muscle (alkaline protease)

VY IC50: 7.1 μM

Dried bonito (Thermolysin)

LKPNM, IC50: 2.4 μM

8 mg/kg BW

LKP IC50: 0.32 μM LKPNM

2.25 mg/kg BW

LKP

30 mg/kg BW

IY, IC50: 2.1 μM

10 mg/kg BW by i.v and 60 mg/kg BW for SOD

Bonito muscle (Thermolysin)

50 mg/kg BW

100 mg/kg BW

IW IC50: 5.1 μM IKP, IC50: 1.6 μM

Yellowsole Mackerel

Tuna Sea breams Salmon

IWH, IC50: 3.5 μM IVGRPR, IC50: 300 μM LKPNM IC50: 2.4 μM IWHHT IC50: 5.8 μM IVGRPRHQG IC50: 2.4 μM MIFPGAGGPEL 10 mg/kg BW IC50: 28.7 μg/ml Hydrolysate 10 mg/kg BW IC50: 0.1 mg/ml Hydrolysate IC50: 0.63 mg/ml Hydrolysate IC50: 0.57 mg/ml Hydrolysate IC50: 27 μg/ml

500–2,000 mg/kg SHR SOD BW

Fujita and Yoshikawa 1999

SBP—30 mmHg SBP—50 mmHg

SBP—28–38 mmHg +4 h

Fujita et al. 2000

Jung et al. 2006 Itou and Akahane 2004 Astawan et al. 1995 Fahmi et al. 2004

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Part 3 Examples of Food Proteins and Peptides with Biological Activity

administration, and the technique by which blood pressure was measured (Meisel et al. 2006).

5. Antioxidant Peptides Derived from Fish Proteins 5.1. Introduction In addition to antihypertensive properties of bioactive fish peptides, there are also numerous studies on the antioxidant behavior of hydrolysates and peptides from many sources, including fish. Most of the studies investigate antioxidant properties in oxidizing lipid systems. Lipids from plant and animal sources include fatty acids, triacylglycerols, steroids, phospholipids, sphingolipids, plasmalogens, eicosanoids, waxes, and terpenes. Lipids function as structural components of biological membranes, energy reserves, and precursors in vitamin and hormone synthesis and contribute to texture and flavor in food materials and products. Fatty acids may be saturated or unsaturated with 1–6 double bonds. Although mono- and polyunsaturated fatty acids are reported to impart health benefits (Mozaffarian et al. 2006), they are also prone to lipid oxidation, resulting in rancidity and the formation of toxic free radicals and other reactive oxygen species and aldehydes that cause protein crosslinking and DNA damage in cells (Alghazeer and Howell 2008). Lipid oxidation is a risk factor for cardiovascular, neurological, immune and eye diseases, as well as cancer and diabetes (Willcox et al. 2004; Halliwell 2000; Halliwell and Whiteman 2004; Collins 2005; Saeed et al. 2006). In food systems, lipid oxidation causes deterioration in food quality due to rancidity and formation of toxic compounds. Lipid oxidation in food products can be controlled by reducing metal ions and minimizing exposure to light and oxygen using packaging, as well as by employing correct levels of natural and synthetic antioxidants. Common food antioxidants include naturally occurring tocopherols, ascorbic acid and phenolic compounds and synthetic butylated hydroxytoluene (BHT), butylated hydroyanisole (BHA), propyl gallate, and ethoxyquin. Because of potential toxic effects of synthetic

antioxidants, natural antioxidants are preferred by consumers and processors. Therefore, additional sources of natural antioxidants including protein hydrolysates, peptides, and amino acids from plant and animal sources are currently being investigated. This chapter will focus on antioxidant activity and mechanism of in vivo and in vitro inhibition by peptides derived from fish proteins.

5.2. Mechanism of Lipid Oxidation Lipids and in particular unsaturated fatty acids are oxidized by autoxidation, photooxidation, and enzymes. In the initiation stage of autoxidation, highly reactive free radicals (hydroperoxide •HO2, hydroxide •OH, peroxide ROO•, alkoxy RO•, alkyl R•) abstract hydrogen from the methylene group of an unsaturated fatty acid, thus generating further free radicals. The alkyl radical lipid is stabilized by a molecular rearrangement to form a conjugated diene. In the presence of molecular oxygen, the alkyl radical forms peroxyl radicals, ROO. In the propagation stage, the peroxyl radical abstracts a hydrogen atom from another lipid molecule, causing an autocatalytic chain reaction. The peroxyl radicals combine with hydrogen to yield a lipid hydroperoxide (or peroxide). The primary oxidation products are transformed into secondary products like aldehydes, ketones, and hydroxides. Unsaturated aldehydes and ketones oxidize further to tertiary compounds including propanol, hexane, and pentane (Frankel 1998). Free radical oxidative reactions are terminated when two radical species (peroxyl, alkoxy, or alkyl) interact with each other forming nonradical adducts. Free radical reactions can also be terminated when one of the lipid radicals reacts with an antioxidant. In this reaction the abstraction of hydrogen by a peroxide radical from the antioxidant molecule produces an inert antioxidant radical (Howell and Saeed 1999).

5.3. Enzymatic Oxidation Enzymes including lipoxygenase (LOX) catalyze reactions between oxygen and unsaturated fatty

Chapter 14 Bioactive Peptides and Proteins from Fish Muscle and Collagen

acids to form hydroperoxides at specific positions. LOX reacts with more than one methylene carbon on the substrate molecule to yield double oxygenation site. The fatty acid peroxy free radical abstracts hydrogen from another unsaturated fatty acid molecule to form a conjugated hydroperoxy diene that results in further oxidation of lipids, proteins, and other compounds, which can themselves interact with the enzyme-substrate complex (Hultin 1994). Soybean LOX-1 is specific for linoleic acid and is involved in the enzymatic catalysis of free fatty acid oxidation, whereas LOX-2 catalyzes the oxidation of triglycerides, carotenoids, and vitamins (Frankel 1998). LOX present in mammals is categorized based on positional specificity of oxygen insertion into the substrate; for example, arachidonate LOX includes 5-, 8-, 12- and 15-lipoxygenase.

6. Food Antioxidants 6.1. Natural Antioxidants Vegetables, fruits, herbs, spices, teas, oilseeds, nuts, cereals, and legumes are rich sources of natural antioxidants (Yanishlieva-Maslarova 2001). These dietary antioxidants include vitamins (vitamin E, C, and β-carotene), plant phenols (flavanoids, other phenolic compounds), and essential minerals (selenium, zinc, manganese, and copper) that form important antioxidant enzymes (Beutner et al. 2001; Hall 2001). Natural antioxidants inhibit lipid oxidation reactions by radical (ROO·, RO·, HO·, 1O2) scavenging, protein (enzymes and their metal-binding sites) complexing, and synergistic effects by reducing oxidized antioxidants and metal chelation (Frankel 1999). At high concentrations, phenolic antioxidants may become prooxidative and initiate lipid oxidation (Gordon 1990). Besides their free radical–scavenging ability, flavonoids exhibit biological activity as protein kinase inhibitors and transcriptional regulators (Moskaug 2001). The mechanism of tocopherol antioxidant capacity is based on their ability to impede the formation of hydroperoxides and inhibit the rearrangement of cis, trans peroxyl radicals to trans, trans isomers (Porter et al. 1995). They also

215

inhibit the decomposition of peroxide (Hopia et al. 1996; Mäkinen and Hopia 2000) and the β-scission of alkoxyl radicals (Frankel 1998). Ascorbic acid is a strong reductant with respect to singlet oxygen, superoxide, H2O2, hydroperoxyl radicals, hydroxyl radicals, and hypochlorous acid at low concentrations, in the presence of transition metal ions (Fe, Cu, Co) and especially in aqueous lipid systems (e.g., milk, mayonnaise, sauces, margarine, and butter). However, ascorbic acid may also act as a prooxidant or react synergistically with vitamin E (Howell and Saeed 1999) to improve antioxidant behavior.

6.2. Synthetic Antioxidants The most common synthetic antioxidants utilized in food systems possess phenolic group(s) and include compounds such as butylated hydroxytoluene, butylated hydroxyanisole, butylhydroquinone, and propyl gallate. The antioxidant mechanisms involve the formation of resonance-stabilized phenolic radicals. The formed radical neither catalyzes the oxidation of other molecules nor reacts with oxygen to form antioxidant peroxides that autoxidize. Synthetic phenolic radicals react with each other in a manner similar to that of tocopherols. Two phenolic radicals react to form a hydroquinone and a regenerated phenolic as well as phenolic dimers. The phenolic radicals also react with other peroxyl radicals formed in termination reactions resulting in the formation of phenolic-peroxyl species. Although synthetic antioxidants are very effective in food systems, their application in the food industry has declined due to consumer safety concerns and demand for all-natural products.

6.3. Antioxidant Fish Peptides Proteins from various plant and animal sources and their hydrolysis products, individual peptides, and amino acids are documented to exhibit antioxidant activity against the peroxidation of lipids and fatty acid. From animal sources, these include hydrolyzed milk proteins (Pihlanto 2006), fish proteins (Je et al. 2005; Wu et al. 2003), egg proteins (Davalos et al.

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2004; Sakanaka et al. 2004), and pork protein (Carlsen et al. 2003; Saiga et al. 2003). Plant proteins possessing antioxidant activity include oilseed proteins (Rhee et al. 1979), wheat gliadin (Iwami et al. 1987), maize zein (Wang et al. 1991; Kong and Xiong 2006), and soybean protein (De Mejia and De Lumen 2006; Chen et al. 1995). Proteins, peptides, and amino acids have been utilized both in in vitro and in vivo systems to inhibit lipid oxidation. In linoleic acid and methyl linoleate model systems, some amino acids showed strong antioxidant activity (Marcuse 1960). The oxidation of corn oil was inhibited by a mixture of yeast and soybean protein hydrolysates (Bishov and Henick 1975). A combination of tryptophan and lysine was efficient in inhibiting the oxidation of butterfat (Merzametov and Gadzhieva 1976). Several amino acids have been shown to have antioxidant activity in vegetable oils: proline in sardine oil (Revankar 1974), methionine in vegetable oils (Sims and Fioriti 1977), and histidine, threonine, lysine, and methionine in sunflower oil (Riison et al. 1980). Tyrosine, methionine, histidine, tryptophan, and proline are reported to exhibit antioxidant activity except at high concentrations, when they behaved as prooxidants (Jung et al. 1995; Marcuse 1960). Antioxidative activity, in vivo, is implied for taurine, hypotaurine, carnosine, and anserine (Aruoma et al. 1988, 1989) and for modified amino acids like S-nitrosocysteine, produced during the curing of meat (Kanner 1979). Peptides derived from enzymatic digestion of various fish proteins are reported to possess antioxidant activity (Table 14.3) based on the nature, size, and composition of the different peptide fractions and the protease specificity. Chen et al. (1996) showed antioxidative activity by soy peptides composed of 3–16 amino acids including hydrophobic amino acids, valine, or leucine at the N-terminal positions, and proline, histidine, or tyrosine in the sequence. Peptides containing histidine demonstrated metal-ion chelation, active-oxygen quenching, and hydroxyl radical-scavenging activities as the modes of inhibiting lipid oxidation. The antioxidant behavior of peptides in general relies on the sequence of the amino acids in the peptide and in the concentration. Some nonfish studies

throw light on the mechanisms involved. Maize zein hydrolyzates made with alcalase or papain possesss strong radical-scavenging and Cu2+ chelation ability and reducing power, which depends on the hydrolysis time. The antioxidant activity of alcalasehydrolyzed zein was comparable to that of butylated hydroxyanisole, α-tocopherol, and ascorbate and was greater than that of intact zein (Kong and Xiong 2006). Interestingly, the ability of milk caseins to inhibit lipoxygenase-catalyzed lipid autoxidation and the quenching of free radicals by oxidation was due to casein rather than free amino acid residues (Laakso 1984). A peptide, Tyr-Phe-Tyr-Pro-GluLeu (YFYPEL), isolated from a peptic digest of casein has superoxide anion radical-scavenging activity attributed to the C-terminal dipeptide GluLeu (EL) sequence (Suetsuna et al. 2000). Kawashima et al. (1979) found that some synthetic peptides with branched-chain amino acids (Val, Leu, ILe) showed antioxidative activity. These branched amino acids are also present in antioxidant peptides derived from yellowfin sole protein (Jun et al. 2004), Alaska pollack protein (Je et al. 2005), shellfish (Jung et al. 2007), and the skin of hoki (Je et al. 2005; Mendis et al. 2005) Chen and Decker (1994) postulated that peptides containing basic amino acids accept electrons from radicals formed during the oxidation of unsaturated fatty acids and terminate the reaction. Thus the presence of lysine and histidine in the peptide derived from giant squid muscle (Rajapakse et al. 2005) and hoki skin (Je et al. 2005; Mendis et al. 2005), respectively, may contribute to the antioxidant behavior. The antioxidant activity of peptides may also be due to the presence of tyrosine at the N-terminal. Tyrosine is a potent hydrogen donor, a unique attribute for radical-scavenging antioxidants as observed in zein hydrolysates (Kong and Xiong 2006). Tyrosine increases the interaction between the peptide and the lipids, thereby effecting antioxidant behavior (Jun et al. 2004; Saeed et al. 2006). Yellowfin sole frame protein hydrolysates (YFPHs) were fractionated and resulted in an antioxidative peptide composed of 10 N-terminal amino acid residues, Arg-Pro-Ser-Phe-Ser-Leu-Glu-Pro-Pro-Tyr (RPSFSLEPPY), with molecular mass 13 kDa

Chapter 14 Bioactive Peptides and Proteins from Fish Muscle and Collagen

217

Table 14.3. Antioxidant peptides derived from fish proteins. Fish type

Source of Peptide

Capelin

Protein

Mackerel Alaska pollack

Protein Skin gelatin

Tuna Herring Mackerel

Cooking juice Protein

Yellowfin sole Alaska Pollack Hoki

Protein Protein Skin gelatin

Giant squid

Muscle

Chum salmon

Cartilage and skin Skin gelatin

Properase, pepsin

Mw < 2 kDa

Muscle protein Meat

Gastrointestinal enzymes Alcalase 2.4 L

LVGDEQAVPAVCVP

Jumbo flying squid Shell fish Yellow stripe trevally

Round scad

Grass carp Smooth hound (shark) Sole

Peptide Sequence

Scavenging Activity

Reference

Hydrolysates Repeating motif—GPP

N lipoxygenase (LOX)

Amarowicz and Shahidi 1997 Chuang et al. 2000 Kim et al. 2001

4–8 amino acid residues Hydrolysates

Carbon centered

Hydrolysates Protease N Alcalase, pronase E, collagenase Protease XXIII Protease N

Walleye Pollack Tuna

Hydrolytic Enzyme

Pepsin and MICE* MICE* Commercial enzymes Pepsin, trypsin chymotrypsin

Scavenging and reducing ability RPDFDLEPPY LPHSGY HGPLGPL NADFGLNGLEGLA NGLEGLK

Hydrolysates

Backbone protein Muscle

GI proteases, pronase, thermolysin Pepsin

Hydrolysates

VKAGFAWTANQQLS

Alcalase

Hydrolysates

Muscle Muscle

Alcalase 2.4 L GI proteases

PSKYEPFV Mw < 3.5 kDa

Skin gelatin

Alcalase

Mw < 6.5 kDa

Hydroxyl radical Superoxide, carbon-centered Radicals carboncentered, hydroxyl and superoxide Superoxide, hydroxyl, carbon centered Superoxide and hydroxyl Hydroxyl, super-oxide and carbon-centered Radical-scavenging, reducing, metal chelating Superoxide and hydroxyl radicals

Jao and Ko 2002 Sathivel et al. 2003 Wu et al., 2003 Jun et al. 2004 Je et al. 2005 Je et al. 2005; Mendis et al. 2005 Rajapakse et al. 2005 Nagai et al. 2006 Lin and Li 2006 Jung et al. 2007 Klompong et al. 2007 Nagai et al. 2007

Radical scavenging, superoxide Radical scavenging, reducing power, and Fe2+ chelating Hydroxyl Radical scavenging

Je et al. 2007

Metal chelating, Fe3+ reducing, radical scavenging

Giménze et al. 2009

Thiansilakul et al. 2007 Ren et al. 2008 Bougatef et al. 2009

* Mackerel intestine crude enzyme.

(Jun et al. 2004). Alaska pollack frame protein, hydrolyzed with mackerel intestine crude enzyme (MICE) and further purified to give a peptide LeuPro-His-Ser-Gly-Tyr (LPHSGY) with molecular weight 672 Da, also demonstrated hydroxyl radicalscavenging activity (Je et al. 2005). Other studies using proteolytic enzyme from Aspergillus oryzae

to hydrolyze tuna cooking juice resulted in seven antioxidative peptides comprising 4–8 amino acid residues and including Val, Ser, Pro, His, Ala, Asp, Lys, Glu, Gly, or Tyr. The isolated peptides demonstrated carbon-centered radical-scavenging effects in a 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging model (Jao and Ko 2002).

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Low molecular weight ultrafiltered peptides obtained from giant squid (Dosidicus gigas) muscle protein, namely Asn-Ala-Asp-Phe-Gly-Leu-AsnGly-Leu-Glu-Gly-Leu-Ala (NADFGLNGLEGLA) (1307 Da) and Asn-Gly-Leu-Glu-Gly-Leu-Lys (NGLEGLK) (747 Da), acted as chain-breaking antioxidants by inhibiting radical-mediated peroxidation of linoleic acid, similar to BHT. Electron spin trapping studies revealed that the peptides were potent scavengers of free carbon-centered, hydroxyl, and superoxide radicals, probably involving the hydrophobic amino acid content (more than 75%) (Rajapakse et al. 2005). Sathivel et al. (2003) showed that herring fish protein hydrolysate (FPH) obtained from alcalase hydrolysis of whole herring had antioxidative activity greater than the hydrolysate from the head and tail. A low molecular weight peptide with potent antioxidative activity was obtained from Mytilus coruscus muscle protein using an in vitro gastrointestinal digestion system. The potent antioxidant peptide was identified as Leu-Val-Gly-Asp-GluG l n - A l a - Va l - P r o - A l a - Va l - C y s - Va l - P r o (LVGDEQAVPAVCVP) (1.59 kDa) and exhibited higher protective activity against polyunsaturated fatty acid (PUFA) peroxidation than ascorbic acid and α-tocopherol. In a free radical–scavenging assay, the peptide quenched hydroxyl, superoxide, and carbon-centered radicals (Jung et al. 2007). A peptide with molecular weight 1.4 kDa obtained from the hydrolysis of mackerel (Scomber austriasicus) with Protease N inhibited the in vitro oxidation of linoleic acid and lipoxygenase (LOX)-catalyzed oxidation of arachidonic acid through radical-scavenging and reducing activities (Chuang et al. 2000; Wu et al. 2003). Antioxidative activity of protein hydrolysates from yellow stripe trevally (Selaroides leptolepis) muscle, hydrolyzed by Alcalase 2.4 L showed that the hydrolysate exhibited antioxidant activity through the radical-scavenging, reducing, and metal-chelating mode of lipid oxidation (Klompong et al. 2007). Enzymatic hydrolysates from walleye pollack (Theragra chalcogramma) containing large amounts of threonine and lysine exhibited strong antioxidative activities, scavenging activities against super-

oxide anion and hydroxyl radicals (Nagai et al. 2007). The antioxidative activity of the hydrolysates from round scad (Decapterus maruadsi) muscle involved radical-scavenging activity, reducing power, and Fe2+ chelating ability (Thiansilakul et al. 2007). Three peptide fractions from protein hydrolysates of capelin (Malfotus villosus) exhibited antioxidant activity in a β-carotene-linoleate model system. The fractions exhibited a maximum absorption at 254 nm and 260 nm, indicating that the fractions had reducing properties and may belong to the amine or thiol group of compounds (Shahidi and Amarowicz 1996; Amarowicz and Shahidi 1997). In addition to muscle proteins, only a few investigations are reported on collagen peptides. Gelatin extracted from Alaska pollack skin produced two peptides composed of 13 and 16 amino acid residues, respectively, that showed potent antioxidative activity on peroxidation of linoleic acid and enhanced cell viability. Both peptides contained a Glycine residue at the C-terminus and the repeating motif Gly-Pro-Hyp (Kim et al. 2001). Hoki (Johnius belengerii) skin gelatin hydrolyzed with commercial enzymes produced a peptide, His-Gly-Pro-Leu-GlyPro-Leu (HGPLGPL) (797 Da), and exhibited the high scavenging activities on superoxide and carbon-centered radicals assessed by electron spin resonance (ESR) spectroscopy against linoleic acid peroxidation similar to the synthetic antioxidant BHT (Je et al. 2005; Mendis et al. 2005). Studies with peptides containing histidine have demonstrated that these peptides can act as metal-ion chelators, active-oxygen quenchers, and hydroxyl radical scavengers (Pihlanto 2006). The presence of the amino acid histidine may be the reason for the antioxidant behavior of the peptide derived from the skin of hoki. Jumbo flying squid skin hydrolyzed with proteases and its peptide fraction (Mw < 2 kDa) showed scavenging effects on superoxide and hydroxyl radicals (Lin and Li 2006).

7. Conclusion Research in the field of food proteins and peptides to produce functional and neutraceutical products

Chapter 14 Bioactive Peptides and Proteins from Fish Muscle and Collagen

with highly specific activities like antihypertensive and antioxidant properties is growing rapidly. Included in this research activity are investigations to produce gelatin from fish that can match the gelling properties of beef and pork gelatin. Collagen, gelatin, and muscle proteins from a diverse range of fish species also offer the possibility of a vast array of bioactive peptides that may provide health benefits. While there have been numerous attempts to relate the structure of peptides to their activity, it is difficult to draw clear conclusions at present as a number of factors relating to peptide size, amino acid sequence, and concentration appear to be important. The ultimate goal would be to identify a peptide pattern for a particular purpose rather than produce peptides by trial and error.

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Chapter 14 Bioactive Peptides and Proteins from Fish Muscle and Collagen

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Chapter 15 Animal Muscle-Based Bioactive Peptides Jennifer Kovacs-Nolan and Yoshinori Mine

Contents 1. Introduction, 225 2. Animal Muscle-Based Bioactive Peptides, 225 3. Biological Activity and Therapeutic Applications, 226 3.1. Antioxidative Activity, 226 3.2. Antiglycation Effects, 227 3.3. Anti-inflammatory and Immune-Modulating Activity, 227

1. Introduction Animal muscle is a valuable source of protein, as well as important micronutrients such as iron, selenium, vitamins A and B12, and folic acid, that are either not present in plant-derived food or have poor bioavailability (Biesalski 2005). However, a number of bioactive substances have also been identified in animal muscle, including conjugated linoleic acid (CLA), carnosine, anserine, L-carnitine, glutathione, taurine, coenzyme Q10, and creatine, and have been shown to exert numerous health benefits (Arihara and Ohata 2008). These bioactive substances have potential applications for the development of novel nutraceuticals or functional meat-based products, and studies have shown that the muscle composition of these bioactive compounds can be altered simply by changing the feeding conditions

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

3.4. Antihypertensive and Cardiovascular Effects, 227 3.5. Neurological Effects, 228 3.6. Wound Healing, 228 3.7. Antiaging, 228 4. Conclusion, 228 5. References, 229

of the animals (Krajcovicova-Kudlackova et al. 2000). This chapter focuses on the physiological roles of animal muscle-based bioactive peptides and provides a brief overview of their potential benefits to human health.

2. Animal Muscle-Based Bioactive Peptides The histidyl dipeptides carnosine (β-alanyl-Lhistidine) and anserine (β-alanyl-1-methyl-Lhistidine) were first identified in extracts of skeletal muscles of vertebrates around the turn of the century (Quinn et al. 1992) and have been suggested to play roles in cellular homeostasis and maintenance. Skeletal muscle is susceptible to oxidative deterioration due to a combination of lipid oxidation catalysts and membrane lipid systems that are high in unsaturated fatty acids, therefore several endogenous antioxidant systems are present in muscle tissue (Chan and Decker 1994). These naturally occurring dipeptides are the most abundant antioxidant compounds 225

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in meats. The concentration of carnosine in meat ranges from 500 mg/kg of chicken thigh to 2,700 mg/ kg of pork shoulder, while anserine is particularly abundant in chicken muscle (Arihara and Ohata 2008). While these peptides are best known for their antioxidant activity, they have also been associated with a number of other health benefits, including exerting antiglycating, antihypertensive, anti-inflammatory, immunomodulating, and anticonvulsive activities and playing a role in wound healing and antiaging. L-carnitine (β-hydroxy-gamma-trimethyl-amino butyric acid), which is involved in the production of biological energy via the transport of long-chain fatty acids across inner mitochondrial membranes, occurs naturally in the muscle tissues of various animals and is most abundant in beef, where the concentration is around 1,300 mg/kg of the thigh (Arihara 2006). In addition to producing energy, L-carnitine has also been shown to possess additional biological activities. It was found to lower cholesterol levels and blood pressure (Seccombe et al. 1987; Shimura and Hasegawa 1993), prevent skeletal muscle apoptosis and muscle myopathy in heart failure (Vescovo et al. 2002), and it showed potential for treating fatigue in chronically ill patients (Harris 2008).

3. Biological Activity and Therapeutic Applications 3.1. Antioxidative Activity Muscle-based bioactive peptides are perhaps best known for their antioxidant activity, owing to their ability to chelate divalent metal ions, such as copper (Brown 1981) or to act as free radical scavengers (Dahl et al. 1988). The consumption of antioxidant-rich foods has been shown to have a preventative effect on oxidative damage in the body (Arihara 2006). It has been reported that reactive oxygen metabolites play important roles in the pathogenesis of a number of diseases including atherosclerosis, inflammation, neurodegenerative diseases, and diabetes (Derin et al. 2004). As such, the effects of muscle-based bioactive peptides on

oxidation-related disease states have been studied extensively. Carnosine is capable of preventing the accumulation of oxidized products derived from the lipid components of biological membranes (Dupin et al. 1984; Decker and Faraji 1990), and both carnosine and anserine have been found to potentiate the effects of lipid soluble antioxidants such as αtocopherol (Quinn et al. 1992; Chan et al. 1994). In addition, it has been shown that carnosine possesses superoxide dismutase activity and appears to regulate lipoxygenase activity (Guliaeva 1987). In vitro, carnosine and anserine were shown to inhibit the fragmentation of human serum albumin and the formation of hydroxyl radicals induced by peroxy radicals or hydrogen peroxide (Kang et al. 2002a, 2002b). Likewise, a combination of carnosine and anserine, purified from chicken extract, prevented protein degradation and human erythrocyte deformation caused by reactive oxygen species (Yanai et al. 2008). In rats, oral administration of L-carnitine strengthened the gastric mucosal barrier and prevented the development of mucosal lesions due to stress-induced lipid peroxidation (Izgut-Uysal et al. 2001). Similarly, pretreatment with carnitine protected gastric mucosa from ischemia-reperfusion injury by decreasing the effects of lipid peroxidation in rats (Derin et al. 2004). More recently, glycosidic derivatives of carnosine and anserine, derivatized with beta-cyclodextrin, were synthesized and found to possess higher antioxidant activity than their free dipeptide counterparts and resistance to carnosinase hydrolysis, indicating the potential of muscle-based peptide derivatives as antioxidant nutraceuticals (Bellia et al. 2008). Combinations of histidine-containing dipeptides at near-physiological concentrations have been found to result in synergistic antioxidant activity (MacFarlane et al. 1991); and recently an extraction of chicken breast obtained by hot water extraction, containing carnosine and anserine, was found to increase brain concentrations of both of these dipeptides, promote the activities of antioxidant enzymes, and protect the liver from oxidative stress in rats (Peng and Lin 2004; Tomonaga et al. 2007; Sato et al. 2008).

Chapter 15 Animal Muscle-Based Bioactive Peptides

3.2. Antiglycation Effects The carbonyl groups of reducing sugars react with side-chain amino groups of proteins to give a variety of products. Further glycoxidative degradation of these glycated proteins leads to the formation of protein-protein crosslinks, also known as advanced glycation end products (AGEs). Evidence suggests that AGEs may be involved in the pathological progression of age-related diseases such as diabetes, cataracts, atherosclerosis, and Alzheimer ’s disease (Reddy et al. 2005). Carnosine has been shown to have AGE inhibitory action, as protein-protein crosslinks induced by methylglyoxal, an oxoaldehyde formed during the Maillard reaction, were found to be eliminated in the presence of carnosine (Hobart et al. 2004). In vitro, Hipkiss et al. (1998) showed that carnosine could protect cultured human fibroblasts, lymphocytes, and rat brain endothelial cells against the effects of AGEs. Furthermore, carnosine has shown promise for the ophthalmic treatment of cataracts, in both rats (Yan et al. 2008) and humans, in clinical trials (Babizhayev and Kasus-Jacobi 2009), by inhibiting the formation of AGEs; and it may have therapeutic potential for preventing diabetes-induced atherosclerosis by inhibiting the glycation of low-density lipoproteins (LDL), which can result in the cellular accumulation of cholesterol in macrophages (Rashid et al. 2007).

3.3. Anti-inflammatory and Immune-Modulating Activity There is a growing body of evidence that suggests that chronic inflammation, which is often linked to defects in the innate immune system, may be at the root of many chronic diseases, including heart disease, diabetes, metabolic syndrome, and cancer (Haffner 2006; Lin and Karin 2007). Son et al. (2005) found that carnosine could suppress the production of the pro-inflammatory cytokine interleukin (IL)-8 by intestinal epithelial cells stimulated with hydrogen peroxide or tumor necrosis factor (TNF)-α, suggesting that carnosine may be a novel anti-inflammatory agent that downregu-

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lates the inflammatory response in intestinal epithelial cells. Carnosine and anserine increased in vitro IL-1β production, a key cytokine involved in innate immunity, by human neutrophils (Tan and Candlish 1998); and L-carnitine increased neutrophil function, T-cell proliferative responses, and antibody concentrations in aged rats (Thangasamy et al. 2008), as well as reduced apoptosis, suggesting the capacity of these peptides to enhance innate and adaptive immune functions and regulate immune homeostasis. Carnosine also inhibited oxygen-glucose deprivation-induced degranulation and histamine release from cultured mast cells (Shen et al. 2008), indicating that carnosine can protect mast cells from degranulation and may play a role in antiallergy processes. Furthermore, polaprezinc, a compound consisting of carnosine and zinc, reduced neutrophil accumulation and expression of the inflammatory cytokine TNF-α in rats with aspirin-induced mucosal injury (Naito et al. 2001); and it attenuated Helicobacter pylori–induced gastric mucosal leukocyte activation and IL-8 production (Suzuki et al. 2001; Handa et al. 2002), further suggesting its therapeutic potential to treat inflammatory conditions.

3.4. Antihypertensive and Cardiovascular Effects One of the first reported observations of the biological effects of muscle-based peptides was the hypotensive effect of carnosine in mammals (Quinn et al. 1992). More recently, it has been shown that the administration of L-carnitine reduces blood pressure and attenuates the inflammatory process associated with arterial hypertension (Miguel-Carrasco et al. 2008). Ririe et al. (2000) reported that carnosine caused vascular relaxation in isolated arterial vessels by a nonendothelial-dependent cyclic GMP (cGMP) mechanism and suggested that carnosine supplementation may be an effective strategy for reducing blood pressure for the treatment of hypertension. Consistent with these findings, Miller and O’Dowd (2000) observed that in vitro, carnosine caused relaxation of vascular smooth muscle, although this

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Part 3 Examples of Food Proteins and Peptides with Biological Activity

effect was dependent on the presence of specific histamine receptors. Angiotensin-converting enzyme (ACE), a zinccontaining metaloprotease, is one of the key enzymes involved in regulating blood pressure. Carnosine and anserine have both demonstrated dose-dependent ACE-inhibitory activities (Hou et al. 2003), which may in part be due to their ability to bind zinc ions (Miller and O’Dowd 2000).

3.5. Neurological Effects It has been suggested that another possible role for carnosine and anserine may be as putative neurotransmitters in the olfactory bulbs, where they may play a modulatory role or function as co-transmitters (Urazaev et al. 1998). Carnosine and anserine are known to enhance neuromuscular transmission, and both dipeptides are localized at the neuromuscular junction (Urazaev et al. 1998). In vitro carnosine protected brain neurons (Boldyrev and Abe 1999; Tabakman et al. 2002) and nonneuronal cells (Kang et al. 2002c; Boldyrev et al. 2004) against oxidative injury, and carnosine has demonstrated neuroprotective effects in rodent models of stroke (Min et al. 2008; Yasuhara et al. 2008). Carnosine has been shown to possess anticonvulsant effects (Wu et al. 2006; Zhu et al. 2007; Kozan et al. 2008), which may be due to its chelating effects or downstream metabolites of carnosine (i.e., histamine), which plays a key role in the regulation of seizure activity and therefore may be useful in the treatment of epilepsy. Carnosine supplementation was also shown to be a promising treatment for children with autistic spectrum disorders, where it significantly improved the receptive speech, socialization, and behavior in children with autism (Chez et al. 2002). Moreover, carnosine has demonstrated antidepressant-like effects in rats (Tomonaga et al. 2008).

3.6. Wound Healing Another reported property of muscle-based dipeptides is their ability to accelerate the healing of

wounds and ulcers (Quinn et al. 1992). The ability of carnosine to stimulate the epithelialization of gastric and duodenal ulcers was first demonstrated more than 70 years ago (Frolov et al. 1936). More recently polaprezinc was shown to enhance the repair of gastric lesions and stabilize gut integrity, via the upregulation of insulin-like growth factor I and stimulation of epithelial cell restoration (Seto et al. 1999; Kato et al. 2001; Mahmood et al. 2007). Carnosine also enhanced expression of transcription factors involved in cell differentiation and formation of periodontal tissues (Ito-Kato et al. 2004), and it has been suggested that it may ameliorate irradiation-induced lung injury in cancer patients (Guney et al. 2006). Furthermore, carnosine has been shown to stimulate the expression of vimentin in cultured rat fibroblasts, which is thought to play a fundamental role in maintaining cell structure and integrity (Ikeda et al. 1999).

3.7. Antiaging Levels of histidine dipeptides in muscle have been shown to decrease with age (Johnson and Hammer 1992). Carnosine has been shown to suppress cell senescence (McFarland and Holliday 1994), reverse protein-aldehyde crosslinking, induce rejuvenating effects (McFarland and Holliday 1999), and protect against telomere shortening (Shao et al. 2004) in cultured human fibroblasts. Moreover, it was found to extend the lifespan of senescence-accelerated mice (Yuneva et al. 1999), indicating the antiaging effects of muscle-based bioactive peptides.

4. Conclusion Muscle-based bioactive peptides have been shown to possess many health benefits, and are therefore promising candidates as nutraceutical or functional food ingredients. Since meat and meat products are important in the diet in most developed countries, functional meat products would contribute to human health (Arihara and Ohata 2008). Emphasizing the biological activities of muscle-based peptides is one possible approach for improving the health image of

Chapter 15 Animal Muscle-Based Bioactive Peptides

meat and developing functional meat products (Arihara 2006). Further studies will be required to demonstrate the clear benefits of muscle-derived bioactive peptides and their potential nutraceutical applications to benefit human health.

5. References Arihara K. 2006. Strategies for designing novel functional meat products. Meat Sci 74:219–229. Arihara K, Ohata M. 2008. Bioactive compounds in meat. In: F Toldra, ed., Meat Biotechnology, 231–249. New York: Springer. Babizhayev MA, Kasus-Jacobi A. 2009. State of the art clinical efficacy and safety evaluation of N-acetylcarnosine dipeptide ophthalmic prodrug. Principles for the delivery, self-bioactivation, molecular targets and interaction with a highly evolved histidyl-hydrazide structure in the treatment and therapeutic management of a group of sight-threatening eye diseases. Curr Clin Pharmacol 4(1):4–37. Bellia F, Amorini AM, La Mendola D, Vecchio G, Tavazzi B, Giardina B, Di Pietro V, Lazzarino G, Rizzarelli E. 2008. New glycosidic derivatives of histidine-containing dipeptides with antioxidant properties and resistant to carnosinase activity. Eur J Med Chem 43(2):373–380. Biesalski H-K. 2005. Meat as a component of a healthy diet—Are there any risks or benefits if meat is avoided in the diet? Meat Sci 70:509–524. Boldyrev A, Abe H. 1999. Metabolic transformation of neuropeptide carnosine modifies its biological activity. Cell Mol Neurobiol 19(1):163–175. Boldyrev A, Bulygina E, Leinsoo T, Petrushanko I, Tsubone S, Abe H. 2004. Protection of neuronal cells against reactive oxygen species by carnosine and related compounds. Comp Biochem Physiol B Biochem Mol Biol 137(1):81–88. Brown CE. 1981. Interactions among carnosine, anserine, ophidine and copper in biochemical adaptation. J Theor Biol 88(2):245–256. Chan KM, Decker EA. 1994. Endogenous skeletal muscle antioxidants. Crit Rev Food Sci Nutr 34(4):403–426. Chan WK, Decker EA, Chow CK, Boissonneault GA. 1994. Effect of dietary carnosine on plasma and tissue antioxidant concentrations and on lipid oxidation in rat skeletal muscle. Lipids 29(7):461–466. Chez MG, Buchanan CP, Aimonovitch MC, Becker M, Schaefer K, Black C, Komen J. 2002. Double-blind, placebo-controlled study of L-carnosine supplementation in children with autistic spectrum disorders. J Child Neurol 17(11):833–837. Dahl TA, Midden WR, Hartman PE. 1988. Some prevalent biomolecules as defenses against singlet oxygen damage. Photochem Photobiol 47(3):357–362. Decker EA, Faraji H. 1990. Inhibition of lipid oxidation by carnosine. J Amer Oil Chem Soc 67:650–652.

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Derin N, Izgut-Uysal VN, Agac A, Aliciguzel Y, Demir N. 2004. L-carnitine protects gastric mucosa by decreasing ischemiareperfusion induced lipid peroxidation. J Physiol Pharmacol 55(3):595–606. Dupin AM, Boldyrev AA, Arkhipenko YV, Kagan VE. 1984. Carnosine affords protection of Ca2+ transport in lipid peroxidation induced damage. Bull Exp Biol Med USSR 97:186–188. Frolov PF, Gikik AS, Normark PR, Umanskaja SB, Dikanskaja EM, Marcus IM, Vorobjev AM, Boguslavskaja EF, Sokdovskaja NI, Taratushkina KK, Rozencveig BK, Ioselevich NI. 1936. Treatment of stomach and duodenal ulcer by Ca2+ionophoresis and diathermia alone or in combination with carnosine. Exp Med (UkSSR) N2:67–78. Guliaeva NV. 1987. Superoxide-scavenging activity of carnosine in the presence of copper and zinc ions. Biokhimiia 52(7): 1216–1220. Guney Y, Turkcu UO, Hicsonmez A, Andrieu MN, Guney HZ, Bilgihan A, Kurtman C. 2006. Carnosine may reduce lung injury caused by radiation therapy. Med Hypotheses 66(5):957–959. Haffner SM. 2006. The metabolic syndrome: Inflammation, diabetes mellitus, and cardiovascular disease. Amer J Cardiol 97(2A):3–11A. Handa O, Yoshida N, Tanaka Y, Ueda M, Ishikawa T, Takagi T, Matsumoto N, Naito Y, Yoshikawa T. 2002. Inhibitory effect of polaprezinc on the inflammatory response to Helicobacter pylori. Can J Gastroenterol 16(11):785–789. Harris JD. 2008. Fatigue in chronically ill patients. Curr Opin Support Palliat Care 2(3):180–186. Hipkiss AR, Preston JE, Himsworth DT, Worthington VC, Keown M, Michaelis J, Lawrence J, Mateen A, Allende L, Eagles PA, Abbott NJ. 1998. Pluripotent protective effects of carnosine, a naturally occurring dipeptide. Ann NY Acad Sci 854:37–53. Hobart LJ, Seibel I, Yeargans GS, Seidler NW. 2004. Anticrosslinking properties of carnosine: Significance of histidine. Life Sci 75(11):1379–1389. Hou WC, Chen HJ, Lin YH. 2003. Antioxidant peptides with angiotensin converting enzyme inhibitory activities and applications for angiotensin converting enzyme purification. J Agric Food Chem 51(6):1706–1709. Ikeda D, Wada S, Yoneda C, Abe H, Watabe S. 1999. Carnosine stimulates vimentin expression in cultured rat fibroblasts. Cell Struct Funct 24(2):79–87. Ito-Kato E, Suzuki N, Maeno M, Takada T, Tanabe N, Takayama T, Ito K, Otsuka K. 2004. Effect of carnosine on runt-related transcription factor-2/core binding factor alpha-1 and Sox9 expressions of human periodontal ligament cells. J Periodontal Res 39(3):199–204. Izgut-Uysal VN, Agac A, Derin N. 2001. Effect of carnitine on stress-induced lipid peroxidation in rat gastric mucosa. J Gastroenterol 36(4):231–236. Johnson P, Hammer JL. 1992. Histidine dipeptide levels in ageing and hypertensive rat skeletal and cardiac muscles. Comp Biochem Physiol B 103(4):981–984.

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Kang JH, Kim KS, Choi SY, Kwon HY, Won MH, Kang TC. 2002b. Protection by carnosine-related dipeptides against hydrogen peroxide–mediated ceruloplasmin modification. Mol Cells 13(1):107–112. ———. 2002a. Protective effects of carnosine, homocarnosine and anserine against peroxyl radical-mediated Cu,Znsuperoxide dismutase modification. Biochim Biophys Acta 1570(2):89–96. Kang KS, Yun JW, Lee YS. 2002c. Protective effect of L-carnosine against 12-O-tetradecanoylphorbol-13-acetate- or hydrogen peroxide–induced apoptosis on v-myc transformed rat liver epithelial cells. Cancer Lett 178(1):53–62. Kato S, Tanaka A, Ogawa Y, Kanatsu K, Seto K, Yoneda T, Takeuchi K. 2001. Effect of polaprezinc on impaired healing of chronic gastric ulcers in adjuvant-induced arthritic rats— Role of insulin-like growth factors (IGF)-1. Med Sci Monit 7(1):20–25. Kozan R, Sefil F, Bagirici F. 2008. Anticonvulsant effect of carnosine on penicillin-induced epileptiform activity in rats. Brain Research 1239:249–255. Krajcovicova-Kudlackova M, Simoncic R, Bederova A, Babinska K, Beder I. 2000. Correlation of carnitine levels to methionine and lysine intake. Physiol Res 49(3):399–402. Lin WW, Karin M. 2007. A cytokine-mediated link between innate immunity, inflammation, and cancer. J Clin Invest 117(5):1175–1183. MacFarlane N, McMurray J, O’Dowd JJ, Dargie HJ, Miller DJ. 1991. Synergism of histidyl dipeptides as antioxidants. J Mol Cell Cardiol 23(11):1205–1207. Mahmood A, FitzGerald AJ, Marchbank T, Ntatsaki E, Murray D, Ghosh S, Playford RJ. 2007. Zinc carnosine, a health food supplement that stabilises small bowel integrity and stimulates gut repair processes. Gut 56(2):168–175. McFarland GA, Holliday R. 1999. Further evidence for the rejuvenating effects of the dipeptide L-carnosine on cultured human diploid fibroblasts. Exp Gerontol 34(1):35–45. ———. 1994. Retardation of the senescence of cultured human diploid fibroblasts by carnosine. Exp Cell Res 212(2):167–175. Miguel-Carrasco JL, Mate A, Monserrat MT, Arias JL, Aramburu O, Vazquez CM. 2008. The role of inflammatory markers in the cardioprotective effect of L-carnitine in L-NAME–induced hypertension. Amer J Hypertens 21(11):1231–1237. Miller DJ, O’Dowd A. 2000. Vascular smooth muscle actions of carnosine as its zinc complex are mediated by histamine H1 and H2 receptors. Biochem (Moscow) 65(7):938–948. Min J, Senut MC, Rajanikant K, Greenberg E, Bandagi R, Zemke D, Mousa A, Kassab M, Farooq MU, Gupta R, Majid A. 2008. Differential neuroprotective effects of carnosine, anserine, and N-acetyl carnosine against permanent focal ischemia. J Neurosci Res 86(13):2984–2991. Naito Y, Yoshikawa T, Yagi N, Matsuyama K, Yoshida N, Seto K, Yoneta T. 2001. Effects of polaprezinc on lipid peroxidation, neutrophil accumulation, and TNF-alpha expression in rats with aspirin-induced gastric mucosal injury. Dig Dis Sci 46(4):845–851.

Peng HC, Lin SH. 2004. Effects of chicken extract on antioxidative status and liver protection under oxidative stress. J Nutr Sci Vitaminol (Tokyo) 50(5):325–329. Quinn PJ, Boldyrev AA, Formazuyk VE. 1992. Carnosine: Its properties, functions and potential therapeutic applications. Mol Aspects Med 13(5):379–444. Rashid I, van Reyk DM, Davies MJ. 2007. Carnosine and its constituents inhibit glycation of low-density lipoproteins that promotes foam cell formation in vitro. FEBS Lett 581(5): 1067–1070. Reddy VP, Garrett MR, Perry G, Smith MA. 2005. Carnosine: A versatile antioxidant and antiglycating agent. Sci Aging Knowledge Environ 2005(18):pe12. Ririe DG, Roberts PR, Shouse MN, Zaloga GP. 2000. Vasodilatory actions of the dietary peptide carnosine. Nutrition 16(3):168–172. Sato M, Karasawa N, Shimizu M, Morimatsu F, Yamada R. 2008. Safety evaluation of chicken breast extract containing carnosine and anserine. Food Chem Toxicol 46(2):480–489. Seccombe DW, James L, Hahn P, Jones E. 1987. L-carnitine treatment in the hyperlipidemic rabbit. Metabolism 36(12): 1192–1196. Seto K, Yoneta T, Suda H, Tamaki H. 1999. Effect of polaprezinc (N-(3-aminopropionyl)-L-histidinato zinc), a novel antiulcer agent containing zinc, on cellular proliferation: Role of insulinlike growth factor I. Biochem Pharmacol 58(2):245–250. Shao L, Li QH, Tan Z. 2004. L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts. Biochem Biophys Res Commun 324(2):931–936. Shen Y, Zhang S, Fu L, Hu W, Chen Z. 2008. Carnosine attenuates mast cell degranulation and histamine release induced by oxygen-glucose deprivation. Cell Biochem Funct 26(3):334– 338. Shimura S, Hasegawa T. 1993. Changes of lipid concentrations in liver and serum by administration of carnitine added diets in rats. J Vet Med Sci 55(5):845–847. Son DO, Satsu H, Shimizu M. 2005. Histidine inhibits oxidative stress- and TNF-alpha-induced interleukin-8 secretion in intestinal epithelial cells. FEBS Lett 579(21):4671–4677. Suzuki H, Mori M, Seto K, Miyazawa M, Kai A, Suematsu M, Yoneta T, Miura S, Ishii H. 2001. Polaprezinc attenuates the Helicobacter pylori–induced gastric mucosal leucocyte activation in Mongolian gerbils—a study using intravital videomicroscopy. Aliment Pharmacol Ther 15(5):715–725. Tabakman R, Lazarovici P, Kohen R. 2002. Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia. J Neurosci Res 68(4):463–469. Tan KM, Candlish JK. 1998. Carnosine and anserine as modulators of neutrophil function. Clin Lab Haematol 20(4):239– 244. Thangasamy T, Subathra M, Sittadjody S, Jeyakumar P, Joyee AG, Mendoza E, Chinnakkanu P. 2008. Role of L-carnitine in the modulation of immune response in aged rats. Clin Chim Acta 389(1–2):19–24.

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Tomonaga S, Hayakawa T, Yamane H, Maemura H, Sato M, Takahata Y, Morimatsu F, Furuse M. 2007. Oral administration of chicken breast extract increases brain carnosine and anserine concentrations in rats. Nutr Neurosci 10(3–4):181– 186. Tomonaga S, Yamane H, Onitsuka E, Yamada S, Sato M, Takahata Y, Morimatsu F, Furuse M. 2008. Carnosine-induced antidepressant-like activity in rats. Pharmacol Biochem Behav 89(4):627–632. Urazaev A, Naumenko NV, Nikolsky EE, Vyskocil F. 1998. Carnosine and other imidazole-containing compounds enhance the postdenervation depolarization of the rat diaphragm fibres. Physiol Res 47(4):291–295. Vescovo G, Ravara B, Gobbo V, Sandri M, Angelini A, Della Barbera M, Dona M, Peluso G, Calvani M, Mosconi L, Dalla Libera L. 2002. L-Carnitine: A potential treatment for blocking apoptosis and preventing skeletal muscle myopathy in heart failure. Amer J Physiol Cell Physiol 283(3):C802– 810. Wu XH, Ding MP, Zhu-Ge ZB, Zhu YY, Jin CL, Chen Z. 2006. Carnosine, a precursor of histidine, ameliorates

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pentylenetetrazole-induced kindled seizures in rat. Neurosci Lett 400(1–2):146–149. Yan H, Guo Y, Zhang J, Ding Z, Ha W, Harding JJ. 2008. Effect of carnosine, aminoguanidine, and aspirin drops on the prevention of cataracts in diabetic rats. Mol Vis 14:2282–2291. Yanai N, Shiotani S, Hagiwara S, Nabetani H, Nakajima M. 2008. Antioxidant combination inhibits reactive oxygen species mediated damage. Biosci Biotechnol Biochem 72(12): 3100–3106. Yasuhara T, Hara K, Maki M, Masuda T, Sanberg CD, Sanberg PR, Bickford PC, Borlongan CV. 2008. Dietary supplementation exerts neuroprotective effects in ischemic stroke model. Rejuvenation Res 11(1):201–214. Yuneva MO, Bulygina ER, Gallant SC, Kramerenko GG, Stvolinsky SL, Semyonova ML, Boldyrev AA. 1999. Effect of carnosine on age-induced changes in senescence-accelerated mice. J Anti-Aging Med 2:337–342. Zhu YY, Zhu-Ge ZB, Wu DC, Wang S, Liu LY, Ohtsu H, Chen Z. 2007. Carnosine inhibits pentylenetetrazol-induced seizures by histaminergic mechanisms in histidine decarboxylase knock-out mice. Neurosci Lett 416(3):211–216.

Chapter 16 Processing and Functionality of Rice Bran Proteins and Peptides Rashida Ali, Frederick F. Shih, and Mian Nadeem Riaz

Contents 1. Introduction, 233 2. Stabilization of Rice Bran, 234 3. Extraction and Isolation, 235 3.1. Alkali Extraction, 236 3.2. Enzymatic Extraction, 236 3.3. Miscellaneous Methods, 236 4. Functional Properties, 237 5. Enzymes Isolated from Rice Bran, 238 6. Rice Bran Proteins in Product Development, 238 6.1. The RBPs as Flavor Enhancers, 238 6.2. RBPs in Baby Foods, 239 6.3. RBPs in Bakery Products, 239 6.4. RBPs as Food-Color Carriers, 239

1. Introduction Cereals are the most widely grown crops of the world and the kernel or caryopsis consists of bran, embryo, and endosperm. The components of bran are largely pericarp, germ, testa, and nuclear and aleurone layers (Figure 16.1). The bran is composed of fibers (pentosans, hemicelluloses, β-glucans, cellulose, legnin, and glucofructans), ash, enzymes, vitamins, and storage proteins. The abrasive milling of rice (oryzae sativum) to finally produce white rice

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

6.5. 6.6. 6.7. 6.8. 6.9. 7. 7.1. 7.2. 7.3. 8. 9. 10.

RBPs in Meat Products, 239 RBPs in Animal Feeds, 239 Rice Bran as Fat Replacers, 239 Rice Bran as Substrate for Microbial Growth, 240 Miscellaneous Applications, 240 Health Benefits of Rice Bran Proteins, 240 Role of RBPs in Prevention and Control of Cancers, 241 Rice Bran Lectins, 241 RBPs as Antioxidants, 242 Antinutritional Behavior of RBPs, 242 Future Trends/Conclusion, 242 References, 243

provides valuable by-products such as hull and the outer brown layer bran. Approximately 617 million metric tons of rice are produced annually worldwide, and more than half of the world production is from Asia (IRRI 2005). In the United States, about 0.52 million tons of rice bran are produced from 6.5 million tons of rice. The bran comprises approximately 10% of the rough rice kernel, and its germ is a rich source of oil, while defatted bran consists of proteins, minerals, vitamins, and fibers or the indigestible carbohydrates. The composition of native and defatted rice bran (RB) is given in Tables 16.1 and 16.2. The quality and quantity of bran components, color, and nutritive value vary with the degree of milling (DOM). The hardness increases from outer to inner 233

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layer, while the yellow and red pigments decrease. About 61% of the minerals are located in bran with DOM < 9% (Lamberts et al. 2007). The rice bran proteins (RBPs) have shown great potentials for use in various foods (Prakash 1996). Cereal proteins provide half of the daily per capita requirement of the human population. Cereal

beard

hull

aleurone layer

grain proteins, in general, are rich in the sulfurcontaining amino acids including cystine, cysteine, and methionine, with lysine as the limiting amino acid. Although rice has the lowest protein mass (7.3%) among the common cereals such as wheat (10.6%), corn (9.8%) barley (11.0%), millet (11.5%), and oat (12.6%), lysine content of rice proteins is highest (3.8 g/16 g of N) as compared to other cereal proteins such as wheat and corn (2.3 and 2.5 g/16 g of N, respectively). Rice bran provides a substantial amount of protein ranging from 12 to 20%, which is rated as high-quality protein for many of the explored and unexplored potentials in value additions (Saunders 1990). Recently the chemical composition of various fractions of rice bran has been reported (AbdulHamid et al. 2007). However, it is observed that there is huge variation in the crude protein as well as amino acid content in rice bran (Madsen 2008).

2. Stabilization of Rice Bran

starchy endosperm

During the rice milling process, the oil and lipase enzyme present in the rice bran come into mutual contact, resulting in the rapid hydrolysis of the triglycerides of the oil into glycerol and free fatty acids

germ

Table 16.1. Composition of rice bran and rice flour (Shih et al. 1999). aleurone layer

Bran Composition

starchy endosperm

hull bran polish Figure 16.1. Parts of rice kernel (www.riviana.com/images/ img_rf_04.jpg).

Flour [g/100 g]

Protein (N × 5.95) Carbohydrate Oil Moisture Ash Crude fiber

12.6 40.0 12.8 10.5 14.5 7.8

8.1 80.9 0.4 10.1 0.6 0.4

Table 16.2. Proximate composition and caloric content of defatted rice brans (Saunders 1990.)

Defatted bran Defatted parboiled bran (without calcium carbonate)

Moisture (%)

Protein (%)

Fat (%)

Fiber (%)

Ash (%)

Calories (oz)

6–9 6.9

15–20 23–27

0.5–1.5 0.5–1.5

10–15 16–20

9–12 11–14

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Chapter 16 Processing and Functionality of Rice Bran Proteins and Peptides

(Carrol 1990). The lipase must be denatured in the rice bran before its contact with the oil to avoid rancidity in foods (Saunders 1990). Once rice bran has been stabilized it can be stored for 30–60 days without a significant increase in free fatty acid level (Randall et al. 1985). Different thermal-processing methods can be used to stabilize the rice bran, including the use of extruders, pellet cookers, and expanders. One method of stabilizing the rice bran is dry extrusion that utilizes a low-cost extruder that does not require high moisture and is operated with high mechanical energy input (Riaz 2000). Other methods of rice bran stabilization include the use of a twin-screw extruder, the universal pellet cooker, a pellet mill, and the expander cooker. Wet extrusion utilizes steam generated from water added and the bran requires drying prior to oil extraction. The pellet cooker is used for the pelleted feed industry, allows the rice bran to be processed at low moistures (14–17%), and requires medium temperatures (110–130°C) to inactivate the enzymes (Thomas 2005). An expander cooker subjects the rice bran to high-pressure cooking, utilizing live steam and water, to elevate the moisture and temperature and cause the agglomeration of the fine particles into porous collets (Williams 1988). In general, the expander produces an unshaped pellet, while the extruder produces a shaped pellet (Lucht 2007). By comparing the different methods of rice bran stabilization, it was found that rice bran stabilized with pellet cooker exhibited superior results regarding the stability, oil extraction, and nutrient destruction (Riaz et al. 2008).

3. Extraction and Isolation Cereal proteins in early stages were classified on the basis of solvent extraction using water, salt, and alcohol or weak acid/alkali, according to the Osborne classification, as albumin, globulins, prolamins, and glutelins, respectively (Osborne 1924). The remaining insoluble portion is referred as residual proteins that later on became functionally indispensable in baking, frying, and extrusion products. The concentration of various RBPs is represented in Figure 16.2.

235

Figure 16.2. Proteins (%) consecutively solubilized with water, salt, alcohol, acetic acid, and sodium hydroxide from ether-defatted brans of several rice cultivators. Insoluble residue protein is the portion of residue protein not soluble in 0.1 M NaOH. (Hamada et al. 1997.)

Solubilization is the initial step for the utilization of RBPs. Exploring effective reproducible, economical, and efficient methods for extraction of RBPs has been a challenge for investigators in view of the poor solubility of bran proteins because of the presence of extensive disulfide linkages, aggregations, and entanglements. Although a number of methods for isolation are reported, alkaline and enzymatic extractions are widely used followed by isoelectric precipitation. The hydrophobicity of insoluble glutelins may be decreased by use of several dissociating agents such as sodiumdodecyl sulfate (SDS), sodium stearate, and cetyltrimethylammonium bromide or other surfactants (Hamada 1997). Apart from hydrophobic domains present in glutelins, the insolubility of RBPs is also attributed to repeated disulfide bonding, glycosylation, and numerous noncovalent interactions (Wen and Luthe 1985). The rice proteins were extracted as early as 1956 (McIntyre and Kymal 1956), and a comprehensive account of the extraction methods for rice bran proteins from defatted rice bran is documented about 4 decades earlier (Chen and Houston 1970). The removal of fat from bran before extraction has revealed certain advantages, such as higher solubilization of proteins in view of their mild hydrolysis, better stability, and superior quality, as the color/ phonolics were eliminated. However, some investigators have preferred extraction of proteins from whole bran. The complete extraction of RBPs is

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fairly impossible. It seems that applying the molecular bases of solubility is more practical to promote the isolation of proteins for value addition, innovation, and utilization of rice bran in food processing (Hamada 1997).

3.1. Alkali Extraction The extraction using alkaline medium is the most primitive method and is quite conventional. In spite of its limitations, it has been used widely to obtain better yield of the proteins (Lew et al. 1975). The alkali extraction induces conformational changes and nutritional losses due to protein reactions, as discussed later in this chapter. The RBP concentrates produced by alkaline extraction had increased nitrogen solubility at higher and lower pH than its isoelectric point (pH 4.5). It has been shown that nitrogen solubility index (NSI) is also related to hydrophobicity and emulsifying capacity, which increases with increased NSI (Bera and Mukherjee 1989). The alkaline extraction and isoelectric precipitation followed by papain hydrolysis produced the peptides with superior solubility, emulsion activity index (EAI), emulsion stability index (ESI), foaming capacity, and foaming stability (Bandyopadhyay et al. 2008). The quantity and performance of the peptides depend on the method of isolation and the enzymes used. It seems that a combination of several methods is more successful.

3.2. Enzymatic Extraction The use of enzymes to enhance RBP extraction was first introduced by Grosman and co-workers in 1980 (Grosman et al. 1980). Hydrolyzing carbohydrates in bran released the protein in solution. The commercial carbohydrases have been used successfully in degrading the cell wall carbohydrates composed of hemicelluloses, celluloses, and xylans (Ansharullah et al. 1997). Although starch is a component of endosperm, it could be present in bran depending on the process of separation. The combined treatment of α-amylase, hemicellulase, and cellulase resulted in 10–27% extraction of proteins. The approach of using a diversified range of carbo-

hydrases was therefore found to be more productive (Shih et al. 1999), providing 25% of the quality proteins. Similar studies show that three different kinds of enzymes including cell-wall breaking enzymes, amylases, and proteases have been found effective in increasing the yield of proteins during extraction. Some of the enzymes used include celluclast, hemicellulase, pectinase ultra SPL, viscozyme L, alcalase, and papain (Hanmoungjai et al. 2002). Multiple enzyme application resulted in the greater production of small peptides and free amino acids, which may cause adverse textural changes. The potentials of applying limited proteolysis for bran protein extraction have been evaluated by Hamada (2000a, 2000b, 1999). The endoprotease mixture consisting of trypsinchymotrypsin was used to serve dual purposes; that is, hydrolyzing the endogeneous lipases responsible for hydrolytic-oxidative damage to bran oil by producing free fatty acids that induce rancidity, while simultaneous release of increased quantity of partially hydrolyzed proteins was also recorded (Parrado et al. 2006). That may be helpful in product development. Tang et al. (2003) used food-grade pectinase II and protease P to prepare protein isolate consisting of 90% protein with nutraceutical properties.

3.3. Miscellaneous Methods A novel method of colloidal milling and homogenization was developed to disperse the rice bran particles to ease the protein extraction from defatted (DF) full-fat-stabilized (FFS) and full-fat-unstabilized bran (FFU). The highest protein extraction was obtained in FFU, where an increase of 21.8–33% was possible applying colloid milling, which if followed by homogenization, further enhanced the quantity of proteins up to 38.2%. The process overall increased protein extractability by 75% (Anderson and Guraya 2001). Further treatment with amylase or a mixture of amylase and protease increased the protein extractability by 25.6–33.9% and 54.0–57.8%, respectively (Tang et al. 2002, 2003). Recently the application of

Chapter 16 Processing and Functionality of Rice Bran Proteins and Peptides

isoelectric precipitation of isolated protein fractions has further increased the quantity (Adebiyi et al. 2007). Many other procedures have been reported to prepare the concentrates and isolates for the enrichment of proteins and bran product development (Lynn 1969; Landers 1992; Prakash and Ramanathan 1994; Chen 2000; Chen and Houston 1970). A novel technique of protein extraction from rice bran involving supercritical water as the solvent has been introduced recently. The protein solubility in water and radical-scavenging activity were increased at a higher temperature; however, total protein content was decreased. The quality of protein hydrolysate improved with increased temperature (Wiboonsirikul et al. 2007; Watchararuji et al. 2008; Sereewattanawut et al. 2008). The proteins were estimated using bicinchoninic acid and other techniques (Hanmoungjai et al. 2002; Chan and Wasserman 1993).

4. Functional Properties Protein solubility serves as a useful parameter for its broad application in processed foods, and it is well established that increased solubility enhances emulsification ability (Kinsella 1976). Rice bran protein isolates (RBPIs) or concentrates (RBPCs), more appropriately termed as rice bran proteolysate, have greater functional properties than the native proteins in view of the size reduction. Accordingly, RBPIs and RBPCs may be extensively used as valueadding ingredients. The freeze-dried and spray-dried rice bran proteins (FD-RBPs and SD-RBPs) consisting of only a few types of proteins with MW from 6.5 to 66.2 kD as determined by SDS-PAGE were found to display better hydrophobicity, nitrogen solubility, emulsifying, and foaming capacities. The functional properties of RBPCs vary with the type of treatment provided, as partial hydrolysis and denaturation are possible during processing (Prakash and Ramanathan 1995). For example, the use of proteases as compared to the use of sodium sulfite is superior in increasing functional properties, as the former is responsible for reduction in the size of proteins due to proteolysis, while in the latter case

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some of the disulfide bridges may split but the protein size is still great enough. Commercial exoand endoproteases have been used for characterization and improvement of protein functionality (Hamada 2000a, 2000b). Rice bran dehydrin proteins (RBDPs) are a distinctive class of RBPs with cryoprotective activities and show potential in food preservation, especially in frozen foods. Dehydrin proteins belong to the late embryogenesis-abundant (LEA) group of proteins, which accumulate in seeds at the time of maturation. Dehydrins are protective proteins and perform specific functions in a plant’s survival at times of abiotic stress, such as desiccation or freezing. RBDPs are like antifreeze proteins and their cryoprotective activity on lactate dehydrogenase was demonstrated in a protein separated by 2-D electrophoresis and identified by immunoblotting assay (Momma 2005). The methods used to improve functionality of food proteins by slight structural modifications such as the deamidation either by chemical or enzymatic techniques have found many applications. The conversion of basic amides into acidic groups enhanced the functional properties such as the solubility, emulsifying ability, and other physical behaviors. As RBPs are rich in glutamines, the application P-Gase shows potential in future utilization of RB for improved functionality at industrial scale (Hamada 1991). The functional properties of RBPs are comparable to those of casein, a well-recognized protein for its utility in food processing. The water-binding capacity of RBPs ranges from 3.87 to 5.60 g/g as compared to that of casein (2.48 g/g), leaf proteins (2.66 g/g), and soyprotein isolate (4.3 g/g); oilabsorption capacity of RBPs varies between 3.74 and 9.18 g/g and bulk density for a kind of Basmati rice was found to be 0.21 g/ml, which also shows good foam stability having a half-life of 42.6 hours at 15% sugar concentration (Chandi and Sogi 2007). The foaming or emylsifying capacity and stability of RBPs increased with increased temperature during extraction (Khuwijitjaru et al. 2007). Elevated temperature released proteins from the complexes with fiber, other carbohydrates, and minerals. Viscozyme is another enzyme used for producing

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RBP peptides for higher emulsifying capacity and stability (Park et al. 2001).

5. Enzymes Isolated from Rice Bran A variety of enzymes have been isolated from RB, but lipases have been especially focused in view of the rapid deterioration of rice bran oil by hydrolytic followed by oxidative reactions, producing free fatty acids. A thermally stable lipase (EC 3.1.1.3) with lipolytic activity toward triolein as substrate was first isolated from RB. The enzyme had MW 9.5 kD with optimum pH and temperature at 11 and 80°C, respectively. It is a glycoprotein showing phospholipase activity simultaneously and exhibited no positional specificity against triacyglycerol (Bhardwaj et al. 2001). The lipases capable of hydrolyzing phospholipids are especially important in clinical nutrition. Lipase yield and activity is highest if bran is predefatted followed by five cycles of aqueous extraction using 50 mM potassium phosphate buffer of pH 7. Addition of glycerol as stabilizer increased its shelf life. The enzyme hydrolyzed tributyrin at a higher rate; its substrate specificity shows potential for use in the designing of structured lipids or triglycerides with identified desired ester linkages (Prabhu et al. 1999). A dietary fiber hydrolyzing enzyme (1→3)–β-Dglucan endohydrolase (EC 3.2.1.39) with an apparent MW 29,000 kD and an isoelectric point of 4.0 has been isolated from RB using a series of techniques such as ammoniumsulfate fractional precipitation, chromatofocusing, an ion exchange and size-exclusion chromatography, and hydrophobic interaction chromatography. The enzyme shows active linear hydrolytic specificity and reduced action toward branched β-D glucan substrates, which are an integral part of the fungal cell wall. It plays a preemptive role in protection of ungerminated grain against pathogen attack and may be of importance in food preservation (Akiyama et al. 1997).

6. Rice Bran Proteins in Product Development Protein solubilization is essential for improving utility of RBPs in food industries and is a prerequi-

site for product development, since it is directly proportional to emulsifying and other properties. Investigators have emphasized the importance of the nitrogen solubility index as an indicator for its utilization in the food system (Hernandez et al. 2000). Parboiling is almost universally applied to improve the edible qualities. However, the extent of improvement depends on the type and quantity of the proteins present, and the protein molecular properties could be altered both in the rice endosperm and bran proteins during parboiling (Reza et al. 2005).

6.1. The RBPs as Flavor Enhancers RBPs have been used frequently in product development. Protein hydrolysates have long been used as flavor promoters, especially with glutamic acid and its salts as monosodium glutamate (MSG). However, MSG has been banned in several countries because of health concerns. Glutamines and asparagines are present in large amounts in RBPs; if deaminated they may serve as excellent flavor enhancers in a variety of food systems. However, the peptides are also known to induce bitterness, and careful production of proteolysates plays a significant role in flavor development (Lemieux and Simard 1992). Partial hydrolysis of RBPs using 0.5 N HCl produced free amino acids and peptides that were aroma-producing compounds and may act as potent flavor precursors. They were formed through the Strecker degradation. The peptides or proteins present in the hydrolysate may also react with reducing sugars to form Maillard compounds that are components of pigments like malonoidins (Jarunrattanasri et al. 2007). The enzymatic hydrolysis has certain disadvantages as well, including the development of bitter taste due to the formation of polypeptides of definite chain length with hydrophobic peptides at the ends of the peptide chain (Alder-Nissen 1979). The debittering of protein hydrolysates at low (10–20%) or high (50% and above) degree of hydrolysate (DH) has been achieved by the use of Flavourzyme, which shows the ability to cleave such hydrophobic ends of the peptides, and the resulting end products do not produce a bitter taste (Pommer 1995).

Chapter 16 Processing and Functionality of Rice Bran Proteins and Peptides

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The bran proteins have shown prospects in producing flavor or flavor enhancers. A variety of peptides obtained after 7.6% DH using commercial proteases turned out to be potent flavor enhancers in view of their richness in glutamic acid.

tryptic digestibility of color-bound proteins is least affected, showing that bioactive sites of neither the enzyme, nor the substrate, are involved in binding (Badaruddin et al. 2007; Abdullah et al. 2008).

6.2. RBPs in Baby Foods

6.5. RBPs in Meat Products

The RBPs with high nutritional values and hypoallergenic characteristics may be used as protein supplements in baby foods (Helm and Burks 1996).

The rice bran consisting of a high amount of protein, fiber, and fat was found suitable for 10% bran substitution in preparation of pork meatballs. The textural profile analysis (TPA) was promising, as hardness, gumminess, and chewiness decreased by addition of the bran. It was interesting to relate that the particle size of the meat products and RB were important in forming a smooth surface because of the molecular interactions of hydrocolloids such as xylans, hemicelluloses, and proteins, which have shown high water-absorption capacity (Huang et al. 2005).

6.3. RBPs in Bakery Products The RBPC obtained by alkaline extraction at pH 11 for 45 minutes produced about 8% RBPC with 69% protein content. The substitution of 1% RBPC in bread production enhanced protein/fiber contents and improved dough behavior; however, adding more RBPC was undesirable because of decreased consumer acceptance (Jiamyangyuen et al. 2005). The water-soluble rice bran enzymatic extract (RBEE) produced with 38.1% protein content was found to comprise 6% of sulfur-containing amino acids. The RBEE consisted of peptides of MW 10 kD or less and free amino acids. The nutritive value was enhanced in view of the solubilization and facilitated water absorption. Rice bran as a whole was added up to 10–15% in developing biscuits of nutraceutical importance that were found excellent in sensory evaluation. The experiments in our lab have shown that 10–12% RB may be used successfully in Chapatti (flat bread) and cookies and also in Parotha (oily flat bread) without any adverse sensory effects (Saeed et al. 2009). It is apparent that RBPs mostly contributed to the functionality for dough development and the adverse effects of flavor were partially overcome by the presence of dietary fibers, which form complexes with proteins. Proteinfiber conjugates appear to be formed during baking.

6.4. RBPs as Food-Color Carriers RBPs, like other proteins, form stable color conjugates and act as carriers for homogeneous distribution of food colors in food systems. Moreover, the

6.6. RBPs in Animal Feeds The demand for soy proteins is increasing day by day with the constant increase in their cost as well. The RBPs are found to be effective replacements for soy proteins in the future. Experiments have shown that in the feeding formula for laying hens consisting of 23% soy meal, if replaced by 30% corn distiller ’s grain or 20% rice bran the formula gives excellent performance in egg production (Popescu and Ciurascu 2003; Samli et al. 2006). The alteration in diet was highly cost-effective. The proteinrich rice bran has successfully been used up to 10% in the diet of laying hens without any adverse effect on performance.

6.7. Rice Bran as Fat Replacers Fat in foods contributes to softness, structural stability, lubricity, and mouth feel that may also be gained by inclusion of additives such as RBPs. Rice bran has been found to effectively replace fat in production of low-fat franks because of its superior emulsifying and foaming properties (Bloukas and Paneras 1996). The RBPs may be promising fat replacers

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in a wide range of food commodities, especially in bakery products.

6.8. Rice Bran as Substrate for Microbial Growth Bateriocins have wide applications as food preservatives, and nisin is commercially used in food industries. The variety of microbes including lactic acid bacteria (LAB) grow well on rice bran as culture in view of its protein contents and minerals apart from other nutrients (Yamamoto et al. 2003). RB is therefore an inexpensive media for producing bacteriocins through LAB. Similarly RB is used as media for production of other useful compounds as vinegar, phytates, and so forth.

6.9. Miscellaneous Applications Stabilized or parboiled rice bran has appealing characteristics to be used in food formulation. For example, the granulated powder of bran having a cream color with tasteless and odorless properties may be useful as a thickening, bulking, or flavoring agent in product formation. It is mostly used in bakery products, possibly because of functional properties of the proteins, which may improve rheological behavior of the dough as desired in the enduse quality. An emulsifier of excellent surface activity for commercial application in food processing was isolated from rice bran (Yun and Hong 2007). The extracting solvent used contains 70:30 W/W ethanol/water mixture, which shows the possibility of the presence of prolamines. The edible protein coating for preservative and cosmetic purposes is drawing significant attraction from both processors and consumers, as it adds value to appearance and shelf life. Protein enrichment, although not intentional in film coating, is another point in value addition. Recently RBPs were found to produce edible films of high quality using glycerol as the plasticizer. The functional properties of the film such as puncture strength were comparable to those of the soy protein–based films (Adebiyi et al. 2008).

7. Health Benefits of Rice Bran Proteins The bran consisting of about 12–20% protein, with lysine content as 3–4% of the total protein (highest among the cereal proteins), is a valuable by-product of multiple health benefits. The RBPs are only next to oat proteins in terms of quality of the proteins. The soluble RBPs have been regarded as high in nutraceutical value in view of their amino acid profile, PER, NPU, and so forth (Saunders 1990). Rice proteins are best known for their high hypoallergenicity (Helm and Burks 1996), and the peptide domain for hypoallergenic activity is already documented (Adachi et al. 1993). The bran proteins are rich in those essential amino acids that lack in the endosperm protein, and whole grains are thus recommended for frequent consumption (Jones and Gersdorff 1925). The amino acid composition of RBP is given in Table 16.3. Rice proteins are easily digestible (73.0%) and so are absorbable with net protein utilization (NPU) of 73.8 as compared to wheat (53.0), corn (58.0), barley (62), and millet (56.0). Protein efficiency ratio ranges from 1.6 to 1.9; however, it increases to 2.0–2.5 in protein isolates and concentrates, Table 16.3. Amino acid composition of RBPs (Rukimini 2003). Amino acid profile

%

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Cystein Tryptophan Ammonia

1.36 0.36 0.97 1.90 0.63 0.80 0.96 0.56 0.25 0.34 0.91 0.44 0.55 0.38 0.68 1.08 0.32 0.21 0.20

Chapter 16 Processing and Functionality of Rice Bran Proteins and Peptides

which is equivalent to casein (Juliano 1985; Bean and Nishita 1985). The alkaline process of protein extraction has multifactual adverse effects such as nutritional losses, off flavor, textural alterations, and reduced functionality (Kinsella 1981). Nutritional deterioration has been observed in RBPs if exposed to extreme alkaline conditions that promote the conversion of cysteine and serine to toxic lysinoalanine (Otterburn 1989), which may damage kidney as observed in the case of rats (Woodward and Short 1973). However, reactions with only certain amino acids induce toxicity; otherwise specific proteases for optimal hydrolysis releasing the desired amino acid chain and the presence of some amino acids at C- or N-terminal play beneficial roles as the nutrients or flavor enhancers in food products. Some value-added peptides were separated from RBPs using preparative HPLC (Hamada et al. 1998). The RBPCs, being a rich source of protein (69.16%), are suitable for protein supplementation. Including a substantial amount of RBPC in formulation is highly suitable for nutritional enrichment of various foods, as the bran is also rich in fat, dietary fibers, minerals, and vitamins (Jiamyangyuen et al. 2005). The phytonutrients of RB and their nutraceutical advantages have been discussed in detail by Rukimini (2003). The nutritional status of RBPs is reported much higher than rice endosperm protein or proteins from any other cereal or legumes (Juliano 1994).

7.1. Role of RBPs in Prevention and Control of Cancers The RBP isolates have been attributed to disease control (Cagampang et al. 1966). The anticancer properties of RBPs have been discussed earlier (Kawamura and Muramoto 1993). A lipoprotein fraction isolated from RBPs exhibited some antitumor properties as the protein inhibited the growth of cultured human endomaterial adenocarcinoma cells like Swano (Fan et al. 2000). An indirect way of actually controlling the cancer is to make the immune system stronger by feeding a diet rich in antioxidants and other bioactive ingredients. The

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immunoactive RBPs and antitumorigenic peptides have been reported; however, detailed studies related to immunomodulatory properties are still missing (Kawamura and Muramoto 1993). The antigenic properties of RBPs, particularly of the water- and salt-soluble proteins, that is, albumins and globulins, respectively, have also been cited (Landers and Hamaker 1994). The adhesive interactions of cancer cells with extracellular matrix promote metastasis that leads to cell growth; therefore, antiadhesion activity at the cellular level is one of the indications for preventive measure of cancer cell growth. A glycoprotein rich in hydroxyproline and proline (45% of the total amino acids) of MW 57-kD was isolated from RB using affinity chromatography, as the protein binds with fibronectin immobilized on agrose column. The adhesive activity may be related to arabinose, which represents 46.8% of the total carbohydrates linked to protein molecules. The RBP was found to be adhesive for murine Lewis lung carcinoma cells (Shoji et al. 2001). It seems that RBPs apart from other components of rice bran, such as oil, carbohydrates, and dietary fibers including β-glycans, may play a constructive role in prevention and control of cancer.

7.2. Rice Bran Lectins Lectins are glycoproteins with strong specific carbohydrate-binding properties that play an important role in nutrition, pathogenesis, and toxicity. They are widely distributed in foods and in the human body. The serum level of the lectins may be indicative of certain diseases. A rice germ lectin was isolated, identified, and studied for its biological properties decades ago (Shen et al. 1984). A lectin of MW 37,000 was isolated from rice bran by ultracentrifugation, which is a dimmer. The dimmer on reduction and carboxy methylation was dissociated into two subunits of MW 11,000 and 8,000; however, they lost their hemagglutinin activity after separation. The lectin is metabolically active, as it increased glucose oxidation and reduced lipolysis stimulated by epinephrine in mouse (Tsuda 1987). An N-acetyl glucosamine-specific rice bran lectin (RBL) with its epitope integrity present at the

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disulphide linkage was identified by ELISA and two mouse monoclonal antibodies (1 gM). However, the binding capacity was abolished after treating the protein with 2-mercaptoethanol, providing a definite proof for position of the disulfide bridge at the epitope (Kolberg 1992). Lectin as glycoproteins, and in view of their ability to form glycoconjugate, they are important in immunomodulation. Lectins may be harmful as well; RBL-2 was found to be more toxic than RBL-1, as revealed by brine shrimp nauplii mortality rate assay (Kabir et al. 2001).

7.3. RBPs as Antioxidants The nutritional value of rice protein is superior to casein or soy protein isolate in terms of amino acid profile (Wang et al. 1999). The isolates/concentrates of RBPs are even better. The focus in nutrition during the last few decades has been constantly increasing on evaluating antioxidants in foods. The proteins displaying the radical-savaging activity have been isolated (Hata et al. 2008). The research reveals that hypocholestremic effect and improvement of the lipid profile activity should not be attributed only toward rice bran oil, but that RBPs may also be responsible for such health benefits. The water-soluble oryzanol enzyme extract (WSOEE) with strong antioxidant activity has shown effective nutraceutical capacity by inhibiting the protein oxidation process and lipid peroxidation in rat brain homogenate. Moreover, its oxidationinhibition capacity was almost similar to Trolox in controlling lipid oxidative damage induced by cumen hydroperoxide. The brain protein activity loss may be attributed to functional proteins such as enzymes, glycol receptors, transporters, or barriers that are overcome naturally by the presence of melatonin. The antioxidative function of WSOEE was equivalent to melatonin (Parrado et al. 2003). We strongly feel that the antioxidant ability of WSOEE is not only due to oryzanol (which is more soluble in fat), but also due to the fact that RBPs may contribute as antioxidants, firstly because of their considerable hydroxal radical-scavenging activity (previously reported) and secondly because of the high protein content, as reported by the authors.

8. Antinutritional Behavior of RBPs All rice bran products including proteins are reported as nontoxic by the Cosmetic Ingredient Review (CIR) expert panel (Anonymous 2006). The allergenic proteins are present in rice endosperm, such as 14–16 kDa protein inhibiting serum IgE-binding activity. These proteins are present in albumin fraction and belong to the wheat/barley α-amylasetrypsin inhibitor family (Tsuji et al. 2001). However, allergens are not yet reported in bran, perhaps because of the fact that RBPs are usually extracted with alkali or enzymes, which may affect their structural integrity. The quantity of proteins depends on whether the rice bran is heat stabilized (HSRB) or nonheat stabilized rice bran (NSRB). The HSRB has higher hydrophobicity than NDRB, as heat denatures proteins and hydrophobic amino acids are then exposed (Gnanasambandam and Hettiararachchy 1995; Betschart et al. 1977). It has been pointed out that protein interactions with other components resulted in toxin production that deteriorated the nutritional status of rice bran (De Groot and Slump 1969). The other RB toxins, including trypsin inhibitors, hemagglutinins, peroxides, and lipases, are denatured and detoxified during processing treatment as heat, acid/alkaline, or enzyme treatment (Khan et al. 2005). Rats on a diet consisting of 10% RB were found to have higher serum albumin level, indicating liver sufficiency. The lipolytic activity that may be harmful was controlled by pan or microwave heating (Ahmed et al. 2007).

9. Future Trends/Conclusion Presently RB is sold as low-cost feed for animals. Unfortunately, its nutritive and bioactive components, such as RBPs and their hydrolysates, are completely overlooked. The various fractions of rice bran such as oil, carbohydrates, and dietary fibers (β-glucans, phytates, tannins) including RBPs have demonstrated their ability in prevention of diseases. Some of them are found to have potent anticancer and antioxidant activity.

Chapter 16 Processing and Functionality of Rice Bran Proteins and Peptides

Recent developments in food proteomics have shown tremendous opportunities for application in food industry and identification as nutritional supplements (Carbonaro 2004). The RBPs have hardly been explored in the field of proteomics, although it shows great potential in identification of crops for their quality functions. The RBPs have been characterized by applying the proteomic approach, and the technique has been found useful to classify between five aromatic and nine nonaromatic varieties of rice (Trisiriroj et al. 2004). Some of the second-generation by-products of rice bran, that is, RBP concentrates and isolates, show tremendous potential for future industrial utilization in view of their multifactorial functionality, including their medicinal value. The residual proteins being high MW with considerable water-binding capacity may be used as hydrocolloids, which have demonstrated health benefits in improving lipid profile, controlling hyperglycemia, and lowering glycemic index of foods. Rice bran has been used successfully as media for microbial growth and is a suitable substrate for production of lactic acid, vinegar, phytase, and so forth in countries where bran is the bulk leftover after rice milling (Gao et al. 2008). Fat substitutes are focal in present food research from a health, economical, and functional point of view. Rice-based fat substitutes with high gelling capacity and rising rates of G′/G″ to improve rheological properties were found suitable to replace fat in baked products (Yang and Xu 2007). Proteins form gel first to produce a firm network, followed by maltrodextrin (produced by amylases) to form matrix. RBPs with superior gelling, emulsifying, and foaming properties are suitable for fat substitute and nutraceutical product development.

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Adachi T, Izumi H, Yamada T, Tanaka K, Takeuchi S, Nakamura R, Matsuda T. 1993. Gene structure and expression of rice seed allergenic proteins belonging to the α-amylase/trypsin inhibitor family. Plant Mol Biol 21:239–248. Adebiyi AP, Adebiyi AO, Jin D-H, Ogawa T, Muramoto K. 2008. Rice bran protein-based edible films. J Food Sci & Technol 43(8):476–483. Adebiyi AP, Adebiyi AO, Ogawa T, Muramoto K. 2007. Preparation and characterization of high-quality rice bran proteins. J Sci Food Agric 87:1219–1227. Ahmed F, Platel K, Vishwanatha S, Puttaraj S, Sirinivasan K. 2007. Improved shelf-life of rice bran by domestic heat processing and assessment of its dietary consumption in experimental rats. J Sci Food Agric 87:60–67. Akiyama T, Shibuya N, Hrmova M, Fincher GB. 1997. Purification and characterization of a (1→3)-β-Dglucan endohydrolase from rice (Oryza sativa) bran. Carbohydrate Research 297:365–374. Alder-Nissen J. 1979. Determination of the degree of food proteins by trinitrobenzenesulfonic acid. J Agric Food Chem 27:1258–1262. Anderson AK, Guraya HS. 2001. Extractability of protein in physically processed rice bran. JAOCS 78(9):969–972. Anonymous. 2006. Amended final report on the safety assessment of oryza sativa. Intl J Toxicology 25(2):91–120. Ansharullah J, Hourigar A, Chesterman CF. 1997. Application of carbohydrases in extracting protein from rice bran. J Sci Food Agric 74:141–146. Badaruddin M, Abdullah SU, Sayeed SA, Ali R, Riaz MN. 2007. Sunset yellow: A food color for protein staining with SDSPAGE. Cereal Foods World 52(1):12–14. Bandyopadhyay K, Misra G, Ghosh S. 2008. Preparation and characterization of protein hydrolysates from Indian defatted rice bran meal. J Oleo Sci 57(1):47–52. Bean MM, Nishita KD. 1985. Rice flours for baking. In: BO Juliano, ed., Rice Chemistry and Technology, 2nd ed., 539– 556. St. Paul, MN: AACC International. Bera MB, Mukherjee RK. 1989. Solubility, emulsifying, and foaming properties of rice bran protein concentrates. J Food Sci 54(1):142–145. Betschart AA, Fong RY, Saunders RM. 1977. Rice by-products: Comparative extraction and precipitation of nitrogen from U.S. and Spanish bran and germ. J Food Sci 42(4):1088–1093. Bhardwaj K, Raju A, Rajasekharan R. 2001. Identification, purification, and characterization of a thermally stable lipase from rice bran. A new member of the (Phospho) lipase family. Plant Physiology 127:1728–1738. Bloukas JG, Paneras ED. 1996. Quality characteristics of low-fat frankfurters manufactured with potato starch, finely ground toasted bread and rice bran. J Muscle Foods 7:109–129. Cagampang GB, Cruz LJ, Espiritu SG, Santiago RG, Juliano BO. 1966. Studies on the extraction and composition of rice proteins. Cereal Chem 43:145–155. Carbonaro M. 2004. Proteomics: Present and future in food quality evaluation. Trends in Food Science & Technol 15:209–216.

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Carrol LE. 1990. Functional properties and applications of stabilized rice in bakery products. Food Technol 74–76. Chan KY, Wasserman BP. 1993. Rapid solid-phase determination of cereal protein using bicinchoninic acid. Cereal Chem 70:27–28. Chandi GK, Sogi DS. 2007. Functional properties of rice bran protein concentrates. J Food Engineering 79:592–597. Chen L, Houston DF. 1970. Solubilization and recovery of protein from defatted rice bran. Cereal Chem 47:72–79. Chen Z. 2000. Extraction of rice bran protein concentrate and its functional evaluation. China Oils Fats 25(1):47–48. De Groot AP, Slump P. 1969. Effects of severe alkali treatment of proteins on amino acid composition and nutritive value. J Nutr 98:45–48. Fan H, Morioka T, Ito E. 2000. Induction of apoptosis and growth inhibition of cultured human endomaterial adenocarcinoma cells (Sawano) by an antitumor lipoprotein fraction of rice bran. Gynecologic Oncology 76:170–175. Gao M-T, Kaneko M, Hirata M, Toorisaka E, Hano T. 2008. Utilization of rice bran as nutrient source for fermentative lactic acid production. Bioresource Technol 99:3659–3664. Gnanasambandam R, Hettiararachchy NS. 1995. Protein concentrates form nonheat-stabilized rice bran: Preparation and properties. J Food Sci 60:1066–1069. Grosman MV, Rao CS, Da Saliva RSF. 1980. Extraction of protein from buckwheat bran: Application of enzymes. J Food Biochem 4:181. Hamada JS. 2000a. Characterization and functional properties of rice bran proteins modified by commercial exoproteases and endoproteases. J Food Science 65(2):305–310. ———. 1997. Characterization of protein fractions of rice bran to devise effective methods of protein solubilization. Cereal Chem 74(5):662–668. ———. 1991. Peptidoglutaminase deamidation of proteins and protein hydrolysates for improved food use. JAOCS 68(7):459– 462. ———. 2000b. Ultrafilteration of partially hydrolyzed rice bran protein to recover value-added products. JAOCS 77(7):779– 784. ———. 1999. Use of protease to enhance solubilization of rice bran proteins. J Food Biochem 23:307–321. Hamada JS, Spanier AM, Bland JM, Diack M. 1998. Preparative separation of value-added peptides from rice bran proteins by high-performance liquid chromatography. J Chromatography 827:319–327. Hanmoungjai P, Pyle DL, Niranjan K. 2002. Enzyme-assisted water-extraction of oil and protein from rice bran. J Chem Technol Biotechnol 77:771–776. Hata S, Wiboonsirikul J, Maeda A, Kimura Y, Adachi S. 2008. Extraction of defatted rice bran by subcritical water treatment. Biochemical Engineering J 40:44–53. Helm RM, Burks AW. 1996. Hypoallergenicity of rice protein. Cereal Foods World 41:839–843. Hernandez N, Rodriguez-Alegria ME, Gonzalez F, LopezMunguia A. 2000. Enzymatic treatment of rice bran to improve processing. J Amer Oil Chem Soc 77:177–180.

Huang SC, Shiau CY, Liu TE, Chu CL, Hwang DF. 2005. Effects of rice bran on sensory and physico-chemical properties of emulsified pork meatballs. Meat Sci 70:613–619. International Rice Research Institute (IRRI). 2005. World Rice Statistics. Jarunrattanasri A, Theerakulkait C, Cadwallader KR. 2007. Aroma components of acid-hydrolyzed vegetable protein made by partial hydrolysis of rice bran protein. J Agric Food Chem 55(8):3044–3050. Jiamyangyuen S, Srijesdaruk V, Harper WJ. 2005. Extraction of rice bran protein concentrate and its application in bread. J Sci Technol 27(1):55–64. Jones DB, Gersdorff CEF. 1925. Proteins of wheat bran. II. Distribution of nitrogen, percentages of amino acids and of free amino nitrogen: A comparison of the bran proteins with the corresponding proteins of wheat endosperm and embryo. J Biological Chem 64:241–251. Juliano BO. 1985. Polysaccharide, proteins and lipids of rice grain composition. In: BO Juliano, ed., Rice Chemistry and Technology, 2nd ed., 59–174. St. Paul, MN: AACC International. ———. 1994. Production and utilization of rice. BO Juliano, ed., Rice Chemistry and Technology, 2nd ed., 1–16. St. Paul, MN: AACC International. Kabir SR, Hossain SE, Absar N. 2001. Reinvestigation on the purification and characterization of rice-bran lectin. Pakistan J Biological Sciences 4(2):197–200. Kawamura Y, Muramoto M. 1993. Anti-tumorigenic and immunoactive protein and peptide factors in food stuff. 2. Antitumorigenic factors in rice bran. In: KW Waldron, IT Johnson, LR Fenwick, eds., Food and Cancer Prevention: Chemical and Biological Aspects, 331–401. Cambridge: Royal Society of Chemistry. Khan AD, Saeed S, Ahmad R, Shahzad K. 2005. Inactivation of rice bran toxins. Proc Pakistan Acad Sci 42(2):183–188. Khuwijitjaru P, Naulchan P, Adachi S. 2007. Foaming and emulsifying properties of rice bran extracts obtained by subcritical water treatment. Silpakorn U Science & Tech J 1(1):7–12. Kinsella JE. 1981. Functional properties of proteins: Possible relationships between structure and function in foams. Food Chem 7:273–288. ———. 1976. Functional properties of proteins of proteolytic enzyme modified soy protein isolates. J Agric Food Chem 38:651–656. Kolberg J. 1992. Monoclonal antibodies against rice bran lectin. Biol Chem Hoppe Seyler 373(2):77–80. Lamberts L, Bie ED, Vandeputte GE, Veraverbeke WS, Derycke V, de Man W, Delcour JA. 2007. Effect of milling on color and nutritional properties of rice. Food Chem 100:1496– 1503. Landers PS. 1992. Production and evaluation of rice bran protein concentrate as a potential human food source. PhD dissertation, University of Arkansas, Fayetteville. Landers PS, Hamaker BR. 1994. Antigenic properties of albumin, globulin, and protein concentrate fractions from rice bran. Cereal Chem 71:409–411.

Chapter 16 Processing and Functionality of Rice Bran Proteins and Peptides

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Chapter 17 Bioactive Proteins and Peptides from Egg Proteins Jianping Wu, Kaustav Majumder, and Kristen Gibbons

Contents 1. Introduction, 247 2. Antimicrobial Proteins and Peptides from Eggs, 249 2.1. Lysozyme, 250 2.2. Antimicrobial Peptides from Lysozyme, 251 2.3. Ovotransferrin, 252 2.4. Antimicrobial Peptides from Ovalbumin, 252 2.5. Other Antimicrobial Proteins and Peptides, 253

1. Introduction Nature forms an avian egg not for human food but to produce a chick. Birds invest in the reproductive process up front, transferring all the nutrients and energy needed to allow the embryo to reach the point of hatching. This concentration of highly available nutrients also makes the egg an ideal nutritional food source; eggs are one of the few foods that are consumed throughout the world. The main components of the egg are water (75%), proteins (12%), and lipids (12%), as well as carbohydrates, vitamins, and minerals (Cotterill and Geiger 1977). Shell eggs consist of about 9.5% shell (including shell membrane), 63% albumen, and 27.5% yolk (Cotterill and Geiger 1977) (Table 17.1). The shell, the outermost part of the egg, consists of different

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

3. 4. 5. 6. 7. 8. 9.

Immunomodulating Proteins and Peptides, 254 Anticancer Proteins and Peptides, 255 Antioxidant Proteins and Peptides, 255 Antihypertensive Peptides, 256 Protease Inhibitors, 258 Conclusion, 258 References, 259

kinds of inorganic and organic materials. It provides the embryo with good protection from predators as well as from soil invertebrates and harmful bacteria. Egg white, or albumen, is the cytoplasm of the egg, consisting of nearly 90% water and 10% proteins. The dominant proteins in egg white are ovalbumin (54%), ovotransferrin (12%), ovomucoid (11%), ovomucin (3.5%), lysozyme (3.4%), and globulin (8%) (Li-Chan et al. 1995); new minor proteins have been discovered recently that are thought to play important roles with respect to the primary biological action of egg white, that is, embryo protection and development (GuérinDubiard and Nau 2007). As the second line of defense after the eggshell, many of the egg white proteins possess antimicrobial activities that function to prevent the growth and spreading of microorganisms attempting to invade the egg. Therefore, it is generally accepted that the biological functions of the egg white proteins are to prevent the movement of microorganisms into the egg yolk and to provide the growing embryo with nutrients (Ibrahim 247

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Table 17.1. Major chemical compositions of different parts of egg. Constituent

Composition (%)

Proteins (%, dry weight)

Lipids (%, dry weight)

Water (%)

Eggshell

9.5 (including eggshell membrane)

6.4

0.03

1.7

Egg white

63.0

0.03

85.0

Egg yolk

27.5

Crude protein (10.6) — Ovalbumin (54.0) — Ovotransferrin (12) — Ovomucoid (11) — Ovomucin (3.5) — Lysozyme (3.4) — Ovomacroglobulin (0.5) — Flaboprotein (0.8) — Avidin (0.05) Crude protein (15.6–16.0) — High-density lipoprotein (36) — Low-density lipoprotein (40) — Phosvitin (13.4) — Livitin (9.3)

Total fat (32.05–35.0) — Triglycerols (66.0) — Phosphatidylcholine (24.0) — Sphingomyelin (0.6) — Cholesterol (5.0)

51.1

Other compounds (%, dry weight) Inorganic salts (91.87) — Calcium carbonate (98.4) — Magnesium carbonate (0.8) — Tricalcium phosphate (0.8) Carbohydrates (0.4–0.9) Ash (0.5–0.6)

Carbohydrates (0.2–1.0) Ash (1.1)

Source: Adapted from Cotterill and Geiger 1977; Kovacs-Nolan et al. 2005.

1997). The yolk contains both lipid and protein in a ratio of ∼2:1; the major proteins in egg yolk are low-density lipoproteins, high-density lipoproteins, phosvitin, and livetin. In addition to providing antibodies for the new organism, egg yolk is the essential source of vital nutrients and energy in the embryogenesis of the bird. Egg proteins are well known for their excellent digestibility; they are also considered to have the highest nutritional quality of all food source proteins, providing all the essential amino acids in amounts that closely match human requirements for essential amino acids (FAO protein value = 100) (Mann and Truswell 1998). Egg proteins are therefore used as the nutritional standard against which all other proteins are compared. Traditionally, dietary protein recommendations have been based on preventing deficiency and growth as opposed to promoting optimal health. However, recent research has indicated links between high-protein intake and weight management as well as reduced incidence of diabetes and cardiovascular disease (Layman 2003).

Although the optimum protein intake for weight loss diets remains unknown, there is increasing evidence that protein intakes above the current recommended daily allowance (RDA) may be beneficial for weight loss. It is thought that the increase in dietary protein resulting in increased plasma levels of leucine is consistent with increased protein synthesis in skeletal muscle and stimulation of the glucose-alanine cycle (Parker et al. 2002; Layman 2003). These findings are also consistent with other reports of a metabolic advantage for weight loss associated with diets containing reduced levels of carbohydrates and increased levels of high-quality protein (Feinman and Fine 2003). For example, in a randomized crossover study, an egg breakfast was shown to have a greater satiating effect compared to a bagel breakfast (Vander Wal et al. 2008). In the present U.S. diet, the percentages of total food energy derived from the three major macronutrients are as follows: carbohydrate (51.8%), fat (32.8%), and protein (15.4%). However, studies of hunter-gatherers showed an elevated dietary protein

Chapter 17 Bioactive Proteins and Peptides from Egg Proteins

intake accounting for ∼30% of total energy (Eaton and Konner 1985). A large egg provides approximately 6 grams of protein (3.6 g in the white and 2.7 g in the yolk); one serving of eggs provides about 13.3 grams of high-quality protein, which represents about 27% of the recommended daily intake (RDI) for adults. Egg proteins may be particularly useful in the diets of ovo-vegetarians, who could experience an insufficient intake of essential amino acids due to low protein digestibility and the poor biological protein value of plant proteins (Millward 2004). Egg proteins are also an ideal protein source for children and elderly people due to their excellent essential amino acid profile, high digestibility, and ease of preparation. Furthermore, egg proteins are reported to have physiological benefits, which have been extensively discussed (Huopalahti et al. 2007; Kovacs-Nolan et al. 2005; Mine 2007). The purpose of this chapter is to summarize recent research development in egg proteins and their derived peptides.

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2. Antimicrobial Proteins and Peptides from Eggs Eggs have both physical and chemical properties that protect the embryo against invasion by microorganisms (Table 17.2). The eggshell and shell membrane form the first physical barrier to obstruct invasion of microorganisms. As a calcitic bioceramic, the avian eggshell possesses unique mechanical properties that are derived from its complex porous polycrystalline structure. Recent studies have demonstrated that several chemical antimicrobial components are specifically and intimately integrated into the calcified eggshell (as matrix proteins) and are also specifically associated with the shell membranes (Hincke et al. 2000; Gautron et al. 2001). The most wellknown antimicrobial protein, lysozyme, has been discovered in the membrane, it circumscribes the egg white and forms the innermost layer of the shell membranes. Lysozyme is also present in the shell

Table 17.2. Examples of antimicrobial proteins in eggs. Proteins

Antimicrobial Functions

Ovotransferrin

Reversibly binds iron

Ovomucin

Maintains viscosity and structure of egg white and prevents the spread of bacteria Lyses the peptidoglycan layer of some Gram-positive organisms

Lysozyme

Avidin

Cystatin Ovomacroglobulin

Binds the water-soluble vitamin biotin and makes it unavailable to some Gram-positive and some Gram-negative bacteria that require it Stops growth of bacteria that produce cysteine proteinases Protease inhibitor

Examples of Bacteria the Protein Is Effective Against Pseudomonas spp., Escherichia coli, Streptococcus mutans Helicobacter pylori

Bacillus stearothermophilus, Clostridium tyrobutyricum, and Clostridium thermosaccharolyticum, Streptococcus mutans Escherichia coli K-12, Klebsiella pneumonia, S.marcesens, P. aeruginosa, S. aureus, and Staphylococcus epidermis Poryphyromonas gingivalis S. marcesens P. aeruginosa

References Charter and Lagarde 2004; Kovacs-Nolan et al. 2005; Valenti et al. 1983 Ibrahim 1997; Kovacs-Nolan et al. 2005; Watanabe et al. 2004; Kodama and Kimura 1999 Charter and Lagarde 2004; Losso et al. 2000; Guérin-Dubiard et al. 2005

Charter and Lagarde 2004, Korpela et al. 1984; Guérin-Dubiard et al. 2005

Ibrahim 1997; Kovacs-Nolan et al. 2005; Baron and Rehault 2007 Wu et al. 2001; Kovacs-Nolan et al. 2005; Nielson et al. 1994; Miyagawa et al. 1994; Molla et al. 1987

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membranes and in the matrix and cuticle of the calcified shell (Hincke et al. 2000). Another antimicrobial protein, ovotransferrin, has been identified in the calcified mammillae and particularly in the eggshell membranes, where it functions as a bacteriostatic filter to reinforce inhibition of Salmonella growth in egg white (Gautron et al. 2003). A novel eggshell-specific protein, ovocalyxin-36 (OCX-36), has been reported (Gautron et al. 2007). OCX-36 is associated only with the eggshell and shell membranes and is not present in the egg white. OCX-36 is the first example of an eggshell-specific protein that may participate in the natural chemical defense of the egg against bacteria and may provide protection for the egg contents during shell formation in the distal oviduct. Although the chicken eggshell is a very effective protection system, bacteria sometimes penetrate the egg or can be already present in the uterus via retrograde movement from the cloaca before eggshell formation. Egg albumen presents an efficient, natural barrier to bacterial contamination of the egg contents. Antimicrobial protection is a function that has been most commonly ascribed to numerous egg white proteins that possess antimicrobial properties (Messens et al. 2005). The antimicrobial mechanisms of eggs include bacterial cell lysis, metal binding, and vitamin binding (Li-Chan and Nakai 1989).

2.1. Lysozyme Lysozyme is the most well-known antimicrobial protein in egg white. Hen egg is the richest source of lysozyme, accounting for 3.5% of total egg white proteins (Charter and Lagarde 2004). Lysozyme is a basic protein consisting of 129 amino acids and has a molecular weight of 14.3 kDa and an isoelectric point of 10.7. As a muramidase enzyme, its antimicrobial activity is linked to its cleavage activity on the glycosidic bonds between C-4 of N-acetylglucosamine (NAG) and the C-1 of Nacetylmuramic acid (NAM) of the bacterial peptidoglycan cell wall (Lesnierowski and Kijowski 2007). Without the barrier of the cell wall, the cell will swell and burst if the lytic action of lysozyme is in

a low osmotic strength media or if the rate of synthesis and polymerization in making the cell wall is slower than the lysozyme-catalyzed degradation (Baron and Rehault 2007). Lysozyme is effective against Gram-positive bacteria such as Bacillus stearothermophilus, Clostridium tyrobutyricum, and Clostridium thermosaccharolyticum (Losso et al. 2000). Gram-negative bacteria have an extra “outer membrane,” a barrier that functions as a permeability barrier to protect the peptidoglycan layer from the lytic action of lysozyme (Baron and Rehault 2007). Therefore, the antimicrobial activity of lysozyme shows little to no effect against Gram-negative organisms, which cause about one-third of bacterial infections. Previous studies have shown that modification of lysozyme can broaden the spectrum of its action. Coupling lysozyme with hydrophobic peptides, or fatty acids at lysine residues, enhances penetration and disruption of the bacterial membrane, which helps to broaden the activity of lysozyme against Gram-negative bacteria, including Escherichia coli K12 (Ibrahim et al. 1993, 1994). Glycosylation can also extend its antimicrobial activity to Gramnegative bacteria (Nakamura et al. 1997). Adding ethylene di-amine tetra acetic acid (EDTA) (Charter and Lagarde 2004), organic acids, nicin (Losso et al. 2000), trypsin, or lactoferrin (Baron and Rehault 2007) helps to destabilize the outer membrane of Gram-negative organisms, broadening the spectrum of lysozyme activity. EDTA chelates the bivalent cations, disorganizing the lipopolysaccharide portion of the outer membrane, thus allowing lysozyme to penetrate through the surface (Baron and Rehault 2007). Compounds lethal to the bacterial membrane, such as phenolic aldehydes, can also be coupled with lysozyme to protect against spoilage/ pathogenic microorganisms (Ibrahim et al. 2002). Lysozyme denatured by heat, reduced by DTT (Ibrahim et al. 1996; Düring et al. 1999), or modified by site-directed mutagenesis (Ibrahim et al. 2001) results in a wider spectrum of bacteriocidal activity against some Gram-negative and Gram-positive bacteria. It has been clearly demonstrated that the polymerization of lysozyme during heating treatment is the reason for its broader spectrum of

Chapter 17 Bioactive Proteins and Peptides from Egg Proteins

antimicrobial activity (Ibrahim et al. 1994, 1996). Similarly, high-pressure homogenization promotes structural changes at the lysozyme active sites and increases antimicrobial activity against Lactobacillus plantarum (Vannini et al. 2004). Lysozyme is a safe protein that has been approved for use in some foods and in some pharmacological and therapeutic applications (Cunningham et al. 1991). It is estimated that over 100 tons of lysozyme are used each year for these purposes (Scott et al. 1987). Lysozyme is used in the food industry as a natural food preservative in cheese, wine, and beer. In cheese making, it is used to prevent the growth of Clostridium tyrobutyricum, which causes offodors and late “blowing” (unwanted fermentation) in some cheeses (Proctor and Cunningham 1988). It is used in wine and beer production to control lactic acid bacteria such as Lactobacillus spp., which is a cause of spoilage (Gerbaux et al. 1997; Daeschel et al. 1999). It can be used to control bacteria in meat products such as sausage, salami, pork, beef, or turkey (Hughey et al. 1989; Bolder 1997). Lysozyme is used in oral health care products such as toothpaste, mouthwash, and chewing gum (Sava 1996; Tenuovo 2002). Lysozyme has been found to be effective against the cariogenic bacteria Streptococcus mutans (Tenuovo 2002) and against perodontitisassociated bacteria (Sava 1996), which explains its effects on oral health.

2.2. Antimicrobial Peptides from Lysozyme As discussed in the previous section, the antimicrobial activity of lysozyme is limited to some Grampositive bacteria through its muramidase activity. Peptides released from lysozyme’s primary sequence by protease digestion demonstrated bacteriostatic activity independent of its enzymatic activity (Düring et al. 1999; Ibrahim et al. 2001; Mine et al. 2004). The helix and net positive charge of a peptide seem to play important roles in the antimicrobial activity (Mine et al. 2004). Many of these peptides share a common structural design such as the helix-loophelix or helical hairpin (Ibrahim et al. 2001); they often contain 40 residues or less and vary in chain length, charge, and hydrophobicity (Mine et al.

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2004). The helix-loop-helix pattern (87-114) is located at the upper lip of the active site of lysozyme. The N-terminal helix exerts antimicrobial activity against Gram-positive bacteria, while the C-terminal helix is necessary to broaden the antimicrobial activity toward Gram-negative bacteria (Ibrahim et al. 2001). The helical hairpin antimicrobial peptides are located in the C-terminal and act against both Gramnegative and Gram-positive bacteria by targeting the cytoplasmic membrane. The helix-loop-helix peptide is able to kill Gram-negative bacteria by crossing the outer membrane through self-promoted uptake and causing damage to the inner membrane through channel formation (Ibrahim et al. 2001). Peptides corresponding to the amino acid residues 98–112 (Ile-Val-Ser-Asp-Gly-Asn-GlyMet-Asn-Ala-Trp-Val-Ala-Trp-Arg) digested from lysozyme with clostripain work against some Grampositive and Gram-negative bacteria such as E. coli K12 (Ibrahim et al. 2001; Mine et al. 2004). These peptides exhibit bactericidal activity but do not exhibit the muramidase activity of native lysozyme (Pellegrini et al. 1997). If the asparagine 106 is replaced by the positively charged amino acid arginine, the bactericidal activity of the peptide increases. Substituting the amino acid in the peptide produces a sequence of Arg-Ala-Trp-Val-Ala-Trp-Arg, which corresponds to the amino acid sequence of 106–112 (Pellegrini et al. 1997). The D-enantiomer of the peptide sequence is as active against bacteria as the L form but is less active against Serratia marcescens, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lentus. If tryptophan 111 is replaced by tyrosine, the peptide loses its antimicrobial activity. Pellegrini et al. (1997) found that the amino acid residues in positions 106–112 participate in antimicrobial activity, whereas positions 98–105 do not exhibit antimicrobial activity. Mine et al. (2004) reported two new antimicrobial peptides: His-Gly-Leu-Asp-Asn-TyrArg (15–21) and Ile-Val-Ser-Asp-Gly-Asp-GlyMet-Asn-Ala-Trp (98–108); both of these peptides interact with the bacterial surface and destroy membrane integrity in a direct manner. LzP, a lysozyme derived peptide, completely inhibits some Bacillus spp., such as Bacillus subtilis

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and Bacillus coagulans (Abdou et al. 2007). LzP is produced from the protein lysozyme by partial enzymatic hydrolysis using pepsin (Abdou et al. 2007). LzP is commercially available as a natural food preservative. It seems that the bactericidal potency of lysozyme is not only due to its muramidase activity, which amounts to only about 11% of the lytic activity of the whole lysozyme protein, but is also due to its cationic and hydrophobic properties (Pellegrini et al. 1997; Hancock and Chapple 1999; Ibrahim et al. 2002).

2.3. Ovotransferrin Ovotransferrin plays an important role in preventing the growth of Gram-negative spoilage organisms in egg white (Baron and Rehault 2007). Belonging to the transferrin family, ovotransferrin contains 686 amino acids, has a molecular weight of 77.90 kDa, and has an isoelectric point of 6.38. Ovotransferrin is composed of two lobes linked by an alpha helix (Charter and Lagarde 2004); each lobe has the capability to reversibly bind one Fe3+ ion. The presence of a bicarbonate ion in the molecule is reported to enhance the iron binding of the molecule (Charter and Lagarde 2004). Its antimicrobial activity is due to its iron-binding ability, which makes the iron unavailable for microorganisms (Bullen et al. 1978). Ovotransferrin acts as an antimicrobial agent toward bacteria species such as Pseudomonas spp., Escherichia coli, and Streptococcus mutans (Valenti et al. 1983). This activity is largely bacteriostatic, being reversed by the addition of ferric ions; if the protein is saturated with iron, the bacteriostatic effect it has on Gram-negative bacteria is overcome (Baron and Rehault 2007). In other studies, however, it has been suggested that the antimicrobial activity of ovotransferrin is not simply due to the deprivation of iron but involves a more complex mechanism. For example, ovotransferrin or metal-saturated ovotransferrin shows an antimicrobial activity dependent on direct interaction with the bacterial surface (Valenti et al. 1987). It has also been demonstrated that ovotransferrin, in apo- or in holo-form, exerts a bactericidal effect against Staphyococcus aureus. Therefore, the bactericidal activity of ovotransferrin

that is unrelated to its iron-binding ability is exerted by both the apo- and holo-forms. It has also been suggested that direct binding to the Candida surface may be responsible for the observed antifungal activity of ovotransferrin (Valenti et al. 1985). The bactericidal activity of ovotransferrin remains separate from the iron-depriving properties of the N-lobe (residues 1–332). Ibrahim et al. (1998) identified an antimicrobial peptide from ovotransferrin by using specific cleavage at aspartyl residues in a diluted acid solution. This 92 amino acid residue sequence, OTAP-92, was also identified from the hydrolysate of ovotransferrin with trypsin and chymotrypsin (Pellegrini 2003). This peptide produces its bactericidal effect by damaging the biological function of the bacterial cytoplasmic membrane. It is able to kill Gram-negative bacteria by crossing the outer membrane through a self-promoted uptake mechanism (Ibrahim et al. 2000). It has a wider antibacterial spectrum than the whole original protein. In addition to acting against Staphylococcus aureus and Escherichia coli K-12 (Ibrahim et al. 2000), this peptide was reported to inhibit Pseudomonas spp., Streptococcus mutans (Valenti et al. 1983), Bacillus cereus (Abdullah and Chahine 1999), and Salmonella enteritidis (Baron et al. 2004).

2.4. Antimicrobial Peptides from Ovalbumin Ovalbumin, the first egg white protein to be discovered in the shell (Hincke 1995), may play a role as an antimicrobial agent. Several antimicrobial peptides have been reported, including Ser-Ala-Leu-Ala-Met (residues 36–40), Ser-AlaLeu-Ale-Met-Val-Tyr (residues 36–42), Tyr-ProIle-Leu-Pro-Glu-Tyr-Leu-Gln (residues 111–119), Glu-Leu-Ile-Asn-Ser-Trp (residues 143–148), AsnVal-Leu-Gln-Pro-Ser-Ser (residues 159–165), AlaGlu - Glu - Arg - Tyr- Pro - Ile - Leu - Pro - Glu - Tyr- Leu (residues 107–118), Gly-Ile-Ile-Arg-Asn (residues 155–159), and Thr-Ser-Ser-Asn-Val-Met-Glu-GluArg (residues 268–276) (Pellegrini et al. 2004). The most effective bactericidal peptides against B. subtilis were Gly-Ile-Ile-Arg-Asn (residues 155–159)

Chapter 17 Bioactive Proteins and Peptides from Egg Proteins

and Thr-Ser-Ser-Asn-Val-Met-Glu-Glu-Arg (residues 268–276), both of which are produced by chymotrypsin digestion. It was thought that, as in many other antimicrobial peptides, the positive charge and hydrophobicity are mainly responsible for making these peptides effective as bactericides against Gram-negative bacteria, but this does not always correlate with the activities of these peptides (Wilde et al. 1989; Bangalore et al. 1990; Pellegrini et al. 1992, 1997; Ibrahim et al. 2002). For example, the peptide with the sequence 155–159 is the only peptide found to have a positive charge (Pellegrini et al. 2004), while the peptide with the sequence 268–276 is the least hydrophobic but exhibits the highest spectrums of antimicrobial action.

2.5. Other Antimicrobial Proteins and Peptides Ovomucin comprises only about 3.5% of total egg white proteins but is the primary contributor to the gel-like nature of egg white (Hiidenhovi 2007). It was reported that ovomucin consists of a carbohydrate-poor subunit (α-subunit, 220 kDa, ∼10–15% carbohydrate) and a carbohydrate-rich subunit (β-subunit, 400 kDa, ∼50–65% carbohydrate) (Hiidenhovi 2007). The primary amino acid sequences of α-ovomucin and β-ovomucin have been deduced from cloned cDNA (Hiidenhovi 2007). The physical function of ovomucin is to maintain the structure and viscosity of the egg white albumen. This function prevents the spread of microorganisms (Ibrahim 1997). Moreover, recent studies have reported that ovomucin has antiviral activity (Tsuge et al. 1996). The adhesion of various microorganisms to host tissues is the first step in infection, which generally takes place after the interaction between the surface of the microorganisms and the carbohydrates present on the surface of the host tissue (Sharon and Ofek 2002). Previous studies suggested that a conjugation of oligosaccharides and glycol can competitively inhibit microorganismcarbohydrate adhesion on intestinal cells, thus helping to prevent microbial infection (Kobayashi et al. 2004). It has been observed that ovomucinderived ovomucin glycopeptides (OGPs) possess

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antimicrobial activity against E. coli O157:H7 through specific binding sites consisting of sialic acid (Kobayashi et al. 2004). Sialic acid–containing oligopeptides from yolk proteins can bind to Salmonella enteritidis and E. coli and prevent Salmonella infection when orally administered to mice (Sugita-Konishi et al. 2002). Ovomacroglobulin is a potent protease inhibitor that inhibits proteases such as serine, cysteine, thiol, and metalloprotease (Kitamoto et al. 1982; Molla et al. 1987; Miyagawa et al. 1994; Wu et al. 2001). It makes up nearly 0.50% of total egg white proteins and is composed of 1,454 amino acid residues. The molecular weight of the protein is 164.10 kDa, and its isoelectric point is 5.75. Miyagawa et al. (1994) found that ovomacroglobulin is capable of inhibiting stromal degradation from Pseudomonas aeruginosa and Serratia marcescens keratitis. Ovomacroglobulin inhibits proteases elastase and alkaline proteases of Pseudomonas aeruginosa and the 56-KDa protease (56KP) of Serratia marcescens in vitro. These Gram-negative pathogens cause severe corneal infections. Ovomacroglobulin had inhibitory effects on keratitis caused by these bacteria (Molla et al. 1987; Miyagawa et al. 1994). Ovomacroglobulin was also found to suppress P. aeruginosa and Vibrio Vulnificus septicemia due to the inhibition of kinin-generating proteases (Maeda et al. 1993; Maruo et al. 1998). Phosvitin is a phosphoglycoprotein that represents about 12% of all yolk proteins. Phosvitin contains 6% carbohydrate and is the most highly phosphorylated protein (Ito and Fujii 1962). Egg posvitin is made up of two polypeptides: α-phosvitin and β-phosvitin, which contain 3% and 12% phosphorus, respectively, and account for about 80% of total protein phosphorus in egg yolk (Joubert and Cook 1958). One of the special features of this protein is its high serine content: nearly 50% of the amino acids in the protein are serine, 96% of which are present in the form of phosphoserines. Because of this characteristic, phosvitin possesses very strong metal-chelating properties. It was reported that 95% of the total iron content of the egg is bound to phosvitin (Albright et al. 1984), and phosvitin is known to also be able to bind calcium and

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magnesium (Taborsky 1983). The high iron-binding capacity is responsible for the antimicrobial properties of phosvitin (Sattar Khan et al. 2000), which can work against Escherichia coli under thermal stress by causing DNA leakage due to disruption of the cells (Choi et al. 2004). Avidin is a homotetrameric protein making up 0.05% of total egg white proteins. It contains 152 amino acid residues, has a molecular weight of 16.77 kDa, and has an isoelectric point of 10.35. Avidin can be easily recovered as a by-product in lysozyme separation (Durance and Nakai 1988). Avidin is best known for its high biotin-binding ability and shows a bacteriostatic effect on bacteria that require biotin (Korpela et al. 1984; Charter and Lagarde 2004). Egg cystatin is a small protein (12.7 kDa) containing no carbohydrates and belonging to cystatin family II (Fossum and Whitaker 1968; Keppler 2006). The antimicrobial activity of cystatin may be attributed to the inhibition of the cysteine proteinase required by the bacteria (Ibrahim 1997). Cystatin shows antibacterial activity against group A Streptococcus (Bjorck 1990) and Salmonella typhimurium (Nakai 2000). It has also been found to inhibit the action of poliovirus protease, effectively inhibiting virus replication in vitro (Korant et al. 1986; Ebina and Tsukada 1991), and oral consumption of cystatin may help in prevention of human rotavirus infection (Ebina and Tsukada 1991).

3. Immunomodulating Proteins and Peptides The immune system includes all parts of the body that help in the recognition and destruction of foreign materials. In mammals, the transfer of maternal antibodies can take place after birth; in chickens, however, the maternal antibodies must be transferred to the developing embryo, to give acquired immunity to the chick. Egg yolk antibodies may find wide applications as previously reviewed (Kovacs-Nolan and Mine 2004; Sunwoo et al. 2006). Several other egg proteins have been reported to have immunomodulating properties.

Ovalbumin is classified as the major allergen in egg white (Hoffman 1983). An active egg albumen product was obtained by fermenting with Saccharomyces cerevisiae (yeast) and spray drying (Araki et al. 1992); oral administration of this product improves the nonspecific activity of neutrophils. Immunogenic peptides were reported from ovalbumin (Vidovic et al. 2002; He et al. 2003). Two peptides, Ser-Val-Asn-Val-His-Ser-Ser-Leu (77–84 amino acid residues) and Tyr-Arg-Gly-Gly-LeuGlu-Pro-Ile-Asn (126–134 amino acid residues), were reported to increase phagocytic activity in macrophages and are currently being studied for immune response in cancer immunotherapy (Goldberg et al. 2003). Ovotransferrin is structurally similar to the hen serum transferrins. Previous research suggested that ovotransferrin can modulate macrophage and heterophil functions in chickens, allowing it to act as an immunomodulator (Hang et al. 2002). Ovotransferrin was successfully used in inhibiting the proliferation of mouse spleen lymphocytes (Otani and Odashima 1997). It has also been shown that ovotransferrin can enhance the phagocytic response of peripheral blood mononuclear cells and polymorphonuclear cells in dogs (Hirota et al. 1995). Ovomucin-derived peptides show macrophagestimulating activity under in vitro conditions (Tanizaki et al. 1997). Synthetic ovomucoid peptides have been studied and have been found to display immunomodulating activity by inducing T-cell secretion of the cytokines interleukin (IL)-4, IL-10, IL-13, and IL-6, as well as in interferon (Holen et al. 2001). Lysozyme activity relies mainly on its effects on monocytes and lymphocytes. Lysozyme is used in antigen-specific immunotherapy; thus, it helps to improve chronic sinusitis (Asakura et al. 1990) and to normalize the humoral and cellular response in patients with chronic bronchitis (Gavrilenko et al. 1992). It was reported that lysozyme can reduce the occurrence of post-transfusion hepatitis in patients who have already undergone a long period of whole blood transfusion (Sato et al. 1981). Previous research suggested that lysozyme can increase antibody production in hybridomas and lymphocytes.

Chapter 17 Bioactive Proteins and Peptides from Egg Proteins

There is evidence that the antimicrobial effect of lysozyme may occur via simulation of macrophage and phagocytic functions (Li-Chan and Nakai 1989). It has also been suggested that lysozyme hydrolysate may act as an adjacent immunomodulator (Herzog et al. 2005). Cystatin was reported to possess immunomodulating activity through inducing the synthesis of TNF-α and IL-10 (Das et al. 2001). Furthermore, cystatins can elevate the production of IL-6 by human gingivial fibroblast cells and murine splenocytes.

4. Anticancer Proteins and Peptides Cancer is a major cause of mortality, with incidence rates increasing throughout the world. It is estimated that 159,900 new cases of cancer and 72,700 deaths will occur in Canada in 2007 (CCS 2007). The World Health Organization predicts that, over the next decade, the number of cases of cancer will increase by 73% in the developing world and by 29% in the developed world. Although the underlying causes of many cancers have not yet been fully defined, a wealth of evidence points to diet as one of the most important modifiable determinants of the risk of developing cancer, possibly due to the presence of potentially bioactive food components that are protective at different stages of cancer formation (Milner 2004). There is tremendous interest in discovering novel peptides that may act as anticancer agents because of their multifunctionality, high sensitivity, and stability characteristics (Lee et al. 2005; Leng et al. 2005). Anticancer peptides have been reported recently in soy proteins and milk proteins (Damiens et al. 1999; Kim et al. 2000). Anticancer properties have been indicated in several egg proteins as well. In previous studies, lysozyme has played a role as an anticancer agent through the host-mediated immune response and probably through the inhibition of tumor formation and growth (Sava et al. 1989; Kovacs-Nolan et al. 2005). It has been established that avidin, an egg white protein, exhibits anticancer activity (Gasparri et al. 1999). The activity of avidin as an antitumor agent may involve changes in the host-tumor relationship or the host-mediated antitu-

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mor response. The great rebirth of interest in cystatin research stems from its inhibition of cysteine proteinases, which has led to an attempt to develop novel anticancer agents, especially after the unsuccessful exploration of serine- and metallo-protease inhibitors as anticancer agents in the last 30 years (Coussens et al. 2002). Cysteine proteases have been implicated in multiple steps of tumor progression, including early steps of immortalization and transformation, intermediate steps of tumor invasion and angiogenesis, and late steps of metastasis and drug resistance (Keppler 2006). Cystatin inhibited tumor invasion in epithelial cells (Premzl et al. 2001) and was found to reduce the activity of the key proteolytic enzymes responsible for the growth of gastric cancer in vitro (Muehlenweg et al. 2000). In addition, egg cystatin was reported to affect the host’s immune response through the cytokine network, which stimulates nitric oxide production, contributing to its anticancer properties (Verdot et al. 1999). Ovomucin was reported to have the potential to inhibit the growth of cancer cells by limiting angiogenesis (Oguro et al. 2001). Recently, a 70 kDa fragment from pronase-treated ovomucin has also demonstrated anticancer properties in a doublegrafted tumor system (Watanabe et al. 1998).

5. Antioxidant Proteins and Peptides Reactive oxygen species (superoxides, hydrogen peroxides, and hydroxyl radicals) and other free radicals cause oxidative damage in DNA, proteins, and other macromolecules. Reactive oxygen species cause degenerative diseases, including diabetes, cancer, and other cardiovascular diseases (Ames et al. 1993). Antioxidants can repair cell or DNA damage and can remain in the body for long periods of time. Antioxidant compounds in food play important roles in protecting health. Scientific studies suggest that antioxidants reduce the risk of chronic diseases, including cancer and heart disease. Primary sources of naturally occurring antioxidants include whole grains, fruits, and vegetables. Most of the antioxidant compounds in a typical diet are derived from plant sources and belong to various classes of compounds with a wide variety of physical and

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chemical properties. In addition to phytochemicals and vitamins, egg proteins have been recognized as an excellent source of antioxidants. Phosvitin has been reported to play an important role as an antioxidant in the yolk by inhibiting ironcatalyzed lipid oxidation due to its high iron-binding capacity (Lu and Baker 1986). Its affinity for inhibiting lipid peroxidation reactions is known to be decreased by high-temperature treatment such as autoclaving (Lu and Baker 1986) but could be improved by Maillard-type protein-polysaccharide conjugates (Nakamura et al. 1998). Phosvitin is a strong inhibitor of hydroxyl radical formation by the Fenton reaction (Ishikawa et al. 2004); it showed higher antioxidative activity than did various ironbinding proteins such as ferritin and transferrin, which indicates that phosphoprotein may be a better iron storage protein to prevent iron-dependent oxidative damage than ferritin in avian eggs. Furthermore, the tryptic digest of phosvitin was as efficient an antioxidant as phosvitin in inhibiting Fe(II)-catalyzed •OH formation. Oligophosphopeptides with molecular weights of 1,000–3,000 daltons, prepared by partial alkaline dephosphorylation and tryptic hydrolysis of egg yolk phosvitin, exhibited antioxidant effects by suppressing the inflammatory response, acting as lipid peroxidation inhibitors, inhibiting lipid oxidation in a linoleic acid system, and exhibiting radical-scavenging activities on 2,2diphenyl-1-picrylhydrazyl (DPPH) free radicals (Xu et al. 2007). Katayama et al. (2007) reported that the antioxidative effects of the peptides are related to the elevation of intracellular antioxidants (glutathione) and the enhancement of antioxidative enzymes (glutathione reductase, glutathione S-transferase and catalase). Although the major peptide sequences have not been characterized in the hydrolysates, it was reported that a fraction denoted as PPP-3, having a large moiety of phosphorus, showed a significantly higher antioxidative activity than that of a PPP-1 fraction without phosphorus. Moreover, the PPP-3 fraction contained half as much His as PPP-1, whereas PPP-3 was not rich in Lys, Pro, Glu, or Ala compared with PPP-1 (Katayama et al. 2006). Ovotransferrin was reported recently as a superoxide dismutase (SOD) mimic protein with high

specificity to dismutate Oi− 2 anions (Ibrahim et al. 2007). The isolated half-molecules of ovotransferrin exhibited higher scavenging activity than did the intact molecule, whereas the N-lobe showed much greater activity. They also showed that metal-bound ovotransferrin exhibited greater Oi− 2 anion dismutation capacity than did the apo-protein. Interestingly, enhanced antioxidative activity in metal-bound ovotransferrin was indicated to be related to a number of smaller polypeptide fragments, resulting from self-cleavage action (Ibrahim et al. 2007). Moreover, peptides from digested ovotransferrin showed significantly increased antioxidative properties. Our laboratory has shown that the ORAC value of ovotransferrin hydrolysate is 2.24 ± 0.01 μmol of Trolox equivalents/mg, compared to 0.19 ± 0.04 μmol of Trolox equivalents/mg in intact ovotransferrin (unpublished data); further work is underway to characterize the antioxidant peptides from the hydrolysate. Lysozyme can protect against acute oxidative stress (OS) as well as confer resistance to chronic OS against milder oxidants. Given these properties, structurally and functionally distinct oxidants may be able to share aspects of intracellular mechanisms through signaling pathways involving key stress response genes, which can be regulated by lysozyme (Liu et al. 2006). Three antioxidant peptides were previously isolated from egg white albumin (Tsuge et al. 1991). Several angiotensin-converting enzyme inhibitory peptides showed antioxidative activities (Davalos et al. 2004); Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu exhibited the highest radical-scavenging activity and delayed low-density lipoprotein lipid oxidation. Two peptides, Leu-Met-Ser-Tyr-Met-Trp-Ser-ThrSer-Met and Leu-Glu-Leu-His-Lys-Leu-Arg-SerSer-His-Trp-Phe-Ser-Arg-Arg, were isolated from egg yolk (Park et al. 2001).

6. Antihypertensive Peptides Hypertension is one of the major causes of morbidity and mortality in Western countries, affecting 27.6% of the population in North America. Angiotensin-converting enzyme (ACE) is the key

Chapter 17 Bioactive Proteins and Peptides from Egg Proteins

enzyme that is responsible for regulating blood pressure through the renin-angiotensin system. ACE catalyzes the formation of angiotensin II, a potent vasoconstrictor, from angiotensin I and inactivates bradykinin, a vasodilator (Ondetti et al. 1977). Elevated ACE activity could lead to a higher level of angiotensin II and therefore cause high blood pressure or hypertension. Inhibition of ACE is the therapeutic target for antihypertensive drug development; currently, ACE inhibitory drugs are widely used as the first-line therapy for hypertension. However, these synthetic ACE inhibitory drugs, such as captopril and enalapril, are reported to inevitably cause adverse side effects such as cough and angioedema (Atkinson and Robertson 1979). The incidence of cough has been estimated at 5–20% of patients and may result in the discontinuation of treatment; angioedema affects 0.1–0.5% of patients but can be life-threatening. Given the adverse side effects and increasing cost associated with drug therapy, there has been great interest in the search for natural ACE inhibitors as alternatives to synthetic ones. Many ACE inhibitory peptides have been reported from various protein sources (FitzGerald et al. 2004; Miguel et al. 2005). Various antihypertensive peptides have been identified from ovalbumin. Ovokinin, an octapeptide Phe-Arg-Ala-Asp-His-Pro-Phe-Leu, is a welldocumented vasorelaxing peptide from ovalbumin pepsin digest (Fujita et al. 1995). Ovokinin significantly lowered the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs), when orally administrated in solution at a dose of 100 mg/ kg, or at a dose of 25 mg/kg in the form of an emulsion in 30% egg yolk (Fujita et al. 1995). A peptide lacking the N-terminus Phe residue of ovokinin, Arg-Ala-Asp-His-Pro-Phe-Leu, was isolated from the ovalbumin pepsin hydrolysate (Miguel et al. 2004). This peptide showed weaker in vitro ACE inhibitory activity (IC50 = 6.2 μM) than that of ovokinin (IC50 = 3.2 μM) but was demonstrated to have a significant antihypertensive activity in SHR (Miguel et al. 2005). A hexapeptide corresponding to the 2-7 fragment of ovokinin was identified from a chymotrypsin digest of ovalbumin and shown to induce nitric oxide–dependent vasorelaxation in an

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isolated mesenteric artery as well as an antihypertensive effect in SHRs (Matoba et al. 1999). Because these peptides are weak ACE inhibitors, the observed antihypertensive activity they produce is not mediated through the ACE inhibitory mechanism (Miguel and Aleixandre 2006). Tyr-Ala-Glu-Glu-Arg-TyrPro-Ile-Leu is another potent ACE inhibitory peptide (IC50 = 4.7 μM) that was identified from pepsin ovalbumin hydrolysate and showed significant antihypertensive activity in SHR at doses of 2 mg/kg (Miguel et al. 2004, 2005). However, this peptide was totally degraded by simulated gastrointestinal digestion, and its ACE inhibitory activity decreased by approximately 100-fold; surprisingly, one of its main fragments, YPI (IC50 > 1,000 μM), could significantly decrease SBP in SHR at a dose of 2 mg/ kg (Miguel and Aleixandre 2006). We have recently shown that cooking methods had a significant effect on the ACE inhibitory activity under simulated gastrointestinal digestion. Fried egg digests showed more potent activity than those of boiled egg digests; the fried whole egg digest had an IC50 value of 0.009 mg protein/mL. Seven peptides were identified by liquid chromatography-mass spectrometry (LC-MS/MS), and their IC50 values were predicted by using our previously reported structure and activity models (Wu et al. 2006; Majumder and Wu 2009) as follows: Val-Asp-Phe (predicted IC50: 6.59 μM), Leu-Pro-Phe (10.59 μM), Met-Pro-Phe (17.98 μM), Tyr-Thr-Ala-Gly-Val (23.38 μM), Glu-Arg-Tyr-Pro-Ile (8.76 μM), IlePro-Phe (8.78 μM), and Thr-Thr-Ile (24.94 μM). The characterization of several tripeptides from in vitro simulated gastrointestinal egg digest indicates a possible high absorption of these peptides to exert in vivo antihypertensive activity, although in vivo study is needed to confirm this assumption. Antihypertensive peptides were reported mostly from ovalbumin in literature (López-Fandiño et al. 2007). The discovery of many ACE inhibitory peptides from food proteins has greatly advanced our knowledge of the structure and activity relationships of peptides. Through quantitative structure and activity relationship (QSAR) study, we have determined the structural requirements of ACE inhibitory peptides (Wu et al. 2006); by using an integrated

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in silico digestion and QSAR study, several novel ACE inhibitory peptides, Ile-Arg-Trp (IC50: 0.6 μM) and Leu-Arg-Trp (IC50: 2.8 μM), were discovered from other egg white proteins (Majumder and Wu 2010). It should be noted that these tripeptides may have a greater chance to escape a gastrointestinal enzyme attack and therefore reach the site of action, although in vivo study is needed.

7. Protease Inhibitors Egg white is a rich source of protease inhibitors, including ovalbumin, ovostatin, ovomucoid, ovoinhibitor, and cystatin C (Saxena and Tayyab 1979). In egg white, protease inhibitors are thought to play a role in embryogenesis (Saxena and Tayyab 1979; Colella et al. 1989). However, the natural targeted proteases of these egg white inhibitors have not been identified, and their physiological role remains unclear (Rehault 2007). Based on sequence homologies, ovalbumin is classified in the serpin (serine proteinase inhibitor) family, although it lacks any protease inhibitory activity in its native form (Hunt and Dayhoff 1980; Stein et al. 1990). Ovalbumin may migrate into the developing embryo by changing its form to a lessordered S-form structure fitted to transportation, and therefore it may play a role in developing embryonic cells (Sugimoto et al. 1999); however, some have suggested that it is simply a storage protein (Whisstock et al. 1998). Mellet et al. (1996) showed that heating ovalbumin for 30 minutes at 97°C transformed it into a potent reversible competitive inhibitor; but the inhibitory ovalbumin obtained so far differs from active serpins in its inability to form irreversible complexes with proteinases. The ovalbumin primary sequence thus contains the information making it possible for the first step of the serpin-proteinase interaction to occur, but it does not contain the information needed for stabilizing this initial complex. Ovostatin has been shown to inhibit several metalloproteases and, with a lesser degree of efficiency, serine proteases, cysteine proteases, and aspartyl proteases (Saxena and Tayyab 1979; Kitamoto et al. 1982; Molla et al. 1987; Miyagawa

et al. 1994; Wu et al. 2001). Ovomucoid is a serine protease inhibitor (Kato et al. 1987) and is particularly useful for the oral delivery of protein/peptide therapeutics (Shah and Khan 2004). Both cystatin and ovoinhibitor are serine protease inhibitors (Tomimatsu et al. 1966). Ovoinhibitor is a 48 kDa glycoprotein, containing seven Kazal-like domains involving 21 disulfide bonds (Scott et al. 1987; Yet and Wold 1990). Domain I is a potent inhibitor of trypsin but is devoid of inhibitory activity against chymotrypsin, elastase, or proteinase K (Galzie et al. 1996). Domains II to IV are thought to inhibit trypsin-like enzymes; domain V possesses antichymotrypsin activity; domains VI and VII inhibit chymotrypsin and elastase (Scott et al. 1987).

8. Conclusion From the beginning of civilization, the hen egg has been used as an essential and important food throughout the world. Eggs are sometimes described as “nature’s perfect food.” Unquestionably, eggs are the most unique food product and have attracted extensive research for many years. Recent studies have uncovered various roles of egg proteins that go well beyond their basic nutrient value. Indeed, egg proteins may gain increasing recognition in the future in human health, as well as in disease prevention and treatment. However, this value can only be captured through the development of industrially viable technologies that are essential for successfully harvesting valuable egg components for commercial use. Lysozyme and antibody technologies are two such examples of extracted components. More recently, proteomic analysis of egg proteins has revealed new minor proteins with several novel biological properties (Guérin-Dubiard and Nau 2007). The presence of biologically active proteins, especially minor egg proteins, has raised interests in developing novel agents from eggs against chronic diseases, such as cancer. Furthermore, accumulated evidence has also pinpointed that eggs are an excellent source of bioactive peptides, such as antihypertensive property and antioxidant, antimicrobial, and immunomodulating properties. Eggs contain more than 60 various types of proteins; the potential of

Chapter 17 Bioactive Proteins and Peptides from Egg Proteins

developing novel bioactive peptides deserves further study.

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Chapter 17 Bioactive Proteins and Peptides from Egg Proteins

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Chapter 18 Soy Peptides as Functional Food Materials Toshihiro Nakamori

Contents 1. Introduction, 265 2. Peptide Transport System, 265 3. Development of Di- and Tri-enriched Peptides from Soy, 266 4. Manufacturing Process for Soy Peptides, 266

1. Introduction Matthews (1975) reported that digestion of proteins to free amino acids in the intestinal lumen was recognized in 1865 by Kollinker and Muller. Early trials of peptides research started in the 1960s, and much attention has been paid to food-derived peptides concerning their absorbability compared with amino acids. Craft et al. (1968) revealed that di- and triglycine peptides are absorbed rapidly compared with the same amount of free glycine in human intestine. Thereafter, many beneficial characters of a peptide transporter were widely recognized (Ziegler et al. 2002), and beneficial effects of soy peptides were showed (Bamba et al. 1989; Nakabou et al. 1990; Chun et al. 1996). On the other hand, soy protein is one of the vegetable proteins examined extensively for lipid-lowering effects in humans and in experimental animals (Yashiro et al. 1985; Erdman 2000). Although soy

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

5. Efficacy of Soy Peptides in Sports Compared with Soy Protein, 267 6. Efficacy of Soy Peptides on Lipid Metabolism Compared with Soy Protein, 267 7. Conclusion, 269 8. References, 270

protein contains a certain amount of bioactive peptides that have distinct physiological activities in lipid metabolism, it is not clear which peptides are responsible for these effects (Aoyama et al. 1996, 2000; Tamaru et al. 2007). This chapter describes the nutritional benefits and the biochemical function of soy peptides.

2. Peptide Transport System In the 1970s, Adibi (Adibi and Mercer 1973; Adibi et al. 1974; Adibi and Soleimanpour) revealed peptide characters of absorptive events in the gastrointestinal tract. The rapid absorption of small peptides compared to amino acids is mainly due to the differences in the absorption system. Adibi (1997) revealed the presence of the peptide transport in 1997. The substrate-specific amino acid transport system located in the brush border membrane is available for the transport of amino acids. Di- and tripeptides are absorbed by peptide form using the peptide transport system and then hydrolyzed into amino acids by intracellular peptidases (Figure 18.1). 265

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Figure 18.1. Amino acid transport system and peptide transport system on small intestine cell.

3. Development of Di- and Tri-enriched Peptides from Soy It was known that peptide transporters have low substrate selectivity, and thus the peptide transport system in the small intestine is an ideal transport system because it uniformly absorbs many types of amino acids (Abe et al. 1987). A few studies using normal and intestinal disorder animal models have studied the possibility that blood amino acid level increases rapidly after ingestion of soy peptides compared with ingestion of soy protein and amino acid mixture (Nakabou et al. 1989, 1990; Ihara et al. 1990). The speed of absorption ratio of amino acids taken into the body varies according to the types of hydrolyzed dietary protein. Chun et al. (1996, 1999) reported on the influence of chain length of soy peptides on intestinal absorption in the intestinal perfusion model (using an intestinal everted sac) of the rat small intestine. Maebuchi et al. (2007) undertook basic studies and clinical application to design the best absorptive soy peptides that hydrolyzed soy protein. Preliminary clinical trials of 11S globulin (a major component of soy protein), peptides prepared from 11S globulin, and an amino acid mixture of equivalent composition were examined in 12 human subjects. It was shown that the ingestion of soy peptides results in faster and more efficient absorption than the consumption of protein or amino acid mixtures in

Figure 18.2. Serum essential amino acid levels in peripheral blood vessel after ingestion of the test beverages (n = 12). Source: Maebuchi et al. 2007.

healthy adult men. In other words, these results may suggest that the peptide transport system efficiently absorbs amino acids into the human body in the form of di- and tripeptides (Figure 18.2).

4. Manufacturing Process for Soy Peptides On the basis of Maebuti and their preliminary clinical trials, our attempt to develop commercial peptides containing numerous dipeptides and tripeptides was successful on an industrial scale. We launched a new product, “HINUTE-AM,” in 2007. Practical commercial soy peptides “HINUTEAM” is produced with following processes: A starting material uses the defatted soybean flake. Water is added to the defatted soy flake, and soymilk is made by removing Okara, which is a fibrous residue of the protein curd. Then, a protein is precipitated by acidification. Soy protein is then hydrolyzed by a number of commercial enzymes in suitable conditions for several hours, the undigested fraction is separated and further purified from the enzyme hydrolysis, and the hydrolysate is evaporated, dried, and packed (Figure 18.3). A hydrolysate produced from dietary protein is generally extremely bitter and has an undesirable taste (Pedersen 1994); however, “HINUTE-AM,” which contains a high degree of di- and tripeptides, does not taste bitter (Table 18.1).

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Figure 18.3. The scheme of production of soy peptides.

Table 18.1. Typical analysis of HINUTE-AM. Crude protein/dry (%) Content of di- and tripeptides Content of free amino acids Ash/dry (%) pH of solution

>90% >65% 5%> 6.5%> 5.5–6.5

5. Efficacy of Soy Peptides in Sports Compared with Soy Protein The protein synthesis and consumption in our body is stimulated by exercise. The protein also helps to repair damaged muscle structure after exercise. A requirement for protein in strength-trained athletes was commonly assumed to be higher than daily protein intakes levels for the general population. Masuda et al. (2007) compared the effectiveness of placebo, soy protein, and soy peptides regarding serum growth hormone (GH) and serum creatine phosphokinase (CPK) levels, which were measured at both 30 minutes and 18 hours later on exerciseinduced muscle damage. The CPK level did not change at 30 minutes after an exercise, though the level at 18 hours was significantly higher. The difference between 8 gram soy protein ingestion and 4 gram soy peptides ingestion was not significant.

Growth hormone levels were higher at 30 minutes, and the difference between placebo and 8 gram soy peptides was significant. A similar trend was observed for 8 gram soy protein ingestion and 4 gram soy peptides ingestion. These results suggest that 4 gram soy peptides had the same efficacy as 8 gram soy protein (Figure 18.4). On the other hand, Miura et al. (1995) compared the availability between soy peptides and soy protein with 24 male power-lifters for 6 months. All participants had three meals a day and did weight training for 2 hours. They drank test beverages before and after exercise. In terms of total muscle strength index, both groups of soy peptides and soy protein significantly increased for 6 months. However, results showed that groups of soy peptides decreased the body fat significantly (Figure 18.5).

6. Efficacy of Soy Peptides on Lipid Metabolism Compared with Soy Protein It has been suggested that soy peptides stimulate a fatter metabolism than soy protein and have more antiobesity effect. Saito (1989, 1991) showed that soy peptides stimulated the thermogenic activity of brown

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Figure 18.4. Comparison of placebo, soy protein, and soy peptides on GH and CPK levels measured at 30 minutes and 18 hours after soy peptides ingestion (N = 41). Source: Masuda et al. 2007.

Figure 18.5. Effect of ingestion for over 6 months in the body composition change (N = 24). Source: Miura et al. 1995.

adipose tissue (BAT), which suggested an increase in energy expenditure. Aoyama et al. (1996, 2000a, 2000b) showed that body fat-reducing effects of soy protein and soy peptide groups were observed using dietary obese rats and genetically obese mice. The soy peptide group tended to have lower levels than

the soy protein group in several parameters, such as body weight, body fat, plasma lipids, and plasma glucose (Figures 18.6 and 18.7). Furthermore, Tamaru et al. (2007) observed a reduction of liver and serum triglyceride (TG) after ingesting soy peptide compared with soy protein and

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Figure 18.6. Effects of soy protein and animal protein on body fat ratio of obese rat and genetically obese mice. Source: Aoyama et al. 2000.

Figure 18.7. Blood lipid level of obese rat. Source: Aoyama et al. 2000.

casein. In the liver, β-oxidation was stimulated and fatty acid synthesis was suppressed. On the other hand, Komatsu et al. (1988, 1989, 1990, 1991, 1992) demonstrated clinical experiments on soy peptides with obese subjects. Their experiments revealed that soy peptides intake helps maintain (or increase) body protein and basal energy expenditure and may stimulate lipid metabolism. Compared to other nitrogen sources, soy peptides increased basal energy expenditure. The antiobesity effect of soy peptides is thought to involve lipid metabolism. Although soy peptides contain a certain amount of bioactive peptides that have distinct physiological activities in lipid metabolism, it is not clear which peptides are responsible for these effects. We think that it is necessary for

hypolipidemic peptides to be isolated and identified in the future.

7. Conclusion Soy peptides prepared from soy protein could well serve as an excellent nutritional supplement with improved absorption properties. The ingestion of soy peptides decreases the muscle damage induced after exercise and shows double effectiveness as compared with soy protein. On the other hand, it was revealed that soy peptides stimulate hypolipidemic events and accelerate antiobesity effects. It is important to conduct more research on active components in the future. Soy peptides may have the ability to prevent some lifestyle diseases.

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8. References Abe M, Hoshi T, Tajima A. 1987. Characteristics of transmural potential changes associated with the protein-peptide cotransport in toad small intestine. J Physiol (Lond) 394:481– 499. Adibi SA. 1997. The oligopeptide transporter (Pept-1) in human intestine: Biology and function. Gastroenterology 113:332– 340. Adibi SA, Fogel MR, Agrawal RM. 1974. Comparison of free amino acid and dipeptide absorption in the jejunum of sprue patients. Gastroenterology 67:586–591. Adibi SA, Mercer DW. 1973. Protein digestion in human intestine as reflected in luminal, mucosal, and plasma amino acid concentrations after meals. J Clin Invest 52:1586– 1594. Adibi SA, Soleimanpour MR. 1974. Functional characterization of dipeptide transport system in human jejunum. J Clin Invest 53:1368–1374. Aoyama T, Fukui K, Nakamori T, Hashimoto Y, Yamamoto T, Takamatsu K, Sugano M. 2000b. Effect of soy and milk whey protein isolates and their hydrolysates on weight reduction in genetically obese mice. Biosci Biotechnol Biochem 64(12): 2594–2600. Aoyama T, Fukui K, Takamatsu K, Hashimoto Y, Yamamoto T. 2000a. Soy protein isolate and its hydrolysate reduce body fat of dietary obese rats and genetically obese mice (Yellow KK). Nutrition 16:349–354. Aoyama T, Fukui K, Yamamoto T. 1996. Effect of various forms of force-fed nitrogen sources on gastric transit time in rats. J Jpn Soc Nut Food Sci 49(1):46–51. Bamba T, Chikamochi N, Fuse K, Hosoda T. 1989. Usefulness of soy peptide in the patients with Crohn’s disease. Nutr Sci Soy Protein Jpn 10(1):117–121. Chun H, Sasaki M, Fujiyama Y, Bamba T. 1996. Effect of peptide chain length on absorption and intact transport of hydrolyzed soybean peptide in rat intestinal everted sac. J Clin Biochem Nutr 21:131–140. ———. 1999. Small peptide of soybean hydrolysate is absorbed more rapidly than large peptide in the perfused rat small intestine. J Clin Biochem Nutr 26(3):183–191. Craft IL, Geddes D, Hyde CW, Wise IJ, Matthews DM. 1968. Absorption and malabsorption of glycine and glycine peptides in man. Gut 9:425–437. Erdman JW Jr. 2000. Soy protein and cardiovascular disease: A statement for healthcare professionals from the nutrition committee of the AHA. Circulation 102:2555–2559. Ihara M, Miyanomae T, Kido Y, Kishi K. 1990. Effects of soy protein peptide on nutritional state and small intestinal function in short-bowel and methotrexate treated rats. Nutr Sci Soy Protein Jpn 11(1):87–94. Komatsu T, Komatsu K, Matsuo M, Nagata M, Yamagishi M. 1990. Comparison between effects of energy restricted diets supplemented with soybean peptide and lactalbumin on

energy, protein and lipid metabolisms in treatment of obese children. Nutr Sci Soy Protein Jpn 11:89–103. ———. 1989. Effects of nitrogen supplement using soybean peptide on diet therapy with energy restriction to obese children. Nutr Sci Soy Protein Jpn 10:84–88. Komatsu T, Komatsu K, Nagata M, Yamagishi M. 1991. Effects of diet containing soybean peptide or lactalbumin on basal energy expenditure of obese children reducing body weight and on thermogenesis after meal. Nutr Sci Soy Protein Jpn 12:80–84. Komatsu T, Komatsu K, Yamagishi M. 1992. Effects of soypeptide consumption on diet induced thermogenesis. Nutr Sci Soy Protein Jpn 13:53–58. Komatsu T, Yamagishi M, Komatsu K. 1988. Studies on clinical application of soy peptide. Nutr Sci Soy Protein Jpn 9:61– 65. Maebuchi M, Samoto M, Kohno M, Ito R, Koikeda T, Hirotsuka M, Nakabou Y. 2007. Improvement in the intestinal absorption of soy protein by enzymatic digestion to oligopeptide in healthy adult men. Food Sci Tech Res 13:45– 53. Masuda K, Maebuchi M, Samoto M, Ushijima Y, Uchida Y, Kohno M, Hirotsuka M. 2007. Effect of soy-peptide intake on exercise-induced muscle damage. J Japanese Society of Clinical Sports Medicine 15:228–235. Matthews DM. 1975. Intestinal absorption of peptides. Physiol Rev 55:537–608. Miura K, Takenaka S, Okuno M, Kohara N. 1995. Effect of soy-bean peptide and soy-bean isolated protein intake over 6 months on work capacity of power-lifters. Bul Fac Edu Okayama Univ 100:139–150. Nakabou Y, Suzuki Y, Okamoto M, Kuzuhara Y, Hagihira H. 1990. Nutritional evaluation of oligopeptide mixture prepared from soy protein isolate (SPI) in rats resected two-thirds of the small intestine. Nutr Sci Soy Protein Jpn 11(1):108– 112. Nakabou Y, Utsunomiya R, Suzuki T, Watanabe M, Hagihira H, Matsuo T, Kimoto M, Ohshima Y. 1989. Demineralization of enzymic hydrolysate of SPI (Hinute PM) and absorption of desalted preparation in rat small intestine in vitro. Nutr Sci Soy Protein Jpn 10(1):76–80. Pedersen B. 1994. Removing bitterness from protein hydrolysates. Food Technol 48(10):96–99. Saito M. 1989. Availability of soy protein peptides for total enteral nutrition. Nutr Sci Soy Protein Jpn 10(1):81– 83. ———. 1991. Effects of soy peptides on energy metabolism in obese animals. Nutr Sci Soy Protein Jpn 12(1):91– 94. Tamaru S, Kurayama T, Sakano M, Fukuda N, Nakamori T, Furuta H, Tanaka K, Sugano M. 2007. Effects of dietary soybean peptides on hepatic production of ketone bodies and secretion of triglyceride by perfused rat liver. Biosci Biotechnol Biochem 71(10):2451–2457.

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Yashiro A, Oda S, Sugano M. 1985. Hypercholesterolemic effect of soybean protein in rats and mice after peptic digestion. J Nutr 115(10):1325–1336. Ziegler TR, Fernandez-Estivariz C, Gu LH, Bazargan N, Umeakunne K, Wallace TM, Diaz EE, Rosado KE, Pascal

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RR, Galloway JR, Wilcox JN, Leader LM. 2002. Distribution of the H+/peptide transporter PepT1 in human intestine: Up-regulated expression in the colonic mucosa of patients with short-bowel syndrome. Amer J Clin Nutr 75:922– 930.

Chapter 19 Bioactivity of Proteins and Peptides from Peas (Pisum sativum, Vigna unguiculata, and Cicer arietinum L) Bo Jiang, Wokadala C. Obiro, Yanhong Li, Tao Zhang, and Wanmeng Mu

Contents 1. Introduction, 273 2. Antioxidant Effects, 274 3. Hypocholesterolemic Effects, 275 4. Anticancer Effects due to Bowman-Birk Inhibitors, 278 5. Angiostensin I-Converting Enzyme Inhibitory Effects, 280 6. Antimicrobial Effects due to Peptides, 281 6.1. Antimicrobial Peptides from Garden Pea (Pisum sativum), 281

1. Introduction Legume proteins have been established as significant sources of therapeutic, nutraceutical, and pharmaceutical agents. In this chapter we present recent findings on biological activity of proteins and peptides from legumes commonly referred to as peas (garden/green pea, Pisum sativum; cowpea, Vigna unguiculata; and chickpeas Cicer arietinum). Legumes are important sources of dietary proteins. There has been increased interest in their beneficial effects in terms of several health claims that are professed. The molecular basis for the

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

6.2. Cowpea (Vigna unguiculata) Antimicrobial Proteins/Peptides, 282 6.3. Chickpea (Cicer arietinum) Antimicrobial Proteins/Peptides, 283 6.4. Commercial Utilization Potential of Pea Antimicrobial Proteins/Peptides, 283 7. Future Prospects, 284 8. References, 284

observed biological activities, however, has been elucidated to a limited extent (Scarafoni et al. 2007). It is recommended that multidisciplinary research, including characterization at the molecular level, be done to enable optimization of the functional/ therapeutic properties of the proteins/peptides. In recent years there has been an increase in the application of plant proteins and peptides as supplements and functional components in food products for liquid foods or high-energy beverages, production of hypoallergenic foods, and development of medical diets for the treatment of specific illnesses (Clemente 2000). Biologically active peptides, apart from the naturally occurring peptides, could be active in the original protein but exert their effects best when released from the parent protein by gastrointestinal digestion, food processing, or controlled hydrolysis. Several 273

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bioactivities have been reported from legume proteins such as opiate, immunomodulation, angiotensin I-converting enzyme (ACE) inhibiting, antithrombotic, antioxidant, and antimicrobial activities (Sirtori et al. 2008). Antimicrobial activities have also been shown to be derived from several naturally occurring legume peptides. These serve defensive purposes against predators and pathogens for the legumes (Ng 2004). Some of these peptides have been reported to be able to resist digestion, to be absorbed, and thereby to reach peripheral organs for beneficial effects. Although soybean is the most used source of vegetable proteins for special nutritional formulations, other legumes such as those from peas like chickpeas (Cicer arietinum), garden/green peas (Pisum sativum), and cowpeas (Vigna unguiculata) have picked up formidable interest recently. Data regarding their purported beneficial effects and the underlying mechanisms is limited compared to soybean proteins. There has also been considerable growing interest in use of genetic manipulation to produce recombinant proteins of high nutritional/ therapeutic value based on these proteins through fermentative commercial production and solid state methods. So far, genetic modification of organisms is still far from being applied in the nutraceutical/ therapeutics sphere, primarily because little is known about the role and mechanisms of action of the best part of bioactive proteins/peptides and about the molecular requirements for their proper administration.

2. Antioxidant Effects Studies on antioxidant peptides from the peas have shown their potential as sources of antioxidant agents. Published works, however, have been limited mainly to chickpeas (Cicer arietinum) and garden/ green peas (pisum sativum). The antioxidant activity of proteins and their resultant hydrolysates mostly depends on their amino acid composition. Particular amino acids such as aromatic amino acids like tyrosine, phenylalanine, and tryptophan and the sulfurcontaining amino acid, cysteine, present activity due to their ability to donate protons to free radicals

(Arcan and Yemenicioglu 2007). The position of the amino acids in the protein sequence, however, is also a very important factor effective for antioxidant activity (Arcan and Yemenicioglu 2007). One major advantage of using the pea proteins and their peptides would be their additional functionalities such as gelation properties (Papalamprou et al. 2009; Zhang et al. 2007), emulsifying properties (Zhang et al. 2009), and rheological properties (Kaur and Singh 2007; Ragab et al. 2004; Shand et al. 2007). Arcan and Yemenicioglu (2007) demonstrated that nonhydrolyzed chickpea (Cicer arietinum) proteins had antioxidant activity that was improved by thermal treatment. They showed that the activity was higher than that of a similar extract from white kidney bean (Phaseolus vulgaris). In addition they showed that ammonium sulfate precipitation was not effective for selective precipitation of antioxidant proteins but improved the free radicalscavenging capacity of lyophilized protein extracts from thermally processed chickpeas and white beans by almost 25% and 100%, respectively. Nonhydrolyzed pea protein antioxidant properties were also demonstrated by Wolosiak and Worobiej (1999) in pisum sativum protein isolates. The particular proteins had the ability to decrease the number of hydroperoxides during oxidation of linoleic acid. Hydrolysis significantly increased the antioxidant activity. In their case, however, although they showed differences in the electophorectic patterns of the hydrolysates, there was no significant influence by aromaticity and hydrophobicity on the antoxidative activity. Recently we demonstrated four fractions (Fra.I, Fra.II, Fra.III, and Fra.IV) from chickpea protein hydrolysates that had significant antioxidant activity as measured using reducing power, inhibition of linoleic acid autoxidation, and 1,1-diphenyl-2-pycrylhydrazyl (DPPH)/superoxide/ hydroxyl radical-scavenging assay (Li et al. 2008a). Relative to standard antioxidants, Fra.IV (81.13%) with the strongest activity was closer than αtocopherol (83.66%) but lower than that of BHT (99.71%) in the linoleic acid oxidation system. This fraction had the highest total hydrophobic amino acid content (38.94% THAA) and hydrophobicity

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Table 19.1. Effect of chickpea protein hydrolysate on serum antioxidant activity of aged mice. Group

MDA (nmol/ml)

SOD (U/ml)

Normal Model CPH I CPH II CPH III Vitamin E

5.87 ± 0.72 8.60 ± 0.87 7.97 ± 0.60 6.16 ± 0.22a 5.66 ± 0.72a 5.91 ± 0.89a

190.74 ± 9.95 165.87 ± 18.42 178.30 ± 5.99 188.38 ± 12.21 231.01 ± 16.63a 189.92 ± 15.28a

a

GSH-PX (U/ml) a

263.75 ± 11.25a 144.59 ± 16.52 188.29 ± 14.81 229.57 ± 10.49a 259.81 ± 11.50a 240.33 ± 12.94a

P < 0.01 compared with the model group. Source: Adapted from Li et al. 2008b.

a

(125.62 kcal/mol amino acid residue) (Li et al. 2008a). To assess the in vivo activity, the study checked for the effects of different doses of similar hydrolysates on antioxidant abilities in serum, liver, and heart of aged mice (induced with D-galactose) (Li et al. 2008b). The chickpea protein hydrolysates in different doses evidently increased the SOD and GSH-Px activities and reduced the MDA level in serum, liver, and heart in the treated mice (Table 19.1). In a study on the effects of different proteolytic treatments on the physiochemical and bitterness properties of pea (Cicer arietinum) protein hydrolysates, Humiski and Aluko (2007) showed that the type of enzyme and hydrolysis conditions will significantly affect the amino acid composition and peptide size and hence the antioxidant activity (Figure 19.1). In the study, papain hydrolysates contained high molecular weight peptides (10– 178 kDa), while hydrolysates from the other four proteases contained predominantly low molecular weight peptides (≤23 kDa). DPPH (1,1-diphenyl-2picrylhydrazyl) free radical–scavenging activity of the Flavourzyme hydrolysate was significantly (P < 0.05) the highest while alcalase and trypsin hydrolysates were the lowest. The limited studies carried out so far indicate that the pea group proteins have potential as sources of antioxidant agents for nutraceutical/therapeutic or pharmaceutical purposes. To enable industrial production using methods such as solid state peptide synthesis, there is need for greater characterization of the peptides, especially regarding the amino acid

Figure 19.1. Free radical (DPPH)–scavenging activity of enzymatic pea protein hydrolysates. Adapted from Humiski and Aluko 2007.

composition of active peptides. Studies on the antioxidant activity of cowpea proteins (isolates or hydrolysates) are also deficient.

3. Hypocholesterolemic Effects Application of vegetable proteins as hypocholesterolemic agents has been recognized as a potent measure through several animal and human studies. In wide studies, it was shown among Chinese women (Zhang et al. 2003) and in Japan that a reduction of total low-density lipoprotein (LDL)cholesterol and that ischemic and cerebral events would occur with a higher soy protein intake (>6 g/d) from 0.5 g/d) (Nagata et al. 1998; Sirtori et al. 2008).

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Studies with results to the same effect from the peas have only recently been conducted (Sirtori et al. 2008). Yang et al. (2007) showed chickpea (Cicer arietinum) feeding induced a favorable plasma lipid profile reflecting decreased TAG, LDL cholesterol (LDL-C), and LDL-C:high-density lipoprotein (HDL) cholesterol levels (P < 0·5). HFD-fed rats had higher TAG concentration in muscle. Zulet et al. (1999) showed a 34% reduction in blood cholesterol levels in a 16-day study on rats fed on chickpeas (Cicer arietinum). Similar results were reported by Pittaway et al. (2006, 2007) in human studies. Other whole pea seeds such as garden pea (Pisum sativum) (Alonso et al. 2001; Dabai et al. 2007; Martins et al. 2004) and cowpea (Vigna unguiculata) (Mahadevappa and Raina 1983) have also been shown to have similar cholesterol and blood lipidemia reducing effects. These hypocholesteremic effects of whole pea seeds are probably a resultant combination of several component effects, namely, the saponins, fiber, and proteins (Zulet and Martínez 1995). The saponins and fiber function mainly through bile binding, which leads to excretion of higher amounts of bile salts in the feces, hence inducing the liver to produce more bile from cholesterol. This reduces the total cholesterol in the body. Protein isolates with undigestible impurities such as saponins, isoflavones, and fibers have been suggested to partly attain their hypocholestoremic effect through this mechanism due to their impurities. Highly pure protein isolates, on the other hand, have been suggested to achieve their effect through some other mechanisms, although they have also been attributed to intestinal bile sequestering (Spielmann et al. 2008). Spielmann et al. (2008) reported comparing rats fed with either 200 g/kg of casein to those fed purified pea protein (Pisum sativum) for 16 days. They found that concentrations of triacylglycerols in liver, plasma, and lipoproteins did not differ between both groups of rats. However, rats fed the pea protein diet had a lower concentration of total cholesterol in the liver and very-low-density lipoproteins (VLDL) fraction than rats fed the casein diet (p < 0.05). Cholesterol concentration in plasma, LDL, and HDL did not differ between the groups. Rats fed pea

protein, moreover, had an increased mRNA concentration of cholesterol-7α-hydroxylase in the liver and an increased amount of bile acids excreted via feces compared with rats fed casein (p < 0.05). Concomitantly, mRNA concentrations of sterol regulatory element-binding protein (SREBP)-2 and its target genes, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and LDL receptor in the liver, were increased in rats fed pea protein (p < 0.05). This suggested that pea protein stimulates formation and excretion of bile acids, which leads to a reduced hepatic cholesterol concentration and a reduced secretion of cholesterol via VLDL (Spielmann et al. 2008). On the other hand, several other hypotheses have been put forward to explain the hypocholesteremic effect of legume protein such as in the pea studies mentioned above. The mechanism is not clearly understood. Most hypotheses put forward have hinged on the unique amino acid composition of the proteins, especially soybean proteins. Low methionine levels in particular have been shown to be a major underlying factor, although other researchers suggested a role played by other constitutive amino acids like arginine and lysine. Sugiyama et al. (1997) demonstrated that about 40% of the hypocholesterolemic action of soybean protein is due to the low methionine content of the protein and might be associated with alteration of plasma total cholesterol through a significant decrease in LDL cholesterol (Figure 19.2). The decrease was accompanied by a drop in phosphatidylcholine (PC) to phosphatidylethanolamine (PE) ratio of liver microsomes. They suggested that the decrease was likely to have arisen from a depression of PE N-methylation due to lower methionine contents of these proteins. Supporting observations were made by Giroux et al. (1999), who noted that enrichment of the diet with lysine and methionine (with or without glycine) significantly increased liver levels of phosphatidylcholine and the ratio of phosphatidylcholine to phosphatidylethanolamine, apparently through increased enzymatic conversion. These changes were consistent with higher lipoprotein levels and thus could explain the hypercholesterolemia that was also observed. Low methionine has also been directly

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Figure 19.2. The hypercholesterolemic effect of dietary methionine. Adapted from Sugiyama et al. 1997.

implicated since it is required in biosynthesis of cholesterol via the formation of the methyl donor S-adenosyl methionine (SAM). SAM is functional in the synthesis of choline for phosphatidyl choline or lecithin, which is the principal phospholipid associated with LDL (Kingman 1991). Higher methionine levels were shown by Oda (2006) to be associated with higher apo A-1 gene activity, hence higher cholesterol levels. A high level of arginine was also suggested to have an effect since it raises the glucagon/insulin ratio; a ratio that is known to depress HAG-CoA reductase, the rate-limiting enzyme for the synthesis of cholesterol (Giroux et al. 1999). Lasekan et al. (1995) suggested that arg/lys or arg/met ratio in pea proteins may partly contribute to reducing

cholesterol in rats since the golden peas are high in ag, arg/lys ratio but low in met; but other mechanisms of action could not be ruled out. According to Kingman (1991), although some studies have shown the anticholestermic effect of pea proteins in animal studies, the underlying mechanism needs to be further elucidated based on human studies. This is significant because there is a less consistent hyper-hypo-response phenomenon that occurs in humans consuming cholesterol-rich diets. The hyperlipidemias observed in man may have their origins in quite different metabolic defects from those deliberately induced in animals, and it is by no means certain that the effects of dietary components will be the same in both cases. In addition, the potential hypocholesteremic effects of the peas

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are far from over since studies on the effects of chickpea and cowpea are limited.

4. Anticancer Effects due to Bowman-Birk Inhibitors Although Bowman-Birk protease inhibitors (BBIs) were extensively known for their antinutritional effects such as reduced protein utilization and pancreatic trophy (Grant et al. 1993; Pusztal et al. 2007), they have recently become a research issue because of their potential as preventive agents against a wide range of human cancers and other ailments. The basis of their potential lies in the inhibition of proteases, which are essential for the pathogenesis of those conditions (Armstrong et al. 2000; Chen et al. 2005; Gran et al. 2006; Kennedy 1998; Qi et al. 2005; Saito et al. 2007). Research has been conducted mainly on the soybean BBIs. Recent studies have shown BBIs from some of the peas (garden peas, Pisum sativum) to be similar to the soybean BBIs. There are yet to be reports on chickpea and cowpea BBIs. Plant serine protease inhibitors fall into a number of structurally distinct families: the “woundinduced” inhibitors I and II of potato and tomato, which are specifically expressed in response to insect attack, or mechanical wounding, the Kunitz type inhibitors. The Bowman-Birk type inhibitors are among the best characterized of the class (Hilder et al. 1989). They are small polypeptides that are typically found in the seeds of various members of the Leguminosae. The inhibitors are double headed,

while those of monocots are single headed (Prakash et al. 1996; Qi et al. 2005). The two heads are independent, nonoverlapping sites for trypsin and chymotrypsin inhibition (Li de la Sierra et al. 1999; Vartak et al. 1980). Some more recent examples have clearly demonstrated their anticancer effects with attempts to explain the underlying mechanisms in both human and animal trials with some involving oral application. These are supported by studies that show that the BBIs resist oral digestion, hence allowing for easy delivery in the gastrointestinal tract (Clemente et al. 2008). Von Hofe et al. (1991) showed that BBI can effectively inhibit NMBzA-induced esophageal tumors when given in tablet form separate from the regular diet. The BBIs were also found to be capable of partially blocking the development of lung tumors in mice (Witschi and Kennedy 1989). In a study comparing the cancer chemopreventive effects of pea (Pisum sativum) BBIs and soybean BBIs, Clemente et al. (2005) used two variant recombinant pea seed protease inhibitors, rTI1B and rTI2B, homologous to BBI but differing in inhibitory activity, on the growth of human colorectal adenocarcinoma HT29 cells in vitro (Table 19.2). They found a significant and dose-dependent decrease in the growth of HT29 cells using all protease inhibitors, with rTI1B showing the largest decrease (IC50 = 46 μM). They suggested that the relative effectiveness of rTI1B and rTI2B may correlate with a variant amino acid sequence within their respective chymotrypsin inhibitory domain, in agreement with a chymotrypsin-like protease as a potential target.

Table 19.2. Specific inhibitory activity of trypsin (T) and chymotrypsin (C) of BBI and recombinant PP1.

T11BC1 T12BC1 BB1

Amino acid sequence of inhibitory sites

Specific inhibitory activity (U/mg protein)

T

C

T

C

CLCTKSNPPTC CLCTKSDPPTC CACTKSNPPQC

CICAYSNPPKC CICALSYPPQC CICALSYPAQC

2926 ± 289 2596 ± 132 4396 ± 159

4973 ± 290 3260 ± 270 3827 ± 336

P1-P1 are the reactive peptide bond sites, marked in bold text. Specific activities and Ki values represent means ± SD from at least six determinations. Source: Adapted from Clemente et al. 2004.

Chapter 19 Bioactivity of Proteins and Peptides from Peas

The potential exploitation of a certain BBI in human health-promotion programs will depend on elucidation of the molecular basis for the variation in biological activity among variant forms. Several classes of distinct plant BBI have been identified. For the BBIs to be utilized as human health-promoting agents, the molecular basis for variation of biological activity among variant forms must be addressed (Clemente et al. 2004). The chymotrypsin head has been associated with the cancer preventive effects, while the trypsin head has been suggested as the main focus for crop resistance development (Chen et al. 2005). Li de la Sierra et al. (1999) determined the crystal structure of the isoform PsTIIVb of the pea BBIs by molecular replacement at 2.7 Å resolution using the x-ray coordinates of the soybean inhibitor as a search model. They noted that the inhibitor crystallized with a nearly perfect twofold symmetric dimer in the asymmetric unit. Although the overall structure was very similar to that seen in other BBIs, there were notable new structural features. Unlike the previously reported x-ray structures of BBIs, the structure of PsTI-IVb included the C-terminal segment of the molecule. The C-terminal tail of each subunit is partly βstranded and interacts with the two-fold symmetryrelated subunit, forming a β-sheet with strands A and B of this subunit. The dimer was mainly stabilized by a large internal hydrogen-bonded network surrounded by two hydrophobic links. Fluorescence anisotropy decay measurements showed that residues Tyr59 and Tyr43 are mobile in the picosecond time scale with large amplitude. Their results were in accordance with De Freitas et al. (1997), who studied the secondary structure of blackeyed pea (Vigna unguiculata) BBI by circular dichroism and reported the protein to be nonhelical with a deep negative band at 201 nm due to the disulfide bonds and small positive ellipticity values at wavelengths above 218 nm. They also reported that the IR spectrum of blackeye pea BBI, in a solid film, in the amide I-II and V regions suggested the occurrence of the unordered, antiparallel β-strand. The inhibitory mechanism of the BBIs has been elucidated by several researchers mainly employing crystallographic methods with computer modeling

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for cowpea BBI (Barbosa et al. 2007), soybean BBI (Gladysheva et al. 2002; Pletnev et al. 2000), and Pisum sativum BBI (Li de la Sierra et al. 1999). Generally it is concluded that the reactive site is located at unique exposed surfaces formed by a disulfide-linked β-sheet loop that is highly conserved, rigid, and mostly composed of nine residues (Qi et al. 2005). They interact with the enzymes via an exposed surface loop that adopts the canonical proteinase inhibitory conformation that is typical of the Bowman-Birk inhibitor family of serine proteinase inhibitors. A noncovalent complex results, which renders the proteinase inactive. The interaction for the BBIs specifically, however, involves a particularly well-defined disulfide-linked short b-sheet region of the interacting loop (McBride and Leatherbarrow 2001). Research on production of recombinant pea BBIs from peas using fermentative systems that can pave the way for commercial large-scale production has already been reported. Clemente et al. (2004) investigated the properties of variant pea seed protease inhibitors, homologous to the anticarcinogenic BBI from soybean but differing most significantly in amino acid sequences at the two independent sites of protease inhibition. The pea protease inhibitors were expressed, using Aspergillus niger, with yields of up to 23 mg secreted recombinant protein per liter of media. The recombinant proteins showed protease inhibitory activity and were deduced to be disulphide-bonded correctly, and limited posttranslational processing had occurred at the aminoterminal ends of all proteins. Differences in trypsin and chymotrypsin specific inhibitory activities, and in inhibition constants, were observed in studies of the two recombinant variants and BBI (Clemente et al. 2004). They concluded that the Aspergillus niger expression system allowed the recovery of active PPI without problems of either solubility, as often encountered with E. coli expression of extensively disulphide-bonded proteins, or the lengthy re-folding protocols that allow only small amounts of active protein to be recovered. They also reported that the yield of active PPI was much higher than had been the case with E. coli. Similar reports have been made using soybean BBI with the Gram-positive

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bacterium Bacillus subtilis (Vogtentanz et al. 2007) and the yeast Pichia pastoris (Yakoby and Raskin 2004) for production of the recombinant soybean Bowman-Birk protease inhibitors (sBBI). Although solid state methods based on the active site canonical nine residue loop of BBIs/SFRI-1 have been used for production of the soybean BBIs (Brauer et al. 2001), they have not yet been tried out for pea (chick, cow, and garden peas) BBIs. All in all, there is still a significant gap of knowledge as regards the BBIs, especially those from chickpeas and cowpeas, which have been relatively less studied, and it is necessary to further assess the potential effects of the recombinant pea BBIs on cancers in animal and human trials.

5. Angiostensin I-Converting Enzyme Inhibitory Effects Angiostensin I-converting enzyme hydrolyzes the decapeptide angiotensin I, yielding the potent vasoconstrictor octapeptide angiotensin II. In addition, ACE hydrolyzes the peptide bradyquinin, which is a potent vasodilator. This results in a decrease in blood pressure (Yust et al. 2003). ACE inhibition originated from studies on peptides rich in proline obtained from invertebrates. Although synthetic products such as captopril were later developed, contemporary research in food sources has been focused on determining ACE-inhibitory peptides directly (Sirtori et al. 2008). Efficient inhibitors of ACE-inhibitory short peptides have been obtained from various food sources for regulation of blood pressure including milk (FitzGerald and Meisel 2007), rice dregs (Chen et al. 2007), beef (Jang and Lee 2005), and soybean protein (Lo and Li-Chan 2005). The peas have also been assessed for production of ACE-inhibitory peptides in some studies, although results from animal tests or clinical trials are yet to be reported. More research attention has been focused on garden pea (Pisum sativum) and cowpea (Vigna unguiculata) proteins relative to those of chickpeas. Yust et al. (2003) demonstrated that hydrolysates of chickpea legumin obtained by treatment with alcalase are a good source of peptides with ACE-

Figure 19.3. Angiostensin-converting enzyme inhibitory activity of enzymatic pea protein hydrolysates. Adapted from Humiski and Aluko 2007.

inhibitory activity. Legumin is the main storage protein in chickpea seeds. Treatment of legumin with alcalase yielded a hydrolysate that inhibited ACE with an IC50 of 0.18 mg/ml. Fractionation of this hydrolysate by reverse phase chromatography gave six inhibitory peptides with IC50 values ranging from 0.011 to 0.021 mg/ml. Pedroche et al. (2002) also showed that chickpea proteins are a good source of ACE-inhibitory peptides when hydrolyzed with the protease alcalase. They found the highest degree of inhibition was found in a hydrolysate obtained by 30 minutes of treatment with alcalase. After Biogel P2 gel filtration chromatography and HPLC C18 reverse phase chromatography, they purified four peptides with ACE-inhibitory activity from the hydrolysate. Two of the peptides were competitive inhibitors of ACE, while the other two were uncompetitive inhibitors. In the study by Humiski and Aluko (2007) previously mentioned (Figure 19.3), DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical– scavenging activity of the Flavourzyme hydrolysate was significantly (P < 0.05) the highest, while alcalase and trypsin hydrolysates were the lowest. Inhibition of ACE activity was significantly higher (P < 0.05) for papain hydrolysate, while Flavourzyme hydrolysate had the least inhibitory activity. They showed that for a given hydroysis treatment, a balance needs to be struck between the physical

Chapter 19 Bioactivity of Proteins and Peptides from Peas

chemical properties, especially bitterness, and the inhibitory activity. Vermeirssen et al. (2005), during fractionation of pea (Pisum sativum) and whey protein, isolated using in vitro gastrointestinal digestion produced high ACE-inhibitory activity with IC50 values of 0.070 and 0.041 mg protein ml−1, respectively. They demonstrated the need for carefully planned fractionation in that the resultant peptides were highly dependent on the fractionation step used. Ultrafiltration/centrifugation using a membrane with a molecular weight cutoff of 3,000 Da decreased the IC50 value to 0.055 mg protein ml−1 for the pea permeate and 0.014 mg protein ml−1 for whey permeate. Further fractionation by reverse phase HPLC gave IC50 values as low as 0.016 mg protein ml−1 for pea and 0.003 mg protein ml−1 for whey. Consequently, the purification steps enriched the ACE-inhibitory activity of the pea digest more than 4 times and that of the whey digest more than 13 times. The activity in this study was also attributed to the presence of short and more hydrophobic peptides. Digestion has been suggested as still the most significant process for peptide derivation from peas as opposed to other methods like fermentation and controlled hydrolysis. In their study on a combined process involving fermentation by Lactobacillus helvicus and Saccharomyces cerevisiae, Vermeirssen et al. (2003) found that after fermentation, the ACEinhibitory activity (%) increased by 18–30% for all treatments, except for the fermentations of whey protein with Saccharomyces cerevisiae at 28°C, where no significant change was observed. After digestion, however, both fermented and nonfermented samples reached maximum ACE-inhibitory activity. It has been established that such hydrolysates and enriched fractions from peas (Pisum sativum) would resist in vivo gastrointestinal digestion after oral administration (Vermeirssen et al. 2005). Studies to examine the effect of oral application ACE-inhibitory pea hydrolysates in humans are yet to be conducted. Further advances in the detection and selection of ACE-inhibitory peptides from peas have been made. Dziuba et al. (2003) reported a versatile general method for assessment of proteins as poten-

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tial precursors of bioactive peptides by a computeraided analysis. Using pea (Pisum sativum) proteins, Vermeirssen et al. (2004) successfully showed in silico identification by two different priorities, high ACE-inhibitory activity and peptide size. There was a significant correlation between the developed method and the activity of in vitro digestion peptides.

6. Antimicrobial Effects due to Peptides There are various peptides and proteins produced by peas (Pisum sativum, Vigna unguiculata, Cicer arietinum) and other plants as defense mechanisms against pathogens and insects. They make up a diverse group of mainly structurally and molecularly dissimilar peptides that have been characterized by several researchers (Ng et al. 2002). Some of these compounds have shown activity against human pathogens and an ability to inhibit human immunodeficiency virus (HIV) transcriptase. For that purpose a potential therapeutic role has been suggested. Selitrennikoff (2001) described 13 classes of antifungal proteins: PR-1 proteins, (1,3) β-glucanases, chitinases, chitin-binding proteins, thaumatin-like (TL) proteins, defensins, cyclophilin-like protein, glycine/ histidine-rich proteins, ribosome-inactivating proteins (RIPs), lipid-transfer proteins (LTPs), killer proteins (killer toxins), protease inhibitors, and other proteins. The classification of the peptides is based primarily on either their mechanism of action (e.g., glucanases), their structure (e.g., cysteine-rich), or their similarity to a known “type” of protein; that is, there is no standard nomenclature. The classification is further complicated by the fact that many of the peptides/proteins can be classified into more than one group (Selitrennikoff 2001).

6.1. Antimicrobial Peptides from Garden Pea ( Pisum sativum) Several antifungal peptides with varying activities have been reported in Pisum sativum. Lam et al. (1998) reported two ribosome inactivating proteins designated α- and β-pisavins isolated from seeds of the garden pea Pisum sativum var. arvense Poir

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with a procedure involving affinity chromatography on Affi-gel Blue gel, immobilized metal ion affinity chromatography on iminodiacetic acid-agarose, cation exchange chromatography on Resource-S, and gel filtration on Superose 12. The α- and βpisavins were nonglycoproteins with molecular weights of 20.5 kDa and 18.7 kDa, respectively. They both showed activity toward tRNA. They acted on ribosomal RNA through its RNA N-glycosidase activity to release an Endo’s fragment and converted the conformation of DNA from supercoiled and circular form into a linear one. The researchers suggested that the two pisavins belong to ribosome inactivating proteins (RIPs) based on sequence identity and their anti-cell-free translation inhibition assays. Ye et al. (2000a) and Ye and Ng (2003) extracted two antifungal peptides, sativin and pisumin form Pisum sativum var. macrocarpon cv sugar snap. Sativin exhibited a molecular weight of 38 kDa in SDS-polyacrylamide gel electrophoresis. It exerted antifungal activity against Fusarium oxysporum, Coprinus comatus, and Pleurotus ostreatus but not against Rhizoctonia solani. It possessed an N-terminal amino acid sequence that showed similarity to those of miraculin and pisavin (a ribosomeinactivating protein from Pisum sativum var. arvense Poir manifesting similarity to miraculin). Unlike pisavin, however, sativin demonstrated negligible ribonuclease activity and inhibited translation in a rabbit reticulocyte lysate system with a very low potency (IC50 = 5.14 μM). Pisumin had a similar antifungal specificity to that of sativin although at varying levels of activity. It had a smaller molecular weight (31 kDa) than that of sativin; and using the same extraction methods, it gave a yield that was almost double that of sativum (Ye and Ng 2003). Two cysteine-rich polypeptides (Psd1 and Psd2) with anti-Aspergillus niger activity were extracted from Pisum sativum by Almeida et al. (2000). The two were localized primarily in vascular bundles and epidermis tissues of pea pods and exhibited high antifungal activity toward several fungi, displaying IC50 values ranging from 0.04 to 22 βg/ml. The two peptides could not be easily assigned to any established group of plant defensive protein although their primary sequences showed homology with

other plant defensins. Later on, using electrostatic surface potential topologies, it was suggested that Psd1 exhibited its activity through potassium channel inhibition (Almeida et al. 2002). A small unassigned peptide with high antifungal activity and a molecular mass of 11 kDa was reported by Wang and Ng (2006) in Pisum sativum. In addition to antifungal activity against Physalospora piricola (IC50 of 0.62 μM) and Fusarium oxysporum and Mycosphaerella arachidicola, the peptide inhibited human immunodeficiency virus type 1 reverse transcriptase with an IC50 of 4.7 μM. Another unassigned lectin with high agglutinating activity and antifungal activity was obtained from Egyptian seeds of Pisum sativum (Sitohy et al. 2007). It exhibited a distinct molecular weight of 50 kDa using gel filtration chromatography and inhibited the growth of Aspergillus flavus, Trichoderma viride, and Fusarium oxysporum.

6.2. Cowpea ( Vigna unguiculata) Antimicrobial Proteins/Peptides Cowpeas (Vigna unguiculata) have also been demonstrated as a good source of defensive bioactive peptides and proteins. Ye et al. (2000b) presented evidence of the existence of multiple proteins with antifungal and antiviral potency in cowpea seeds. They showed two proteins, designated α- and βantifungal proteins in accordance with their order of elution from the CM-Sepharose column, which were capable of inhibiting HIV reverse transcriptase and one of the glycohydrolases associated with HIV infection, α-glucosidase, but not β-glucuronidase. The α- and β-antifungal proteins had molecular weights of 28 kDa and 12 kDa, respectively. Based on N-terminal sequencing α-antifungal protein was characterized as a chitinases-like protein, while the β-antifungal protein exhibited a sequence not related to any previously known. Another cowpea antifungal protein capable of inhibiting human immunodeficiency virus-1 reverse transcriptase and the glycohydrolases α- and β-glucosidases was reported by Ye and Ng (2001). The protein, assigned unguilin, was devoid of lectin activity, and data indicated that unguilin was neither a ribosome-inactivating

Chapter 19 Bioactivity of Proteins and Peptides from Peas

protein nor a lectin. It possessed a molecular weight of 18 kDa and an N-terminal sequence resembling that of cyclophilins and the cyclophilin-like antifungal protein from mung beans. It exerted an antifungal effect toward fungi including Coprinus comatus, Mycosphaerella arachidicola, and Botrytis cinerea. An antibacterial peptide with promise for use in production of novel antibacterial drugs was purified and characterized by Franco et al. (2006) from cowpeas. The novel γ-thionin from cowpea seeds (Vigna unguiculata) was named Cp-thionin II and had bactericidal activity on both Gram-positive and Gram-negative bacteria. They later illustrated another new thionin from cowpea (Vigna unguiculata) with proteinase inhibitory activity that inhibited trypsin, but not chymotrypsin, binding with a stoichiometry of 1:1 (Melo et al. 2002). A lipid transfer protein (10 kDa) from cowpea seeds and a defensin (6.8 kDa) have also been demonstrated (Carvalho et al. 2001). By assessing the development of the two, it was concluded that Vigna unguiculata contains defensin and lipid transfer proteins (LTPs) that inhibit the early growth of several fungi and cause many hyphal morphological alterations for fungi growing in the presence of these peptides as compared to growth on control medium. Similar growth-protecting peptides were shown to be released by cowpea seeds during the first hour of water imbibition that happens prior to germination (Rose et al. 2006).

6.3. Chickpea (Cicer arietinum) Antimicrobial Proteins/Peptides Four novel antimicrobial proteins from chickpeas have been isolated and characterized, namely cicerin (Ye et al. 2002), arietin (Ye et al. 2002), cicerarian (Chu et al. 2003), and cyclophilin-like protein (CLAP) (Ye and Ng 2002). Cicerin and arietin exhibited a molecular weight of approximately 8.2 and 5.6 kDa, respectively. Cicerin was the more potent of the two, exhibiting higher translationinhibiting activity in a rabbit reticulocyte lysate system and higher antifungal potency toward Mycosphaerella arachidicola, Fusarium oxysporum, and Botrytis cinerea. Both were devoid of mitogenic and

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anti-HIV-1 reverse transcriptase activities. Another peptide (cicerarian) with a molecular weight of 8 kDa (SDS-PAGE) was isolated from chickpeas by Chu et al. (2003). It exhibited heat-stable antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, and Physalospora piricola. The antifungal activity was preserved after exposure to 100°C for 15 minutes. Unlike cicerin and arietin, cicerarian was able to inhibit HIV-1 RT with an IC50 of 87.5 μM. A chickpea CLAP isolate by Ye and Ng (2002) also showed HIV-1 reverse transcriptase inhibition on top of stimulating a mitogenic response from mouse splenocytes. It had a molecular weight of 18 kDa (SDS-PAGE) and an N-terminal sequence homologous to cyclophilins.

6.4. Commercial Utilization Potential of Pea Antimicrobial Proteins/Peptides Commercial utilization of peptides requires stable production to secure the required amounts of adequate quality and purity. In principle, both chemical synthesis and recombinant production systems can be utilized. However, owing to the relatively large size of defensins (40–60 amino acids), their highly ordered tertiary structure, and presence of 3–4 disulfide bridges, only recombinant production is commercially viable. For research purposes, antifungal peptides have been heterologously expressed in diverse hosts, such as bacteria, yeasts, fungi, and plants (Thevissen et al. 2007). Since fungal expression systems such as those of Saccharomyces cerevisiae, Pichia pastoris, and Aspergillus spp. provide a potential for high-level production (multiple grams per liter), secreted production facilitating downstream processing and purification, and correctly matured and folded product with formation of the essential disulfide bridges, they are recommendable for commercial production. Present examples of commercially viable production of antifungal peptides from plants include the successful expression of the Pisum sativum Psd1 in Pischia pastoris by Cabral et al. (2003) and Almeida et al. (2001). After purification and productivity improve as was demonstrated by these reports, then application of the plant antifungal and antiviral peptide will assume

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commercial appeal. There remains a great deal of research to be conducted along the way before there is full utilization in humans as therapeutic or antimicrobial agents.

7. Future Prospects Chickpea (Cicer arietinum), garden/green peas (Pisum sativum), and cowpea (Vigna unguiculata) proteins provide another dimension to the utilization of legume proteins as sources of beneficial biological activities. Some aspects, however, need to be addressed before their full potential can be utilized. These include 1. Characterization of the mechanisms of the various reported activities at the molecular level. The activities may not necessarily follow the same mechanisms as soy proteins, and studies would also provide further insight into the soy protein mechanisms. 2. More extensive animal and human studies to assess the in vivo profiles of the reported activities or give insights into the mechanisms in man. 3. Research into improved fermentative and/or solid state synthesis production of the identified peptides to facilitate commercial utilization.

8. References Almeida MS, Cabral KMS, Kurtenbach E, Almeida FCL, Valente AP. 2002. Solution structure of Pisum sativum defensin 1 by high resolution NMR: Plant defensins, identical backbone with different mechanisms of action. J Mol Biol 315(4):749–757. Almeida MS, Cabral KS, Neves de Medeiros L, Valente AP, Almeida FCL, Kurtenbach E. 2001. cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris. Arch Biochem Biophys 395(2):199–207. Almeida MS, Cabral KMS, Zingali RB, Kurtenbach E. 2000. Characterization of two novel defense peptides from pea (Pisum sativum) seeds. Arch Biochem Biophys 378(2):278– 286. Alonso R, Grant G, Marzo F. 2001. Thermal treatment improves nutritional quality of pea seeds (Pisum sativum L.) without reducing their hypocholesterolemic properties. Nutr Res 21(7):1067–1077. Arcan I, Yemenicioglu A. 2007. Antioxidant activity of protein extracts from heat-treated or thermally processed chickpeas and white beans. Food Chem 103(2):301–312.

Armstrong WB, Kennedy AR, Wan XS, Taylor TH, Nguyen QA, Jensen J, Thompson W, Lagerberg W, Meyskens FL. 2000. Clinical modulation of oral leukoplakia and protease activity by Bowman-Birk inhibitor concentrate in a phase IIa chemoprevention trial. Clin Cancer Res 6(12):4684–4691. Barbosa J, Silva LP, Teles RCL, Esteves GF, Azevedo RB, Ventura MM, de Freitas SM. 2007. Crystal structure of the Bowman-Birk inhibitor from Vigna unguiculata seeds in complex with beta-trypsin at 1.55 A resolution and its structural properties in association with proteinases. Biophys J 92(5):1638. Brauer ABE, Kelly G, McBride JD, Cooke RM, Matthews SJ, Leatherbarrow RJ. 2001. The Bowman-Birk inhibitor reactive site loop sequence represents an independent structural βhairpin motif. J Mol Biol 306(4):799–807. Cabral KMS, Almeida MS, Valente AP, Almeida FCL, Kurtenbach E. 2003. Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: Overcoming the inefficiency of the STE13 protease. Protein Expression Purif 31(1):115–122. Carvalho AO, Machado OLT, Da Cunha M, Santos IS, Gomes VM. 2001. Antimicrobial peptides and immunolocalization of a LTPin Vigna unguiculata seeds. Plant Physiol Biochem 39(2):137–146. Chen Q, Xuan G, Fu M, He G, Wang W, Zhang H, Ruan H. 2007. Effect of angiotensin I-converting enzyme inhibitory peptide from rice dregs protein on antihypertensive activity in spontaneously hypertensive rats. Asia Pac J Clin Nutr 16(1):281– 285. Chen YW, Huang SC, Lin-Shiau SY, Lin JK. 2005. BowmanBirk inhibitor abates proteasome function and suppresses the proliferation of MCF7 breast cancer cells through accumulation of MAP kinase phosphatase-1. Carcinogenesis 26(7):1296. Chu KT, Liu KH, Ng TB. 2003. Cicerarin, a novel antifungal peptide from the green chickpea. Peptides 24(5):659–663. Clemente A. 2000. Enzymatic protein hydrolysates in human nutrition. Trends Food Sci Technol 11(7):254–262. Clemente A, Gee JM, Johnson IT, MacKenzie DA, Domoney C. 2005. Pea (Pisum sativum L.) Protease inhibitors from the Bowman-Birk class influence the growth of human colorectal adenocarcinoma HT29 cells in vitro. J Agric Food Chem 53(23):8979–8986. Clemente A, Jimenez E, Marin-Manzano MC, Rubio LA. 2008. Active Bowman-Birk inhibitors survive gastrointestinal digestion at the terminal ileum of pigs fed chickpea-based diets. J Sci Food Agric 88(3):513–521. Clemente A, MacKenzie DA, Jeenes DJ, Domoney C. 2004. The effect of variation within inhibitory domains on the activity of pea protease inhibitors from the Bowman-Birk class. Protein Expression Purif 36(1):106–114. Dabai FD, Walker AF, Sambrook IE, Welch VA, Owen RW, Abeyasekera S. 2007. Comparative effects on blood lipids and faecal steroids of five legume species incorporated into a semipurified, hypercholesterolaemic rat diet. Br J Nutr 75(04):557– 571.

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De Freitas SM, de Mello LV, da Silva MCM, Vriend G, Neshich G, Ventura MM. 1997. Analysis of the black-eyed pea trypsin and chymotrypsin inhibitor-α-chymotrypsin complex. FEBS Lett 409(2):121–127. Dziuba J, Iwaniak A, Minkiewicz P. 2003. Computer-aided characteristics of proteins as potential precursors of bioactive peptides. Polimery 48(1):50–53. FitzGerald RJ, Meisel H. 2007. Milk protein–derived peptide inhibitors of angiotensin-I-converting enzyme. Br J Nutr 84(S1):33–37. Franco OL, Murad AM, Leite JR, Mendes PAM, Prates MV, Bloch C. 2006. Identification of a cowpea γ-thionin with bactericidal activity. FEBS J 273(15):3489–3497. Giroux I, Kurowska EM, Freeman DJ, Carroll KK. 1999. Addition of arginine but not glycine to lysine plus methionineenriched diets modulates serum cholesterol and liver phospholipids in rabbits. J Nutr 129(10):1807–1813. Gladysheva IP, Popykina NA, Zamolodchikova TS, Larionova NI. 2002. Study on interaction between duodenase-protease with dual specificity and inhibitors of Bowman-Birk family. Protein Peptide Lett 9(2):139–144. Gran B, Tabibzadeh N, Martin A, Ventura ES, Ware JH, Zhang GX, Parr JL, Kennedy AR, Rostami AM. 2006. The protease inhibitor, Bowman-Birk inhibitor, suppresses experimental autoimmune encephalomyelitis: A potential oral therapy for multiple sclerosis. Multiple Sclerosis 12(6):688. Grant G, Dorward PM, Pusztai A. 1993. Pancreatic enlargement is evident in rats fed diets containing raw soybeans (Glycine max) or cowpeas (Vigna unguiculata) for 800 days but not in those fed diets based on kidney beans (Phaseolus vulgaris) or lupinseed (Lupinus angustifolius). J Nutr 123(12):2207. Hilder VA, Barker RF, Samour RA, Gatehouse AMR, Gatehouse JA, Boulter D. 1989. Protein and cDNA sequences of BowmanBirk protease inhibitors from the cowpea (Vigna unguiculata Walp.). Plant Mol Biol 13(6):701–710. Humiski LM, Aluko RE. 2007. Physicochemical and bitterness properties of enzymatic pea protein hydrolysates. J Food Sci 72(8):605–611. Jang A, Lee M. 2005. Purification and identification of angiotensin converting enzyme inhibitory peptides from beef hydrolysates. Meat Sci 69(4):653–661. Kaur M, Singh N. 2007. Characterization of protein isolates from different Indian chickpea (Cicer arietinum L.) cultivars. Food Chem 102(1):366–374. Kennedy AR. 1998. The Bowman-Birk inhibitor from soybeans as an anticarcinogenic agent. Amer J Clin Nutr 68:1406–1412S. Kingman SM. 1991. The influence of legume seeds on human plasma lipid concentrations. Nutr Res Rev 4(01):97–123. Lam SSL, Wang H, Ng TB. 1998. Purification and characterization of novel ribosome inactivating proteins, alpha-and betapisavins, from seeds of the garden pea Pisum sativum. Biochem Biophys Res Commun 253(1):135–142. Lasekan JB, Gueth L, Khan S. 1995. Influence of dietary golden pea proteins versus casein on plasma and hepatic lipids in rats. Nutr Res 15(1):71–84.

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Pittaway JK, Ahuja KDK, Robertson IK, Ball MJ. 2007. Effects of a controlled diet supplemented with chickpeas on serum lipids, glucose tolerance, satiety and bowel function. J. Am Coll Nutr 26(4):334. Pletnev VZ, Zamolodchikova TS, Pangborn WA, Duax WL. 2000. Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin-like specificities. Proteins: Struct Funct Gen 41(1):8–16. Prakash B, Selvaraj S, Murthy MRN, Sreerama YN, Rajagopal Rao D, Gowda LR. 1996. Analysis of the amino acid sequences of plant Bowman-Birk inhibitors. J Mol Evol 42(5):560–569. Pusztal A, Grant G, Brown DJ, Stewart JC, Bardocz S, Ewen SWB, Gatehouse AMR, Hilder V. 2007. Nutritional evaluation of the trypsin (EC 3.4. 21.4) inhibitor from cowpea (Vigna unguiculata Walp.). Br J Nutr 68(3):783–791. Qi RF, Song ZW, Chi CW. 2005. Structural features and molecular evolution of Bowman-Birk protease inhibitors and their potential application. Acta Biochim Biophys Sinica 37(5): 283–292. Ragab DDM, Babiker EE, Eltinay AH. 2004. Fractionation, solubility and functional properties of cowpea (Vigna unguiculata) proteins as affected by pH and/or salt concentration. Food Chem 84(2):207–212. Rose TL, Conceiçao AS, Xavier-Filho J, Okorokov LA, Fernandes KVS, Marty F, Marty-Mazars D, Carvalho AO, Gomes VM. 2006. Defense proteins from Vigna unguiculata seed exudates: Characterization and inhibitory activity against Fusarium oxysporum. Plant Soil 286(1):181–191. Saito T, Sato H, Virgona N, Hagiwara H, Kashiwagi K, Suzuki K, Asano R, Yano T. 2007. Negative growth control of osteosarcoma cell by Bowman-Birk protease inhibitor from soybean; involvement of connexin 43. Cancer Lett 253(2):249– 257. Scarafoni A, Magni C, Duranti M. 2007. Molecular nutraceutics as a mean to investigate the positive effects of legume seed proteins on human health. Trends Food Sci Technol 18(9):454– 463. Selitrennikoff CP. 2001. Antifungal proteins. Applied Environmental Microbiol 67(7):2883–2894. Shand PJ, Ya H, Pietrasik Z, Wanasundara P. 2007. Physicochemical and textural properties of heat-induced pea protein isolate gels. Food Chem 102(4):1119–1130. Sirtori CR, Galli C, Anderson JW, Arnoldi A. 2008. Nutritional and nutraceutical approaches to dyslipidemia and atherosclerosis prevention: Focus on dietary proteins. Atherosclerosis 203(1):8–17. Sitohy M, Doheim M, Badr H. 2007. Isolation and characterization of a lectin with antifungal activity from Egyptian Pisum sativum seeds. Food Chem 104(3):971–979. Spielmann J, Stangl GI, Eder K. 2008. Dietary pea protein stimulates bile acid excretion and lowers hepatic cholesterol concentration in rats. J Anim Phys Anim Nutr 92(6):683– 693. Sugiyama K, Yamakawa A, Kumazawa A, Saeki S. 1997. Methionine content of dietary proteins affects the molecular

species composition of plasma phosphatidylcholine in rats fed a cholesterol-free diet. J Nutr 127(4):600–607. Thevissen K, Kristensen HH, Thomma B, Cammue BPA, Francois I. 2007. Therapeutic potential of antifungal plant and insect defensins. Drug Discovery Today 12(21–22):966– 971. Vartak HG, Rele MV, Jagannathan V. 1980. Proteinase inhibitors from Vigna unguiculata subsp. cylindrica. III. Properties and kinetics of inhibitors of papain, subtilisin, and trypsin. Arch Biochem Biophys 204(1):134–140. Vermeirssen V, Van Camp J, Decroos K, Van Wijmelbeke L, Verstraete W. 2003. The Impact of fermentation and in vitro digestion on the formation of angiotensin-I-converting enzyme inhibitory activity from pea and whey protein. J Dairy Sci 86(2):429–438. Vermeirssen V, Van Camp J, Verstraete W. 2005. Fractionation of angiotensin I converting enzyme inhibitory activity from pea and whey protein in vitro gastrointestinal digests. J Sci Food Agri 85(3):399–405. Vermeirssen V, van der Bent A, Van Camp J, van Amerongen A, Verstraete W. 2004. A quantitative in silico analysis calculates the Angiotensin I Converting Enzyme (ACE) inhibitory activity in pea and whey protein digests. Biochimie 86(3):231– 239. Vogtentanz G, Collier KD, Bodo M, Chang JH, Day AG, Estell DA, Falcon BC, Ganshaw G, Jarnagin AS, Kellis JT. 2007. A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor. Protein Expression Purif 55(1):40–52. von Hofe E, Newberne PM, Kennedy AR. 1991. Inhibition of N-nitrosomethylbenzylamine-induced esophageal neoplasms by the Bowman-Birk protease inhibitor. Carcinogenesis 12(11):2147. Wang HX, Ng TB. 2006. An antifungal protein from the pea Pisum sativum var. arvense Poir. Peptides 27(7):1732–1737. Witschi H, Kennedy AR. 1989. Modulation of lung tumor development in mice with the soybean-derived Bowman-Birk protease inhibitor. Carcinogenesis 10(12):2275. Wolosiak R, Worobiej E. 1999. Antioxidant activity of pea protein isolate and hydrolysates. Zywnosc. Nauka. Technologia. Jakosc (Poland). Yakoby N, Raskin I. 2004. A simple method to determine trypsin and chymotrypsin inhibitory activity. J Biochem Biophys Methods 59(3):241–251. Yang Y, Zhou L, Gu Y, Zhang Y, Tang J, Li F, Shang W, Jiang B, Yue X, Chen M. 2007. Dietary chickpeas reverse visceral adiposity, dyslipidaemia and insulin resistance in rats induced by a chronic high-fat diet. Br J Nutr 98(04):720–726. Ye XY, Ng TB. 2002. Isolation of a new cyclophilin-like protein from chickpeas with mitogenic, antifungal and anti-HIV-1 reverse transcriptase activities. Life Sci 70(10):1129–1138. ———. 2003. Isolation of pisumin, a novel antifungal protein from legumes of the sugar snap pea Pisum sativum var. macrocarpon. Comp Biochem Physiol, C: Toxicol Pharmacol 134(2):235–240.

Chapter 19 Bioactivity of Proteins and Peptides from Peas

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Chapter 20 Wheat Proteins and Peptides Hitomi Kumagai

Contents 1. Wheat Production and Consumption, 289 2. Wheat Classification, 289 3. Wheat Protein, 290 3.1. Wheat Protein Classification, 290 3.2. Albumin, 291 3.3. Prolamin (Gliadin), 291 3.4. Glutelin (Glutenin), 293 3.5. Gluten, 295 4. Wheat Allergy, 295 4.1. Baker ’s Asthma, 296

1. Wheat Production and Consumption World wheat production has been accounting for about 30% of the world cereal production, about 587 million tons in 2006 according to the annual report of the United States Department of Agriculture (USDA) Foreign Agricultural Service. Wheat production and consumption are the highest in the European Union (EU) countries, followed by China and India (Figure 20.1). In contrast to rice that is mainly produced and consumed in Asian countries, wheat is produced and consumed widely all over the world. In Japan, approximately 0.8 million tons of wheat have been produced and 5.5 million tons have been imported to cover Japanese wheat consumption, and Japanese people consume about 88 grams

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

4.2. Wheat-Dependent Exercise-Induced Anaphylaxis (WDEIA), 296 4.3. Celiac Disease, 297 4.4. Epitope Structure of Wheat Protein, 297 4.5. Reduction of Wheat Allergenicity, 297 5. Prevention of Diabetes by Wheat Albumin, 298 6. Prevention of Hypertension by Wheat Peptide, 298 7. Conclusion, 299 8. References, 299

of wheat per day, which is about one-third of their daily cereal consumption according to data summarized by the Ministry of Agriculture, Forestry and Fisheries in Japan.

2. Wheat Classification Wheat belongs to the genus Triticum in the family Poaceae, consisting of about 20 species. Wheat is classified by chromosome number (ploidy), sowing season, grain color, and grain hardness. There are diploid (2n = 2x = 14), tetraploid (2n = 4x = 28), and hexaploid (2n = 6x = 42) species in wheat (Table 20.1). Among them, T. aestivum, T. durum, and T. compactum are the major cultivated species. T. aestivum is known as bread wheat or common wheat, being used for making bread. T. durum is too hard to be ground into flour and is milled into 0.25 and 0.75 millimeters in diameter; its coarse milling grade is called semolina or farina. The durum semolina is used for making pasta 289

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Figure 20.1. World wheat production in 2006. Source: Report of the Ministry of Agriculture, Forestry and Fisheries in Japan.

The grain color of wheat differs according to the cultivation area and species. The wheat with brownish grain color is called red wheat, while the one having light color is called white wheat or amber wheat. White wheat is said to have a sweeter, milder flavor than red wheat. The wheat cultivated in the United States and in Japan is mostly red wheat, and that cultivated in Australia is mostly white wheat. The grain hardness of wheat depends on its protein content. Hard wheat contains 11–15% protein and is suitable for making bread and pasta, while soft wheat contains 6–10% protein and is suitable for making cakes and pastries. Protein content, particularly gluten content, is crucial for the processing properties of wheat.

3. Wheat Protein Table 20.1. Major cultivated species of wheat. Diploid

Tetraploid

Hexaploid

T. monococcum (Einkorn wheat) T. aegilopoides (T. boeoticum) T. urartu

T. durum (Durum wheat) T. dicoccum (Emmer wheat) T. dicoccoides T. polonicum T. turgidum T. orientale (T. turanicum)

T. aestivum (Bread wheat) T. compactum (Club wheat) T. spelta T. macha T. sphaerococcum T. vavilovii

Sources: Kihara 1954; Nagao 1998.

products. T. compactum is called club wheat and contains less gluten than T. aestivum and T. durum; it is suitable for making cakes, pastries, and cookies. There are two sowing seasons for wheat. The wheat whose seeds are sowed in spring and harvested in autumn is called spring wheat, while the one whose seeds are sowed in autumn and harvested in summer is called winter wheat. The yield of spring wheat is approximately two-thirds that of winter wheat because of the shorter cultivation period. Therefore, three-fourths of wheat grown in the United States is winter wheat. However, if compared at the same protein content, spring wheat is often superior to winter wheat for bread making.

3.1. Wheat Protein Classification Proteins are classified into four groups according to their solubility in solvents: water-soluble albumin, salt-soluble globulin, 60–70% aqueous alcoholsoluble prolamin, and dilute acid- or alkali-soluble glutelin. Wheat prolamin is called gliadin and wheat glutelin is called glutenin. Wheat flour is made from the endosperm part of the wheat kernel (berry), where gliadin and glutenin are biosynthesized (Shani et al. 1992). Therefore, gliadin and glutenin are the most abundant in wheat flour, albumin content being about 9%, globulin content being about 5%, prolamin (gliadin) content being about 40%, and glutelin (glutenin) content being about 46%. The protein composition of wheat is similar to that of corn, which has about 4% albumin, 2% globulin, 55% prolamin (zein), and 39% glutelin. Wheat protein has a peculiar amino acid composition, being rich in glutamine and proline and poor in lysine. Its nutritional value is estimated to be about 40% of egg protein. Gliadin has about 40– 55% of glutamine residues, about 20–30% of proline residues, and only about 0.3–0.7% of lysine residues (Kasarda et al. 1983; Shewry et al. 1986; DuPont et al. 2000). Similarly, glutenin has about 29% of glutamine residues and about 12% of proline residues, and only 2% of lysine residues. In addition

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291

Figure 20.2. Amino acid sequence of α-amylase inhibitor in Triticum aestivum. Source: Wang et al. 2006.

to the unique amino acid composition, gliadin and glutenin have a series of glutamine-rich tandem amino acid sequences, which contribute to the characteristic properties of wheat protein.

coded by the genes of the Hardness locus on chromosome 5DS, the soft (Ha) and hard (ha) alleles being present in hexaploid bread wheat (Triticum aestivum).

3.2. Albumin

3.3. Prolamin (Gliadin)

Wheat albumin contains α-amylase and trypsin inhibitors (Singh et al. 2001; Shewry et al. 1984; Buonocore et al. 1985), a peroxidase precursor, serine carboxypeptidase (Singh et al. 2001), serpins (Østergaard et al. 2000), and puroindolines (Morris 2002; Evrard et al. 2008). The molecular masses of α-amylase inhibitors are 12, 24, and 60 kDa, with subunits of 12 kDa (Deponte et al. 1976; Buonocore et al. 1985). αAmylase inhibitors of the 12 kDa family (0.28, 0.32, 0.35, 0.39, and 0.48 inhibitors) are monomers (Silano et al. 1973), while those of the 24 kDa family (0.19, 0.36, 0.38, and 0.53 inhibitors) are dimmers of 12 kDa subunits (Choudhury et al. 1996). The 0.19 and 0.53 inhibitors of the 24 kDa family are located on the short arms of chromosomes 3D and 3B, respectively (Sánchez-Monge et al. 1986). The α-amylase inhibitors of the 24 kDa family have 10 cysteine residues in each subunit (Wang et al. 2006) (Figure 20.2). Serpins are serine proteinase inhibitors, inhibiting chymotrypsin and cathepsin G (Østergaard et al. 2000). Puroindolines are basic cysteine-rich proteins of 13 kDa with 5 disulfide bridges (Charnet et al. 2003) having tryptophan-rich domain (Evrard et al. 2008). Both puroindolines a and b work for grain texture (Morris 2002) and have antimicrobial activity (Dubreil et al. 1998; Capparelli et al. 2005; Evrard et al. 2008). When either puroindolines a or b is absent or mutated, the grain texture becomes hard. As durum wheat lacks both puroindoline a or b, the grain texture is very hard. Puroindolines are

Wheat gliadins are monomeric proteins and are separated into 35–50 components (Kasarda et al. 1984; Scheets and Hedgcoth 1988). They are further classified into αβ-, γ-, and ω-gliadins (Woychik et al. 1961), each being divided into subcomponents of αβ1–αβ11, γ1–γ6, ω1–3, and ω5 (Kasarda et al. 1983; Dieterich et al. 2006). Gliadins are coded by loci on the short arms of groups 1 and 6 chromosomes (Gli-1 loci and Gli-2 loci) (Masci et al. 1993), αβ-gliadins being on the short arms of group 6 chromosomes (Gli-2 loci) and γ-, and ω-gliadins on those of group 1 chromosomes (Gli-1 loci) (Kasarda et al. 1984; Masci et al. 1991; Sabelli and Shewry 1991; Anderson et al. 1997, 2001). The genes of γ- and ω-gliadins encoded by Gli-1 loci are tightly linked to those of LMW glutenin encoded by Glu-3 loci (Liu and Shepherd 1995; Hsia and Anderson 2001). αβ-Gliadins contain about 150 different copies (Anderson et al. 1997), γ-gliadins about 16–39 copies, and ω-gliadins about 15–18 copies (Sabelli and Shewry 1991). The molecular masses of αβ-gliadins are about 28–40 kDa and γ-gliadins about 30–46 kDa with intermolecular disulfide bonds (Okita 1984; Okita et al. 1985; Shewry et al. 1986; Scheets and Hedgcoth 1988), while those of ω-gliadins are about 50–75 kDa without disulfide bonds (Kasarda et al. 1983; Okita 1984; DuPont et al. 2000). αβ-Gliadins have 6 cysteine residues (Okita et al. 1985; Kasarda and D’Ovidio 1999; Arentz-Hansen et al. 2000b) (Figure 20.3), and γgliadins have 7–9 cysteine residues (Okita 1984; Okita et al. 1985; Rafalski 1986; Sugiyama et al.

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Figure 20.3. Amino acid sequence of αβ-gliadin in Triticum aestivum. Under solid bar: PQPQPFP; under solid dot bar: sequence similar to PQPQPFP; upper solid bar: PSSQVSFQ; under double bar: QQLQQQQQQQ; under double dot bar: sequence similar to QQLQQQQQQQ; upper double bar: QQQQFP. Source: Okita et al. 1985.

Figure 20.4. Amino acid sequence of γ-gliadin in Triticum aestivum. Under solid bar: QPQQPFPQ; under dot bar: sequence similar to QPQQPFPQ; upper solid bar: LQQQL. Source: Sugiyama et al. 1986.

1986; Scheets and Hedgcoth 1988; Arentz-Hansen et al. 2000b; von Büren et al. 2000; Piston et al. 2006; Gao et al. 2007) (Figure 20.4). Therefore, αβ- and γ-gliadins are called sulfur-rich prolamins, and ω-gliadins are called sulfur-poor prolamins. Both αβ-gliadins and γ-gliadins have a high degree of sequence homology with tandem repeat region, polyglutamine region, and high and low proline regions in their structure (Okita et al. 1985; Sugiyama et al. 1986). These regions contribute to the characteristics of gliadins. αβ-Gliadins have 2 repeats of the motifs of PQPQPFP, PSSQVSFQ, QQQQFP, and QQLQQQQQQQ (Figure 20.3), while γ-gliadins have 8 repeats of the motif QPQQPFPQ (Okita et al. 1985; Sugiyama et al. 1986; Scheets and Hedgcoth 1988) and 2 repeats of the motif LQQQL in the N-terminal side (Figure 20.4). As ω-gliadins are not able to form sulfide bonds, their role in dough during gluten formation is not clarified yet. Most of the sequences in ω-

gliadins are tandem repeat motifs. They have 28 repeats of the motif QFPQQ and 9 motifs that have similar sequence to QFPQQ. When considering a little longer motif including QFPQQ, there are 22 repeats of the motif QQFPQQQ. ω-Gliadins also have 13 repeats of the motif QIPQQ and 3 repeats of the motif QQLPQQQ. When the motifs of long sequences are considered, there are 2 repeats of the motifs of PQQQIPQQHQIPQQPQQFPQQQQF, QPQQIPQQQQIPQQPQQFPQQQFPQQQ, QQFPQQQFPQQQQFPQQQ, QQIPQQQQIPQQPQQ, and QQLPQQQQIPQQ each (Figure 20.5). ω-Gliadins are further fractionated by acidPAGE into ω1-gliadins, ω2-gliadins (slow-moving gliadins), and ω5-gliadins (fast-moving gliadins or 1B-type ω-gliadins) (Kasarda et al. 1983). αβ- and γ-Gliadins correspond to prolamin B, having similar characteristics to hordein B in barley and secalin B in rye, while ω-gliadins correspond to prolamin C, having similar characteristics to hordein C in barley

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293

Figure 20.5. Amino acid sequence of ω-gliadin in Triticum aestivum. Under solid bar: QFPQQ; under dot bar: sequence similar to QFPQQ; upper solid bar: QIPQQ; upper dot bar: sequence similar to QIPQQ; upper double bar: QQLPQQQ; upper dot double bar: sequence similar to QQLPQQQ. Source: Matsuo et al. 2005a.

and secalin C (ω-secalins) in rye (Kasarda et al. 1983; Tatham and Shewry 1991).

3.4. Glutelin (Glutenin) Glutenin subunits are bound to each other through disulfide bonds (Field et al. 1983) that enforce their conformation. Glutenin subunits consist of two types of molecules: high-molecular-weight (HMW) glutenin subunits and low-molecular-weight (LMW) glutenin subunits. HMW glutenin subunits (A subunits) have more than 15 subunits of molecular masses of about 95– 140 kDa being coded by genes on the long arms of group 1 chromosomes (Glu-1 loci), more specifically either chromosomes 1A, B, or 1D (Payne et al. 1980; Shewry et al. 1986). Most of the locus consist of two genes encoding an x-type subunit of around 83– 88 kDa and a y-type subunit of around 67–74 kDa, 1Bx, 1Dx, and 1Dy subunits existing in every cultivar (Payne et al. 1980; Shewry and Tatham 1990; Shewry et al. 1992). The x-type subunits have 4–5 cysteine residues: 1 in the C-terminal end, 3 in the N-terminal end, and sometimes 1 in the repetitive domain, while the y-type subunits have 7 cysteine residues: 1 in the C-terminal end, 5 in the N-terminal end, and 1 in the repetitive domain (Shewry et al. 1992). These x-type and y-type subunits often form dimmers, although some are dimmers of 2 x-type subunits. Glutenin subunits also have abundant repeat motifs in their structure. The repetitive domain in the

middle part is estimated to form β-sheet structure, and N-terminal and C-terminal regions are mostly α-helix structure (Shewry et al. 2002). The x-type HMW glutenin subunits have 20 repeats of the motif QGQQPGQGQ, 18 motifs similar to QGQQPGQGQ, 7 repeats of the motif GQGQPGYYPTS, and 8 motifs similar to GQGQPGYYPTS (Figure 20.6), while the y-type HMW glutenin subunits have 8 repeats of the motif GQGQQGYYPTS, 3 motifs similar to GQGQQGYYPTS, 4 repeats of the motif SQQQPGQGQQG, 10 motifs similar to SQQQPGQGQQG, 2 repeats of the motif SLQQPGQGQQI, 4 motifs similar to SLQQPGQGQQI, 5 repeats of the motif QQSGQG, and a motif similar to QQSGQG (Figure 20.7). LMW subunits are further divided into B, C, and D subunits of about 30–50 kDa. The B and C LMW glutenin subunits are coded by genes on the short arms of group 1 chromosomes mainly at Glu-3 loci but some at Gli-1 loci, while the D LMW glutenin subunits are coded by genes on the short arms of chromosomes 1B and 1D (Masci et al. 1993). The molecular masses of the B LMW glutenin subunits are about 40–50 kDa, and those of the C LMW glutenin subunits are about 30–40 kDa. As the amino acid sequences of the D LMW glutenin are similar to those of 1D-coded ω-gliadins, the D LMW glutenin subunits are assumed to be mutated ω-gliadins (Masci et al. 1999). LMW glutenin subunits have 7 repeats of the motif SQQQQPPF and 13 motifs similar to

Figure 20.6. Amino acid sequence of x-type high-molecular-weight glutenin subunit in Triticum aestivum. Under solid bar: QGQQPGQGQ; under dot bar: sequence similar to QGQQPGQGQ; upper solid bar: GQGQPGYYPTS; upper dot bar: sequence similar to GQGQPGYYPTS. Source: Gu et al. 2006.

Figure 20.7. Amino acid sequence of y-type high-molecular-weight glutenin subunit in Triticum aestivum. Under solid bar: SQQQPGQGQQG; under dot bar: sequence similar to SQQQPGQGQQG; under double bar: SLQQPGQGQQI; under double TOT bar: sequence similar to SLQQPGQGQQI; upper solid bar: GQGQQGYYPTS; upper dot bar: sequence similar to GQGQQGYYPTS; upper double bar: QQSGQG; upper double dot bar: sequence similar to QQSGQG. Source: Gu et al. 2006.

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Figure 20.8. Amino acid sequence of low-molecular-weight glutenin subunit in Triticum aestivum. Under solid bar: SQQQQPPF; under dot bar: sequence similar to SQQQQPPF. Source: Özdemir and Cloutier 2005.

SQQQQPPF in the N-terminal side in addition to polyglutamine sequences (Figure 20.8). Although glutenin is dilute acid or alkali-soluble glutelin, LMW glutenin dissolves in 70% alcohol. In that sense, LMW glutenin can be classified into prolamin.

3.5. Gluten When wheat flour is mixed with water, gliadin and glutenin form a complex called gluten. This gluten has a polymerized structure and a viscoelastic property that provide firm texture to noodles when boiled and allow dough to expand when heated. Therefore, wheat is suitable for making pasta and bakery products such as bread and cakes. Gluten consists of about 50 proteins of monomeric gliadins and polymeric glutenins (Sabelli and Shewry 1991). More specifically, gluten proteins are divided into ω1,2-gliadins (4%), ω5-gliadins (5%), HMW glutenin subunits (9%), a complex of αβ- (32%) and γ-gliadins (23%), and LMW glutenin subunits (25%) (Wiesser 2000; Sabelli and Shewry 1991). The property of gluten mainly depends on the structure of glutenin polymerized through the disulfide-sulphydryl exchanges and the formation of hydrogen bonds and hydrophobic interactions. Gliadins are not so elastic, but extensible and viscous, while glutenins are rather elastic. These differences are probably attributed to their different aggregative states (Masci et al. 1991). The elastic property of glutenin is estimated to be due to the β-sheet struc-

ture of repeat motifs and disulfide bonds between glutenin subunits that form the frame of the large networks. Within these large glutenin networks, gliadins provide a plasticizer function (Wrigley 1996). The dough property becomes more elastic with the higher molecular weight of polymerized glutenin (MacRitchie 1984). As the highly polymerized glutenin is not extracted either by acid solution or SDS buffer, the percentage of unextractable polymeric protein (%UPP) can be used to estimate the elasticity of dough.

4. Wheat Allergy Allergies are abnormal inflammatory responses of the immune system to substances such as food, pollen, and dust. Patients suffering from allergies are estimated to be about 0.3–10% of human population, those suffering from wheat allergy comprising around 30% of them (Hughes and Mills 2001). In contrast to milk or egg allergies, wheat allergy is less likely to be outgrown. Wheat allergy is mainly classified into three types: (1) baker ’s asthma, a respiratory disorder caused by inhalation of wheat flour, (2) wheatdependent exercise-induced anaphylaxis (WDEIA), an immediate hypersensitive reaction caused by wheat intake and physical exercise, and (3) celiac disease, a genetic digestive disorder caused by gluten intake. Baker ’s asthma and WDEIA are mediated by IgE responses, while celiac disease is an IgE-independent immunologic reaction. Common symptoms of wheat allergy include atopic dermatitis

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(eczema), urticaria (hives), diarrhea, asthma, rhinitis, and nausea. Among sera of patients presenting a wide spectrum of clinical symptoms such as atopic dermatitis, asthma, urticaria, or anaphylaxis, 67% react with albumin/globulin fraction, 60% with αβ-gliadins and LMW glutenin subunits, 55% with γ-gliadins, 48% with ω-gliadins, and 26% with HMW glutenin subunits (Battais et al. 2003). As for allergens in albumin/globulin fraction, allergenicity is high for the proteins of 16, 18, 22, 35, 60, and 75 kDa (Handoyo et al. 2008) and of 20 and 47 kDa (Jones et al. 1995), those of 16 and 18 kDa corresponding to α-amylase inhibitors, 35 kDa to a peroxidase precursor, and 60 kDa to a serine carboxypeptidase (Battais et al. 2003). α-Amylase inhibitors are also allergens in rice (Alvarez et al. 1995; Nakase et al. 1996). In neutral and acidic soluble fraction, the proteins of 26, 28, and 69 kDa bind to IgE of patients with atopic dermatitis (Varjonen et al. 1995). N-terminal amino acid sequences of some allergens in albumin/globulin fraction have been determined (Singh et al. 2001; Watanabe et al. 2001a). As for LMW glutenin subunits, a protein of 42 kDa is a major allergen in patients with gastrointestinal symptoms after wheat ingestion (Simonato et al. 2001).

4.1. Baker’s Asthma Baker ’s asthma is an occupational respiratory disorder that occurs by inhaling wheat flour and is a serious problem for those working in the food industries. Major allergens exist in water-/salt-soluble fraction (Mittag et al. 2004) that contains α-amylase inhibitor of 12–18 kDa (Gómez et al. 1990; SánchezMonge et al. 1992; Posch et al. 1995; James et al. 1997; Amano et al. 1998; Simonato et al. 2001), peroxidase of 36 kDa (Sánchez-Monge et al. 1997; Yamashita et al. 2002), glycerinaldehyde-3phosphate dehydrogenase, serpin (Sander et al. 2001), acyl-CoA oxidase of 27 kDa (Posch et al. 1995; Weiss et al. 1993, 1997), and fructosebisphosphate aldolase of 39 kDa (Weiss et al. 1997). α-Gliadins, fast γ-gliadins, and LMW glutenin subunits in wheat (Sandiford et al. 1997; Maruyama

et al. 1998) and glycerinaldehyde-3-phosphate dehydrogenase and triosephospate isomerase in water-/salt-soluble fraction from barley (Hordeum vulgare) (Sander et al. 2001) are also the allergens of baker ’s asthma.

4.2. Wheat-Dependent Exercise-Induced Anaphylaxis (WDEIA) Food-dependent exercise-induced anaphylaxis (FDEIA) is a severe allergic reaction induced by physical exercise after the injection of causative food, but it is also triggered by fatigue, stress, and the intake of drugs such as aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) or alcohol, instead of physical exercise. Among various foods, wheat is the most frequent causative food (57%) followed by shrimp (18%), cuttlefish (5%), crab (3%), grape (2%), nuts (2%), buckwheat (2%), and fish (1%) for Japanese patients (Morita et al. 2007). Allergens for WDEIA are mainly ω5-gliadins (fast ω-gliadins) (Palosuo et al. 2001; Lehto et al. 2003; Morita et al. 2003), γ-gliadins of 65 kDa (Palosuo et al. 1999; Morita et al. 2000, 2001; Mittag et al. 2004), and α-gliadins of 40 kDa (Palosuo et al. 1999); but α-amylase inhibitor, lipid transfer proteins, and LMW glutenin subunits are also reported as allergens (Pastorello et al. 2007). Patients suffering from WDEIA usually have IgE antibodies. After wheat intake and physical exercise, serum gliadinpeptide level usually increases (Matsuo et al. 2005c). The enhancement in antigen absorption is observed even in healthy subjects, although no allergic symptoms are exhibited. The mechanism of exerciseinduced enhancement of intestinal absorption of wheat into the blood is assumed to be the endocytotic uptake of the antigen into the enterocytes and its rapid transcytosis into the lamia propria, followed by the increase in antigen flux through tight junctions by a paracellular route after mast cell activation by crosslinking of IgE bound to FcεFI receptors (Morita et al. 2007). Because gliadins are resistant to gastric enzymes and exhibit low solubility in gastric and duodenal fluids (Mittag et al. 2004), this may be one of the reasons of their high allergenicity. Cross-reactivity is observed with γ-gliadins, slow

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ω-gliadins (ω1- and ω2-gliadins) (Morita et al. 2003), γ70- and γ35-secalins in rye, and γ3-hordein in barley (Lehto et al. 2003).

4.3. Celiac Disease Celiac disease is an autoimmune disorder of the small intestine caused by wheat intake. It is considered to be a genetic disease because 90% of patients inherited genes encoding human leucocyte antigen (HLA)-DQ2 and/or -DQ8, the majority of them carrying HLA-DQ2 (Sjöström et al. 1998). Incidence of celiac disease is high in European countries, and about 1 out of 300 European people are suffering from it (Arentz-Hansen et al. 2000a). Chronic inflammatory disorder of the small intestine is derived from the binding of antigens to HLA-DQ2 or -DQ8, and the resultant CD4 T-cell stimulation is potentiated when glutamine residues are deamidated by tissue transglutaminase (tTG) in acidic pH. tTG also catalyzes the crosslinking reaction between glutamine and lysine residues and makes gliadins bound to interstitial collagen types I, III, and VI, immobilizing gliadins in the intestinal extracellular matrix to produce neoepitopes (Dieterich et al. 2006). Celiac disease is not IgE antibody-mediated (Mittag et al. 2004), and patients suffering from celiac disease have high levels of serum IgA antibodies to gliadins because α-, γ-, and ω-gliadins are the potential substrates of tTG (Dieterich et al. 2006), α-gliadins being major initiators of celiac disease (Anderson and Greene, 1997).

4.4. Epitope Structure of Wheat Protein IgE-binding epitopes for WDEIA are QQIPQQQ, QQLPQQQ, QQFPQQQ, QQSPEQQ, QQSPQQQ, QQYPQQQ, PYPP (Matsuo et al. 2004), QQFHQQQ, QSPEQQQ, YQQYPQQ, and QQPPQQ (Matsuo et al. 2005b) in ω5-gliadins, and QQPGQ, QQPGQGQQ, and QQSGQGQ in HMW glutenin subunits (Matsuo et al. 2005b). Among QQIPQQQ and QQFPQQQ in ω5-gliadins, QXXPQQQ (Matsuo et al. 2004) or QQXPQQQ (Battais et al. 2005) are critical structure, while among QQPGQGQQ and QQSGQGQ in HMW glutenin subunits,

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QQPGQXQX and QXSGQXQ are critical structure for IgE binding. Based on these findings, synthesized peptides of KPQQQSPQQQFPQQQIPQQQ and PTSPQQSGQGQQPGQGQQ are found to be a useful tool for diagnosis of WDEIA (Matsuo et al. 2005b). As for IgE-binding epitopes for atopic dermatitis, QQPFP and PQQPF in gliadins (Tanabe 2004), and QQQPP motif in LMW glutenin subunits are identified (Watanabe et al. 1995; Tanabe et al. 1996). Most patients suffering from celiac disease are HLA-DQ2 positive (Sjöström et al. 1998). HLADQ2-restricted α-gliadin epitopes are PFPQPQLPY, PQPQLPYPQ (Arentz-Hansen et al. 2000a), PYPQPQLPY (Arentz-Hansen et al. 2002), and LGQQQPFPPQQPYPQPQPF (Sturgess et al. 1994), while HLA DQ2-restricted γ-gliadin epitopes are PQQSFPQQQ (Sjöström et al. 1998), IIQPQQPAQ (Vader et al. 2002), FPQQPQQPYPQQP, FSQPQQQFPQPQ, LQPQQPFPQQPQQPYPQQPQ (Arentz-Hansen et al. 2002), where bold letters are glutamine residues targeted by tTG. Most of the α-gliadin and γ-gliadin epitopes contain deamidated glutamines and several proline residues (ArentzHansen et al. 2002).

4.5. Reduction of Wheat Allergenicity There are several methods to reduce allergenicity of wheat such as by hydrolyzing or modifying allergens with enzymes (Watanabe et al. 1994, 2000), polishing whole wheat grain (Handoyo et al. 2008), or deamidating glutamine residues (Kumagai et al. 2007). Among proteases, actinase, collagenase, and transglutaminase are effective to decompose the proteinaceous allergens (Watanabe et al. 1994). Treatment with cellulase prior to that with actinase is further efficient because the former hydrolyze the carbohydrate allergens without developing sweetness (Watanabe et al. 2000). The use of transglutaminase that catalyzes the transfer of the acyl groups of glutamine residues on amino groups of lysine residues reduces allergenicity of gliadins (Leszczyn´ska et al. 2006). As allergens are not located in the innermost part, the stepwise polishing of wheat grain can be used to obtain hypoallergenic

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fraction of wheat flour (Handoyo et al. 2008). Since almost all the epitopes of gliadins contain glutamine residues, deamidation conversion of glutamine residues to glutamic-acid residues by cation-exchange resins is effective for reducing their allergenicity (Kumagai et al. 2007). Hydrophilicity of deamidated gliadins makes them susceptible to enzymatic hydrolysis and increases their digestibility in gut, which may further reduce their allergenicity. Intake of hypoallergenic flour prepared by enzymatic hydrolysis (Watanabe et al. 2001b) or deamidated gliadins (Kumagai et al. 2007) induces oral tolerance, and these could be used for wheat-allergy treatment.

5. Prevention of Diabetes by Wheat Albumin Diabetes mellitus is a syndrome that leads to increase in blood glucose level due to disordered metabolism of insulin secretion or insulin action. Diabetes is serious because it often leads to various complications such as retinopathy, nephropathy, and neuropathy or atherosclerosis. Type I (insulin-dependent) diabetes is caused by the deficiency of insulin due to a loss of the insulin-producing beta cells of pancreatic Langerhans islets. It has an inherited genetic predisposition, often being developed in childhood. On the other hand, type II (noninsulin-dependent) diabetes is caused by resistance to insulin or reduction in insulin sensitivity. The prime risk factor of type II diabetes is overweight or obese, and it often develops in people over 40. Among patients suffering from diabetes mellitus, about 90% of them are classified into noninsulin-dependent (type II) diabetics. The number of type II diabetics is estimated to be 215.6 million in 2010 by the World Health Organization (WHO), about half of them being in Asian countries (Kodama et al. 2005). In order to reduce the risk of diabetes, it is necessary to control the blood glucose level. Wheat albumin delays carbohydrate digestion and prevents the increase in postprandial hyperglycemia by inhibiting the activity of α-amylase that converts polysaccharides to disaccharides (Koike et al. 1995; Kodama et al. 2005). There are three

families of α-amylase inhibitors in wheat: those of 12, 24, and 60 kDa. The 12 kDa inhibitors act only on insect amylase, while 24 kDa inhibitors act on mammalian, insect, and bacterial amylase (Silano et al. 1973; Maeda et al. 1985). The function of 60 kDa inhibitors has not yet been clarified. α-Amylase inhibitors of the 12 kDa family are monomers and further divided into 0.28, 0.32, 0.35, 0.39, and 0.48 inhibitors according to their electrophoretic mobility on native gel (Silano et al. 1973), while those of the 24 kDa family are dimmers of 12 kDa subunits and further divided into 0.19, 0.36, 0.38, and 0.53 inhibitors (Choudhury et al. 1996). Among inhibitors of the 24 kDa family, the 0.38 inhibitor exerts the highest inhibitory activity against pancreatic αamylase. The 0.19 inhibitor, the major inhibitor, is more specific to salivary amylase than to pancreatic amylase, and its inhibitory activity is higher than that of the 0.53 inhibitor (Choudhury et al. 1996).

6. Prevention of Hypertension by Wheat Peptide Blood pressure is mainly controlled by the reninangiotensin system. Renin released by the kidneys converts circulating angiotensinogen to angiotensin I. Angiotensin I is then converted to angiotensin II by angiotensin-converting enzyme (ACE), which cleaves His-Leu from the C-terminal of angiotensin I (Matsui et al. 1993; Kawasaki et al. 2000). The produced angiotensin II constricts arteries and veins by binding to type 1 angiotensin II (AT1) receptors on the smooth muscle cells in blood vessels and heart tissues. Therefore, inhibition of ACE blocks the formation of angiotensin II, which produces vasodilatation and reduction of blood pressure. Several peptides from food origin that are essentially composed of hydrophobic and/or aromatic amino acid residues (Matsui et al. 1993) and/or ProPro sequence (Maruyama et al. 1989; Miyoshi et al. 1991) are known to inhibit ACE. These are, for example, Ile-Tyr and Ile-Trp-His-His-Thr from dried bonito (Yokoyama et al. 1992), Val-Pro-Pro and Ile-Pro-Pro from milk (Nakamura et al. 1995), Val-Tyr from sardine muscle (Kawasaki et al. 2000),

Chapter 20 Wheat Proteins and Peptides

and Leu-Arg-Pro, Leu-Ser-Pro, and Leu-Gln-Pro from α-zein (Miyoshi et al. 1991). As for peptides from wheat origin, 12 peptides from wheat germ (Matsui et al. 1999) and Ile-Ala-Pro from wheat gliadin (Motoi and Kodama 2003) inhibit ACE in vitro. Among peptides from wheat germ, Ile-ValTyr is the main contributor to ACE inhibition, and its intake lowers blood pressure due to the combined effect of itself and Val-Tyr that is produced from Ile-Val-Tyr by the action of aminopeptidase in plasma (Matsui et al. 2000).

7. Conclusion Wheat is a major crop that is consumed all over the world in the form of various products such as pasta, bread, cakes, and cookies. The quality of wheat products is affected by protein content because the viscoelastic property of gluten allows dough to expand during heating and provides firm texture to pasta after it is boiled. This characteristic of gluten is mainly attributed to the elastic property of polymerized glutenins and the plasticizing property of gliadins. Glutelin and gliadin account for about 40–46% of total protein, having unique amino acid sequences that are rich in glutamine and proline residues. Although the amount of albumin is not high, it contains α-amylase inhibitor that inhibits amylase activity and retards carbohydrate digestion, preventing the increase in postprandial hyperglycemia. However, α-amylase is resistant to proteolysis by digestive enzymes, and it may become a cause of allergic diseases such as baker ’s asthma. On the other hand, gliadins sometimes become a cause of WDEIA and celiac disease. As epitopes in gliadins for WDEIA and celiac disease are comprised of glutamine-rich tandem repeat motifs, the cleavage or modification of the motifs is effective to reduce their allergenicity. Some peptides such as Ile-Ala-Pro from wheat αβ-gliadin and Ile-Val-Tyr from wheat germ prevent hypertension by inhibiting ACE. As shown here, wheat proteins have various functions. Science needs to be developed to maximize their favorable functions such as prevention of

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diabetes and hypertension and to minimize their adverse functions such as allergenicity.

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Hsia CC, Anderson OD. 2001. Isolation and characterization of wheat ω-gliadin genes. Theor Appl Genet 103(1):37–44. Hughes DA, Mills C. 2001. Food allergy: A problem on the increase. Biologist 48(5):201–204. James JM, Sixbey JP, Helm RM, Bannon GA, Burks AW. 1997. Wheat α-amylase inhibitor: A second route of allergic sensitization. J Allergy Clin Immunol 99(2):239–244. Jones SM, Magnolfi CF, Cooke SK, Sampson HA. 1995. Immunologic cross-reactivity among cereal grains and grasses in children with food hypersensitivity. J Allergy Clin Immunol 96(3):341–351. Kasarda DD, Autran JC, Lew EJL, Nimmo CC, Shewry PR. 1983. N-Terminal amino acid sequences of ω-gliadins and ω-secalins. Implications for the evolution of prolamin genes. Biochim Biophys Acta 747(1–2):138–150. Kasarda DD, D’Ovidio R. 1999. Deduced amino acid sequence of an a-gliadin gene from spelt wheat (spelta) includes sequences active in celiac disease. Cereal Chem 76(4):548– 551. Kasarda DD, Okita TW, Bernardin JE, Baecker PA, Nimmo CC, Lew EJL, Dietler MD, Greene FC. 1984. Nucleic acid (cDNA) and amino acid sequences of α-type gliadins from wheat (Triticum aestivum). Proc Natl Acad Sci USA 81(15):4712– 4716. Kawasaki T, Seki E, Osajima K, Yoshida M, Asada K, Matsui T, Osajima Y. 2000. Antihypertensive effect of valyl-tyrosine, a short chain peptide derived from sardine muscle hydrolyzate, on mild hypertensive subjects. J Hum Hypertens 14(8):519– 523. Kihara H (ed.). 1954. Studies on Wheats, 2nd ed. Tokyo: Yokendo (in Japanese). Kodama T, Miyazaki T, Kitamura I, Suzuki Y, Namba Y, Sakurai J, Torikai Y, Inoue S. 2005. Effects of single and long-term administration of wheat albumin on blood glucose control: Randomized controlled clinical trials. Eur J Clin Nutr 59(3): 384–392. Koike D, Yamadera K, DiMagno EP. 1995. Effect of a wheat amylase inhibitor on canine carbohydrate digestion, gastrointestinal function, and pancreatic growth. Gastroenterology 108(4):1221–1229. Kumagai H, Suda A, Sakurai H, Kumagai H, Arai S, Inomata N, Ikezawa Z. 2007. Improvement of digestibility, reduction in allergenicity, and induction of oral tolerance of wheat gliadin by deamidation. Biosci Biotech Biochem 71(4):977– 985. Lehto M, Palosuo K, Varjonen E, Majuri M-L, Andersson U, Reunala T, Alenius H. 2003. Humoral and cellular responses to gliadin in wheat-dependent, exercise-induced anaphylaxis. Clin Exp Allergy 33(1):90–95. Leszczyn´ska J, Lącką A, Bryszewska M. 2006. The use of transglutaminase in the reduction of immunoreactivity of wheat flour. Food Agric Immunol 17(1–4):1–9. Liu C-Y, Shepherd KW. 1995. Inheritance of B subunits of glutenin and ω- and γ-gliadins in tetraploid wheats. Theor Appl Genet 90(7–8):1149–1157.

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MacRitchie F. 1984. Baking quality of wheat flours. Adv Food Res 29:201–277. Maeda K, Kakabayashi S, Matsubara H. 1985. Complete amino acid sequence of an α-amylase inhibitor in wheat kernel (0.19-inhibitor). Biochim Biophys Acta 828(3):213–221. Maruyama N, Ichise K, Katsube T, Kishimoto T, Kawase S-I, Matsumura Y, Takeuchi Y, Sawada T, Utsumi S. 1998. Identification of major wheat allergens by means of the Escherichia coli expression system. Eur J Biochem 255(3):739–745. Maruyama S, Miyoshi S, Kaneko T, Tanaka H. 1989. Angiotensin I-converting enzyme inhibitory activities of synthetic peptides related to the tandem repeated sequence of a maize endosperm protein. Agric Biol Chem 53(4):1077–1081. Masci S, Egorov TA, Ronchi C, Kuzmicky DD, Kasarda DD, Lafiandra D. 1999. Evidence for the presence of only one cysteine residue in the D-type low molecular weight subunits of wheat subunits of wheat glutenin. J Cereal Sci 29(1):17– 25. Masci S, Lafiandra D, Porceddu E, Lew EJL, Tao HP, Kasarda DD. 1993. D-glutenin subunits: N-terminal sequences and evidence for the presence of cysteine. Cereal Chem 70(5):581– 585. Masci S, Porceddu E, Colaprico G, Lafiandra D. 1991. Comparison of the B and D subunits of glutenin encoded at the Glu-D3 locus in two biotypes of the common wheat cultivar Newton with different technological characteristics. J Cereal Sci 14(1):35–46. Matsui T, Li C-H, Osajima Y. 1999. Preparation and characterization of novel bioactive peptides responsible for angiotensin I-converting enzyme inhibition from wheat germ. J Pept Sci 5(7):289–297. Matsui T, Li C-H, Tanaka T, Maki T, Osajima Y, Matsumoto K. 2000. Depressor effect of wheat germ hydrolysate and its novel angiotensin I-converting enzyme inhibitory peptide, Ile-ValTyr, and the metabolism in rat and human plasma. Biol Pharm Bull 23(4):427–431. Matsui T, Matsufuji H, Seki E, Osajima K, Nakashima M, Osajima Y. 1993. Inhibition of angiotensin I-converting enzyme by Bacillus licheniformis alkaline protease hydrolyzates derived from sardine muscle. Biosci Biotech Biochem 57(6):922–925. Matsuo H, Kohno K, Morita E. 2005a. Molecular cloning, recombinant expression and IgE-binding epitope of ω-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis. FEBS J 272(17):4431–4438. Matsuo H, Kohno K, Niihara H, Morita E. 2005b. Specific IgE determination to epitope peptides of ω-5 gliadin and high molecular weight glutenin subunit is a useful tool for diagnosis of wheat-dependent exercise-induced anaphylaxis. J Immunol 175(12):8116–8122. Matsuo H, Morimoto K, Akaki T, Kaneko S, Kusatake K, Kuroda T, Niihara H, Hide M, Morita E. 2005c. Exercise and aspirin increase levels of circulating gliadin peptides in patients with wheat-dependent exercise-induced anaphylaxis. Clin Exp Allergy 35(4):461–466.

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proteins. Cloning and characterization of five major molecular forms. J Biol Chem 275(43):33272–33279. Özdemir N, Cloutier S. 2005. Expression analysis and physical mapping of low-molecular-weight glutenin loci in hexaploid wheat (Triticum aestivum L.). Genome 48(3):401–410. Palosuo K, Alenius H, Varjonen E, Koivuluhta M, Mikkola J, Keskinen H, Kalkkinen N, Reunala T. 1999. A novel wheat gliadin as a cause of exercise-induced anaphylaxis. J Allergy Clin Immunol 103(5):912–917. Palosuo K, Varjonen E, Kekki O-M, Klemola T, Kalkkinen N, Alenius H, Reunala T. 2001. Wheat ω-5 gliadin is a major allergen in children with immediate allergy to ingested wheat. J Allergy Clin Immunol 108(4):634–638. Pastorello EA, Farioli L, Conti A, Pravettoni V, Bonomi S, Iametti S, Fortunato D, Scibilia J, Bindslev-Jensen C, BallmerWeber B, Robino AM, and Ortolani C. 2007. Wheat IgEmediated food allergy in European patients: α-Amylase inhibitors, lipid transfer proteins and low-molecular-weight glutenins. Allergenic molecules recognized by double-blind, placebo-controlled food challenge. Intl Arch Allergy Immunol 144(1):10–22. Payne PI, Law CN, Mudd EE. 1980. Control by homoeologous group 1 chromosomes of the high-molecular-weight subunits of glutenin, a major protein of wheat endosperm. Theor Appl Genet 58(3–4):113–120. Piston F, Dorado G, Martin A, Barro F. 2006. Cloning of nine γ-gliadin mRNAs (cDNAs) from wheat and the molecular characterization of comparative transcript levels of γ-gliadin subclasses. J Cereal Sci 43(1):120–128. Posch A, Weiss W, Wheeler C, Dunn MJ, Goerg A. 1995. Sequence analysis of wheat grain allergens separated by twodimensional electrophoresis with immobilized pH gradients. Electrophoresis 16(7):1115–1119. Rafalski JA. 1986. Structure of wheat gamma-gliadin genes. Gene 43(3):221–229. Sabelli PA, Shewry PR. 1991. Characterization and organization of gene families at the Gli-1 loci of bread and durum wheats by restriction fragment analysis. Theor Appl Genet 83(2):209– 216. Sánchez-Monge R, Barber D, Mendez E, García-Olmedo F, Salcedo G. 1986. Genes encoding α-amylase inhibitors are located in the short arms of chromosomes 3B, 3D and 6D of wheat (Triticum aestivum L.). Theor Appl Genet 72(1):108– 113. Sánchez-Monge R, García-Casado G, Lopez-Otin C, Armentia A, Salcedo G. 1997. Wheat flour peroxidase is a prominent allergen associated with baker ’s asthma. Clin Exp Allergy 27(10):1130–1137. Sánchez-Monge R, Gómez L, Barber D, Lopez-Otin C, Armentia A, Salcedo G. 1992. Wheat and barley allergens associated with baker ’s asthma. Glycosylated subunits of the alpha-amylase-inhibitor family have enhanced IgE-binding capacity. Biochem J 281(2):401–405. Sander I, Flagge A, Merget R, Halder TM, Meyer HE, Baur X. 2001. Identification of wheat flour allergens by means of

2-dimensional immunoblotting. J Allergy Clin Immunol 107(5):907–913. Sandiford CP, Tatham AS, Fido R, Welch JA, Jones MG, Tee RD, Shewry PR, Newman Taylor AJ. 1997. Identification of the major water/salt insoluble wheat proteins involved in cereal hypersensitivity. Clin Exp Allergy 27(10):1120–1129. Scheets K, Hedgcoth C. 1988. Nucleotide sequence of a γ-gliadin gene: Comparisons with other γ-gliadin sequences show the structure of γ-gliadin genes and the general primary structure of γ-gliadins. Plant Sci 57(2):141–150. Shani N, Steffen-Campbell JD, Anderson OD, Greene FC, Galili G. 1992. Role of the amino- and carboxy-terminal regions in the folding and oligomerization of wheat high molecular weight glutenin subunits. Plant Physiol 98(2):433–441. Shewry PR, Halford NG, Belton PS, Tatham AS. 2002. The structure and properties of gluten: An elastic protein from wheat grain. Philos T R Soc B, Biol Sci 357(1418):133– 142. Shewry PR, Halford NG, Tatham AS. 1992. High-molecularweight subunits of wheat glutenin. J Cereal Sci 15(2):105– 120. Shewry PR, Lafiandra D, Salcedo G, Aragoncillo C, GarciaOlmedo F, Lew EJL, Dietler MD, Kasarda DD. 1984. N-Terminal amino acid sequences of chloroform/methanolsoluble proteins and albumins from endosperms of wheat, barley and related species. Homology with inhibitors of αamylase and trypsin and with 2S storage globulins. FEBS Lett 175(2):359–363. Shewry PR, Tatham AS. 1990. The prolamin storage proteins of cereal seeds: Structure and evolution. Biochemical J 267(1): 1–12. Shewry PR, Tatham AS, Forde J, Kreis M, Miflin BJ. 1986. The classification and nomenclature of wheat gluten proteins: A reassessment. J Cereal Sci 4(2):97–106. Silano V, Pocchiari F, Kasarda DD. 1973. Physical characterization of α-amylase inhibitors from wheat. Biochim Biophys Acta—Protein Struct 317(1):139–148. Simonato B, De Lazzari F, Pasini G, Polato F, Giannattasio M, Gemignani C, Peruffo AD, Santucci B, Plebani M, Curioni A. 2001. IgE binding to soluble and insoluble wheat flour proteins in atopic and non-atopic patients suffering from gastrointestinal symptoms after wheat ingestion. Clin Exp Allergy 31(11): 1771–1778. Singh J, Blundell M, Tanner G, Skerritt JH. 2001. Albumin and globulin proteins of wheat flour: Immunological and N-terminal sequence characterization. J Cereal Sci 34:85–103. Sjöström H, Lundin KE, Molberg Ø, Körner R, McAdam SN, Anthonsen D, Quarsten H, Norén O, Roepstorff P, Thorsby E, Sollid LM. 1998. Identification of a gliadin T-cell epitope in coeliac disease: General importance of gliadins deamidation for intestinal T-cell recognition. Scand J Immunol 48(2):111– 115. Sturgess R, Day P, Ellis HJ, Lundin KE, Gjertsen HA, Kontakou M, Ciclitira PJ. 1994. Wheat peptide challenge in coeliac disease. Lancet 343(8900):758–761.

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Sugiyama T, Rafalski A, Soll D. 1986. The nucleotide sequence of a wheat γ-gliadin genomic clone. Plant Sci 44(3):205–209. Tanabe S. 2004. IgE-binding abilities of pentapeptides, QQPFP and PQQPF, in wheat gliadin. J Nutr Sci Vitaminol 50(5):367– 370. Tanabe S, Arai S, Yanagihara Y, Mita H, Takahashi K, Watanabe M. 1996. A major wheat allergen has a Gln-Gln-Gln-Pro-Pro motif identified as an IgE-binding epitope. Biochem Biophys Res Commun 219(2):290–293. Tatham AS, Shewry PR. 1991. Conformational analysis of the secalin storage proteins of rye (Secale cereale L.). J Cereal Sci 14(1):15–23. Vader W, Kooy Y, Van Veelen P, De Ru A, Harris D, Benckhuijsen W, Pena S, Mearin L, Drijfhout JW, Koning F. 2002. The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. Gastroenterology 122(7):1729–1737. Varjonen E, Vainio E, Kalimo K, Juntunen-Backman K, Savolainen J 1995. Skin-prick test and RAST responses to cereals in children with atopic dermatitis. Characterization of IgE-binding components in wheat and oats by an immunoblotting method. Clin Exp Allergy 25(11):1100–1117. von Büren M, Lüthy J, Hübner P. 2000. A spelt-specific γ-gliadin gene: Discovery and detection. Theor Appl Genet 100(2):271– 279. Wang J-R, Yan Z-H, Wei Y-M, Nevo E, Baum BR, Zheng Y-L. 2006. Molecular characterization of dimeric alpha-amylase inhibitor genes in wheat and development of genome allelespecific primers for the genes located on chromosome 3BS and 3DS. J Cereal Sci 43(3):360–368. Watanabe J, Tanabe S, Sonoyama K, Kuroda M, Watanabe M. 2001a. IgE-reactive 60 kDa glycoprotein occurring in wheat flour. Biosci Biotech Biochem 65(8):1729–1735. Watanabe J, Tanabe S, Watanabe M, Kasai T, Sonoyama K. 2001b. Consumption of hypoallergenic flour prevents gluteninduced airway inflammation in Brown Norway rats. Biosci Biotech Biochem 65(8):1729–1735.

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Part 4 Recent Advances in Bioactive Peptide Analysis for Food Application

Chapter 21 Peptidomics for Bioactive Peptide Analysis Icy D’Siva and Yoshinori Mine

Contents 1. Introduction, 307 2. Methods in Peptidomics, 308 2.1. Affinity Peptidomics, 308 2.2. Combinatorial Peptidomics, 309 2.3. Mass Spectrometry, 309 3. Sample Preparation for Peptidomics, 311 3.1. Sample Collection and Preanalytical Treatment, 311 3.2. Enrichment and Detection of Proteins Useful to Peptidomic Studies, 312 3.3. Peptide Separation, 313

1. Introduction Peptidomics can be defined as the comprehensive multiplex analysis of endogenous peptides contained within a biological sample under defined conditions to describe the multitude of native peptides in a biological compartment (Tammen et al. 2007). Peptidomics or “peptide proteomics” refers to the analysis of all the peptides of a cell or tissue (peptidome), while proteomics refers to the analysis of the proteins (proteome). Peptidomics involves the simultaneous visualization and identification of the peptidome through the use of purification and/or characterization techniques, and the knowledge of the structure and function of proteins in the biologi-

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

3.4. 3.5. 3.6. 3.7. 4. 4.1. 4.2. 4.3. 4.4. 4.5. 5.

Peptide Identification, 314 Peptide Quantification, 315 Posttranslational Modifications, 316 Method Validation, 317 Applications of Peptidomics, 317 Peptides as Biomarkers, 317 Peptide Biomarkers in Disease, 319 Peptides and Diet–Disease Associations, 319 Peptides in the Regulation of Behavior, 320 Peptidomics and Novel Peptides, 320 References, 321

cal or biochemical context of all organisms. Peptides are short stretches of amino acids linked by peptide bonds. The length of the peptides varies from two amino acids (dipeptide) to several amino acids (polypeptides) up to a molecular mass of 20 kDa (Schrader and Selle 2006). Peptides that are biologically active play an important role in the physiological processes of an organism, are tightly regulated by proteolytic control (Sewald and Jakubke 2002), and serve as ideal biomarkers for specific and sensitive diagnosis and therapeutics. Bioactive peptides may be classified as peptide hormones, neuropeptides, cytokines, and growth factors. The completion of the human genome project opened up new opportunities for the rapid identification and functional analysis of peptides (Baggerman et al. 2004). The field of peptidomics is relatively new and is progressing rapidly with the advent of high throughput mass spectrometry (MS)-based technologies, which include matrixassisted laser desorption ionization-time of flight 307

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MS (MALDI-TOF-MS), high-performance liquid chromatography-MS (HPLC-MS), and capillary electrophoresis-MS (CE-MS) (Chen 2008; Kussmann 2009; Mine 2009; Tao 2009). Unlike the importance of two-dimensional gel electrophoresis (2DGE) as well as MS for proteomics, the method of choice for peptidomics is MS since 2DGE does not favor the separation of peptides less than 10 kDa in size. This chapter presents an overview of the advances in peptidomics and applications of peptidomics.

2. Methods in Peptidomics The composition of the peptidome can be determined by a number of methods, such as affinity peptidomics, combinatorial peptidomics, and MS with or without prior reduction in sample complexity (Figure 21.1).

2.1. Affinity Peptidomics In affinity peptidomics, the composition of the peptidome is determined using immobilized antibodies

by directly assaying the range of peptides obtained from protease or chemically digested protein mixture of biological extracts. It has been reported that affinity agents developed against the most hydrophilic peptides resulted in an enhanced performance of the affinity assay, possibly due to most peptides being hydrophilic (Scrivener et al. 2003). The hydrophilicity and immunogenicity of the released peptides, as well as the availability of synthetic peptides as antigens, has facilitated the generation of antibodies for affinity peptidomics. Most human proteins, including hydrophobic proteins, yield sufficiently hydrophilic peptides on tryptic digestion. The chips are immobilized on a solid support with an array of captured antibodies that specifically recognize a region of a target peptide fragment. Identification of captured species may be carried out either by fluorescent scanning or by MS. In fluorescent scanning, peptides are labeled prior to incubation to allow a spatially resolved image of fluorescence on scanning the chip. Although faster scanning and information retrieval is possible with fluorescent scanning, it is unable to verify that the right peptides

Figure 21.1. Schematic representation of the methods of peptidomics. In affinity peptidomics, fluorescence labeled or unlabeled sample peptides are bound to antibodies and detected by fluorescence scanning or mass spectrometry, respectively. In combinatorial peptidomics, sample peptides bound to amino acid filters are either depleted or enriched and detected by mass spectrometry. Sample peptides may be detected directly by mass spectrometry. FS = fluorescense scanning, MS = mass spectrometry

Chapter 21 Peptidomics for Bioactive Peptide Analysis

are captured and it does not help in distinguishing different posttranslational modifications (PTMs) of the polypeptide fragments. MS allows the identification of mass/charge (m/z) of the peptide by desorbing captured species directly from the array, resulting in clear identification of the captured species and in the identification of PTMs. However, some of the disadvantages are that hydrogel pads mounted into silicon are not suitable for MALDI-TOP-MS, quantitative data cannot be obtained directly, and several factors influence peak height. Aside from the cost and availability of antibodies, the stability of the peptides and nonrequirement of labeling of the peptides are some of the advantages of affinity peptidomics.

2.2. Combinatorial Peptidomics Combinatorial peptidomics allows the determination of the composition of the peptidome by assaying peptides directly from crude tryptic digests without using antibodies or any other chromatographic or electrophoretic selection. It relies on the chemical reactivities of the side chains of amino acids and amino acid content of the peptides and is not based on their physical properties. Protein samples are proteolytically digested and the peptide pools are either quantitatively depleted or enriched by chemical crosslinking of a subset of peptides through amino acid side chains to a solid support, reducing complexity to a required degree to make it compatible with MS detection (Soloviev et al. 2003). Like proteins, unmodified peptides generally contain up to eight reactive groups, of which six are chemically reactive amino acid side chains, while the other two reactive groups are the amino (–NH2) and carboxyl (–COOH) terminal groups or epsilon amino groups of lysine and side chains of aspartic acid and glutamic acid. Any of these chemically reactive amino acid side chains can potentially be used to deplete or enrich a sample of peptide, which contains them, in a specific and fully predictable manner with respect to amino acid content. These may be used for amino acid sequence- and amino acid content-independent manipulations through

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chemical radioactive fluorescent or isotopic labeling (Soloviev and Finch 2005). In the depletion method, amino acid filters from one to six different combinations can be used repeatedly or until the amino acid complexity of the peptide pool and the amino acid complexity of the remaining peptides is reduced to the optimum level. Peptides that do not contain an amino acid that is not recognized by any of the amino acid filters remain in the mixture. The depleted pool with decreased amino acid complexity allows direct analysis by TOF-MS and TOF/TOF-MS. The depletion rate varies depending on either the number of amino acid filters used or the length of peptide fragments, longer peptides being more likely to be crosslinked. Incompletely digested peptides are easily eliminated from the samples due to their increased peptide length, which has more probability of being crosslinked (Soloviev et al. 2003). This method is straightforward, faster, and more stable for peptides of 10 amino acids and longer. In the enrichment selection technique, peptides containing the required amino acids are retained. The peptide mixtures are passed through individual or a different combination of selected amino acid filters and these filters form bonds with the peptides containing the respective amino acids. The filter assembly is washed to remove free peptides and the chemical crosslink is cleaved in order to free the bound peptides. The method is more suitable for shorter peptides. Like the depletion technique, the procedure can be repeated using single or multiple combinations of amino acid filters.

2.3. Mass Spectrometry MS is currently one of the most important analytical techniques since it generates information on the structure and mass of the peptide and allows sensitive and precise detection of the total peptide content even in complex mixtures (Yates 1998). It is designed to measure the m/z ratio of ions as well as the number of ions present at each m/z value. Each peak on the spectrum represents an ionized molecule, namely, a peptide with the height proportional to the abundance of the peptide. The complexity of the peptide

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mixture needs to be reduced prior to MS by enriching the protein of interest and enhancing the range and sensitivity of the instrument. MALDI-TOF-MS in combination with liquid chromatography (LC) off-line and electro-spray ionization (ESI)-TOF-MS combined with nanoLC on-line (Clynen et al. 2003) are used extensively in peptidomics. A one-step sampling, extraction, and storage protocol for peptidomics using MALDI-MS-dihydroxybenzoic acid has been reported for signaling peptides using mouse pituitary gland as a model, as well as other neuronal tissues (Romanova et al. 2008). Key characteristics of a modern mass spectrometer are sensitivity (femtomolar to attomolar), mass accuracy (high to low ppm), mass resolution (10,000 to millions), and speed of MS and MS/MS acquisition. MALDI (Tanaka 2003) and ESI (Fenn 2003) are the most popular and powerful methods to produce gas phase ions of proteins and peptides. Analyzers, such as triple quadrupole (QqQ) instruments, are used especially for targeted multireaction monitoring (MRM) experiments to, for example, simultaneously quantify dozens of plasma proteins/peptides (Anderson and Hunter 2006). They may be combined with time of flight systems, such as Q-TOF and Q-ion-trap (Q-IT) instruments. Fourier transform-ion cyclotron resonance (FTICR) mass spectrometers (Nielson et al. 2005) and the more recently introduced orbitrap system (Makarov et al. 2006) represent the high-end proteomics MS. These instruments offer ultimate resolution (>100,000) and low ppm mass accuracy that enables “top-down” analysis of intact proteins as opposed to the more frequently employed “bottomup” approach (Siuti and Kelleher 2007). The top-down approach involves high-resolution mass spectrometric measurement and fragmentation of intact ionized molecules in the gas phase. MALDI is very popular because it is highly sensitive and forms singly charged ions, which facilitate the analysis of complex mixtures (Boonen et al. 2008). Due to its pulsed nature, MALDI is more often coupled to a TOF mass analyzer. The peptidome of a cell, organelle, or organ can be directly analyzed by MALDI-TOF-MS (Clynen et al. 2003). In this method, a small amount of sample is

premixed in solution with a light-absorbing material known as the matrix (alpha-cyano-4hydroxycinnamic acid) and applied to the target plate. After drying, the plate is inserted into the ionization source where a pulse of UV light is directed toward the sample. Ionization of the sample results in singly charged molecules (Ashcroft 2003). MALDI-TOF-MS has many advantages as it analyses mixtures directly, is less susceptible to impurities and salt, and also results in the formation of singly charged molecules. Other ionization methods like ESI result in multiple charged states of larger peptides that interfere in mass spectra data interpretation. MALDI-TOF instruments equipped with an ion selector fragmentation by postsource decay (PSD) help in obtaining structural information. The major drawback of PSD is the time-consuming spectrum acquisition and the low resolution of the ion selectors. This may be problematic when working with complex mixtures like tissue extracts (Baggerman et al. 2004). Recent advances in instrument development, such as MALDI-Q-TOF, which combines the advantages of MALDI ionization and Q-TOF tandem MS analysis, provide an excellent tool for in situ peptide analysis (Verhaert et al. 2001). The combination of MS with miniaturized separation techniques, such as nanoLC ESI-TOF, results in very powerful tools. ESI can be coupled with HPLC (on-line) since it transfers ions from solution directly into the gas phase. The advantage is that the peptides are separated and elute gradually while each peptide is sharply focused and concentrated in a chromatographic peak. Then peptides are analyzed mass spectrometrically as they elute from the column (Clynen et al. 2003). While an important aspect of ESI in favor of proteins is the production of multiple charged ions, which helps in analyzing proteins with masses of up to 100 kDa in a small m/z window, fragmentation of multiple charged ions yields more detectable fragment ions (Boonen et al. 2008). MS data analysis requires sophisticated software to acquire, store, retrieve, process, validate, and interpret the data and to eventually transform it into useful biological information. While peptide and

Chapter 21 Peptidomics for Bioactive Peptide Analysis

protein identification and database search programs like Mascot (Perkins et al. 1999), Sequest (Yates et al. 1995) or Phenyx (Colinge et al. 2004) have a long and successful standing, entirely new software infrastructures for data processing and validation have been built, such as the SBEAMS architecture housing the Trans-Proteomic Pipelene and microarray modules that manage both gene and protein/ peptide expression data. Open databases such as PeptideAtlas (Desiere et al. 2006) have been established to convert data and result in publicly accessible information. The idea behind such resources is to provide the researcher with means to assess the quality of the data in a data-set–dependent manner and to control the trade-off between false positives (specificity) and false negatives (sensitivity) (Urfer et al. 2006) on the basis of extensive statistical evaluation. Apart from the progress in data processing, software platforms enabling cross-correlation with other omics sources and supporting pathway interpretation are emerging and maturing rapidly. The success of peptidomics will largely depend on the ability of converting data into proper biological information.

3. Sample Preparation for Peptidomics Standard operating procedures are necessary to minimize variations due to sample handling. Organs like the brain and fluids like blood and cerebrospinal fluid contain a high concentration of peptides requiring separation prior to MS. Some body fluids and organs contain a very low concentration of peptides that require preconcentration of samples before being subjected to MS analysis (Oosterkamp et al. 1998). Peptide loss during sample drying, which is an important step in peptidomics, is a concern. Pezeshki et al. (2008) found that applying n-nonyl-β-D-glucopyranoside (C9-Glu) as an additive along with choosing the appropriate vial type (polypropylene for lyophilization and glass for centrifugal evaporation) can avoid or diminish peptide loss during the evaporation procedure. Sample preparation may entail a number of steps (Figure 21.2).

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3.1. Sample Collection and Preanalytical Treatment Selection, collection, and preanalytical methods are important for peptidomics. The National Cancer Institute Best Practices of Biospecimen Resources, National Cancer Institute of the National Institutes of Health has established the Office of Biorepositories and Biospecimen Research (OBBR). Tissue samples need to be kept intact with biological function preserved by quickly transferring and preserving in liquid nitrogen. Tissue array technology allows the cells to be deposited on a protein chip or tissue array chip with numerous features followed by target recognition by antibodies of over a hundred kinds. Body fluid samples may be analyzed as whole blood, plasma, serum, peripheral blood mononuclear cells, and circulating tumor cells used as surrogate tissue. Enzyme-linked immunosorbent assays, enzymatic assays, flow cytometry, and transcript analysis help detect biomarkers in fluids. Carriers need to be separated from abundant proteins, and proteins separated from peptides. Identification of posttranslational modifications and splice variants requires specialized analytical techniques and sophisticated database searching. Cultured cells are useful as they allow more accessibility, easier control of variants and experimental conditions, homogeneity, use of many cell biology research tools and resources, obtaining specific biochemical information at spatial and temporal points by confocal microscopy, in vivo polymerase chain reaction (PCR), and other cell biology techniques. While one of the disadvantages of using cells for peptidomic studies is the limited availability of cultivable types of cells, the advantages of using cells is that they can be used to evaluate a new analytical technique and identify variability, reproducibility, and sensitivity of the methods. The data may be evaluated and data from cells and tissues compared. Extraction of proteins from cultured cells with guanidinium hydrochloride, mechanical disruption of tissue blocks, and homogenization of soft tissues result in inconsistent yields. Pressure Cycling Technology Sample Preparation System (PCT SPS), a more robust and better-controlled extraction

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Figure 21.2. Steps in sample preparation for peptidomics. Each step (bold text) involves the use of specific techniques, some of which have been listed (normal text).

method, is used to release DNA, RNA, and proteins (Schumacher et al. 2002; Tao et al. 2006). It provides a rapid, safe, and reproducible approach for processing many types of biosamples for further PCR, RT-PCR, microarray, immunoreaction, SDS PAGE, western blotting, and 2D gels. The use of instrument-controlled processes in sealed single-use tubes facilitates a high level of operational safety and reproducibility and is cross-contamination free. PCT SPS extraction is a powerful technique to extract a variety of molecules from a wide selection of samples. It is a gentle method that can be used to extract subcellular organelles that retain structural

integrity and biological functions, and it is less shearing of DNA. High-molecular-weight proteins and hydrophobic molecules are more efficiently extracted. It is more reproducible than conventional extraction techniques.

3.2. Enrichment and Detection of Proteins Useful to Peptidomic Studies High-quality standardized reagents are indispensable for specificity and reproducibility of peptidomic studies. The target can be enriched from complex mixtures by capturing molecules, such as antibodies

Chapter 21 Peptidomics for Bioactive Peptide Analysis

and aptamers. Lack of qualified standards, reagents, and validated technologies results in lack of development, management, interpretation, and comparison, thus slowing down the progress from discovery to clinical applications. Aptamers show promise as adjuncts to antibodies (Mosing and Bowser 2007). New solid-state protein-binding microarray assays based on conventional immunoassay principles have been developed using a variety of immobilized capture reagents to detect proteins and protein fragments (peptides) extracted from biological samples. The capture reagents are immobilized on a solid support such as glass, synthetic membranes, mini or nano beads, and mass spectrometer plates. Tens of thousands of capture features may be arranged on a grid since the microarray elements can be miniaturized, each element selected being specific for a given protein or peptide. Using this format, multiple assays can be conducted simultaneously. High density of capture reagents permits a high-throughput system for measuring many proteins and their roles in the networks of biochemical and signaling interactions within a sample. Reporter molecules may be needed in detecting the presence of a target in a particular biological sample, and the reporter may sometimes work by modifying target molecules. Reference materials are required for calibrating instruments or comparing different proteomic platform technologies. Membrane proteins can be important diagnostic and prognostic markers and make up 30% of gene-coded proteins, while 60% of the drugs act on membrane proteins. Membrane proteins are responsible and essential for cellular and subcellular metabolisms and hence it is important to detect their structures. They are difficult to obtain in sufficient quantities. It is difficult to mimic their environment and difficult to crystallize them as they function in association with other cellular components, such as organelles or protein complexes. Posttranslational modifications are important for their function. By combining PCT SPS with the novel chemistry provided by the ProteoSolveLRS kit, it is possible to fractionate the lipid and protein components of a sample, resulting in higher protein recovery and greater reproducibility as compared to traditional detergent-based extraction methods.

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Underrepresented proteins in extracts of conventional methods have been successfully extracted by the PCT SPS/ProteoSolveLRS method.

3.3. Peptide Separation Generally, trypsin is used to digest proteins into several peptides. Separation of peptides from proteins >20 kDa is a necessary preparation step to reduce the protein load for chromatography and MS. Subsequent reduction of the complexity of peptide mixtures by separation or concentration prior to MS may be required for optimal peptide analysis. Separation of complex mixtures decreases the negative influence of contaminants, thus improving sensitivity and accuracy, and prevents competitive ionization by detection techniques like MALDI and ESI. Concentration of the analyte determines ionization efficiency because concentrated analytes are more sensitive to detection (Wilm and Mann 1996). The most preferred method of separation of peptides is reversed-phase HPLC, especially a combination of ion-exchange with reversed-phase HPLC (Issaq et al. 2005), which allows the analytes to remain in the liquid phase. Peptide recovery can be possible in an unchanged molecular form, since the majority of peptides do not contain complex secondary and tertiary structures (Schulz-Knappe et al. 2001). Chromatography-based techniques have been developed for protein and peptide preseparation, referred to as multidimensional protein identification technology (MudPIT) (Motoyama et al. 2006), also called “shotgun” proteomics approach (Wu and MacCoss 2002). Ion-exchange followed by reversedphase, that is, two-dimensional chromatography is coupled to ESI-MS, the approach being based on “proteome” digestion upstream in the workflow and peptide-level separation. The most abundant proteins from the proteome-scale complex sample may have to be depleted from the sample by affinity chromatography without affecting the remaining peptide composition, especially when analyzing human plasma (Gong et al. 2006) or serum (Bjorhall et al. 2005). Equalizer technology described by Righetti et al. (2006) consists of a combinatorial library of ligands bound to beads, which reduces the

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concentration differences in human plasma and urine essentially by binding less of the abundant and more of the rare proteins. A complementary strategy is to enrich low abundant proteins, for example, when targeting subproteomes, such as phosphoor glycoproteome. Chemical scavengers, such as immobilized metal affinity capture (IMAC), titanium dioxide resins, or alumina particles, are used to capture phosphopeptides and phosphoproteins. Reinders and Sickannn (2005) thoroughly compared the performance of these techniques. Glycosylated peptides can be enriched by lectin affinity (Vosseller et al. 2006) or by different trapping reactions, such as hydrazide chemistry (Sun et al. 2007). Nandi et al. (2006) have developed a so-called tagging-viasubstrate (TAS) approach for global identification of O-glycosyl enrichment. In combination with MS, capillary electrophoresis allows the separation of peptides ranging from small molecules to large proteins in the same analysis. The advantages of capillary electrophoresis are high efficiency, fast analysis times, and low sample and reagent consumption (Herrero et al. 2008).

3.4. Peptide Identification Identification of peptides based on the separating power of chromatographic techniques carries specific problems. Biological samples usually have a higher concentration of larger proteins as compared to peptides. For example, in blood plasma, albumin content is higher than that of whole protein content (about 40–50 gl/l), which corresponds to almost 1 mmol/l in molecular concentration. However, typical concentrations of peptides in blood vary between micromolar and picomolar concentration for peptide hormones (Heine et al. 2002). MRM assays are established for the quantification of candidate-based protein markers in plasma. It is the gold standard for identification and quantification of drug molecules and metabolites in clinically relevant plasma samples due to its extremely high sensitivity and specificity. For clinical proteomics, a method involving strong cation exchange chromatographic fractionation of peptides and immunoaffinity enrichment on specific anti-

peptide antibodies (Stable Isotope Standards and Capture by Anti-Peptide Antibodies [SISCAPA]) is used. Combined with MRM quantification by MS, SISCAPA generates reliable and reproducible quantitation of signature peptides from protein digests. Other methods for sample fractionation are the use of magnetic beads for capture of peptides, allowing larger surface area-to-volume ratio than flat plate protein chip designs; high-throughput immunoaffinity combined with MS instrumentation and biofabrication methods for integrating and comparing proteomics data from different platforms; microarray platform employing interferometric analysis that offers the potential for label-free high-throughput and sensitive analysis of small amounts of biological fluids leading to the identification of useful antibody reagents and antibody arrays suitable for discovery and clinical proteomics; in vitro phage display to identify high-affinity peptide ligands; and evaluation of methods to monitor the degree of degradation of banked biological samples. Two different ionization procedures that allow the transfer of peptide into the mass analyzer are used in MS. ESI-MS is the method of choice for analysis of tryptic fragments by combining LC and MS. This method is complicated when larger peptides are present in the samples. In this case, complex mixtures resolve better with higher sensitivity by using MALDI-MS technology since it results in singly charged molecules but less often doubly charged molecules (Uttenweiler-Joseph et al. 1998). Further, Schulz-Knappe et al. (2001) introduced the concept of peptide trapping in which peptide analysis was done using MS with subsequent production of MS fingerprints and sequence determination of all detected peptides. Using peptide trapping, they have done large-scale analysis of peptides from human blood and created a peptide bank from more than 100,000 L of blood ultrafiltrate (Schulz-Knappe et al. 1996). The amino acid sequence of peptides, especially peptides up to 9 kDa, is identified with a top-down approach (Kelleher 2004). However, identification of peptides with the same amino acid composition but different sequences is limited, as these peptides have the same molecular weights and generate the

Chapter 21 Peptidomics for Bioactive Peptide Analysis

same MS spectrum of molecular ions. Here, CE and LC coupled with MS is desirable. Using two tripeptides, Gly-Ser-Phe and Gly-Phe-Ser, for separation by CE, Wu et al. (2009) have shown that excellent separation selectivity was achieved. CE allows separation efficiencies up to several million plates per meter due to efficient heat dissipation of the small diameter capillary and the high electric field. In addition, fast analysis time, low sample and reagent consumption, and versatility make it a powerful alternative to HPLC. CE/CEC-ESE-MS methods for analysis are also used. The top-down approach has been successfully applied for the identification of endogenous peptides in cerebrospinal fluid (Heine et al. 2002; Mohring et al. 2005) and murine brain tissue (Mohring et al. 2005).

3.5. Peptide Quantification The samples are first labeled with one of the two related chemicals that differ isotopically. In quantitative peptidomics studies, an important issue is an accurate comparison of the quantitative level of the peptides of interest between two samples. When performing HPLC separations of peptides (LC-MS), the easiest way to compare the relative abundances of peptides is through the use of a UV detector placed between the LC system and the mass spectrometer. However, less reproducibility of peak area/height associated with MS signals is an important drawback to comparison of peptide levels. Nevertheless, for compounds having similar mass and functional groups, the relative ion intensities may correspond to their relative contents (Baggerman et al. 2004). Direct quantification by MS is done by using internal standards. An internal standard is often a copy of the molecule containing one or more heavy stable isotopes. The addition of known concentrations of the internal standard to the biological sample can be used to obtain accurate quantification (Kirkpatrick et al. 2005). However, in peptidomics studies, a large number of peptides are detected in a single analysis. Direct quantification of every peptide present in the tissue would be labor-intensive and too costly. Instead, it is easier to examine the relative level of a peptide in two different samples.

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Absolute peptide quantification was described by Gerber et al. (2003) employing the classical isotopelabeled internal standard approach. The concept of proteotypic peptides takes this strategy to the proteome level by selection of the best flying peptide for each protein as a unique identifier (Mallick et al. 2007). Recently, targeted quantification of more than 50 plasma proteins was demonstrated with this approach (Anderson and Hunter 2006). Design and production of such isotope-labeled reference peptides still needs further improvement for optimal exploitation. Relative peptide quantification can be achieved by incorporating stable isotopes into peptides in a differential manner (heavy versus light isotope) followed by mass spectrometric comparison of the signals derived from the heavy and light isotope labeled sample. This method has been summarized by Julka and Regnier (2004) and assessed in real-life scenarios (Wu et al. 2006) and Kolkman et al. (2005). Isotopic labels can be incorporated into the peptides after their extraction. Each one of the two samples of peptides to be compared is labeled with one of the two isotopic forms of a given reagent (e.g., one sample may be labeled with the natural H-isotopic form of a given reagent, such as the H6acetic anhydride-H6-Ac2O, and the other one with its deuterated form D6-acetic anhydride, D6-Ac2O). Both samples are mixed and subjected to micro-/ nano HPLC-MS analysis. The relative intensity of the MS signals of the heavy and light forms of the labeled peptides reveals their relative amounts in both biological samples. These isotopic-labeling techniques do not provide an absolute quantitation of the petptide and instead only provide a measure of the relative changes in the peptide levels between two samples (Saz and Marina 2008). In another approach, if working with a cell culture system or with an organism, the diet containing enriched isotope can be fed and the label can be incorporated into the peptide during its biosynthesis. This approach has been used for analysis of the rate of peptide biosynthesis in neuroendocrine cell lines (Che et al. 2004). In this method, relative abundance of the peptide in the two samples can be calculated from the ratio of the peak intensity or peak area or

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the two isotopic forms. Introduction of the mass labels can also be achieved by metabolic labeling (Beynon and Pratt 2005; de Godoy et al. 2006), which is advantageous because of its minimal interference with the biological system. Metabolic labeling is routinely performed with cultured cells ranging from bacteria and yeast to mammalian cells. This has been demonstrated in multicellular organisms, such as Caenorhabditis elegans, D. melanogaster (Krijgsveld et al. 2003), and rats (McClatchy et al. 2007). Chemical or enzymatic methods must be applied to label ex vivo recovered tissue or fluids. The chemical tagging concept was introduced under the name Isotope-Coded Affinity Tag (ICAT) (Gygi et al. 1999). The Isotope Tags for Relative and Absolute Quantification (iTRAQ) method (Ross et al. 2004) offers quadruplex analysis that in four conditions can be compared in one experiment. In view of multiple chemical tagging methods typically performed by postdigestion and targeting one amino acid side chain at a time, a new concept was introduced and termed Aniline-Benzoic Acid Labeling (AniBAL), where the same tag is introduced into all amino and carboxyl functions already at the protein level. This approach minimizes sample bias, optimizes proteome coverage, and is based on simple and symmetric chemistry. Bowman and Zaia (2007) have extended the stable isotope concept to quantitative glycomics by developing a quadruplex derivatization scheme amenable to mass spectral read out. Label-free approaches have been developed more recently based on highly reproducible LC-MS conditions, which allow comparative peptide analysis of complex samples (Old et al. 2005; Ono et al. 2006). ICATs that recognize sulfhydryl group of cysteines have been employed in peptidomic studies. Peptides that are extracted from two groups of animals are labeled with either the heavy or light form of ICAT reagent. After enzymatic digestion, the labeled peptides from the two combined samples are purified by affinity chromatography; and then peptides are identified by LC-MS/MS analysis. In both conditions, the relative abundance of peptides can be compared by comparing their relative signal intensities (Gygi et al. 1999). However,

very few peptides contain cysteine residues, and therefore ICAT technology can be used for only cysteine-containing peptides. To avoid such general labeling methods, other reagents that react with amines are introduced. The best-described isotopic labels for peptidomics are the active esters of trimethylammoniumbutyrate (TMAB) containing nine, six, and three deuteriums (heavy) or a light form without deuterium, which allows for multivariate analysis in a single LC/MS analysis, with each isotopically tagged peptide differing in mass by 3 Da per tag incorporated (Che and Fricker 2005; Fricker et al. 2006; Morano et al. 2008). The N-hydroxysuccinimide ester of the TMAB reagent reacts with amines that are present on the N-terminus of the peptide and/or on the side chain of lysine residues. With this quantitative peptidomic method, it is possible to detect, quantify, and identify approximately 100 peptides in extracts of a single mouse hypothalamus (Che et al. 2005a; Pan et al. 2006). Moreover, a large number of peptides are also detected in other brain regions such as prefrontal cortex, striatum, hippocampus, and amygdala (Lim et al. 2006). However, the disadvantage of this method is that TMAB reagent showed instability of labeled peptides on some MS analysis depending on the experimental condition (Che and Fricker 2005). Using a quantitative peptidomics approach, a number of studies have been performed, some of which are relevant to the area of food intake/body weight regulation and/or which involve mice lacking peptide-processing enzymes. On the basis of the quantitative peptidomics analysis, Fricker (2007) showed that increased body weight of the Cpefat/fat mice is due to the reduced levels of peptides that decrease body weight, such as alpha-melanocytestimulating hormone.

3.6. Posttranslational Modifications The covalent processing events that change the properties of a peptide by cleavage or addition of one or more amino acids are called PTMs (Mann and Jensen 2003), and most peptides require a modification to become functionally active or to improve their stability. The commonly involved processing

Chapter 21 Peptidomics for Bioactive Peptide Analysis

enzymes are endopeptidases. In addition to the peptidase cleavage, other enzymes perform additional acetylation, phosphorylation, C-terminal amidation, and sulfation. An important protective PTM in peptides is amidation, which is carried out by peptidyl alpha amidating monooxygenase, a multifunctional enzyme that performs a two-step process of converting C-terminal glycine residues into an amide group (Prigge et al. 2000). Most neuropeptides carry a pyroglutamic acid at their N-terminus, which is formed by the cyclization of an N-terminal glutamine (Schoofs et al. 1997). Some peptides, like melanotropines and beta-endorphins, are acetylated at their N-terminus to increase their stability and to modulate their activity (O’Donohye et al. 1982). Peptides are not always processed the same way in different cell types or even within the same cell type under different conditions. This is because different types of peptides often bind to their receptor targets with different affinities. The posttranslational processing can serve to regulate the resulting bioactive properties of the peptide. An important example is the processing of proopiomelanocortin into adrenocorticotropic hormone in the anterior pituitary and into α-melanocyte-stimulating hormone in the intermediate pituitary (Eipper et al. 1986). Usually PTMs can be identified by comparison of experimental data to a known amino acid sequence, but this requires prior detection of peptide sequence before PTM analysis. In the case of labile modifications such as sulfation and glycosylation, modification will be lost before peptide fragmentation itself. In this condition, the peptide can be sequenced and PTM identified, but the mass increment between the obtained sequence and the parent mass will be the same as that of the PTM. Hence, in this condition the exact location of the PTM cannot be identified (Torfs et al. 2002). If the modification is stable, such as acetylation, methylation, oxidation, N-terminal amidation, or formation of a pyroglutamic acid, the fragmentation spectrum will be similar to that of the unmodified peptide. There is an exception for the modified amino acids, which will have a mass increment or decrement corresponding to the PTM. In this condition, the fragmen-

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tation pattern will allow the exact localization of the modification (Baggerman et al. 2004).

3.7. Method Validation Many factors complicate the data such as age, gender, genetic makeup, race, behavior, habits, physical activities, food/nutrient intakes, environmentally caused mutations, and modifications. Data are more or less determined at the time of sample collection, hence sample collection is the most important. Technological, clinical, and biological validation are necessary. Technological validations are rigorous studies that demonstrate the reproducibility, precision, and sensitivity of the processing methods. Data should have small <5% coefficient of variation and small >0.95 standard deviation covering a large dynamic range of 3–4 logs, which will be challenging given the facts that the number and types of proteins in a sample can be tremendous; for example, serum proteins of the highly abundant and low abundant types can range to greater than 10 billion-fold with variations in the recovery of the high and low proteins by any specific method. Clinical validation is equally challenging and is required for diagnostics and therapeutics. Validated and standardized methods are critical and must be done in an organized and collaborative way. Peptidomic methods are advancing with possible future low costs, better results, novel information, and improved public health with better therapeutics and natural foods. The majority of the sequence information is generated through mass spectrometric methods, followed by database comparison owing to the high-throughput nature of the technique (Aebersold and Mann 2003).

4. Applications of Peptidomics Peptidomics offers a number of applications (Table 21.1).

4.1. Peptides as Biomarkers A biomarker may be defined as a molecule that can be measured and evaluated as an indicator of normal

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Table 21.1. Applications of peptidomics. Application

Example

Reference

Peptides as biomarkers

Plasma Endothelial cells Urine

Tammen et al. 2008 Karagiannis and Popel 2008 Metzger et al. 2009 Balog et al. 2009

Peptide biomarkers in disease

Cancer

Alzheimer ’s disease

Wulfkuhle et al. 2003 Vierhapper et al. 2005 Tammen et al. 2007 Edwards et al. 2008 Ke et al. 2009 Dorsey et al. 2006 Zhang et al. 2008 Nilsson et al. 2009 Westman-Brinkmalm et al. 2009 Wei and Li 2009 Sunderland et al. 2006

Peptides and diet-disease associations

Food and hypothalamus

Che et al. 2005

Peptides in the regulation of behavior

Bee behavior

Brockmann et al. 2009

Peptidomics and novel peptides

Coenorhabditis Tityus serrulatus Agalychnis callidryas Ratus Rana pleuraden

Husson et al. 2008 Rates et al. 2008 Wang et al. 2008 Lee et al. 2008 Yang et al. 2009

Cardiovascular pathogenesis Pancreatic cysts Parkinson’s disease

biologic processes, pathologic processes, or pharmacologic responses to therapeutics. Peptides serve as ideal biomarkers since they have the ability to account for all or most of the variations of the physiological state of the individual. Peptides appear in all body fluids, (blood, plasma, urine, or cerebrospinal fluid) and have the ability to migrate between compartments of an organism. Tammen et al. (2008) elucidated the potential of peptides as markers for in vivo protease inhibition by analyzing plasma samples from animals treated with either the indirect FXa inhibitor FONDAPARINUX or the dipeptidylpeptidase IV inhibitor AB192. They have shown that regulated peptides were either substrates, products, or downstream targets of the inhibited protease, suggesting that in vivo analysis of peptides by peptidomics has the potential to broaden the knowledge of inhibitor-related effects in vivo and that this method may pave the way to development of predictive biomarkers. A systematic methodology for proteome-wide identification of

peptides inhibiting the proliferation and migration of endothelial cells developed by Karagiannis and Popel (2008) provides a guideline for a computational-based peptidomics approach for the discovery of endogenous bioactive peptides. State-of-the-art proteomic platforms have been established or adapted to use urine as a sample source for biomarker discovery (Metzger et al. 2009). The fast and one-step screening method of CE-MS allows resolution of the urinary small molecular weight proteome in high-throughput mode. Reproducible high-throughput peptidomic analysis of urine peptides is often hampered by the inherent variability in factors, such as pH and salt concentration. Balog et al. (2009) developed a generally applicable rapid and robust method for screening a large number of urine samples, resulting in a broad spectrum of native peptides, as a tool to be used for biomarker discovery. Peptide samples were trapped, desalted, pH-normalized, and fractionated on a miniaturized automatic reverse-phase strong cation

Chapter 21 Peptidomics for Bioactive Peptide Analysis

exchange (RP-SCX) cartridge system. Eluted peptides were analyzed using MALDI-TOF, Fourier transform ion cyclotron resonance, and LC-iontrap MS. A selected few urine samples from Schistosoma haematobium–infected individuals were chosen to evaluate clinical applicability. The RP-SCX sample cleanup and fractionation system exhibits a high qualitative and quantitative reproducibility with both BSA standard and urine. Because of the relatively high cartridge-binding capacity, eluted peptides can be measured with high sensitivity using multiple MS.

4.2. Peptide Biomarkers in Disease Peptidomics helps in early detection, characterization, and diagnosis of many diseases such as Parkinson’s disease, Alzheimer ’s disease, and cancer and are crucial for the control and prevention of the diseases (Wulfkuhle et al. 2003; Dorsey et al. 2006; Sunderland et al. 2006; Tammen et al. 2007). Cancer cells exhibit specific changes in protein expression and alteration in proteolytic activities; in turn peptides are capable of reflecting these pathological changes (Tammen et al. 2007). Calcitonin, a 3.5 kDa peptide tumor marker, is expressed primarily by the C-cells (parafollicular or ultimobranchial cells of the thyroid), while serum calcitonin is a sensitive and accurate marker of medullary thyroid carcinoma (Vierhapper et al. 2005). Broad-spectrum screening of cellular protein or peptide complements during cardiovascular pathogenesis is favored by peptidomics and proteomics technologies (Edwards et al. 2008). MALDI-TOF with LC/MS/MS (HPLC-tandem MS) protein identification, 2DGE, GeLC/MS/MS (tryptic digestion of proteins fractionated by SDS-PAGE and identified by LC/MS/MS) were used to identify biomarkers for pancreatic cyst diagnosis and risk stratification (Ke et al. 2009). The latter approach, which requires less than 40 ml of cyst fluid per sample offering the possibility to analyze cysts smaller than 1 cm in diameter, revealed a panel of potential biomarker proteins to facilitate future pancreatic cyst diagnosis and risk-stratification, which correlated with carcinoembryonic antigen (CEA). The biomarkers

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were two homologs of amylase, solubilized molecules of four mucins, four solubilized CEA-related cell adhesion molecules (CEACAMs), and four S100 homologs. Neuropeptides take part in mediating the central nervous system failure associated with Parkinson’s disease. The principal causative pathology of Parkinson’s disease is the progressive degeneration of dopaminergeic neurons in the substantia nigra pars compacta projecting to the striatum in the brain (Nilsson et al. 2009). A thoroughly controlled sample preparation technique together with a peptidomics approach and targeted neuropeptide sequence collections enabled sensitive detection, identification, and relative quantification of a number of endogenous neuropeptides. Previously not recognized alterations in neuropeptide levels were identified in the unilateral lesioned mice with/without subchronic L-DOPA administration, the conventional treatment of Parkinson’s disease. Several of these peptides originated from the same precursor, such as secretogranin-1. Disease-related biotransformation of precursors into individual peptides was observed in the experimental model of Parkinson’s disease. Several previously unreported potentially biologically active peptides were also identified from the striatal samples. There is a need to search for biomarkers that are indicative of neurodegenerative diseases, as the clinical diagnosis of which remains unsatisfactory. MS has been used for studying peptide identities, structures, modifications, and interactions that collectively drive their biological functions. Peptidomics tools are being used extensively to find CSF biomarkers for neurodegenerative diseases (Westman-Brinkmalm et al. 2009). MSbased proteomics technology is well suited for biomarker discovery (Wei and Li 2009). Using quantitative peptidomics, Zhang et al. (2008) have shown that carboxypeptidase E contributes to the production of a majority of neuropeptides.

4.3. Peptides and Diet–Disease Associations Peptidomics helps understand the food-health relationship. Knowledge gained through the study of the food-health relationship will help in providing

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guidance regarding a balanced diet and in learning more about the diseases. Peptidomics helps identify biomarkers through diet-induced peptide changes, to allow a deeper understanding of the role of food components at the cellular and molecular level and to study the mechanisms of diet-disease associations (Che et al. 2005b).

4.4. Peptides in the Regulation of Behavior By combining a recently established quantitiative peptidomic method with compelling behavioral paradigms in bees, Brockmann et al. (2009) have elucidated the function of bioactive brain peptides in the regulation of behavior. Using differential isotope labeling combined with mass spectrometric analysis to quantify 50% of the known bee brain peptides in the context of foraging, Brockmann et al. (2009) found eight peptides that showed robust and dynamic regulation. Other observations were that some bee brain peptides showed quick changes in abundance as a function of experience in nectar and pollen collection for storage rather than ingestion, predisposition of some bees to collect either nectar or pollen but not both, and strongest abundance changes of tachykinin, PBAN, and sNPF peptides in association with nectar and pollen foraging, these peptides being known to be involved in regulating food intake in solitary insects, suggesting an evolutionary connection between food intake in solitary insects and social foraging in bees. Brain peptides play an important role in orchestrating physiological and behavioral processes in animals by functioning as cell-signaling neurohormones, neuromodulators, and neurotransmitters. These molecules are produced from their corresponding precursor genes by cleavage at specific sites followed by additional posttranslational modifications, a complex process that can make bioactive peptides difficult to predict (Li and Sweedler 2008). The honey bee genome sequencing led to a new high-throughput approach for neuropeptide discovery: algorithms that predict cleavage sites in peptide precursors followed by sequencing from brain samples with mass spectrometry leading to the pre-

diction of 36 peptide-encoding genes (Hummon et al. 2006) and confirmation of 100 endogenous peptides in the bee brain, numbers similar to those in the fruit fly and the house mouse (Hewes and Taghert 2001; Svensson et al. 2003). Recent advances in peptidomics have greatly accelerated peptide characterization. However, high-throughput approaches to discover peptide function have been much less common.

4.5. Peptidomics and Novel Peptides Mapping of the peptide content of Caenorhabditis elegans and C. briggsae by LC-MALDI-TOF MS allowed a better annotation of neuropeptideencoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons (Husson et al. 2008). MALDI-TOF and de novo sequencing were used to identify Tityus serrulatus venom peptides. Twenty-eight peptides were identified as fragments from Pape proteins; 10 corresponded to N-terminal fragments of the TsK beta toxin, some were fragments of potassium channel toxins, and novel peptides without sequence similarities to known molecules were also identified (Rates et al. 2008). By integrating systematic peptidome and transcriptome studies of the defensive skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas, Wang et al. (2008) have identified novel members of three previously described antimicrobial peptide families, a 27-mer dermaseptin-related peptide, a 33-mer adenoregulin-related peptide, and a 27-mer caerin-related peptide. A recent study by Yang et al. (2009) reveals the possibility of a novel skin antioxidant system from the skin of Rana pleuraden, the antioxidant system consisting of a number of peptides belonging to 11 groups and some of them having multifunctional properties, such as combined antioxidant, anti-inflammatory, and antimicrobial activities. Lee et al. (2008) applied in vitro phage display technique to identify high-affinity peptide ligands to aquaporin-2-expressing membranes, and library analysis identified proteins having homologous motifs.

Chapter 21 Peptidomics for Bioactive Peptide Analysis

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Pan H, Che FY, Peng B, Steiner DF, Pintar JE, Fricker LD. 2006. The role of prohormone convertase-2 in hypothalamic neuropeptide processing: A quantitative neuropeptidomic study. J Neurochem 98(6):1763–1777. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. 1999. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18):3551–3567. Pezeshki A, Vergote V, Van Dorpe S, Baert B, Burvenich C, Popkov A, De Spiegeleer B. 2008. Adsorption of peptides at the sample drying step: Influence of solvent evaporation technique, vial material and solution additive. J Pharm Biomed Anal 49(3):607–612. Prigge ST, Mains RE, Eipper BA, Amzel LM. 2000. New insights into copper monooxygenases and peptide amidation: Structure, mechanism and function. Cell Mol Life Sci 57(8–9): 1236–1259. Rates B, Ferraz KK, Borges MH, Richardson M, De Lima ME, Pimenta AM. 2008. Tityus serrulatus venom peptidomics: Assessing venom peptide diversity. Toxicon 52(5):611–618. Reinders J, Sickannn A. 2005. State-of-the-art in phosphoproteomics. Proteomics 5(16):4052–4061. Righetti PG, Boschetti E, Lomas L, Citterio A. 2006. Protein equalizer technology: The quest for a “democratic proteome.” Proteomics 6(14):3980–3992. Romanova EV, Rubakhin SS, Sweedler JV. 2008. One-step sampling, extraction, and storage protocol for peptidomics using dihydroxybenzoic acid. Anal Chem 80(9):3379–3386. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ. 2004. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3(12):1154–1169. Saz JM, Marina ML. 2008. Application of micro and nano-HPLC to the determination and characterization of bioactive and biomarker peptides. J Sep Sci 31(3):446–458. Schoofs L, Veelaert D, Vanden Broeck J, De Loof A. 1997. Peptides in the locusts, Locusta migratoria and Schistocerca gregaria. Peptides 18(1):145–156. Schrader M, Selle H. 2006. The process chain for peptidomic biomarker discovery. Dis Markers 22(1–2):27–37. Schulz-Knappe P, Raida M, Meyer M, Quellhorst EA, Forssmann WG. 1996. Systematic isolation of circulating human peptides: The concept of peptide trapping. Eur J Med Res 1(5):223– 236. Schulz-Knappe P, Zucht HD, Heine G, Jürgens M, Hess R, Schrader M. 2001. Peptidomics: The comprehensive analysis of peptides in complex biological mixtures. Comb Chem High Throughput Screen 4(2):207–217. Schumacher RT, Manak M, Garrett P, Miller W, Lawrence N, Tao F. 2002. Automated solution for sample preparation: Nucleic acid and proteins extraction from cells and tissues using pressure cycling technology (PCT). Amer Lab 34(16): 38–43.

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Chapter 22 In silico Analysis of Bioactive Peptides Marta Dziuba and Bartłomiej Dziuba

Contents 1. Introduction, 325 2. Structure and Function of the BIOPEP Database of Proteins and Bioactive Peptides, 326 3. In silico Analysis of Proteins and Bioactive Peptides, 326 4. Release of Bioactive Peptides from Selected Precursor Proteins by Enzymes or Enzyme Conjugates—Proteolysis in silico, 331

1. Introduction Research conducted by numerous scientific centers around the world has contributed to the identification and description of a vast number of biologically active peptides isolated from the body tissues and fluids of many organisms, including bacteria, fungi, plants, and animals (Gill et al. 1996). The investigated peptides are characterized by high chemical diversity. This group of compounds includes simple dipeptides as well as complex, linear, or cyclic oligopeptides and polypeptides that are often modified as the result of post-translational glycosylation, phosphorylation, acetylation, or hydroxylation of amino acid residues. Food proteins are among the precursors of many biologically active peptides. Peptides derived from proteins lower blood pressure; inhibit the activity of proline endopeptidases; stimulate the

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

5. Identification of Selected Biopeptides Released by Trypsin from Milk Proteins, 334 6. Conclusion, 336 7. Acknowledgments, 339 8. References, 339

immune system; exhibit opioid activity; act as opioid antagonists; contract smooth muscles; inhibit platelet aggregation; inhibit HIV proteinase and oxidation; show antibacterial, fungicidal, and surface activity; bond ions; participate in the transport of minerals; determine the sensory properties and improve the nutritive value of food; and control body weight. According to Karelin et al. (1998), in addition to its primary function, every protein may be a reserve source of peptides controlling the life processes of organisms. For this reason, the determination of a new, additional criterion for evaluating proteins as the potential source of biologically active peptides contributes to a more comprehensive and objective definition of their biological value (Dziuba and Iwaniak 2006). Potentially biologically active protein fragments whose structural motifs resemble that of bioactive peptides remain inactive in precursor protein sequences. These fragments, released from the precursor by proteolytic enzymes, may interact with selected receptors and affect the overall condition and health of humans (Schlimme and Meisel 1995; Meisel and Bockelmann 1999). 325

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There is a growing interest in the use of bioactive peptides for therapeutic purposes, in particular for medical treatment involving antibiotics and antifungal drugs, as well as for the treatment of the following diseases: neoplasm, viral infections, immune system disorders, and neurological and cardiologic disorders (Latham 1999). Biologically active peptides are the recommended ingredients of functional food, that is, food designed with the aim of generating the desired functional and biological properties (Arai and Fujimaki 1991; Korhonen et al. 1998). Functional food continues to attract increasing interest worldwide (De Felice 1995). Researchers have also been investigating the option of including bioactive peptides as physiological components of industrially processed food or nutraceutics for the production of phormones. This research focuses primarily on peptides that lower blood pressure in combination with phosphopeptides and immunomodulatory peptides (Gobbetti et al. 2002; Hartmann and Meisel 2002). Biologically active peptides may be the products of protein proteolysis in the gastrointestinal tract (Britton and Koldovsky 1989; Grimble 1994) or of proteolysis as a protein-processing stage. Fermented food, which is gaining increased popularity, may also be a source of bioactive peptides (CampbellPlatt 1994; Dziuba et al. 1999a). The emergence of new peptides from various sources necessitated the creation of a database and the establishment of additional criteria for assessing the value of proteins as precursors of bioactive peptides. The BIOPEP database of proteins and bioactive peptides was developed in 2003 at the Department of Food Biochemistry, University of Warmia and Mazury in Olsztyn, Poland. It is available online at http://www.uwm.edu.pl/biochemia (Dziuba et al. 2003).

classification of proteins according to the developed algorithms, and the design of proteolytic processes for the purpose of acquiring or removing bioactive peptides. The acquired data are used in the process of designing the desired food attributes as well as in contemporary biochemistry, for example, to predict the biological functions of proteins. The main data sheet of the BIOPEP application comprises three interlinked databases of protein sequences, biologically active peptides, and proteolytic enzymes. All three databases are regularly updated and presently comprise 659 and 2,333 protein and peptide sequences and 25 proteolytic enzymes. Protein and peptide amino acid sequences are entered into the application with the use of a one-letter code. Peptides containing 20 most frequently encountered amino acids as well as peptides comprising posttranslational modifications can also be input. The database contains information on 44 bioactive peptides. Proteins are evaluated as bioactive peptide precursors based on automatically set or computed indicators: protein’s potential biological activity profile, frequency of occurrence of bioactive motifs, potential protein activity, and the integrated protein biological activity coefficient (Dziuba et al. 1999b; Dziuba and Iwaniak 2006; Minkiewicz et al. 2008). BIOPEP can be interfaced with global databases investigating protein structure and functions, such as ProDom, PROSITE, TrEMBL, SWISS-PROT, and the PREDICT 7 application. BIOPEP is also used to analyze the possible release or hydrolysis of bioactive peptides by proteolytic enzymes, and the results obtained in silico may be verified by analytical methods (PAGE 2D, SM, HPLC). The produced results contribute to current knowledge in the area of food science, medicine, and pharmacy. The BIOPEP database is regularly expanded to account for data on biologically active peptides and proteins.

2. Structure and Function of the BIOPEP Database of Proteins and Bioactive Peptides

3. In silico Analysis of Proteins and Bioactive Peptides

The BIOPEP database of protein and bioactive peptide sequences facilitates the search for bioactive fragments in polypeptide chains, the hierarchical

In addition to analytical methods, many research laboratories resort to computer-aided techniques of evaluating food components, including proteins.

Chapter 22

The process of modelling the physical and chemical properties of proteins, predicting their secondary structure, or searching for a homology between proteins to identify their functions requires analyses supported by databases of protein sequences or sequence motifs. A complementary part of such research is the strategy of examining proteins and bioactive peptides with a view to obtaining them from defined protein groups, including an evaluation of proteins as a source of biologically active pep-

In silico Analysis of Bioactive Peptides

327

tides based on the identified profiles of potential biological activity and the computed frequency of biopeptide occurrence (parameter A), classification of proteins into families with similar biological activity profiles, modelling processes of protein proteolysis from the richest families—peptide precursors with a given level of biological activity, and verification of the results of computer-aided proteolysis design with a view to releasing bioactive peptides by relying on analytical methods (Figure 22.1).

Figure 22.1. Strategy for research on proteins and bioactive peptides.

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From among 44 types of biological activity characteristic of peptides, as listed in the BIOPEP database, motifs with the following 23 types of activity occur most frequently in protein sequences: antiamnestic, antithrombotic, antihypertensive, immunomodulative, chemotactic, smooth muscle contracting, toxic, embryotoxic, antioxidant, dipeptidyl peptidase-IV inhibiting, opioid, opioid antagonistic, stimulating erythrocyte synthesis, hemolytic, bonding and transporting metals, bacterial permease ligands, anorectic, activating ubiquitindependent proteolysis, controlling ion flow, neuropeptide inhibiting, controlling gastric mucosal function, antibacterial and antiviral, and controlling phosphoinositol function. Proteins listed in the database have been grouped into families of peptide precursors that exhibit a given type of biological activity. This approach has led to the identification of 23 types of protein families—peptide precursors—with the following types of activity: antihypertensive—148 proteins; antithrombotic—82 proteins; antiamnestic—83 proteins; immunomodulative—89 proteins; antioxidant— 79 proteins; toxic—13 proteins; smooth muscles contracting—2 proteins; chemotactic—6 proteins; embryotoxic—3 proteins; opioid—111 proteins; controlling ion flow—41 proteins; controlling gastric mucosal function—79 proteins; dipeptidyl peptidase-IV inhibiting—148 proteins; activating ubiquitin-dependent proteolysis—124 proteins; controlling phosphoinositol function—28 proteins; neuropeptide inhibiting—71 proteins; bacterial permease ligands—80 proteins; stimulating erythrocyte synthesis—2 proteins; hemolytic—1 protein; anorectic—12 proteins; opioid antagonistic—6 proteins; bonding and transporting metals and metal ions—3 proteins; antibacterial and antiviral—10 proteins. Every protein family—peptide precursors showing a given type of biological activity—can be classified into subfamilies based on the frequency of occurrence of active sections in the protein sequence (parameter A) (Dziuba and Darewicz 2007). Based on stemplot analysis, a respective number of proteins has been classified into a given subfamily (interval). Such intervals are known as class inter-

vals, and the difference between the upper and the lower boundaries of the ith class interval is the interval span hi. h i = x1i − x 0 i

(22.1)

where x0i, i x1i (i = 1, … , k) are the boundaries of the ith interval. There are no clear-cut rules for determining the span and the number of intervals. They are usually determined intuitively to ensure that the interval span produces a relatively transparent distribution of a given attribute. The determined subfamilies show significant diversity as regards their size. In some cases, a given subfamily is not represented by any proteins. This implies that the intensity of the investigated attribute (frequency of occurrence of bioactive fragments (A)) for all studied cases (proteins-precursors) does not fit the set class interval. The above is observed during the classification of families characterized by the following types of activity: antithrombotic, antiamnestic, controlling ion flow, controlling gastric mucosal function, and inhibiting neuropeptides. Owing to their infrequent occurrence, subfamilies have not been created for families of peptide precursors showing the following types of activity: toxic, contracting smooth muscles, chemotactic, embryotoxic, stimulating erythrocyte synthesis, hemolytic, anorectic, opioid antagonistic, binding and transporting metals and metal ions, antibacterial, and antiviral. In the subsequent stage of modelling the proteolysis process with a view to releasing peptides characterized by a given type of biological activity, proteins forming the richest subfamily or two richest subfamilies (for the following types of activity: antithrombotic, antiamnestic, antioxidant, opioid, controlling ion flow, controlling gastric mucosal function, dipeptidyl peptidase-IV inhibiting, activating ubiquitin-dependent proteolysis, controlling phosphoinositol function) were analyzed. Proteins belonging to the three richest subfamilies were analyzed to determine the activity of bacterial permease ligands. The richest subfamilies of proteins, precursors of biologically active peptides (Dziuba and Darewicz 2007), are presented in Table 22.1.

Table 22.1. Richest subfamilies of proteins—precursors of peptides with selected activities. Analyzed Proteins Biological Activity

BIOPEP ID

Protein Name

Antihypertensive

1097 ÷ 1199; 1101; 1102 1100; 1103 1111 1112 1113 1111 1112 1113 1115 1077; 1079; 1082

β-casein gen. var. A1; A2; A3; C; E bovine (Bos taurus) β-casein gen. var. B; F, bovine (Bos taurus) collagen α-1(III) chain, bovine (Bos taurus) collagen α-1(I) chain (fragment) bovine (Bos taurus) collagen α-1(I) chain precursor, chicken (Gallus gallus) collagen α-1(III) chain, bovine (Bos taurus) collagen α-1(I) chain (fragment), bovine (Bos taurus) collagen α-1(I) chain precursor, chicken (Gallus gallus) α-lactalbumin, bovine (Bos taurus) α-lactalbumin, human (Homo sapiens); α-lactalbumin, goat (Capra hircus); α-lactalbumin, sheep (Ovis aries) β-casein gen. var. A2; E, bovine (Bos taurus) myoglobin, horse (Equus caballus) flavodoxin mutant: D58P (Clostridium beijerinckii) prolamin precursor (clone PPROL 14), rice (Oryza sativa) prolamin precursor (clone PPROL 7), rice (Oryza sativa) prolamin precursor (clone PPROL 4A), rice (Oryza sativa); monellin chain B (II), serendipity berry (Dioscoreophyllum cumminsii); pancreatic trypsin inhibitor (precursor), bovine aprotinin (Bos taurus) hemoglobin β-chain, major-form, rat (Rattus norvegicus) hemoglobin β-chain, horse (Equus caballus); hemoglobin β-chain, human (Homo sapiens) κ-casein gen. var. A, bovine (Bos taurus) α/β-gliadin precursor (prolamin) (class a-I), wheat (Triticum aestivum) ω-gliadin fragment, wheat (Triticum aestivum) α/β-gliadin precursor (prolamin) (MM1), wheat (Triticum aestivum) α/β-gliadin precursor (prolamin) (class a-II), wheat (Triticum aestivum) α/β-gliadin precursor (prolamin) (class a-III), wheat (Triticum aestivum) α/β-gliadin precursor (prolamin) (clone PW 1215), wheat (Triticum aestivum) α/β-gliadin precursor (prolamin) (class a-IV), wheat (Triticum aestivum) α/β-gliadin precursor (prolamin) (clone PW8142), wheat (Triticum aestivum) α/β-gliadin precursor (prolamin) (class a-V), wheat (Triticum aestivum) α/β-gliadin fragment (prolamin) (clone PTO A10), wheat (Triticum aestivum) ω-gliadin precursor (fragment), wheat (Triticum aestivum) ω-gliadin precursor, wheat (Triticum aestivum); ω-gliadin B precursor, wheat (Triticum aestivum) κ-casein gen. var. A, bovine (Bos taurus) κ-casein, goat (Capra hircus)

Antithrombotic

Antiamnestic

Immunomodulating

1098; 1102 1125 1108 1154 1152 1155; 1169; 1232

Antioxidative

Celiac-toxic

1224 1231; 1234 1117 1177 1189 1179 1178 1180 1182 1183 1184 1181 1186 1187 1147; 1185

Inducing contractions of smooth muscles

1117 1109

A Parameter Value 0.224880 0.220096 0.274238 0.272144 0.212291 0.272392 0.267009 0.210196 0.024590 0.024390 0.023923 0.022222 0.021429 0.020270 0.020134 0.020000

0.047945 0.034247 0.029586 0.041985 0.035714 0.029316 0.027491 0.024823 0.020270 0.020202 0.019169 0.018809 0.005376 0.003984 0.003436 0.011834 0.010417

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Table 22.1. Continued Analyzed Proteins

A Parameter Value

Biological Activity

BIOPEP ID

Protein Name

Chemotactic

1107 1112 1192 1143 1113 1076 1112 1111 1176

1125 1138 1080 1214 1217

elastin precursor (tropoelastin) (Bos taurus) collagen α-1(I) chain (fragment), bovine (Bos taurus) PR-10 protein, white lupine (Lupinus albus) legumin 11S globulin, ginkgo (Ginkgo biloba) collagen α-1(I) chain precursor, chicken (Gallus gallus) connectin fragment (titin), chicken (Gallus gallus) collagen α-1(I) chain (fragment), bovine (Bos taurus) collagen α-1(III) chain, bovine (Bos taurus) α-hordothionin precursor (purothionin II), barley (Hordeum vulgare) α-lactalbumin, rabbit (Oryctolagus cuniculus) wheat glutenin, high molecular weight subunit PW212 precursor (Triticum aestivum) narbonin, vetch (Vicia pannonica) narbonin, narbon bean (Vicia narbonensis) phycocyanin, blue-green bacteria (Cyanobacteria) monellin chain A (I) serendipity berry (Dioscoreophyllum cumminsii) collagen α-1(I) chain precursor, chicken (Gallus gallus) collagen α-1(I) chain (fragment), bovine (Bos taurus) collagen α-1(III) chain, bovine (Bos taurus) epidermal retin acid-binding protein EBP, mouse (Arabidopsis thaliana) collagen α-1(I) chain precursor, chicken (Gallus gallus) collagen α-1(I) chain (fragment), bovine (Bos taurus) kafirin PGK1 precursor, sorghum (Sorghum vulgare) kafirin PSK8 precursor, sorghum bicolor (Sorghum vulgare) lysozyme C precursor, human (Homo sapiens) kafirin PSKR2 precursor, sorghum bicolor (Sorghum vulgare) connectin (titin), fragment, chicken (Gallus gallus) α-lactalbumin, human (Homo sapiens); α-lactalbumin, horse (Equus caballus); α-lactalbumin, goat (Capra hircus); α-lactalbumin, sheep (Ovis aries); α-lactalbumin, guinea pig (Cavia porcellus); α-lactalbumin, arabian camel (Camelus dromedarius) α-lactalbumin, bovine (Bos taurus) α-lactalbumin, red-necked wallaby (Macropus rufogriseus) lysozyme precursor, silk moth (Bombyx mori) major urinary protein 1 precursor, mouse (Mus musculus) wheat glutenin, high molecular weight subunit PW212 precursor (Triticum aestivum) myoglobin, horse (Equus caballus) troponin C, bovine (Bos taurus) α-lactalbumin, rabbit (Oryctolagus cuniculus) hemoglobin α-chains, horse (Equus caballus) hemoglobin α-chain, pig (Sus scrofa)

0.037037 0.052000 0.081970 0.007062 0.007092

1090

α-S2-casein gen. var. A, bovine (Bos taurus)

0.024155

Embriotoxic

Opioid

1080 1110 Regulating ion flow

1191 1190 1126 117

Regulating the functions of the gastric mucosa Dipeptidyl aminopeptidase IV inhibitors

1113 1112 1111 1200

Activating ubiquitinmediated proteolysis

1113 1112 1149 1197 1091 1196

Regulating the action mechanism of phosphoinositol

1118 1077 ÷ 1079; 1082; 1083; 1085

1115 1081

Neuropeptide inhibitors Ligands of bacterial permease Stimulating the synthesis of red blood cells Hemolytic

330

1093 1195 1110

0.023578 0.001284 0.006329 0.002273 0.002095 0.001511 0.001284 0.000923 0.078740 0.081970 0.121359 0.010309 0.010345 0.012346 0.022222 0.210195 0.267009 0.272391 0.181818 0.204609 0.258023 0.033457 0.037453 0.038462 0.041199 0.046154 0.008130

0.008197 0.008264 0.008333 0.009259 0.162621

Chapter 22

In silico Analysis of Bioactive Peptides

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Table 22.1. Continued Analyzed Proteins Biological Activity

BIOPEP ID

Protein Name

Anorectic

1107 1143 1097 ÷ 1103

elastin precursor (tropoelastin), bovine (Bos taurus) legumin 11S globulin, ginkgo (Ginkgo biloba) β-casein gen. var. A1; A2; A3; B; C; E; F, bovine (Bos taurus) collagen α-1(I) chain precursor, chicken (Gallus gallus) collagen α-1(III) chain, bovine (Bos taurus) collagen α-1(I) chain (fragment), bovine (Bos taurus) α-S1-casein gen. var. B; C; D, bovine (Bos taurus) α-S1-casein gen. var. A, bovine (Bos taurus) κ-casein, goat (Capra hircus) κ-casein gen. var. A, bovine (Bos taurus) lactoferrin precursor, bovine (Bos taurus) lactoferrin, bovine (Bos taurus) α-S2-casein gen. var. A, bovine (Bos taurus) lactoferrin, human (Homo sapiens) lactoferrin, goat (Capra hircus) hemoglobin α-chain, bovine (Bos taurus) α-lacatalbumin, rat (Rattus norvegicus) α-lactalbumin, arabian camel (Camelus dromedarius) lactoferrin, bovine (Bos taurus) lactoferrin precursor, bovine (Bos taurus) α-S2-casein gen. var. A, bovine (Bos taurus) α-lactalbumin, bovine (Bos taurus) α-lactalbumin, sheep (Ovis aries)

Opioid-antagonistic

Binding and transporting metals and metal ions Antibacterial and antiviral

1113 1111 1112 1087 ÷ 1089 1086 1109 1117 1236 1212 1090 1121 1213 1233 1084 1085 1212 1236 1090 1115 1082

The richest protein subfamilies are most widely represented by milk proteins, wheat alpha- and betagliadins, and collagen. Proteins that are a source of peptides with antihypertensive, antithrombotic, and antiamnestic activity as well as peptides controlling gastric mucosal function and inhibiting dipeptidyl peptidase-IV are marked by the highest respective values of parameter A.

4. Release of Bioactive Peptides from Selected Precursor Proteins by Enzymes or Enzyme Conjugates—Proteolysis in silico BIOPEP is a tool for the comprehensive analysis of proteins as a source of bioactive peptides. The database is continuously expanded and updated. One of the major upgrades involved an application for a computer-aided simulation of the process of protein

A Parameter Value 0.001387 0.002273 0.004785 0.036313 0.041551 0.044929 0.005025 0.005376 0.010417 0.011834 0.005650 0.005806 0.024155 0.004335 0.005805 0.007092 0.007143 0.008130 0.015965 0.016949 0.019324 0.024590 0.032520

proteolysis by enzymes listed in the built-in database of endopeptidases. BIOPEP contains data on 24 proteolytic enzymes. Every listed enzyme is characterized by the following data: identification number—ID; classification number—EC; recognition sequence—amino acid sequence recognized by a given enzyme; cutting sequence—amino acid residue characteristic of a given enzyme that forms a bond hydrolyzed by that enzyme; C terminus/N terminus activity—the terminus indicates whether the enzyme hydrolyzes the bond on the C-terminal or the N-terminal end of the amino acid. The applied algorithms support the simulation of protein substrate hydrolysis involving one enzyme or several enzymes simultaneously. The application also supports the search for biologically active peptides and facilitates a comparison of the sequences of released fragments with those listed in the database to identify their potential biological activity. When the user

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Part 4 Recent Advances in Bioactive Peptide Analysis for Food Application

selects the analyzed protein and the individual enzyme (or an enzyme conjugate) and chooses the "View the report with the results" option, the program automatically displays a screen listing the sequences and the locations of all fragments released by the enzyme/enzymes. The "Search for active fragments" icon initiates the option of searching the bioactive peptides database. The option produces a list of bioactive fragments released from the precursor protein by a given enzyme/enzymes. A sample diagram of proteolysis simulation in silico is presented in Figure 22.2. The family of short fragments containing frequently repeated glycine (G) and proline (P) residues (e.g., PG, GP, PGP) is a well-researched group of multifunctional peptides. They are characterized by the following types of activity: antithrombotic, antiamnestic, controlling gastric mucosal function, and inhibiting dipeptidyl peptidase-IV (only the GP fragment). The above peptides are often found in the sequence of precursor proteins, and they are the best-represented group of peptides with antithrombotic and antiamnestic activity as well as peptides controlling gastric mucosal function. They are the fragments that are most frequently released by proteolytic enzymes. The results of proteolysis simulation are presented jointly due to the multifunctional nature of PG and GP peptides, the main representatives of antithrombotic and antiamnestic activity and activity controlling gastric mucosal function, and due to the fact that those peptides are most frequently encountered in the sequences of the same proteins. Table 22.2 lists enzymes that release multifunctional peptides with antithrombotic and antiamnestic activity and peptides controlling gastric mucosal function, as well as the number of the resulting fragments (Dziuba et al. 2006). The most frequently released fragments are peptides with a GP and PG sequence. Proteinase K, proline oligopeptidase, and chymotrypsin C release fragments with a GP sequence, while the remaining enzymes release fragments with a PG sequence. Table 22.3 lists enzymes releasing in silico bioactive peptides from precursor proteins (Dziuba et al. 2006). Animal proteins, mainly milk proteins, are most likely to release bioactive proteins from among

all analyzed protein sequences. The resulting fragments are mostly peptides with antihypertensive, opioid, immunomodulative, antithrombotic, ion flow controlling, and antiamnestic action, as well as peptides inhibiting dipeptidyl peptidase-IV. Enzymes specific for milk proteins that produce the highest quantity of the above peptides are elastase, chymotrypsin, and trypsin. Peptides released from all milk proteins are mainly dipeptides and tripeptides, for example, RL, GY, AY, VPL, QP, LW, FY, FP, YL, FGK, RF, AP. These short peptides are most readily absorbed from the gastrointestinal tract into the bloodstream (Siemensma et al. 1993; Dziuba et al. 1996). As demonstrated by Dziuba et al. (2006), peptides that are suitable for use as physiologically active food components—that is, peptides marked by antihypertensive activity, peptides responsible for ion metal transport, peptides with immunomodulative, antibacterial, and antioxidant activity—are generally not hydrolyzed by proteolytic enzymes of the digestive tract. The BIOPEP database lists the main proteolytic enzymes of the gastrointestinal system. They are pepsin, trypsin, chymotrypsin, pancreatic elastase, and proline oligopeptidase. All of the above enzymes hydrolyze peptide bonds formed with the involvement of the C-terminal of the following amino acids: A, G, V, L, Y, F, M, W, E, Q, K, R, and P. A comparison of the specificity of digestive enzymes with the sequence of physiologically important peptides confirms that they are generally resistant to proteolysis. The only exceptions are peptides containing proline (P) inside a sequence motif. Those peptides are hydrolyzed by proline oligopeptidase. Therefore, designers of bioactive peptide ingredients should first investigate whether intermolecular peptide bonds are hydrolyzed by digestive enzymes or not. Plant proteins are generally a less abundant source of bioactive peptides. Peptides released from, for example, wheat gladiadin, show antihypertensive, opioid, and antioxidant activity, and they act as inhibitors of dipeptidyl peptidase-IV. The highest number of bioactive peptides is released by proteinase K, proline endopeptidase, and elastase. Enzymes such as trypsin, clostripaine, collagenase, pronase,

Figure 22.2. Example of proteolysis simulation in BIOPEP.

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Table 22.2. Enzymes releasing peptides with antithrombotic and antiamnestic activity and peptides controlling gastric mucosal function. Number of fragments with antithrombotic, antiamnestic, and regulating stomach mucosal membrane activity released from precursor proteins Enzyme Proteinase K Pancreatic elastase Prolyl oligopeptidase Chymotrypsin C Papain Ficain Bromelain Glycyl endopeptidase

Protein ID 1111

Protein ID 1112

Protein ID 1113

57 48 57 57 55 52 38 9

47 28 34 46 29 31 19 —

64 32 52 70 45 46 30 —

papain, plasmin, thermolysin, cathepsin, and bacterial proteinases do not release any active fragments (Dziuba et al. 1999a). The hydrophilic environment of active sequences in the investigated proteins supports their release. An analysis of the possibility of release of bioactive peptides under the influence of proteolytic enzymes should account for the following factors: the precursor protein’s abundance in bioactive fragments, impact of physical and chemical factors (temperature, pH, enzyme specificity) required for effective hydrolysis, physiological aspects determining the release and absorption of bioactive peptides by the organism (Adler-Nissen 1986; Friedman 1996; Vorob’ev and Goncharova 1998). Research into the release of bioactive peptides may be conducted experimentally or with the involvement of computer-aided methods (in silico). As regards the latter, it should be noted that a computer simulation of proteolysis accounting for factors specific to a given environment, such as the gastrointestinal tract, can be programmed only in some cases (Vorob’ev and Goncharova 1998). In view of the above, the results obtained by the in silico method may partially correspond to the results of laboratory experiments. One of such examples is lactoglobulin β, which, in theory, has many bonds that are susceptible to pepsin-induced hydrolysis, but in practice, it is resistant to this enzyme (Chobert et al. 1997). Lactoglobulin β owes this resistance to its compact globular structure. Peptide bonds susceptible to

pepsin-induced hydrolysis are found inside the molecule and, therefore, are not available to the enzyme. Thermal processing may unfold the protein chain and reveal inaccessible bonds (Dziuba and Minkiewicz 1996; Dziuba and Darewicz 2000).

5. Identification of Selected Biopeptides Released by Trypsin from Milk Proteins Milk proteins are the best-researched precursors of biologically active peptides (Meisel 1998; Korhonen and Pihlanto-Leppälä 2001; Dziuba et al. 1999a). Milk protein sequences comprise fragments with antihypertensive, opioid, opioid antagonistic, immunomodulative, antibacterial, antiviral, antioxidant activity, fragments that bind and transport metals, have antiamnestic activity, act as bacterial permease ligands, contract smooth muscles, have antithrombotic activity, and inhibit dipeptidyl peptidase-IV. The results of proteolysis in silico (Dziuba et al. 2006) indicate that trypsin may release many active fragments from precursor proteins. This enzyme is also capable of releasing a peptide with antiviral activity from lactoferrin and peptides with antihypertensive activity from lactalbumin α, casein κ, and casein β. A profile of the potential biological activity of lactalbumin α is presented in Figure 22.3. Bioactive fragments released in silico by trypsin are marked in red in the presented profile of

Table 22.3. Enzymes releasing bioactive peptides from precursor proteins in silico. Activity

Proteolytic Enzymes

Antithrombotic, antiamnestic, regulating stomach mucosal membrane activity

Proteinase K (EC.3.4.21.14), Pancreatic elastase (EC 3.4.21.36), Prolyl oligopeptidase (EC 3.4.21.26), Chymotrypsin C (EC 3.4.21.2), Papain (EC 3.4.22.2), Ficin (EC 3.4.22.3), Bromelain (EC 3.4.22.4), Glycyl endopeptidase (EC 3.4.22.25) Thermolysin (EC 3.4.24.27)/Chymotrypsin A (EC 3.4.21.1), Thermolysin (EC 3.4.24.27)/Trypsin (EC 3.4.21.4), Thermolysin (EC 3.4.24.27)/Pepsin (EC 3.4.23.1), Thermolysin (EC 3.4.24.27)/V-8 protease (Glutamyl endopeptidase)(EC 3.4.21.19), Thermolysin (EC 3.4.24.27)/Plasmin (EC 3.4.21.7), Thermolysin (EC 3.4.24.27)/Cathepsin G (EC 3.4.21.20), Thermolysin (EC 3.4.24.27)/ Clostripain (EC 3.4.22.8), Thermolysin (EC 3.4.24.27)/Chymase (EC 3.4.21.39), Thermolysin (EC 3.4.24.27)/Leukocyte elastase (EC 3.4.21.37), Thermolysin (EC 3.4.24.27)/Metridin (EC 3.4.21.3), Thermolysin (EC 3.4.24.27)/Trombin (EC 3.4.21.5), Thermolysin (EC 3.4.24.27)/Pancreatic elastase II (EC 3.4.21.71), Thermolysin (EC 3.4.24.27)/Glutamyl endopeptidase II (EC 3.4.21.82), Thermolysin (EC 3.4.24.27)/Oligopeptidase B (EC 3.4.21.83), Leukocyte elastase (EC 3.4.21.37)/ Trypsin (EC 3.4.21.4), Leukocyte elastase (EC 3.4.21.37)/Oligopeptidase B (EC 3.4.21.83), Leukocyte elastase (EC 3.4.21.37)/Cathepsin G (EC 3.4.21.20) Chymotrypsin A (EC 3.4.21.1), Trypsin (EC 3.4.21.4), Pepsin (EC 3.4.23.1), Prolyl oligopeptidase (EC 3.4.21.26), Thermolysin (EC 3.4.24.27), Chymotrypsin C (EC 3.4.21.2), Plasmin (EC 3.4.21.7), Cathepsin G (EC 3.4.21.20), Papain (EC 3.4.22.2), Metridin (EC 3.4.21.3), Pancreatic elastase II (EC 3.4.21.71), Bromelain (EC 3.4.22.4), Oligopeptidase B (EC 3.4.21.83), Chymase (EC 3.4.21.39)/Pancreatic elastase (EC 3.4.21.36), Chymase (EC 3.4.21.39)/Clostripain (EC 3.4.22.8), Chymase (EC 3.4.21.39)/Leukocyte elastase (EC 3.4.21.37) Proteinase K (EC.3.4.21.14), Pancreatic elastase (EC 3.4.21.36), Thermolysin (EC 3.4.24.27), Cathepsin G (EC 3.4.21.20), Papain (EC 3.4.22.2), Ficin (EC 3.4.22.3), Chymotrypsin C (EC 3.4.21.2) Pancreatic elastase (EC 3.4.21.36), Glycyl endopeptidase (EC 3.4.22.25), Chymase (EC 3.4.21.39), Thermolysin (EC 3.4.24.27)/V-8 protease (Glutamyl endopeptidase)(EC 3.4.21.19), Chymase (EC 3.4.21.39)/Glutamyl endopeptidase II (EC 3.4.21.82), Thermolysin (EC 3.4.24.27)/Trypsin (EC 3.4.21.4) Termolysin (EC 3.4.24.27)/Clostripain (EC 3.4.22.8) Trypsin (EC 3.4.21.4), Plasmin (EC 3.4.21.7), Oligopeptidase B (EC 3.4.21.83)

Antihypertensive

Antioxidative

Immunomodulating

Celiac toxic Smooth-muscle contracting Embryotoxic Chemotactic Opioid Regulating ion flow Dipeptydyl-peptidase IV inhibitory Activating ubiquitin mediated proteolysis Regulating phosphoinositole mechanism

Neuropeptide inhibitor Ligand of bacterial permease Stimulating red blond cells creation

Thermolysin (EC 3.4.24.27)/Leukocyte elastase (EC 3.4.21.37) — Pepsin (EC 3.4.23.1), Pancreatic elastase (EC 3.4.21.36), Prolyl oligopeptidase (EC 3.4.21.26), Leukocyte elastase (EC 3.4.21.37) Proteinase K (EC.3.4.21.14), Chymotrypsin C (EC 3.4.21.2), Papain (EC 3.4.22.2), Ficin (EC 3.4.22.3), Bromelain (EC 3.4.22.4) Proteinase K (EC.3.4.21.14), Pancreatic elastase (EC 3.4.21.36), Prolyl oligopeptidase (EC 3.4.21.26), Thermolysin (EC 3.4.24.27), Chymotrypsin C (EC 3.4.21.2), Papain (EC 3.4.22.2), Ficin (EC 3.4.22.3), Leukocyte elastase (EC 3.4.21.37), Bromelain (EC 3.4.22.4) Pancreatic elastase (EC 3.4.21.36), Papain (EC 3.4.22.2), Ficin (EC 3.4.22.3), Leukocyte elastase (EC 3.4.21.37), Bromelain (EC 3.4.22.4) Chymase (EC 3.4.21.39), Calpain (EC 3.4.22.17)/Prolyl oligopeptidase (EC 3.4.21.26), Calpain (EC 3.4.22.17)/V-8 protease (Glutamyl endopeptidase) (EC 3.4.21.19), Calpain (EC 3.4.22.17)/ Plasmin (EC 3.4.21.7), Calpain (EC 3.4.22.17)/Clostripain (EC 3.4.22.8), Calpain (EC 3.4.22.17)/ Leukocyte elastase (EC 3.4.21.37), Calpain (EC 3.4.22.17)/Thrombin (EC 3.4.21.5), Calpain (EC 3.4.22.17)/Glutamyl endopeptidase II (EC 3.4.21.82), Calpain (EC 3.4.22.17)/Oligopeptidase B (EC 3.4.21.83) Chymotrypsin C (EC 3.4.21.2), Thermolysin (EC 3.4.24.27)/Proteinase K (EC.3.4.21.14), Thermolysin (EC 3.4.24.27)/Prolyl oligopeptidase (EC 3.4.21.26) Leukocyte elastase (EC 3.4.21.37) —

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Table 22.3. Continued Activity

Proteolytic Enzymes

Hemolytic Anorectic

— Thermolysin (EC 3.4.24.27)/Chymotrypsin A (EC 3.4.21.1), Thermolysin (EC 3.4.24.27)/Pepsin (EC 3.4.23.1), Thermolysin (EC 3.4.24.27)/Cathepsin G (EC 3.4.21.20), Thermolysin (EC 3.4.24.27)/ Chymase (EC 3.4.21.39), Thermolysin (EC 3.4.24.27)/Metridin (EC 3.4.21.3), Thermolysin (EC 3.4.24.27)/Pancreatic elastase II (EC 3.4.21.71), Thermolysin (EC 3.4.24.27)/V-8 protease (Glutamyl endopeptidase) (EC 3.4.21.19), Thermolysin (EC 3.4.24.27)/Clostripain (EC 3.4.22.8), Thermolysin (EC 3.4.24.27)/Glutamyl endopeptidase II (EC 3.4.21.82), Thermolysin (EC 3.4.24.27)/Oligopeptidase B (EC 3.4.21.83) Trypsin (EC 3.4.21.4), Plasmin (EC 3.4.21.7), Oligopeptidase B (EC 3.4.21.83) Pancreatic elastase II (EC 3.4.21.71) Chymotrypsin A (EC 3.4.21.1), Trypsin (EC 3.4.21.4), Thermolysin (EC 3.4.24.27), Plasmin (EC 3.4.21.7), Chymase (EC 3.4.21.39), Metridin (EC 3.4.21.3), Bromelain (EC 3.4.22.4), Oligopeptidase B (EC 3.4.21.83), Glutamyl endopeptidase II (EC 3.4.21.82)/Pepsin (EC 3.4.23.1), Glutamyl endopeptidase II (EC 3.4.21.82)/Chymase (EC 3.4.21.39), Glutamyl endopeptidase II (EC 3.4.21.82)/Metridin (EC 3.4.21.3), Glutamyl endopeptidase II (EC 3.4.21.82)/Pancreatic elastase II (EC 3.4.21.71), Ficin (EC 3.4.22.3)/V-8 protease (Glutamyl endopeptidase) (EC 3.4.21.19), Ficin (EC 3.4.22.3)/Chymotrypsin C (EC 3.4.21.2), Ficin (EC 3.4.22.3)/Glutamyl endopeptidase II (EC 3.4.21.82), Bromelain (EC 3.4.22.4)/V-8 protease (Glutamyl endopeptidase) (EC 3.4.21.19), Bromelain (EC 3.4.22.4)/Chymotrypsin C (EC 3.4.21.2), Bromelain (EC 3.4.22.4)/Glutamyl endopeptidase II (EC 3.4.21.82)

Opioid antagonist Metal-ion binding Antibacterial and antiviral

potential biological activity of lactalbumin α. They include peptides identified by mass spectrometry. Isolated factions of the analyzed proteins were characterized by chromatography. A chromatogram of bovine lactalbumin α is presented in Figure 22.4. The resulting chromatograms were compared with the mass spectral chromatograms contained in the database at the Department of Food Biochemistry, University of Warmia and Mazury in Olsztyn, Poland. The comparison has shown that the investigated protein was pure. The protein was then hydrolyzed with the use of a Maldi ToF reflectron mass spectrometer. The selected peptide was identified by peptide mass fingerprinting (PMF). The above analysis also provided a formal basis for the identification of precursor proteins. The mass spectrum of lactalbumin α hydrolysate (Figure 22.5) showed a peak with m/z ratio of 1,200 Da. This peak corresponded to fragment 98– 107 of lactalbumin α with sequence VGINYWLAHK. This peptide demonstrates antihypertensive activity. Peaks corresponding to fragments EQLTK and

ALCSEK, in theory also released by trypsin, were not found. The results of computer-aided simulation of protein proteolysis, validated experimentally, indicate that proteins are a potential source of active peptides. Simulation results support the need for the development of further research strategies to investigate bioactive peptide proteins with the involvement of the BIOPEP database, liquid chromatography, two-dimensional electrophoresis, and mass spectrometry.

6. Conclusion Based on the frequency of occurrence of bioactive fragments in the protein sequence and the results of a stemplot analysis, all proteins can be classified into families—peptide precursors with specific biological activity, and, consequently, into subfamilies. The sizes of families are greatly diversified. The richest families are formed by precursors of antihypertensive peptides and dipeptidyl peptidase-IV

Figure 22.3. Profile of the potential biological activity of bovine lactalbumin α (BIOPEP ID 1115). Bioactive fragments released by trypsin are marked in lighter gray type.

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Figure 22.4. Chromatogram (RP-HPLC) of bovine lactalbumin α.

Figure 22.5. Mass spectrum of tryptic hydrolysate of bovine lactalbumin α. 1—mass to charge (m/z) ratio 693.295 DA; 2—m/z = 710.323 Da; 3—m/z = 1091.533 Da; 4—m/z = 1200.674 Da (peptide: VGINYWLAHK); 5—m/z = 1308.645 Da; 6—m/z = 1326.589 Da; 7—m/z = 1699.778 Da; 8—m/z = 1779.851 Da; 9—m/z = 1827.867 Da; 10—m/z = 1892.922 Da; 11—m/z = 2003.804 Da; 12—m/z = 2591.037 Da.

inhibitors, and the least-abundant protein families by precursors of hemolytic peptides. Subject to the type of hydrolyzed protein, double-enzyme conjugates determine the effectiveness of bioactive peptide release. As regards peptides with antihypertensive action; peptides that contract smooth muscles,

control ion flow, activate ubiquitin-dependent proteolysis, and inhibit neuropeptides; peptides with bacterial permease ligand activity and opioid antagonistic activity; peptides binding and transporting metal ions, and the combined effect of two enzymes do not contribute to the effectiveness of proteolysis.

Chapter 22

When applied to peptides with antithrombotic, antiamnestic activity, peptides controlling gastric mucosal function, peptides with antioxidant, immunomodulative, opioid activity, and peptides that act as inhibitors of dipeptidylpeptidase-4, doubleenzyme conjugates enhance the release effectiveness of fragments with the above properties. Doubleenzyme hydrolysis of peptides controlling the phosphoinositol function and peptides with antibacterial and antiviral activity produces fragments with sequences that are not released by a single enzyme. Toxic and embryotoxic peptides are released only by double-enzyme conjugates. The results of a computer-aided simulation of protein proteolysis, verified experimentally for lactoferrin, lactalbumin α, casein β, and casein κ hydrolyzed by trypsin, indicate that they are relatively abundant sources of biologically active peptides. The phrase "relatively abundant sources" relates to the comparison of the results of the computerized simulation of the proteolysis process with experimental results rather than the general number of bioactive motifs in precursor proteins. As demonstrated by Dziuba et al. (2006), many peptides that are suitable for use as physiologically active food ingredients, including peptides with antihypertensive activity, peptides responsible for ion metal transport, and peptides with immunomodulative, antibacterial, and antioxidant activity, are generally not hydrolyzed by proteolytic enzymes of the gastrointestinal tract. Bioinformatics methods applied in biotechnology and biochemistry are becoming increasingly popular due to short waiting times for results, low testing costs, the option of recording results in the form of text files, and, consequently, the recoverability of results, and they continue to be improved as new advances are made in the field of information technology.

7. Acknowledgments This work was supported by the Ministry of Science and Higher Education within research project no. N N312286333 (2007–2010). The authors are grateful to Professor Jerzy Dziuba for inspiration,

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help, and providing background information for this chapter.

8. References Adler-Nissen J. 1986. Enzymatic Hydrolysis of Food Proteins. London: Elsevier Applied Science, 1–19, 57–73. Arai S, Fujimaki M. 1991. Enzymatic modification of proteins with special reference to improving their functional properties. In: PF Fox, ed., Food Enzymology, vol. 2, 83–104. London: Elsevier Applied Science. Britton JR, Koldovsky O. 1989. Development of luminal protein digestion: Implications for biologically active dietary polypeptides. J Pediatr Gastr Nutr 9:144–162. Campbell-Platt G. 1994. Fermented foods—A world perspective. Food Res Intl 27:253–257. Chobert JM, Briand L, Dufour E, Dib R, Dalgalarrondo M, Haertle T. 1997. How to increase β-lactoglobulin susceptibility to peptic hydrolysis. J Food Biochem 20:439–462. De Felice SL. 1995. The nutritional revolution: Its impact on food industry R&D. Trends Food Sci Tech 6:59–61. Dziuba M, Darewicz M. 2007. Food proteins as precursors of bioactive peptides—Classification into families. Food Sci Tech Intl 13(6):393–404. Dziuba J, Darewicz M. 2000. Structural aspects of functional properties of milk proteins. Natur Sc 4:257–272. Dziuba M, Dziuba J, Minkiewicz P. 2006. Design of food proteins proteolysis with a view to obtaining bioactive peptides. Pol J Natur Sci 21(2):999–1020. Dziuba J, Iwaniak A. 2006. Database of protein and bioactive peptide sequences. In: Y Mine, F Shahidi, eds., Nutraceutical Proteins and Peptides in Health and Disease, 543–564. Boca Raton, FL: CRC Press. Dziuba J, Iwaniak A, Niklewicz M. 2003. Database of protein and bioactive peptide sequences—BIOPEP. http://www.uwm. edu.pl/biochemia. Dziuba J, Minkiewicz P. 1996. Influence of glycosylation on micelle-stabilizing ability on biological properties of C-terminal fragments of cow’s κ-casein. Inter Dairy J 6:1017–1044. Dziuba J, Minkiewicz P, Plitnik K. 1996. Chicken meat proteins as potential precursors of bioactive peptides. Pol J Food Nutr Sci 5/46(4):85–95. Dziuba J, Minkiewicz P, Nałecz D. 1999a. Biologically active peptides from plant and animal proteins. Pol J Food Nutr Sci 8/49(I):3–16. Dziuba J, Minkiewicz P, Nałecz D, Iwaniak A. 1999b. Database of biologically active peptide sequences. Nahrung 43(3):190–195. Friedman M. 1996. Nutritional value of proteins from different food sources: A review. J Agr Food Chem 44:6–29. Gill I, López-Fandiño R, Jorba X, Vulfson EN. 1996. Biologically active peptides and enzymatic approaches to their production. Enzyme Microb Tech 18:162–168. Gobbetti M, Stepaniak L, De Angelis M, Corsetti A, Di Cagno R. 2002. Latent bioactive peptides in milk proteins: Proteolytic

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activation and significance in dairy. Crit Rev Food Sci 42(3):223–239. Grimble GK. 1994. The significance of peptides in clinical nutrition. Annu Rev Nutr 14:419–448. Hartmann R, Meisel H. 2002. Cytochemical assessment of phosphopeptides derived from casein as potential ingredients for functional food. Nahrung 46:427–431. Karelin AA, Blishchenko EY, Ivanov VT. 1998. A novel system of peptidergic regulation. FEBS Lett 428:7–12. Korhonen H, Pihlanto-Leppälä A. 2001. Milk protein–derived bioactive peptides—Novel opportunities for health promotion. Bulletin IDF 363:17–26. Korhonen H, Pihlanto-Leppälä A, Rantamäki P, Tupasela T. 1998. Impact of processing on bioactive proteins and peptides. Trends Food Sci Tech 9:307–319. Latham PW. 1999. Therapeutic peptides revisited. Nat Biotechnol 17:755–758.

Meisel H. 1998. Overview on milk protein-derived peptides. Inter Dairy J 8:363–373. Meisel H, Bockelmann W. 1999. Bioactive peptides encrypted in milk proteins: Proteolytic activation and thropho-functional properties. A Van Leeuw 76:207–215. Minkiewicz P, Dziuba J, Iwaniak A, Dziuba M, Darewicz M. 2008. Biopep and other programs processing bioactive peptide sequences. J AOAC Intl 91(4):965–980. Schlimme E, Meisel H. 1995. Bioactive peptides derived from milk proteins: Structural, physiological and analytical aspects. Nahrung 39:1–20. Siemensma AD, Weijer WF, Bak HJ. 1993. The importance of peptide lengths in hypoallergenic infant formulas. Trends Food Sci Tech 4:16–21. Vorob’ev MM, Goncharova A. 1998. Computer simulation of proteolysis. Peptic hydrolysis of partially demasked βlactoglobulin. Nahrung 2:61–67.

Chapter 23 Flavor-Active Properties of Amino Acids, Peptides, and Proteins Eunice C.Y. Li-Chan and Imelda W.Y. Cheung

Contents 1. Introduction, 341 2. Taste Receptors, 342 3. Taste-Active Properties of Amino Acids, 343 3.1. Primary Taste Characteristics of Amino Acids, 343 3.2. Synergistic Interactions Affecting Taste and Flavor, 344 4. Taste-Active Properties of Peptides and Protein Hydrolysates, 344 4.1. Physico-Chemical Properties Related to Bitterness of Peptides and Protein Hydrolysates, 345 4.2. Prediction of Bitterness of Peptides, 347 4.3. Potential Correlation between Bitterness and Bioactive Properties of Peptides, 348

1. Introduction “It is an accepted fact that a purportedly healthy product will fail in the marketplace if it does not taste good” (McGregor 2007). With the increasing demand for incorporating functional proteins and peptides into food ingredients, natural health products, and dietary supplements to enhance health, it is crucial to examine the flavor-active properties of

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

4.4. Approaches for Debittering or Production of Less-Bitter Protein Hydrolysates, 349 5. Taste-Active Properties of Proteins, 351 5.1. Sweet Proteins, 351 5.2. Interaction between Proteins and Sweet-Taste Receptors, 351 5.3. Taste-Modifying Property of Sweet Proteins, 352 6. Interactions of Amino Acids, Peptides, and Proteins with Flavor Ingredients, 352 6.1. Amino Acids, 352 6.2. Peptides and Protein Hydrolysates, 352 6.3. Proteins, 353 7. Conclusion and Research Needs, 354 8. References, 355

these components, which may influence their acceptance by consumers. Most intact proteins inherently possess little or no intrinsic taste (Linden and Lorient 1999). However, the flavor of protein-containing products can be affected through interactions of proteins with flavor compounds or by generation of flavoractive compounds during storage or processing. Furthermore, many flavor-active components can develop during protein hydrolysis to form amino acids and peptides (Linden and Lorient 1999). This occurs not only during the traditional processing to prepare fermented products such as cheese, fish sauce, or soy sauce but also in the production of 341

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functional protein hydrolysates and their derived products with bioactive properties. Three of the five basic taste modalities that are currently recognized, namely sweet, salty, and umami, have historically been associated with foods that contain nutrients important for human health and well-being—energy-containing carbohydrates (sweet), essential minerals (salty), and amino acids (umami). On the other hand, the other two tastes may be linked to protective mechanisms against ingesting compounds that may be harmful to the body, such as spoiled food (sour) or toxic substances (bitter). Despite the medical implications of higher risk of diseases such as hypertension, diabetes, and obesity by overconsumption of salty and sweet foods, these foods are usually considered as desirable and tasty by consumers. Conversely, components with health benefits including phytonutrients, polyphenols, and protein hydrolysates may often not be acceptable due to their bitter taste (McGregor 2007). Recent studies suggest a genetic basis for the diversity of human responses in terms of acceptance or rejection of bitter and pungent foods (Tepper et al. 2003). Since many of the bitter-tasting and pungent components in foods are associated with antioxidant and chemopreventive properties, it is of public health importance to gain an understanding of factors that influence their consumption (Tepper et al. 2003). The objective of this chapter is to explore the flavor-active properties of amino acids, peptides, and proteins that must be considered for successful incorporation and acceptance of components with biologically active properties as ingredients in natural health products, nutraceuticals, and functional foods. The scope of “flavor” as discussed in this chapter primarily focuses on taste-active properties of amino acids, peptides, and protein hydrolysates, including both their inherent taste as well as methods for modifying (enhancing or suppressing) taste, through considering the current state of knowledge of taste reception and sensory coding. In addition, proteins may be involved in binding of aroma compounds, thus the impact on overall flavor of the product is dependent on whether these interactions are reversible or irreversible.

2. Taste Receptors It is generally recognized that the detection of tasteactive stimuli depends on taste receptor cells (TRCs) found in the taste buds of the oral cavity. Tasteactive compounds may interact directly with ion channels causing TRC depolarization and Ca2+ channel activation, or they may activate membranebound G protein-coupled receptors (GPCRs). Specifically, salty and sour tastes involve the sensing by the tongue of sodium ions and protons, respectively, whereas sweet, bitter, and umami tastes are associated with either ionotropic receptors connected to cation channels or GPCRs (Gilbertson et al. 2000). Salty sensation can be triggered both by the epithelial sodium channel ENaC, an Na+-specific salt receptor protein, as well as by TRPV1t, a nonselective cation taste receptor (DeSimone and Lyall 2006). Sour taste may involve several transduction mechanisms and is associated with the lowering of intracellular pH in proton-sensing taste receptors, accompanied by an increase in intracellular Ca2+ concentration, which is associated with the later or tonic part of the response to the stimulus (DeSimone and Lyall 2006). Sweet and umami tastes both depend on the human T1R-receptors, but the sweet GPCR is composed of the heterodimeric hT1R2-hT1R3, whereas the umami or savory GPCR is composed of hT1R1-hT1R3 (Margolskee 2003; Zhao et al. 2003; Maruyama et al. 2006). Artificial sweeteners can also elicit sweet taste through activation of the cation channel by binding to the ionotropic receptor; likewise, some amino acids such as arginine may activate ionotropic receptors leading to TRC depolarization (Gilbertson et al. 2000). Bitter substances, including bitter amino acids and peptides, are recognized by the T2R family of GPCRs (Behrens et al. 2004; Sternini 2007). Of the more than 25 TAS2Rs identified in humans, several (hTAS2R1, hTAS2R4, hTAS2R14, and hTAS2R16) are believed to be responsive to bitter substances, with one (hTAS2R1) receptor being much more sensitive to bitter peptides than other bitter compounds such as denatonium, picrotin, salicin, and caffeine (Maehashi et al. 2008). It has also been reported that

Chapter 23 Flavor-Active Properties of Amino Acids, Peptides, and Proteins

some amphiphilic peptides elicit bitterness by interacting directly with the G-proteins, possibly due to their structural similarity with the G-protein binding site of the receptors (Hofmann et al. 2004). In addition, some compounds trigger a bitter transduction mechanism simply by inhibiting apical potassium ion channels (Gilbertson et al. 2000). It is important to note that “there is more to taste than meets the tongue” (Katz et al. 2000). The information on taste, texture, and temperature that is transduced on the tongue is integrated in the brain with information from the gastrointestinal system, which can modulate the overall taste of food. In addition, research describing the expression of taste signal transduction components, including the T2R family of bitter receptors in the gastric and intestinal mucosa as well as the pancreas, implies that signaling pathways may operate in the enteroendocrine cells to prepare the gut for a response, including whether to absorb or to initiate a protective response against the ingested substances (Sternini 2007). Calcium-sensing receptors that are expressed widely in the gastrointestinal tract have also been identified as GPCRs, and their response to L-amino acids may have functional implications on the control of digestion, absorption, and satiety (Conigrave and Brown 2006). An understanding of these complex processes is important in assessing the potential bioavailability of functional amino acid, peptide, or protein hydrolysate products.

3. Taste-Active Properties of Amino Acids Amino acids may directly contribute to taste by virtue of their inherent taste-active properties, or by acting synergistically with other components in the food or natural health products.

3.1. Primary Taste Characteristics of Amino Acids Table 23.1 shows the predominant taste and detection threshold attributed to the L-form of each of the amino acids. Sour taste is attributed to aspartic and glutamic acids when they are in dissociated form,

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Table 23.1. Taste, detection threshold, and tasteenhancing effects of L-amino acids. Amino Acid

Predominant Taste(s)a,b

Detection Threshold (mg/100mL)b

TasteEnhancing Effectc

Asp Glu Asn Glu-Na Asp-Na Gln His Met Val Arg Ile Phe Trp Leu Tyr Lys Pro Ala Gly Ser Thr Cys

Sour Sour Sour Umami Salty and umami Sweet and umami Bitter Bitter Bitter Bitter Bitter Bitter Bitter Bitter Bitter Sweet and bitter Sweet and bitter Sweet Sweet Sweet Sweet NA

3 5 100 30 100 NA 20 30 40 50 90 90 90 190 NDd 50 300 60 130 150 260 ND

* NAd NA NA NA NA — ** ** — — — NA NA — — ** ** ** ** * **

a

Compiled from Fuke 1994 and Linden and Lorient 1999. Adapted from Linden and Lorient 1999. c Adapted from Fuke 1994; taste-enhancing effect refers to effect of the amino acid on mixtures of 5'-ribonucleotides and sodium salt of Glu or Asp as follows: **P < 0.01; *P < 0.05; — = not significant. d NA = not analyzed; ND = not detected b

but the salt form of these two amino acids is described as umami, savory, or having a meaty or chicken broth taste, sometimes mixed with a sweet taste (Linden and Lorient 1999). Bitterness is associated with several L-amino acids possessing hydrophobic side chains, especially phenylalanine, tyrosine, leucine, isoleucine, and valine. Interestingly, the D-form of these amino acids results in strongly sweet rather than bitter taste. Proline and lysine have been described both as sweet and bitter, while alanine, glycine, serine, and threonine are reported to possess a sweet taste. The sulfur amino acid cysteine does not have any strong taste, while methionine is reported to be bitter.

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Given the low level of these free amino acids typically found in most foods, they are not usually considered direct or major contributors to the taste of food. Nonetheless, the content of free amino acids may be elevated significantly in fermented foods or in enzymatically produced hydrolysates, or even during storage, thus playing an important role in the taste of foods containing them (Kato et al. 1989; Itou et al. 2006). In addition, aside from the free amino acids shown in Table 23.1, which are the building blocks of peptides and proteins, foods may also contain varying amounts of other amino acids such as taurine, hydroxyproline, betaine, sarcosine, and so forth, which can contribute to the overall taste (Kato et al. 1989; Itou et al. 2006).

3.2. Synergistic Interactions Affecting Taste and Flavor Even at low levels, the free amino acids may interact synergistically to enhance taste. A 1 : 1 combination of L-aspartic acid and L-arginine was claimed to enhance the salty flavor of sodium chloride without sour or bitter taste (Lee 1992). In addition, as shown in Table 23.1, some of the free amino acids have an enhancing effect on the umami or savory taste when included in ternary mixtures with 5'-ribonucleotides and the sodium salt of glutamate or aspartate (Fuke 1994). Similar taste was imparted by this ternary synergism, even though the inherent tastes of the individual amino acids differed. Moreover, additional synergistic interactions were observed with other food components such as minerals or sweeteners. For example, inclusion of threshold amounts of sucrose (0.09 g/100 mL) in the mixture containing glycine at its threshold value (0.11 g/100 mL) resulted in much stronger sweetness and palatability (Fuke 1994). Although specific taste receptors for glutamate have been found (Zhao et al. 2003; Maruyama et al. 2006), glutamate on its own as a taste stimulus does not present a highly pleasant taste, nor does it act synergistically with the other four tastes (sweet, salty, bitter, and sour). The umami taste perceived when glutamate is consumed together with consonant odors such as vegetable, fish, and meat is pro-

posed to actually be the result of a flavor-enhancing effect in which glutamate combines supralinearly with consonant odors to activate cortical areas, leading to synergistic effects of a rich, pleasant, and “delicious” flavor that is not achieved by separate activation of the taste and olfactory components (McCabe and Rolls 2007).

4. Taste-Active Properties of Peptides and Protein Hydrolysates Protein hydrolysates and peptide products may be produced from food sources by chemical hydrolysis with acid or base, by autolysis with endogenous enzymes, and by the addition of enzymes from exogenous sources, as well as through the process of fermentation. Protein hydrolysates consisting mainly of shorter peptides and free amino acids are beneficial to patients with disorders related to digestion, malabsorption, or poor amino acid metabolism, as well as those that are hypersensitive to allergic reactions (Clemente 2000). Hydrolysis may also produce desirable techno-functional as well as bioactive properties by releasing specific peptide sequences that had previously remained nascent within the three-dimensional structure of the protein molecule (Dziuba et al. 1999). In addition to considering their nutritive values and potential bioactivity, the organoleptic characteristics of these peptide and protein hydrolysate products must be evaluated for successful incorporation into foods and nutraceuticals. Hydrolysis of proteins can yield desirable sensory attributes. For example, hydrolyzed vegetable protein and autolyzed yeast extract are commercially produced for their flavor-enhancing characteristics (Aaslyng et al. 1999). The contribution of amino acids to the sensory profile of food has been extensively studied in, for example, sake, miso, soy sauce (Kirimura et al. 1969), and fish sauce (Park et al. 2002). Savory peptides have been isolated and identified from products such as fish sauce (Park et al. 2002), as well as hydrolysates from fish protein (Noguchi et al. 1975), soybean, casein, bonito, and chicken protein (Maehashi et al. 1999), while peptides with umami and sweet taste

Chapter 23 Flavor-Active Properties of Amino Acids, Peptides, and Proteins

have also been isolated (Gill et al. 1996). Salty taste has been attributed to basic dipeptides such as ornithyl-β-alanine hydrochloride and ornithyltaurine hydrochloride, which may have potential applications as NaCl substitutes for individuals with hypertension, diabetes, or other diseases (Seki et al. 1990). Production of low-salt soy sauce by partial replacement of NaCl using hydrochloride salts of ornithyltaurine, glycine ethyl ester, or lysine (Kuramitsu 2004), and salt-enhancing formulations composed of various other amino acids or their derivatives and peptides (Hofmann and Dunkel 2007) have also been proposed. Products of the Maillard reaction between peptides from soy protein hydrolysates and various sugars, glucosamine, or galacturonic acid have recently been reported to produce a biphasic effect on human salt taste (Katsumata et al. 2008). Unfortunately in many instances, protein hydrolysis is accompanied by the undesirable attribute of bitterness. For example, hydrolysis of threadfin bream fish muscle by Alcalase produced umami, bitter, and fishy tastes, with the bitter amino acids methionine, valine, isoleucine, phenylalanine, leucine, and tyrosine making up almost one-third of the total amino acid composition of the hydrolysate (Normah et al. 2004). With the discovery of bioactive peptides generated through protein hydrolysis, increasing interest has focused on methods for eliminating, reducing, or masking the bitter taste that is often elicited upon hydrolysis, and on understanding the potential relationships between bioactivity and bitterness of peptides and protein hydrolysates.

4.1. Physico-Chemical Properties Related to Bitterness of Peptides and Protein Hydrolysates Over the years, many scientists have isolated bitter peptides from different food sources, as illustrated in Table 23.2. A comprehensive list of bitter amino acids and peptides reported in the literature has also been compiled (Kim and Li-Chan 2006a). In order to address the bitterness problem commonly found in protein hydrolysates, extensive research has been conducted to elucidate the char-

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acteristics of peptides that are associated with bitter taste. In general, bitterness has been reported to increase with overall hydrophobicity of the molecules. The correlation between bitterness and hydrophobicity was first suggested by Ney, who proposed a “Q value” representing the average hydrophobicity calculated from the free energy for the transfer of amino acid side chains from ethanol to water (Ney 1979; Adler-Nissen 1986). Peptides with Q values greater than 1.400 kcal/mol were hypothesized to exhibit bitterness. Calculation of Ney’s Q values for the bitter peptides listed in Table 23.2 indicates that the majority have Q values greater than 1.400 kcal/mol, thus generally supporting Ney’s hypothesis. Nevertheless, it is also evident from Table 23.2 that there are many exceptions to the Q-rule, including peptides ranging in length from 3 to 13 residues (e.g., SKGL, GAL, EVLN, NENLL, NALKPD, HNIGQT, IYPGCP, SIIDT, VPLGTQYTDAPSF). These exceptions to Ney’s Q-rule can be readily explained since the calculated Q values represent only average hydrophobicity and consider neither the size nor the distribution of hydrophobic residues in the peptide sequences, factors that may be critical in eliciting bitterness. Furthermore, although hydrophobicity is usually recognized as the factor that is most closely associated with bitterness, the contributions of polar uncharged groups and basic bulky groups as well as spatial orientation and proximity of the hydrophobic and polar groups to bitterness and overall taste quality are now also well documented (Kim and LiChan 2006a, 2006b; Kim et al. 2008). Research has consistently shown that most bitter peptides are small, with molecular weight below 5,000 Daltons (Aubes-Dufau et al. 1995; Kim et al. 1999; Cho et al. 2004). Otagiri et al. (1985) reported a progressive increase in bitterness from single amino acid to tripeptides of arginine, proline, and phenylalanine, all comprising a very bulky side chain. Ishibashi et al. (1988b) suggested a minimum number of three carbons in the backbone in order for peptides to exhibit bitterness, while Tamura et al. (1990a) reported that peptides with more than seven amino acids do not show further increase in bitterness intensity. Kim and Li-Chan (2006a)

Table 23.2. Bitter peptides of proteins and protein hydrolysates isolated by different researchers. Sourcea

Peptides 1

Butterkaese Beer yeast residue2

Bovine β–CN (C-terminal)2 Casein1

Bacterial proteinase hydrolysate of casein1 Chymotrypsin hydrolysate of casein1 Papain hydrolysate of casein1 Rennin hydrolysate of casein1

Subtilisin hydrolysate of casein3

Trypsin hydrolysate of casein3 Trypsin hydrolysate of casein1

Tryptic hydrolysate of casein1 Isolated from cheese3

Swiss mountain cheese1 Alkalase hydrolysate of hemoglobin2 Pepsin hydrolysate of hemoglobin4 Soybean proglycinin5

Soy hydrolysate1 Pepsin hydrolysate of soy globulin1

Pepsin hydrolysate of soya protein3

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PFPGPIPNS WF WP LPW RGPFPIIV LGYLEQLL FYPELFR APK RGPPFFIV FYPELF VEVFAPPF AQTQSLVYPFPGPIPNSLPQN-IPPLTQ FLL LRF VPLGTQYTDAPSF PQVF AYFYPEL LRL LVPRYFG RGPPFIV VYPFPPGINH GPFPIV GPFPVI FFVAPFPEVFGK FALPQYLK FFVAPFPQVFGK YLGYLEQLLR EVLN NENLL APFPEVF LW VVYPW VVYPWTQRF NALKPD HNIGQT NAMFVPH NAMFV IYPGCPST IYPGCP SIIDT EF GL LK LF FL YFL QYFL F(I,L2)QGV Pyr-GSAIFVLc F(R,D2,Q2,G,I,L,K2,P,S,T)W(A,R,D,G,V)QYFL RL RLL FIQGV SKGL

Ney’s ΔQ (kcal/mol)b 1.411 2.600 2.400 2.567 1.850 1.775 1.800 1.600 1.800 1.917 1.788 1.441 2.700 2.100 1.277 1.725 1.686 2.300 1.714 1.729 1.570 1.833 1.833 1.733 1.875 1.733 1.690 1.375 1.220 1.729 2.900 2.160 1.756 1.250 0.917 1.557 1.540 1.175 1.350 1.320 1.400 1.450 2.400 2.600 2.600 2.267 1.825 1.929 1.614 1.300 2.000 2.300 1.540 1.250

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Table 23.2. Continued Sourcea 1

Pepsin hydrolysate of zein

Peptides

Ney’s ΔQ (kcal/mol)b

AIA AAL GAL LQL LEL LVL LPFNQL LPFSQL

1.567 1.633 1.300 2.100 2.100 2.667 1.733 1.783

a

Compiled from 1Guigoz and Solms 1976; 2Saha and Hayashi 2001; 3Pedersen 1994; 4Aubes-Dufau et al. 1995; 5Kim et al. 1999. Calculated from hydrophobic effect estimated as side chain burial in kcal/mol taken from Li-Chan 2004. c Pyr refers to pyroglutamyl. b

compiled a database consisting of 224 di- to tetradecapeptides and five amino acids reported in the literature to exhibit bitterness. Although more than half of the reported bitter peptides were di- and tripeptides, it was noted that peptides ranging from 4 to 14 amino acid residues comprised 95 of the bitter peptides in the database, and interestingly, eight of the peptides with the highest bitterness intensity ranged from 6 to 12 amino acid residues in length (Kim and Li-Chan 2006a). In addition to hydrophobicity and size, the spatial arrangement of not only the hydrophobic amino acid residues but also particular polar residues in the peptide sequence also significantly affects the bitterness potency. For example, the presence of a bulky basic group such as arginine at the N-terminal or a hydrophobic amino acid residue such as phenylalanine or tyrosine at the C-terminal have been correlated with stronger bitterness (Otagiri et al. 1985; Ishibashi et al. 1987). Based on these observations, a model has been proposed, describing bifunctional units of peptides exhibiting bitterness, in which two sites are spaced approximately 4.1 Å apart and with a pocket size of 15 Å (Ishibashi et al. 1988a; Tamura et al. 1990a). The primary site, also called the binding unit, consists of hydrophobic side chains with a minimum of three carbons, while the secondary site, referred to as the stimulating site, contains either a bulky basic group or a hydrophobic group, which acts as a determinant site for bitterness (Ishibashi et al. 1988a).

These two sites together with the correct steric configuration can interact with the bitter taste receptor to elicit bitterness. The importance of spatial arrangement of peptides was demonstrated by generating synthetic peptides only differing in the C-terminal residues and evaluating the peptides both sensorially and by structural analysis using computer simulation (Kim et al. 2008). It was concluded that the composition of hydrophobic regions as well as the spatial orientation and proximity between hydrophobic regions and polar groups within the same planar space of the peptides are primary factors determining both type and intensity of bitterness. Furthermore, the findings suggested that bitter molecules generally contain a polar group that may affect bitter taste quality and a hydrophobic group that affects the bitterness intensity (Kim et al. 2008).

4.2. Prediction of Bitterness of Peptides Currently the bitterness of peptides is most commonly evaluated by sensory analysis (Aubes-Dufau et al. 1995; Cho et al. 2004; Humiski and Aluko 2007; Seo et al. 2008). However, sensory evaluation is a time-consuming process and requires extensive training to yield reliable results that can be compared across different studies. Consequently, many scientists have attempted to explore the approach of developing models based on QSAR (quantitative structure-analysis relationship) for the prediction

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of peptide bitterness based on their structural properties. Several studies have employed multivariate data analysis to develop QSAR models with various amino acid descriptors to predict bitterness using the dataset of 48 dipeptides compiled by Asao et al. (1987). For example, principal component analysis (PCA) of 50 physico-chemical variables to characterize the 20 coded amino acids generated 8 principal component scores, collectively named “Vectors of Hydrophobic, Steric, and Electronic” (VHSE), which were used to predict bitterness of the 48 dipeptides (Mei et al. 2005). Similarly, Tong et al. (2008) proposed a novel descriptor, “vector of principal component scores” (VSW) for weighted holistic invariant molecular index (WHIM), which utilized 9 major principal components from a matrix of 99 WHIM indices of the 20 coded amino acids to predict bitterness of the 48 dipeptides. The WHIM descriptors contain information about size, shape, symmetry, and atomic distribution of the whole molecular structure. Furthermore, Kim and Li-Chan (2006a) conducted a QSAR study on 5 amino acids and 224 bitter peptides varying in length from 2 to 14 amino acid residues. Using partial least squares (PLS) regression with full cross-validation, QSAR models that were developed successfully predicted bitterness based on amino acid z-scores representing hydrophobicity, bulkiness/molecular size, and electronic properties considered in conjunction with positional influence, in addition to total hydrophobicity, peptide length, and mass values of the peptides. Performing the QSAR analysis on subsets of the peptides according to peptide length revealed that bulky hydrophobic side chains at the C-terminal and bulky residues at the N-terminal are important for bitterness of shorter (di- and tri-) peptides. For longer peptides with four or more amino acid residues, bitterness was related to C-terminal bulky hydrophobic amino acids with or without basic properties and N-terminal bulky basic amino acids (Kim and Li-Chan 2006a). The potential of Fourier transform (FT)-Raman spectroscopy as an analytical tool for predicting bitterness of peptides and amino acids has also been explored (Kim and Li-Chan 2006b). PLS regression

analysis was performed to generate models for predicting bitterness based on the FT-Raman spectra of amino acids and synthetic peptides composed of phenylalanine, glycine, and proline. A significant correlation was obtained between predicted and reference bitterness values reported in the literature, and the results also confirmed the importance of hydrophobicity and peptide length on bitterness.

4.3. Potential Correlation between Bitterness and Bioactive Properties of Peptides As described throughout this book, extensive research in recent years has demonstrated potent bioactive properties of food protein hydrolysates and peptides. At the same time, as described in the preceding sections of this chapter, there have also been ongoing studies to elucidate the structural and physico-chemical characteristics of amino acids and peptides that are responsible for their taste-active properties. However, despite the critical importance of sensory properties, particularly undesirable attributes such as bitterness, on the consumer acceptance of bioactive peptides and protein hydrolysates as nutraceuticals or functional food ingredients, there is very limited research on sensory evaluation of these bioactive components, and a dearth of knowledge on the possible correlation between biological activity and taste-active properties. Among the diverse bioactive properties attributed to peptides and protein hydrolysates, inhibitory activity against the angiotensin I-converting enzyme (ACE) involved in blood pressure regulation has been the most extensively studied with respect to relating peptide structure to ACE inhibitory activity and its potential correlation with bitterness. Although the structure-activity relationship of food-derived ACE inhibitory peptides is not yet fully elucidated, what is known so far does indicate remarkable commonality with the characteristics identified to be associated with bitterness. Most ACE inhibitory peptides are relatively short sequences that typically possess hydrophobic residues at each of the three C-terminal positions, with proline, tryptophan, and branched aliphatic chains such as isoleucine, valine,

Chapter 23 Flavor-Active Properties of Amino Acids, Peptides, and Proteins

and leucine contributing to higher potency (Murray and FitzGerald 2007). Positively charged side chains of arginine and lysine residues at the peptide terminus are also reported to be important in exhibiting ACE inhibitory activity, probably by their influence on binding of the peptide to the enzyme’s catalytic site. Branched-chain aliphatic amino acids such as valine and isoleucine at the N-terminal enhance inhibitory activity of shorter (i.e., di- and tri-) peptides while spatial configuration and conformation play an important role for longer peptides (Murray and FitzGerald 2007). In fact, very few studies have been reported in the literature that experimentally evaluated both ACE inhibitory activity and bitterness on the same set of peptides. Thus, the investigation of potential correlation between these properties has only been made possible by compiling available data for each property and generating QSAR models to predict one property from the other. Using this approach, Pripp and Ardo (2007) modeled the potential relationship between ACE inhibition and bitter taste. In their dipeptide datasets consisting of 58 ACE inhibitory peptides and 48 bitter peptides, both properties had been experimentally measured for only 15 of the peptide sequences. QSAR modeling using 6 different sets of amino acid descriptors was therefore conducted to predict the ACE-inhibitory or bitter properties of the remaining peptides. For both experimentally observed and QSAR-predicted values, significant correlations were found between ACE inhibition and bitter taste for the dipeptides. Interestingly, the relationship between ACE inhibition and bitterness was less strong for oligopeptides, suggesting that the greater structural variation possibilities in the longer peptides may improve the potential to yield molecules with potent ACE inhibition while being less bitter, that is, sensorially acceptable. Although there is a paucity of research on possible correlations between peptide bitterness and other biological properties, one may speculate about potential candidates on the basis of the primary sequences and sizes of isolated peptides possessing these bioactive properties. For example, peptides high in hydrophobic residues and basic or bulky

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residues at one end of the peptide include the hypocholesterolemic peptides such as LPYPR and IAVPGEVA isolated from soy glycinin and IIAEK from milk β-lactoglobulin, as well as the hypotriglyceridemic peptides VVYP, VYP, and VTL from globin (Erdmann et al. 2008). Similarly, many antioxidative peptides such as those from milk proteins (Pihlanto 2006) or soy proteins and their synthetic derivatives (Chen et al. 1995, 1996) are composed of hydrophobic (aliphatic and/or aromatic) residues, often with hydrophobic bulky residues being dominant at one end, and basic or bulky aliphatic groups at the other end of the peptides. While peptides and protein hydrolysates have excellent potential to serve as antioxidants, the issue of possible bitter off-flavors needs to be addressed (Elias et al. 2008). However, since peptides may serve as antioxidants by multiple mechanisms including reactive oxygen species inactivation, free radical scavenging, transition metal chelation, and hydroperoxide reduction, research leading to a deeper understanding of the peptide structural characteristics for each of these different modes of action may be useful to design effective antioxidative peptides with less bitterness.

4.4. Approaches for Debittering or Production of Less-Bitter Protein Hydrolysates Several methods have been developed to remove, reduce, or mask the level of bitterness in protein hydrolysates. These include treatment with activated carbon (FitzGerald and O’Cuinn 2006), azeotropic extraction or chromatographic separation (Lalasidis and Sjoberg 1978; FitzGerald and O’Cuinn 2006), acetylation of bitter amino acids (Tamura et al. 1990b; Yeom et al. 1994), masking of bitterness by the addition of other compounds (Tamura et al. 1990b; FitzGerald and O’Cuinn 2006), and enzymatic treatment (Minagawa et al. 1989; Izawa et al. 1997; Raksakulthai and Haard 2003). Processes such as treatment with activated carbon, azeotropic extraction, and chromatographic separation target the removal of hydrophobic peptides and amino acids that are associated with

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bitterness (Lalasidis and Sjoberg 1978; Saha and Hayashi 2001; Cheison et al. 2007). These methods not only increase the number of processing steps but are likely to reduce the nutritive values and bioactive properties of the end products. Masking bitterness with the use of α-cyclodextrin, monosodium glutamate, polyphosphates, gelatin, and glycine (Pedersen 1994; Saha and Hayashi 2001) is a fairly simple method that may or may not be acceptable depending on intensity and type of sensory attributes introduced by the masking agents (McGregor 2007). Low or no bitter flavor was detected in peptide mixtures with sweet compounds or 0.3% NaCl (Walters 1996; Park et al. 2002). Enzymatic treatment to further hydrolyze bitter peptides using oligopeptidases or exopeptidases (aminopeptidases and carboxypeptidases) has been suggested to reduce the bitterness of protein hydrolysates (Saha and Hayashi 2001; Raksakulthai and Haard 2003). However, the liberation of free amino acids by this approach may not only introduce other undesirable taste properties but could also alter bioavailability due to different intestinal absorption rates of peptides and free amino acids. Moreover, the loss of key amino acids from the peptide termini may be accompanied by decreased bioactivity. Alternatively, proposals to use enzymes to catalyze peptide bond formation (“plastein” condensation) or transfer reactions have been researched for improving taste (Fujimaki et al. 1977; Suzuki and Kumagai 2004) but are not yet widely used in practice. Several studies have investigated the importance of selecting the appropriate enzymes to produce less- or non-bitter hydrolysates. For example, Deng et al. (2004) developed a poly-enzymatic method to produce nonbitter protein hydrolysates, in which a first stage incorporating simultaneous hydrolysis by alkaline and neutral protease was followed by flavorase treatment. Extensive research has been performed to identify proteases that tend to yield nonbitter protein hydrolysates; for example, papain (Humiski and Aluko 2007), α-chymotrypsin (Humiski and Aluko 2007), Flavourzyme (Nilsang et al. 2005), and Actinomucor elegans peptidases (Li et al. 2008) are among the enzymes that were

reported to be successful in producing protein hydrolysates with no or low bitterness. It should be emphasized that for each application, the structural and functional characteristics of the substrate and the target bioactive peptides, substrate specificity, and activity of the enzyme, as well as conditions of hydrolysis, must all be considered in order to successfully produce bioactive peptides or protein hydrolysates having low bitterness. For example, Spellman et al. (2005) reported less bitterness when enzymatic hydrolysis using Debitrase™ HYW20 was conducted on the substrate (whey protein) at 30% compared to 5% total solids and attributed this to the release of a bitter peptide (VEELKPTPEGDLEIL) corresponding to β-lactoglobulin f(43–57) during hydrolysis at the lower substrate concentration. In general, it is not possible to directly predict characteristics of the hydrolysates that might be produced based on the endoproteinase and exopeptidase complement of crude enzyme preparations (Smyth and FitzGerald 1998). Better predictability might be expected by using highly purified and characterized enzymes for hydrolysis. Given the association of bitterness with peptides bearing hydrophobic residues at their terminal ends, Kodera et al. (2006) isolated a novel cysteine protease “D3” from germinating soybean cotyledons, which preferentially cleaves polypeptide sequences containing hydrophobic amino acids at the P2 position. This substrate specificity is predicted to generate peptides bearing hydrophilic rather than hydrophobic residues at the peptide termini, which are expected to have low or no bitterness. In fact, protein hydrolysates obtained by the action of D3 protease produced in an E. coli expression system were significantly less bitter than hydrolysates obtained by other proteases such as subtilisin, pepsin, trypsin, and thermolysin (Kodera et al. 2006). ACE-inhibitory activity of several peptides identified as being generated from soy and casein proteins by the D3 protease was determined by in vitro assay, with the peptide NWGPLV being the most potent inhibitor (IC50 value of 21 μM); furthermore, the antihypertensive effect of the D3produced hydrolysates was confirmed by in vivo

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assay in spontaneously hypertensive model rats (Kodera and Nio 2006).

5.2. Interaction between Proteins and Sweet-Taste Receptors

5. Taste-Active Properties of Proteins

The structure-activity relationships that result in sweet-taste elicitation are not well understood. Common sequences or tertiary structural features so far have not been identified to distinguish these taste-active proteins from the majority of proteins that do not elicit sweet taste. Research in the past decade has therefore turned to focus on the structure of the heterodimeric T1R2-T1R3 sweet-taste receptors and their interactions with the sweet proteins. Furthermore, recent research has suggested that the sweet receptor is also expressed in the gut and may function as a glucose sensor to regulate peptide hormone secretion (Jang et al. 2007). Therefore, in addition to contributing to greater understanding of sensory perception, investigation of the sweet receptor could provide new insights into potential targets for therapy of obesity and diabetes (Nakajima et al. 2008). Most sweet substances are small molecular mass compounds, while the sweet proteins are macromolecules, for example, ranging in size from the 6.5 kDa monomer of brazzein to the 98.4 kDa tetrameric form of miraculin (Kant 2005). Tancredi et al. (2004) hypothesized that potential “sweet fingers” protruding from the surface of the sweet proteins might function as the active components that interact with the sweet receptor binding site in a similar way to smaller sweet molecules. However, none of the three synthesized cyclic peptides corresponding to loops of brazzein, monellin, and thaumatin, which were speculated to be candidates for the sweet fingers, were found to have a sweet taste. These results suggested that sweet proteins recognize a binding site that differs from the one that binds to small sweeteners (Tancredi et al. 2004). Using homology models for molecular docking of sweet ligands to the sweet-taste receptors together with experimental investigation on the effects of site-directed mutagenesis of the receptors on the sweet response, Cui et al. (2006) concluded that the T1R2-T1R3 receptors possess multiple potential ligand-binding sites related to sweet taste. The amino terminal domain of the T1R2 receptor was

Most proteins have little flavor on their own due to their size and often bulky three-dimensional structure that hinders their effectiveness in binding to the TRCs, which transmit signals to the brain to generate taste perception. Salty and sour tastes are usually generated when small molecules activate the ion channels, while the elicitation of bitterness requires exposure of hydrophobic sites, which are usually buried in the interior of the protein, to interact with the bitter taste receptors. Recently, several sweet proteins have been discovered and a few have also demonstrated taste-modifying ability.

5.1. Sweet Proteins With the increasing prevalence of obesity, diabetes, and metabolic syndrome disorder and the growing demand for more natural products, there has been an urge to reduce the use of sucrose, high fructose corn syrup, and other caloric sweeteners as well as artificial sweeteners such as aspartame, neotame, acesulfame K, sucralose, cyclamate, and saccharin. Sweet proteins could have the potential to fill the need for naturally occurring ingredients providing sweet taste without high caloric contribution. Most of the sweet proteins identified so far have been isolated from plants found in tropical rainforests. These include thaumatin, monellin, brazzein and its dimeric form pentadin, neoculin (previously referred to as curculin), and miraculin from West Africa, as well as mabinlin from China (Kurihara 1992; Kant 2005; Nakajima et al. 2008). Of these, thaumatin and monellin are the most potently sweet, eliciting sweet-taste responses at molar concentrations that are 105-fold lower than that required for sucrose (Masuda and Kitabatake 2006). Lysozyme, an enzyme from hen egg white that has been utilized for its antimicrobial activity, has also been reported to have a sweet taste, with a sweetness threshold value about 200 times higher than thaumatin and monellin (Masuda et al. 2001; Masuda and Kitabatake 2006).

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proposed to exhibit receptor activity toward small dipeptide sweeteners such as aspartame and neotame, while the transmembrane domain of T1R3 was proposed to be responsible for receptor activity toward cyclamate (Cui et al. 2006). Given their large size, it was evident that the sweet proteins could not fit either into the transmembrane domain of T1R3 or within the cleft of the closed form of the amino terminal domain. Instead, a cysteine-rich extracellular domain of T1R3 was postulated to be responsible for binding of the sweet proteins, and the amino terminal domain of the T1Rs was also suggested to be involved (Cui et al. 2006).

5.3. Taste-Modifying Property of Sweet Proteins

2002, 2006). Aldehydes have been reported to participate in both irreversible covalent bonding as well as reversible hydrophobic interactions (Guichard 2002). Factors such as the properties of the flavor compound, amino acids, peptides, and protein and the processing history, as well as the conditions including temperature and solvent composition (ionic strength, pH, the presence of solvents such as ethanol), can affect the extent and nature of the interactions (Kühn et al. 2006; Tromelin et al. 2006). Furthermore, an increase in viscosity or formation of gels can lead to hindered mass transfer and result in a decrease of aroma release and perception (Weel et al. 2002; Reineccius 2006).

6.1. Amino Acids

In addition to their sweetness, the sweet proteins neoculin and miraculin are of particular interest for their taste-modifying property, specifically of being able to convert sourness to sweetness (Kant 2005). Nakajima et al. (2008) quantitatively assessed the pH-dependent, acid-induced sweetness of neoculin using a cell-based assay system, in which the human T1R2-T1R3 sweet receptors were expressed together with G-α proteins in HEK293T cells. They reported that neoculin exhibited weak sweetness at neutral pH but much stronger sweetness at acidic pH. In comparison, when histidyl residues of neoculin were replaced with alanine, the resulting neoculin variant elicited strong sweetness independently of pH. Their data suggested that neoculin acts as an agonist of the sweet-taste receptor at acidic pH but functionally changes into an antagonist at neutral pH (Nakajima et al. 2008).

It has been reported that the amino acids show large variation in their affinity for binding volatile compounds (Luyten et al. 2000), and the adsorption may vary depending on whether the amino acids are in solution or in solid form (Maier and Hartmann 1977). Of the 22 amino acids examined in dry form at 23°C, lysine adsorbed the greatest amount of volatile aldehyde and ketone compounds, and the adsorption appeared to be irreversible (Maier and Hartmann 1977). Next to lysine, cysteine also adsorbed many carbonyl compounds, possibly through formation of thiazolidinecarboxylic acids; in addition, arginine, histidine, phenylalanine, tyrosine, proline, valine, leucine, and isoleucine all showed significant amounts of adsorbed compounds (Maier and Hartmann 1977).

6. Interactions of Amino Acids, Peptides, and Proteins with Flavor Ingredients

Although much research has been performed to optimize and investigate protein hydrolysis for the purpose of producing flavor ingredients such as HVP (hydrolyzed vegetable protein) or soy sauce, relatively little research has been conducted to examine the effects of protein hydrolysis and bioactive peptide generation on the binding of aroma compounds. One would expect that limited hydrolysis would promote unfolding of the protein threedimensional structure and enhance accessibility of

Binding of flavor compounds by amino acids, peptides, and proteins may occur through reversible physical adsorption involving van der Waals interactions, hydrophobic interactions, hydrogen bonding and/or electrostatic linkages, or by formation of irreversible covalent bonds (O’Neill 1996; Guichard

6.2. Peptides and Protein Hydrolysates

Chapter 23 Flavor-Active Properties of Amino Acids, Peptides, and Proteins

hydrophobic groups to interact with nonpolar aroma compounds (Reineccius 2006). Hydrophobic interactions could be reduced by extensive protein hydrolysis conducted with the aim of producing oligo-, tri- or even dipeptides with bioactive activity. However, the possibility of electrostatic interactions through numerous terminal amino and carboxyl groups in these hydrolysates could lead to enhanced binding of hydrophilic taste compounds, in addition to generating peptides with their own taste-active properties. Furthermore, a greater degree of reactivity with carbonyls and more sulfur interchange by hydrolysates than intact protein could be expected to effect flavor release from foods containing these hydrolysates (Reineccius 2006). Since perception of flavor is influenced by texture (Weel et al. 2002), and a longer and more pronounced retronasal aroma release resulting in greater satiation was observed by consuming solid than liquid foods (Ruijschop et al. 2008), it is likely that the binding, release, and transport of flavor compounds in the presence of peptides and protein hydrolysates would differ from protein carriers due to the lessening of mass transfer effects resulting from the decreased viscosity and gel or macromolecular structures usually attributed to proteins (Reineccius 2006). Further research is necessary to assess the impact of incorporating bioactive peptides and hydrolysates on the perception of flavor in food systems.

6.3. Proteins Many proteins may influence flavor of a food system in various ways, through accompanying compounds found, for example, in soy proteins, by binding or adsorption leading to masking or altered release of flavor compounds, or by chemical reactions during storage or process-induced changes (O’Neill 1996; Luyten et al. 2000). Skarra (2004) provided an interesting perspective on the paradigm shift in soy protein applications in food products over the years. Early applications starting in the 1950s and 1960s were based on making use of particular desirable functional properties of soy protein to replace more expensive (e.g., meat) ingredients in food; therefore, these applica-

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tions focused on using the minimum level of soy protein to achieve the intended “techno-functional” effect. In contrast, current applications aim to deliver sufficiently high levels of soy protein to meet health claims and “bio-functional” or bioactive properties. Incorporating high levels of soy proteins can present a challenge in terms of flavor-active properties of the proteins (MacLeod and Ames 1988). In addition to the intrinsic “off-flavor” problems, shifts in overall flavor profiles due to adsorption or changes in solubility or volatility of desirable flavor compounds may arise from interactions with the protein (Moon and Li-Chan 2007a, 2007b). Unlike amino acids, peptides, and protein hydrolysates, binding of aroma or flavor compounds to proteins has been more extensively studied, particularly for the milk whey protein β-lactoglobulin (Guichard 2002, 2006). A minimum of three distinct binding sites have been identified on β-lactoglobulin, with the two major ones being at a hydrophobic pocket in the central cavity of the β-barrel structure and on the outer surface of the protein near the helix (Guichard 2002, 2006; Tromelin et al. 2006). Although both polar and nonpolar compounds may bind to this protein, within a class of chemical structures, the binding affinity appears to be correlated with hydrophobicity of the compounds; and binding occurs mainly through hydrophobic interactions and hydrogen bonding. In contrast to the specific binding sites of β-lactoglobulin, the interactions of other proteins, such as soy protein, bovine serum albumin, and other milk proteins, with flavor compounds appear to be less specific and may involve many binding sites for both polar and nonpolar compounds (MacLeod and Ames 1988; Kühn et al. 2006; Zhou and Cadwallader 2006). Regardless of whether the protein-flavor interactions are specific or nonspecific, important considerations include the number of binding sites and the nature and strength of the interactions (Taylor 2002; Guichard 2006). The final impact on sensory perception will depend not only on binding of the flavor compounds by the proteins but also on their release and transport to the flavor receptors in the mouth and nose in the dynamic conditions found during mastication and consumption.

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The importance of distinguishing between orthonasal and retronasal contributions to aroma perception has led to research studying these chemosensory stimuli in isolation (Negoias et al. 2008). For example, while static headspace analysis indicated that the retention of aldehydes by whey proteins was dependent on the carbon chain length of the aldehyde and pH of the solution, considerably fewer pronounced effects were observed in the release of aldehydes measured in vivo in exhaled air (Weel et al. 2003). It was suggested that a thin film of aldehydecontaining solution remains in the pharynx during swallowing, and free molecules of aldehyde as well as those reversibly bound to whey proteins are released in vivo due to the large flow of air during exhalation after swallowing (Weel et al. 2003). Thus, although orthonasal perception of aroma intensity may be diminished in aldehyde-whey protein solutions, the aroma release during in vivo consumption may in fact not reveal any difference in retronasal perception compared to protein-free aldehyde solutions. Furthermore, the brain’s response to retronasal perception of food aroma has been suggested to be linked with the sensation of satiety, prompting research to understand and control retronasal aroma release as a strategy to trigger perception of satiation and potentially reduce risk of obesity (Ruijschop et al. 2008).

7. Conclusion and Research Needs The growing demand for nutraceuticals and functional foods in our diet to promote health and reduce risk of disease has led to a plethora of research on amino acids, peptides, proteins, and protein hydrolysates with specific biological and physiological functions beyond basic nutritional contributions. Unfortunately, the rapid pace of discovery of these bioactive molecules has not been accompanied by adequate research into their flavor-active properties that will be critical for successful marketing and consumer acceptance. Ongoing research is necessary to develop new methods to improve taste acceptability of bitter amino acids, peptides, and protein hydrolysates (McGregor 2007). Although simple addition of salt or sugar may be used to decrease perception of bit-

terness, this approach is not always successful for highly bitter components, or for the formulation of foods to minimize risk of hypertension or diabetes. Microencapsulation or other forms of coating that block the bitter bioactive component from contacting the taste-receptive cells is another alternative, but research is needed to investigate the potential effects on subsequent release and availability of the bioactive component in the gastrointestinal system. Similarly, while enzymes such as exopeptidases have been investigated for the purpose of debittering, their effect on bioactive properties of functional peptides or protein hydrolysates has not been adequately explored. Production of target peptide sequences with biologically active as well as desirable taste-active properties may be designed based on knowledge gleaned from QSAR studies and using enzymes screened for the required substrate specificity. However, this strategy may not always be successful given the similarities that have been observed in the structural attributes of peptides exhibiting certain biological activity and undesirable taste-active properties, for example, between bitterness and ACE-inhibition (Pripp and Ardo 2007). Greater understanding of QSAR for biological properties and taste-active properties may provide new paradigms for development of functional ingredients, through producing acceptable tasting “prodrug” peptides that are cleaved by proteolytic enzymes during gastrointestinal digestion to yield the active peptide forms in vivo. Due to the limitations of approaches that are based on altering the chemical or physical attributes of the taste-active molecules, much research has appeared in recent years on alternative strategies based on blocking the sensation of undesirable taste sensations such as bitterness, through designing compounds that may activate or inhibit the taste active receptors, to enhance or inhibit specific taste modalities (Gravina et al. 2004; Hofmann et al. 2004; McGregor 2007). In addition to research on the inherent tasteactive properties of biologically active amino acids, peptides, and proteins, there is also a need to investigate impact of these molecules on the binding, release, and perception of flavor compounds as well

Chapter 23 Flavor-Active Properties of Amino Acids, Peptides, and Proteins

as the binding and bioavailability of other bioactive components in the food matrix. For example, even though the beneficial health effects of polyphenolic compounds has been under extensive scrutiny in recent years, there is relatively little research on the flavor-active properties such as bitterness or astringency of polyphenolic compounds consumed within protein or peptide-rich food matrices, despite the widely accepted occurrence of protein-polyphenol interactions. There is also an opportunity to develop tasteactive amino acid, peptide, or protein products as ingredients for foods that will enable reduction of caloric or sodium intake while retaining sweet or salty perception. For example, although synthetic dipeptide sweeteners such as aspartame and neotame are already commercially available and widely used, the available research suggests good potential to develop other sugar replacers, salt enhancers or substitutes, and sour-taste modifiers based on naturally occurring peptides, sweet proteins, or derivatives such as Maillard reactions between peptides, sugar, and other food components (Masuda and Kitabatake 2006; Hofmann and Dunkel 2007; Katsumata et al. 2008). Finally, besides considering the flavor-active amino acids, peptides, and proteins only from the point of view of sensory perception, one should explore the potential of these molecules to impart additional functional properties that can contribute to health enhancement and disease reduction. Further research on the possible links between the gustatory perception of flavor-active components and the gastrointestinal, hormonal, and nervous system responses that signal satiety or trigger different metabolic or absorption responses may reveal novel therapeutic approaches for various conditions including obesity, metabolic disorder, or diabetes (Conigrave and Brown 2006; Jang et al. 2007; Sternini 2007; Nakajima et al. 2008; Ruijschop et al. 2008).

8. References Aaslyng MD, Poll L, Nielsen PM, Flyge H. 1999. Sensory, chemical and sensometric studies of hydrolyzed vegetable protein produced by various processes. Eur Food Res Technol 209(3–4):227–236.

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Jang HJ, Kokrashvili Z, Theodorakis MJ, Carlson OD, Kim BJ, Zhou J, Kim HH, Xu X, Chan SL, Juhaszova M, Berneir M, Mosinger B, Margolskee RF, Egan JM. 2007. Gut-expressed gustducin and taste receptors regulate secretion of glucagonlike peptide-I. Proc Natl Acad Sci USA 104(38):15069– 15074. Kant R. 2005. Sweet proteins—Potential replacement for artificial low calorie sweeteners. Nutr J 4(1):5–10. Kato H, Rhue MR, Nishimura T. 1989. Role of free amino acids and peptides in food taste. In: R Teranishi, RG Buttery, F Shahidi, eds., Flavor Chemistry Trends and Developments, 158–174. Washington, DC: American Chemical Society. Katsumata T, Nakakuki H, Tokunaga C, Fujii N, Egi M, Phan T-HT, Mummalaneni S, DeSimone JA, Lyall V. 2008. Effect of Maillard reacted peptides on human salt taste and the amiloride-insensitive salt taste receptor (TRPV1t). Chem Senses 33(7):665–680. Katz DB, Nicolelis MA, Simon SA. 2000. Nutrient tasting and signaling mechanisms in the gut. IV. There is more to taste than meets the tongue. Amer J Physiol Gastrointest Liver Physiol 278(1):G6–9. Kim H-O, Li-Chan ECY. 2006a. Quantitative structure-activity relationship of bitter peptides. J Agric Food Chem 54(26): 10102–10111. Kim H-O, Li-Chan ECY. 2006b. Application of Fourier transform Raman spectroscopy for prediction of bitterness of peptides. Appl Spectros 60(11):1297–1306. Kim M-R, Choi S-Y, Kim C-S, Kim C-W, Utsumi S, Lee C-H. 1999. Amino acid sequence analysis of bitter peptides from a soybean proglycinin subunit synthesized in Escherichia coli. Biosci Biotechnol Biochem 63(12):2069–2074. Kim M-R, Yukio K, Kim KM, Lee C-H. 2008. Tastes and structures of bitter peptide, asparagine-alanine-leucine-prolineglutamate, and its synthetic analogues. J Agric Food Chem 56(14):5852–5858. Kirimura J, Shimizu A, Kimizuka A, Ninomiya T, Katsuya N. 1969. The contribution of peptides and amino acids to the taste of foodstuffs. J Agric Food Chem 17(4):689–695. Kodera T, Asano M, Nio N. 2006. Characteristic property of low bitterness in protein hydrolysates by a novel soybean protease D3. J Food Sci 71(9):S609–614. Kodera T, Nio N. 2006. Identification of angiotensin I-converting enzyme inhibitory peptides from protein hydrolysates by a soybean protease and the antihypertensive effects of hydrolysates in spontaneously hypertensive model rats. J Food Sci 71(3):C164–173. Kühn J, Considine T, Singh H. 2006. Interactions of milk proteins and volatile flavor compounds: Implications in the development of protein foods. J Food Sci 71(5):R72–82. Kuramitsu R. 2004. Quality assessment of a low-salt soy sauce made of a salty peptide or its related compounds. In: F Shahidi, AM Spanier, C-T Ho, T Braggins, eds., Quality of Fresh and Processed Foods, 227–238. Advances in Experimental Medicine and Biology Vol. 542. New York: Kluwer Academic/ Plenum Publishers.

Chapter 23 Flavor-Active Properties of Amino Acids, Peptides, and Proteins

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Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides Idit Amar-Yuli, Abraham Aserin, and Nissim Garti

Contents 1. Introduction, 359 2. Entrapment and Stability of Proteins and Peptides by Liquid Crystalline Systems, 360

1. Introduction The protein and peptide therapeutics have become an important class of drugs due to advancement in molecular biology and technology. However, an effective and convenient delivery of these drugs in the body remains a challenge. Over the last 10 years an extensive study has been carried out to improve their delivery. Yet there are still a number of limitations due to their intrinsic physico-chemical and biological properties, including poor permeation through biological membranes (large molecular size), short half-life, physical and chemical instability, enzymatic catalysis, aggregation, adsorption, bio-incompatibility, and immunogenicity. This chapter presents the difficulties in delivery of proteins and peptides and discusses the liquid crystal–based controlled delivery systems that were produced and examined to improve delivery. Over the last 2 decades, biopharmaceuticals (therapeutic proteins and peptides, synthetic vaccines,

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals, Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang. © 2010 Blackwell Publishing Ltd. and Institute of Food Technologists

3. Barriers and Mechanisms of Bioavailability of Proteins and Peptides, 366 4. Conclusion, 379 5. References, 379

monoclonal antibody–based products, and nucleic acid–based medicinal products) have become an important new class of therapeutic agents. Globally, about 500 candidate biopharmaceuticals are at various stages of clinical trial evaluations. According to the Pharmaceutical Research and Manufacturers of America, 324 potential protein-based medicines are being investigated to treat cancer, autoimmune disorders, infectious diseases, inflammation, and cardiovascular disease (Holmer 2004; Kumar et al. 2006). Although numerous protein-based therapeutics have been approved or are in advanced clinical testing, the development of more sophisticated delivery systems for this rapidly expanding class of therapeutic agents has not kept pace. Despite the revolutionary progress in large-scale manufacturing of proteins and peptides, effective and convenient delivery of these agents in the body remains a challenge. This is mainly due to their intrinsic physico-chemical and biological properties, including poor permeation through biological membranes (e.g., intestinal mucosa) due to their large molecular size, short half-life (degradation by proteolytic enzymes and neutralization by antibodies), physical and chemical instability, enzymatic catalysis, 359

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aggregation, adsorption, bio-incompatibility, and immunogenicity. These chemical properties prevent the oral administration of this class of drugs (Kumar et al. 2006). Oral administration of therapeutic macromolecules is notoriously difficult as a result of their high molecular weight, hydrophilicity, and susceptibility to enzymatic inactivation in the gastrointestinal tract. Parenteral routes of administration enable total systemic availability and fastest onset of action. The most commonly used parenteral routes are intravenous (I.V.), intramuscular (I.M.), and subcutaneous (S.C.). Intravenous injection offers rapid onset, which is desired for certain drugs such as streptokinase, where the beneficial effect is a function of time. However, administration with needles causes pain and can lead to the transmission of blood-borne pathogens induced by accidental needle pricks or intentional needle reuse. The consequence is reduced patient compliance (Simonsen et al. 1999). To improve therapeutic efficacy, research has focused on finding alternative means of administration, particularly noninvasive routes such as pulmonary, intranasal, or transdermal. The transdermal route has attracted interest as a promising means to advance the delivery of proteins and peptides and to minimize side effects as well as first-pass metabolism. For several decades it has been used successfully for numerous well accepted systems (e.g., hormone replacement therapy, smoking cessation, and pain management). However, there have been challenges in expanding use of the technology to the delivery of peptides, proteins, and other macromolecules. These biopharmaceuticals cannot permeate the skin’s outer stratum corneum layer (the upper skin barrier) at levels or rates that achieve significant therapeutic effects. In order to impede the influx of toxins into the body and minimize water loss, the stratum corneum layer has very low permeability for foreign molecules (Wertz and Downing 1989). Currently, various technologies are used to increase the permeability of macromolecules by exploring iontophoresis, electroporation, ultrasound, and microporation using electrical current/ voltage, radio frequency, and microneedles to open the skin. Although mechanical abrasion and chemi-

cal enhancers may increase drug permeation, their effects on the skin’s inherent rate-controlling properties are difficult to control and they may irritate the skin (Hadgraft 1999). Therefore, in recent years tremendous efforts have been made to improve controlled delivery of proteins and peptides. Liquid crystalline phases, such as lamellar, reverse hexagonal, and cubic phases, present interesting properties for a topical delivery system, and hence were considered and have been studied as delivery vehicles of pharmaceuticals via the skin and mucosa (Carr et al. 1997; Lee and Kellaway 2000a, 2000b). These phases (1) are thermodynamically stable; (2) consist of nano-scaled hydrophilic and hydrophobic domains, which are separated by the surfactant self-assembled layers; (3) contain an extremely large surface and present the ability to incorporate compounds independent of their solubility, to protect them from physical and enzymatic degradation, and to sustain their delivery; and (4) may consist of permeation enhancers as the structure-forming lipid/solvent (Shah et al. 2001; Lara et al. 2005; Amar-Yuli et al. 2008). The success of an innovative delivery system for proteins or peptides will depend on its ability to fulfill some or all of the following parameters and perquisites: 1. 2. 3. 4. 5. 6. 7.

In vitro and in vivo stability Improved systemic availability Prolonged biological half-life (T1/2) Patient convenience and compliance Reduced dosing frequency Local toxicity and safety concerns Product life cycle management

This review will focus on the current knowledge of liquid crystal–based controlled delivery systems at present under investigation, for protein and peptide delivery, mainly in transdermal administration.

2. Entrapment and Stability of Proteins and Peptides by Liquid Crystalline Systems Understanding how a protein or peptide functions requires knowledge of their three-dimensional

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

structures at the atomic level (Liu and Caffrey 2005). Their structures are so diverse that it is difficult to categorize them except broadly on the basis of their secondary structure (Giuliani et al. 2007). Basically, there are four major classes of proteins and peptides: β-sheet, α-helical, loop, and extended proteins and peptides, with the first two classes being the most common in nature. In addition to the natural proteins and peptides, thousands of synthetic variants have been produced that also fall into these structural classes (Giuliani et al. 2007). During the last two decades, proteins and peptides were incorporated into liquid crystalline structures and their entrapment capacities, as well as in some cases crystallization behavior was studied (Ericsson et al. 1983; Razumas et al. 1994, 1996). To date, several membrane proteins have been successfully crystallized from liquid crystal mesophases (termed “in meso” method) and used for highresolution structure determination. These include bacteriorhodopsin (Luecke et al. 1998), halorhodopsin (Kolbe et al. 2000), sensory rhodopsin (Luecke et al. 2001), the sensory rhodopsin/transducer complex (Gordeliy et al. 2002), the photosynthetic reaction center from Rhodobacter sphaeroides (Katona et al. 2003), and gramicidin D (Liu and Caffrey 2005). A mechanism for the “in meso” crystallization process has been proposed by Caffrey (2000), who posits that the purified and detergentsolubilized membrane protein reconstitutes into the lipid bilayer of the hosting liquid crystal mesophase. Precipitants, of which there is a wide variety (McPherson 1999), serve in part to alter the curvature of the membrane and to affect a phase separation of sorts where one of the phases takes the form of a protein crystal (Caffrey 2000). Liu and Caffrey tested pentadecapeptide (gramicidin D), an antibiotic that creates pores in membranes through which water and small cations can pass (Liu and Caffrey 2005). The helical dimer is considered to be the active species that creates such transient holes (Figure 24.1A). An alternative, double-helical conformation can be adopted in a more polar environment (Chen and Wallace 1997). The two conformations can be distinguished by their characteristic electronic circular dichroism (CD)

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spectra in the UV region. Accordingly, CD spectroscopy was used to probe the gramicidin D conformation adopted in the cubic phase membrane containing glycerol monooleate (GMO), compared to methanol, ethanol, molten GMO, and to unilamellar dioleoyl phosphatidylcholine (DOPC) vesicles. When reconstituted into the cubic-Pn3m phase of hydrated GMO or the bilayer of DOPC vesicles, gramicidin adopted the helical dimer conformation (as shown in the lower part of Figure 24.1A). In methanol, ethanol, and molten GMO the spectral properties revealed a double-helix conformation. The DOPC vesicles have a limiting diameter of 300 Å (Huang 1969), in contrast to the cubic phase aqueous channel diameter, which is about one-sixth of this value and much more curved (Figure 24.1B). Nevertheless, the conformation of gramicidin D in these two structures is similar, suggesting that its conformation is not sensitive to the degree of bilayer curvature. The cubic phase itself consists of two interpenetrating highly curved aqueous channels that never contact one another, being separated by a continuous lipidic bilayer. As such, gramicidin D has two possible destinations within the cubic phase into which it can partition—the bilayer and the aqueous channels. Due to its hydrophobic nature, gramicidin D is energetically favorable to be accommodated in the lipid bilayer compartment. It has been further demonstrated by the fluorescence quenching study that the gramicidin D is in the apolar environments within the cubic phase (Figure 24.1B; Liu and Caffrey 2005). Furthermore, Liu and Caffrey claim that part of the surfactant molecule segregates from the lipidic bilayer of the mesophase and selectively envelops and solubilizes the peptide. Given that the channel diameter is of the order of ∼50 Å, such an arrangement is not unreasonable geometrically. However, the conjunction is untenable thermodynamically; at low levels, the cubic phase can accommodate the peptide with little or no effect on phase microstructure. However, at higher concentrations of 10 mol% gramicidin D, it destabilized the cubicPn3m phase and induced HII phase formation (Liu and Caffrey 2005). It has been affirmed that peptides and proteins could be accommodated in liquid crystal phases

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16 Å

A

W 15 W 13

W9 W 11

26 Å W9

W 13

46.4 Å

W 15 B C

35.6 Å

N N

C

Figure 24.1. (A) Upper: Space-filling model of the channel form of gramicidin A. Trypotophans are shown in purple and cyan and are identified by their position in the chain. Viewed from within the lipid bilayer along the dimeric two-fold axis. Nontryptophan hydrogen, carbon, nitrogen, and oxygen atoms are shown. Lower: Cartoon representations of gramicidin displaying the polypeptide backbone fold in the helical dimer (channel or pore) conformation. (B) Cartoon representation of the cubic-Pn3m phase with the channel form of gramicidin reconstituted into its lipid bilayer and dissolved as a mixed micelle in the aqueous channel. Dimensions are based on the cubic phase formed using glycerol monooleate at 39 wt% water and 20°C. The continuous blue zones are the aqueous channels in the cubic phase (Liu and Caffrey 2005).

either in the aqueous channel compartments (watersoluble proteins and peptides) (Ericsson et al. 1983; Razumas et al. 1994; Leslie et al. 1996; Razumas et al. 1996) or in the lipid bilayer constituent (membrane proteins and peptides) (Pebay-Peyroula et al. 1997; Kolbe et al. 2000; Libster et al. 2007, 2008). It should be stressed that water-soluble proteins and peptides, as well as other molecules, are considered solutes with kosmotropic or chaotropic properties in the aqueous phase of the amphiphilic system. Kosmotropic (water-structure makers) and chaotropic (water-structure breakers) solutes have a very significant impact on the properties of liquid crystalline phases by indirect (Hofmeister) interactions with these structures. It was demonstrated that kosmotropic solutes, which are likely to be solubilized

within the bulk water, stabilize the structure of bulk water (Tsvetkova et al. 1991; Koynova et al. 1997; Amar-Yuli et al. 2008). Water structures (kosmotropes) interfere with the tetrahedral network of water and, as a result, dehydration of the surfactant polar heads takes place (“salting-out” effect) and the effective interfacial area per amphiphilic molecule is condensed. Consequently, greater curvature is promoted and formation of inverted cubic and hexagonal phases at the expense of Lα phase is preferred. Alternatively, chaotropes tend to destabilize the structure of bulk water and lead to an expansion of the effective interfacial area per amphiphilic molecule and hence induce formation of the Lα phase in place of the inverted cubic and hexagonal phases (Tsvetkova et al. 1991; Koynova et al. 1997; AmarYuli et al. 2008).

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

For instance, peptides and proteins such as alamethicin, gramicidin, melittin, and cytochrome C have been shown to promote the formation of cubic or hexagonal phases in lipid systems displaying lamellar or cubic liquid crystalline phases, respectively (Keller et al. 1996; Angelova et al. 2000; Liu and Caffrey 2005). These peptides and proteins can be considered kosmotrope surface-active molecules that incorporate into the headgroup region of the membranes and change the monolayer curvature and properties of the lipid bilayers. Fab fragments of IgG1 exhibited the opposite effect, and it can be considered a chaotropic protein. Its addition to the monoolein-based liquid crystalline system decreased the mean packing parameter and shifted the phase sequence toward lamellar phase formation (Angelova et al. 2003). However, the presence of immunoglobulin (mixed with albumin), transferrin, and fibrinogen that were added to a similar system did not modify the lipid bilayer thickness and the aqueous channel sizes, compared to those in a protein-free system (Angelova et al. 2003). Two decades ago, researchers assumed that water-soluble proteins were accommodated in the water channels of the bicontinuous cubic lattices (Ericsson et al. 1983; Mariani et al. 1988). On the basis of new structural information, more current investigations can hardly support the hypothesis that protein macromolecules are placed within the aqueous channels of the liquid crystal structure. There are investigators who also question whether even smaller proteins could be freely located inside the water channels, which appeared to have a diameter of 30–40 Å (Angelova et al. 2000, 2003). For example, a geometrical consideration of the peptide leptin, which has a molecular weight (MW) of only 16 kDa, reveals that the extended state molecular dimensions of this rather small protein (44 × 32 × 31Å3) may exceed the internal pore diameter of the cubic diamond-type channel networks. Hence, it is irrational to suppose that the medium- (MW 48 kDa) and large-size (MW 145–340 kDa) proteins would be regularly located within the pores of the network structures. However, these proteins appeared to be confined within the networks, and despite the fact

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that the proteins are too large to be accommodated inside the pores, it was detected that they do not separate into macroscopic micellar aggregates in the excess water environment of the cubic phase (Angelova et al. 2003). Therefore, a new mechanism of protein entrapment within the structure’s liquid crystals (e.g., cubic media) has been proposed by Angelova et al., considering the bicontinuous cubic phase medium as a nanostructured assembly of cubosome particles (discrete domains of cubic structures). They presumed that the proteins were located at the interfaces between the cubosome nanoparticles (domains) in the system or associated with the surface of the interconnected cubosomal entities, owing to their surface activity and intermolecular interactions (Angelova et al. 2003). A recent report by Kraineva et al. (2007) regarding the confinement and stability of the small protein insulin (dimer: 2 × 5.7 kDa ∼30 Å × 40 Å; Figure 24.2) in GMO-based liquid crystals (cubic and lamellar) reinforces the entrapment mechanism concept suggested by Angelova et al. It is known that the biologically active form of insulin is the monomer. However, in solution, it forms monomers, dimers, tetramers, and hexamers in equilibrium conditions, depending on the pH, cosolvents, and concentration (Kraineva et al. 2007). The phase behavior of empty GMO/water systems and those loaded with 0.1, 0.25, 0.5, 1, 2, and 4 wt% insulin were studied at pH 1.9 where insulin forms

~40 Å Figure 24.2. Schematic representation of the dimeric insulin molecule (Kraineva et al. 2007).

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essentially dimers (Kraineva et al. 2007). Smallangle x-ray scattering (SAXS) measurements of the empty GMO/water system exhibited two phase transitions with increasing temperature: between a lamellar crystalline phase LC (lattice parameter (a) = 49.3 Å) and a lamellar liquid-crystalline phase Lα (a = 43.7 Å) at 22°C and a second one between Pn3m and Ia3d cubic phases at ∼20°C (Figure 24.3a). Upon incorporation of insulin, the lamellar phase was stabilized (instead of LC or Pn3m phases) with a similar range of lattice parameters as the protein-free Lα phase (43–44 Å, Figure 24.3b). Only at ∼31–35°C does a transformation into the cubic Ia3d phase occur. The authors presumed that addition of the protein slightly dehydrated the sample within the water channels, thus stabilizing the lamellar structure (Kraineva et al. 2007).

A further striking difference in the diffraction patterns of the GMO system with incorporated insulin was the appearance of additional broad peaks at lower scattering angles. The intensity of the reflections increased with increasing protein concentration; when insulin concentration was 4 wt%, reflections were well characterized and assigned to a cubic lipid structure with lower order induced by the protein (Pn3m cubic type-X, with main dspacing of ∼70 Å). Additionally, the water channel radius of the Ia3d phase, which was formed at higher temperatures (∼31–35°C), decreased slightly with increasing insulin concentration (9–10 Å). Given that the insulin dimer and monomer have radii of gyration (Rg) of 14.9 Å and 11.6 Å, respectively, the size of the water channels in Ia3d would hardly be compatible with the size of the monomer.

Figure 24.3. Small-angle x-ray scattering patterns and lattice constants a of the lamellar and cubic phases of pure GMO (a) and GMO loaded with 4% insulin (b) in 20 wt% water at pH 1.9 as a function of temperature (Kraineva et al. 2007).

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

Furthermore, the dimers could be accommodated only at the three-way junctions of the Ia3d labyrinth structure. Hence, the insulin was assumed to be located merely within the newly formed cubic structure X. Protein and peptide drugs are known to be inactivated by physical instability processes such as aggregation, precipitation, denaturation, and surface adsorption (Manning et al. 1989). The aggregation process resulting in protein/peptide precipitation has been a fundamental obstacle to the development of long-term delivery devices (Brange and Langkjaer 1993). Various classic approaches have been used to protect protein and peptide drugs from physical inactivation, one of which has been the use of stability-enhancing additives (Manning et al. 1989). In the case of insulin, surfactants, dicarboxylic amino acids, and phenol have been used to prevent its agitation-induced aggregation (Chawla et al. 1985). In addition, compounds such as polysaccharides, polyols, amino acids, and salts are known to stabilize proteins in solution by the excluded solute mechanism (Carpenter and Crowe 1988; Fágáin 1995). Sadhale and Shah examined GMO/water cubic phase for the protection of insulin from agitationinduced aggregation (1999a). Insulin was chosen as a model protein since it is known for its proclivity to aggregate and precipitate upon agitation and high temperature, leading to a drastic reduction in its biological potency (Sadhale and Shah 1999a). Furthermore, its precipitation process can be easily followed spectroscopically (Chawla et al. 1985; Brange and Langkjaer 1993). The protection against aggregation was studied by comparing GMO-based cubic phases (GMO/ insulin; 70/30 wt/wt) with three insulin solutions, Humulin®, Regular Iletin I®, and Regular Iletin II®, at a concentration of 30 U/gr subsequent to agitation at 100 oscillations/min (at 37°C). Aggregation of insulin was monitored as a function of an increase in optical density of the three systems and the three solutions at 600 nm. The optical density values were plotted against time to obtain aggregation profiles (Figure 24.4A–C; Sadhale and Shah 1999a). The authors showed a slow initial rise in optical density

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Figure 24.4. Aggregation profiles of the solutions: (A) Humulin®, (B) Regular Iletin I®, and (C) Regular Iletin II® at pH 7.4 (䊏), and in cubic phase containing the corresponding insulin solutions (䊉) agitated at 100 oscillations/minute at 37°C (Sadhale and Shah 1999a).

aggregation profile of each of the three solutions, due to an increase in turbidity resulting from the formation of increasing amounts of insulin aggregates. It was assumed that the initial slow rise was due to the

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time necessary to obtain stable fibril nuclei, which are able to initiate subsequent aggregation (Brange and Langkjaer 1993). When sufficient quantities of fibril nuclei were accumulated, there was an increase in the rate of aggregation, as seen by the rather sharp rise in optical density from the second day. This was followed by a steep rise that gradually ends in a plateau, indicating completion of the aggregation process (Figure 24.4A–C). In contrast, optical density of the cubic phases stayed around the initial low optical density value similar to that of a blank cubic phase (without insulin). This indicates that insulin had not aggregated in the cubic phase even upon agitation, implying that the cubic phase is able to protect insulin from agitation-induced aggregation at 37°C. Although the cubic liquid crystals appeared to have protected the insulin from agitation-induced aggregation, it was not clear whether the native conformation of insulin was still intact; therefore, the investigators further examined its secondary structure by circular dichroic (CD) spectroscopy. The CD spectra of insulin in solution and liquid crystalline cubic phase during the agitation study, compared to that of control insulin solution, are shown in Figure 24.4A–B. The CD spectrum of freshly prepared control insulin solution (Figure 24.5a–b) in the far UV region (185–250 nm) showed maxima at 196, 209, and 222 nm with ellipticity values crossing over to the negative side of the X-axis at around 204 nm. The maximum at 196 nm resulted from a contribution by the β-conformation in addition to the optical activity associated with other conformations, while the maxima at 209 and 222 nm were attributed to the α-helical structure (∼20–40%) in insulin (Sadhale and Shah 1999a). The CD spectra of insulin in agitated solutions confirmed the authors’ earlier observations regarding the aggregation behavior of insulin (Figure 24.5a, 1–7 days). On the third day a slight decrease in the magnitude of the three maxima was detected, indicating the beginning of the aggregation process, also seen by a rise in optical density at 600 nm (Figure 24.4). The further decrease in the magnitude of the maxima in the spectrum on the fourth day and the blue shift of the maximum at

196 nm and the X-axis crossover point was explained by the researchers as the beginning of the chain unfolding process and the loss of unfolded and aggregated insulin from solution. This was followed by a further decrease in the magnitude of the three maxima. The complete disappearance of the three maxima in the spectra on the sixth and seventh days was indicative of total loss of native conformation that could have led to aggregation and loss of insulin from solution (Figure 24.5a). The CD spectra of insulin in the liquid crystalline system that displayed the three maxima at 196, 209, and 222 nm (Figure 24.5b) demonstrated that insulin was unaffected by agitation. It should be noted that Sadhale and Shah also reported similar CD spectra obtained from insulin in cubic phase that had been agitated for 2 months (Sadhale and Shah 1999a). This research determined that GMO-based liquid crystal was able to protect the incorporated protein from undergoing conformational changes due to its exposure to water-air interfaces. Furthermore, the high viscosity of the structure that reduced the insulin mobility may have led to reduced collision frequencies, thus preventing aggregation by association. Sadhale and Shah (1999b) further studied whether this agitated insulin in solutions or entrapped in liquid crystal phase was still biologically active. Thus, the ability of insulin in the nonagitated, as well as agitated, cubic phase to lower blood glucose levels in vivo was tested in an additional study (Sadhale and Shah 1999b). Administration of both the nonagitated and the agitated insulin entrapped in the liquid crystalline structure resulted in lowering of blood glucose levels in rats. However, administration of the agitated insulin solution had no effect on the blood glucose levels (data not shown). These results evidenced that agitation led to a total loss of the activity of insulin in solution while insulin in the cubic phase was unaffected.

3. Barriers and Mechanisms of Bioavailability of Proteins and Peptides Ever since the seminal research of Ericsson et al. (1991) on the subject of cubic phases as delivery systems by intramuscular (parenteral) route for

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

50

(a) Day2 Day1 Day4 Day3 Day7 Day6 Control Insulin

40 30 Ellipticity (millidegrees)

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20 10 0 180

190

200

210

220

230

240

250

260

250

260

–10 –20 Wavelength (nanometers) –30 50

(b) Day2 Day1 Day4 Day3 Day7 Day6 Control Insulin

Ellipticity (millidegrees)

40 30 20 10 0 180

190

200

210

220

230

240

–10 –20

Wavelength (nanometers)

–30 Figure 24.5. Changes in the circular dichroic (CD) spectra of insulin in (a) solution and (b) cubic liquid crystal, compared with control insulin, when both were agitated at 100 oscillations/minute at 37°C for 7 days. The CD spectrum of control insulin (Humulin®) consists of three maxima at 196, 209, and 222 nm (Sadhale and Shah 1999a).

peptides, the study of liquid crystals as delivery systems has advanced the fields of oral and transdermal administrations (in vitro and in vivo reports) (Lee and Kellaway 2000b; Ericsson et al. 1991; Lopes et al. 2006a, 2006b). The primary progress was achieved in the field of transdermal delivery using cubic and hexagonal liquid crystalline phases (Lopes et al. 2006a, 2006b; Kim et al. 2008).

Topically administrated drugs must permeate the skin and penetrate into the deeper skin layer. The outermost layer of the skin, the stratum corneum, determines this barrier function (Wertz 2000; Trommer and Neubert 2006). There are two general methods for permeation of the stratum corneum by drug molecules: the transepidermal route and the route via pores (Figure 24.6; Trommer and Neubert

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Transepidermal intercellular

transcellular

Corneocytes

Via pores transglandular

transfollicular

Hair papilla Gland

Figure 24.6. Schematic illustration of drug penetration routes across the stratum corneum (Trommer and Neubert 2006).

2006). The transepidermal route can be divided into the transcellular and the intercellular routes. The more direct route is transcellular, where the drug has to cross the skin by directly passing through both the lipid structures of the stratum corneum and the cytoplasm of the dead keratinocytes. Although this is the shortest route, the substances encounter significant resistance to permeation due to the requirement for crossing both lipophilic and hydrophilic structures. The more common path for drugs to permeate the skin is the intercellular route, passing between the corneocytes (Hadgraft 2004). Recently, it was shown that follicular penetration (although occupying only 0.1% of the total skin surface) may also be an important pathway for the penetration of topically administered substances (Trommer and Neubert 2006). The follicular apparatus of hair follicles, the sweat glands, and microlesions in the interfollicular horny layer were introduced as theoretical vertical pathways for percutaneous penetration (Schaefer and Lademann 2001). Several compounds, that is, penetration enhancers, are known to promote active transporting across the skin barrier. The influence of penetration enhancers on both the lipophilic and the hydrophilic pathways of drug penetration is illustrated in Figure 24.7. One possibility for the variety of mechanisms for penetration enhancement is the interaction of the

enhancers with the polar headgroups of the lipids (Trommer and Neubert 2006). Thus, the lipid-lipid headgroup interactions and the packing order of the lipids are disturbed and the diffusion of the hydrophilic drug is facilitated (Walker and Smith 1996). Water is one of the most effective and safest penetration enhancers. By stratum corneum hydration, the penetration of most drugs can be increased. The water content in the stratum corneum is normally 5–10% and can be increased up to 50% under occlusive conditions (e.g., by use of impermeable foil or by application of occlusive vehicles) (Trommer and Neubert 2006). By the increased content of free water molecules between the bilayers, an augmentation of the cross-section for polar drug diffusion is obtained, which results in enhancement of drug penetration (Williams and Barry 2004). The headgroup disturbance of lipids by polar enhancer substances can also indirectly affect the hydrophobic parts of the lipids, lead to rearrangements in these bilayer areas, and thus improve lipophilic drug passage (Magnusson et al. 2001). Interactions of lipophilic enhancers with the hydrocarbon chains of the bilayer lipids may increase fluidization of the hydrocarbon chains resulting in packing order disturbance, thereby facilitating penetration of lipophilic drugs. These changes also influence the order of the polar headgroups, thus the penetration of hydrophilic drugs can be enhanced as well (Trommer and Neubert 2006).

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

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Polar headgroup interaction

Aqueous region Polar enhancers 1. Hydrophilic pathway

Skin penetration of drugs

Action of penetration enhancers

2. Lipophilic pathway

Hydrophobic region

Long chained, less polar enhancers Lipid chain interaction

Figure 24.7. Hydrophilic and lipophilic pathways of drug penetration and mode of action of penetration enhancers (Trommer and Neubert 2006).

Nevertheless, most of the chemical enhancers require suitable vehicles to reach the polar lipid parts and exert their functions. Lopes et al. (2006a) investigated cubic and hexagonal phases containing chemical enhancers as delivery vehicles for cutaneous penetration of cyclosporin A in vitro (using porcine ear skin) and in vivo (using hairless mice). Cyclosporin A (CysA) was selected as a model peptide since it is a cyclic and highly lipophilic peptide that presents extremely poor skin penetration, unless a chemical or physical strategy is used (Duncan et al. 1990; Lopes et al. 2005). Additionally, this peptide is an immune-suppressant agent and has therapeutic potential in the treatment of skin inflammatory disorders (Duncan et al. 1990). The cubic and hexagonal phases based on GMO/water and GMO/oleic acid/water, respectively, are well studied and were shown to have the ability to sustain the release of incorporated compounds (Bender et al. 2005; Lee and Kellaway 2000b; Borné et al. 2001). Moreover, each component constructing either the cubic or the hexagonal phase is a penetration enhancer by itself. GMO is known to promote

ceramide extraction and enhancement of lipid fluidity in the stratum corneum, and oleic acid is considered to increase epidermal permeability via a mechanism involving perturbation of the stratum corneum lipid bilayers and lacunae formation (Trommer and Neubert 2006). The penetration of CysA in the skin and its percutaneous delivery were assessed in an in vitro model of porcine ear skin using a Franz diffusion cell, with the stratum corneum facing the donor compartment and the dermis facing the receptor (Lopes et al. 2005, 2006a). After 3–12 hours, the skin was separated to the stratum corneum (SC), epidermis (E), and dermis (D); and each skin section was analyzed. The quantity of CysA detected in SC and in [E + D] was indicative of drug penetration into the skin, whereas the amount of drug in the receptor phase was indicative of its percutaneous delivery. The in vitro skin penetration and percutaneous delivery of the cubic and hexagonal liquid crystalline phases compared to the control formulation (olive oil) containing CysA is depicted in Figure 24.8. Incorporation of CysA in both liquid crystalline formulations

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Figure 24.8. Time course of in vitro skin penetration and percutaneous delivery of CysA incorporated in cubic and hexagonal liquid crystalline phases, compared to olive oil as a control formulation (Lopes et al. 2006a).

enhanced the in vitro penetration of the peptide in the skin, probably due to the action of monoolein (in combination with oleic acid in the case of hexagonal phase) as penetration enhancer (Carr et al. 1997). Indeed, monoolein has been demonstrated to be released from liquid crystalline phases and to influence the buccal and skin permeation of the incorporated compounds (Carr et al. 1997; Lee and Kellaway 2000a).

Several differences between the two liquid crystalline phases were detected (Figure 24.8). The cubic phase increased the penetration of CysA in the SC and [E + D] but did not influence the percutaneous delivery of the peptide compared with the control formulation. The hexagonal phase increased the penetration of CysA in the [E + D] as well as its percutaneous delivery. No enhancement of the peptide concentration in the SC was observed when the hexagonal phase was used, since the increased penetration of CysA was into deeper skin layers and through the skin. Whereas the cubic phase formulation favored retention of CysA in the skin in vitro, the hexagonal phase favored its penetration into deeper skin layers and its percutaneous delivery. The cubic and hexagonal phases used in this study differ in their internal structure and composition, since oleic acid was added (5%) to the hexagonal phase. Thus, the authors attribute the differences observed in the in vitro skin penetration of CysA with the difference of constituents of both phases. Addition of oleic acid, a penetration enhancer, which has already been shown to increase the skin absorption of several drugs, including peptides, resulted in greater CysA penetration into deeper skin layers and through this tissue (Williams and Barry 2004). Another study regarding membrane permeation in buccal delivery (oral delivery) showed that oleic acid was released from a system of cubic phase containing PEG 200 (5% or 10%) and the permeation of the incorporated peptide was enhanced (Lee and Kellaway 2000b). The researchers believe that the structure of the delivery system can also influence the skin penetration of CysA based on nonpeptidic compound tests (Gabboun et al. 2001). In this matter, the release of nonpeptidic compounds was shown to be influenced by the structure of the delivery system, drug/system interactions, and system physico-chemical characteristics (Gabboun et al. 2001). On the basis of FT-IR measurements (data not shown), similar interactions between CysA and monoolein were observed in both cubic and hexagonal phases; thus, they should not contribute to the differences in skin penetration. Alternatively, cubic and hexagonal phases present different internal structures, viscosities, and

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

Figure 24.9. In vivo skin penetration of CysA incorporated in cubic and hexagonal liquid crystalline phases, compared to olive oil as a control formulation (Lopes et al. 2006a).

drug-release properties (including for CysA) that might influence their interactions with SC and thus the delivery of CysA to the skin (Lopes et al. 2006a). The penetration of CysA, incorporated into the same three formulations, into the SC and [E + D] was further evaluated using an in vivo mouse model (Lopes et al. 2006a). Once more, the entrapment of CysA in the liquid crystalline phases led to increased skin penetration of the peptide in vivo at 6 hours postapplication (Figure 24.9). Even though the skin models used are different (mouse skin used in the in vivo experiment is more permeable than the porcine skin), the results obtained in vivo confirmed the in vitro observation that monoolein-based delivery systems enhanced skin penetration of CysA. Taking the in vitro and in vivo models together, Lopes et al. (2006a) concluded that the penetration enhancement that was caused by structural alteration of the SC can further induce changes in the deeper skin layers depending on the penetration enhancer used and its concentration. To obtain liquid crystalline systems that are expected to cause lower skin irritation with larger surface area to interact with the skin, higher fluidity,

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and ability to be incorporated into other product formulations, Lopes et al. (2006b) also tested the hexagonal dispersion. The penetration properties of the dispersion of the hexagonal phase discussed earlier were studied by dispersing this liquid crystalline phase in excess water in the presence of a dispersing agent (i.e., Poloxamer) (Gustafsson et al. 1996; Amar-Yuli et al. 2007). Dispersions of cubic phase have been suggested as suitable systems for percutaneous delivery of small molecules such as indomethacin (Esposito et al. 2005). Lopes et al. probed whether the hexagonal phase (GMO/oleic acid/water) nanodispersion had the ability to increase skin penetration of the relatively large peptide CysA. Its penetration in the skin and its percutaneous delivery were assessed in a similar in vitro procedure as discussed earlier regarding the hexagonal phase (Lopes et al. 2005, 2006a). Correspondingly, after 6–12 hours, the skin was separated into three sections and the quantity of CysA detected in SC and [E + D] was indicative of drug penetration in the skin, whereas the amount of drug in the receptor phase was indicative of its percutaneous delivery (Figure 24.10). Compared to the control formulation (olive oil), the hexagonal nanodispersion significantly enhanced skin penetration of CysA in the SC and [E + D] at 6 and 12 hours postapplication (Figure 24.10). Using the hexagonal phase nanodispersion revealed delivery of 7.12 and 3.85% of the applied dose/cm2 of CysA to the SC and [E + D], respectively, after 6 hours, and 13.10 and 5.06% of the applied dose/cm2 to the SC and [E + D] 12 hours after administration. The maximum concentrations were ∼two-fold greater than those obtained using the control formulation. It should be noted that in vivo skin penetration experiments were carried out as well and confirmed the in vitro observations. Similar penetration experiments of CysA were studied by Verma and Fahr (2004) using liposomes containing ethanol (10%). They observed delivery of 4.079% CysA of the applied dose/cm2 to the SC after 6 hours; 0.042% was detected in deeper skin layers. It can be concluded that the hexagonal dispersion provided a more efficient delivery of CysA, especially to deep skin layers than that delivered by other methods.

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Figure 24.11. The effect of drug radius of gyration on transport from the cubic phase of hydrated monoacylglycerol. Dependence of the aqueous diffusion coefficient (Daq) on the reciprocal of the radius of gyration. The line of best fit to the data is Daq = 9.58 (1/Rg) [R2 = 0.99], where Daq has units cm2/s (Table 24.1) (Clogston and Caffrey 2005).

Figure 24.10. In vitro penetration of CysA in the SC and [E + D] at 6 and 12 hours following its topical application using the hexagonal phase nanodispersion or the control (olive oil) formulation. SC = stratum corneum, [E + D] = epidermis (without SC) plus dermis (Lopes et al. 2006b).

Clogston and Caffrey (2005) believed that the properties of the liquid crystalline structures or the nature of the entrapped molecule can be tuned to regulate the rate of drug release. They took a systematic approach toward understanding how cubic phase transport is controlled by phase identity and microstructure and by the size, shape, and charge of the drug itself. The size of the drug ranged from a single amino acid to a larger molecule as a multisubunit protein consisting of 4,176 residues and a 7 μm-long stretch of nuclear DNA. For a given cubic phase, after loading with a size-range series of drugs (including peptides and proteins, Table 24.1), the release of each drug was measured by a Fickian diffusion model (Clogston and Caffrey 2005). For reference, the diffusion characteristics of all of the additives, with the exception of the 10-mer and nuclear DNA, were measured in

the absence of the cubic phase. The corresponding release data yielded diffusion coefficients (Daq) that are included in Table 24.1. Given that Daq is reciprocally related to the radius of the diffusing species, one can expect that Daq will decrease as the size of the additive molecule increases (strict for spherical molecules). The linear dependence of Daq on 1/Rg is evident, as shown in Figure 24.11. In the case of small molecules, such as rubipy, tryptophan, and to some extent the 10-mer DNA linker, release from the cubic phase occurred over a period of 1–3 days. For intermediate-sized molecules, such as cytochrome C, lysozyme, and myoglobin, the bulk of the release happened in a week. In contrast, for the large macromolecules ovalbumin, conalbumin, apo-ferritin, and calf-thymus DNA, less than 12% release took place in a period of 3 weeks. When the same release experiment was repeated, this time with the additive dissolved in the solvent creating the aqueous channels of the cubic phase, the release profiles were dramatically slower. For example, in the case of myoglobin, only 60% of the additive had been released during the first week. It was apparent that the additive size and shape had an impact on the transport rates. Some of the molecules are spherical while others are distinctly rod-shaped as with ovalbumin, conalbumin, and the two DNA samples. For molecules that are free to tumble, the transport was dependent on the effective size described by Rg relative to the diameter of the

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

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Table 24.1. Molecular and transport characteristics in bulk aqueous medium of the additive molecules.a Additive

PDB identification code

Tryptophan His6Trp Rubipy 10-mer Cytochrome c Lysozyme Myoglobin Ovalbumin Conalbumin Apo-ferritin DNA

— — — 1DNT 1HRC 1BWI 1YMB 1OVA 1AIV 1MFR —

MW (kDa) 0.204 1.03 0.585 3.02 11.7 14.4 15.3 42.7 75.8 460 6164

Dimensions (Å) — — sphere, 12 24 × 24 × 34 sphere, 34 23 × 31 × 44 25 × 44 × 44 43 × 44 × 74 55 × 70 × 106 sphere, 120 24 × 24 × > 66,000

Rg (Å)

Daq(× 107 cm2/s)

4.3 — 4.7 18.6 13.2 15.2 16.3 22.9 30.7 46.5 —

21.8 (4.4) — 21.2 (–) — 7.0 (0.7) 5.4 (1.2) 5.3 (0.1) 3.6 (0.7) 3.5 (0.3) 2.4 (–) —

Source: Clogston and Caffrey 2005. Standard deviations are shown in parentheses.

a

aqueous channel (dw). Surprisingly, in the case of apo-ferritin with a diameter of 107 Å, entrapped in a cubic phase with dw = 73 Å, while the release was slow, it was observed (data not shown). The authors explained this contradiction on the basis that the cubic phase is a liquid crystal with inherent flexibility. The amplitude of this flexible motion, which amounts to a molecular breathing or peristalsis, is sufficient to create transient and random sections of channels that are large enough for the oversized additive to pass through. Assuming that transport is through the aqueous channels, probably the large molecule snakes its way through the channel network and eventually into the sink compartment (Clogston and Caffrey 2005). Another possible reason for retention, raised by Clogston and Caffrey (2005), concerns the fact that the cubic phase is composed of randomly oriented domains with a cubic symmetry. It is not clear what envelops each domain and what is at the interface between adjacent domains. If domains are considered to be surrounded by one or several continuous bilayers, then transfer between domains is expected to be very slow involving the breaching of at least one bilayer. Alternatively, if the domains are locally connected by a highly distorted “cubic phase” (e.g., sponge phase), transport between domains might be retarded by the unusual properties of the bilayer and/ or the aqueous channels.

The effect of the aqueous channel’s size on the rate of release was stressed by testing three monoacylglycerols—monoolein (labeled as 9.9 MAG), monopalmitolein (9.7 MAG), and monovaccenin (11.7 MAG)—producing Pn3m cubic phase with full hydration at 20°C. The 9.9, 11.7, and 9.7 MAG produced cubic phases under these conditions with channel diameters of 47, 66, and 60 Å, respectively. The impact of the water channels on the rate of transfer was more pronounced on the diffusion characteristics of a macromolecule whose size approached that of the channel. Biochemical enhancers are a novel approach to increasing skin permeability for transdermal drug delivery, and the use of pore-forming peptide to increase transdermal transport is limited to just a small number of studies (Chen et al. 2006). The following recent and novel study by Kim et al. provides insight into the mechanism by which magainin peptides increase skin permeability. Moreover, their revealed transdermal-pH dependence offers the opportunity to modulate or trigger transdermal delivery rates by increasing or decreasing pH (Kim et al. 2008). Magainin has a net charge up to +4 and binds to negatively charged lipid membranes by way of electrostatic interactions. Its secondary structure is not well defined in a neutral aqueous environment; however, it forms an α-helical structure when

Part 4 Recent Advances in Bioactive Peptide Analysis for Food Application

adsorbed onto a negatively charged membrane, such as the surface of a bacterium (Matsuzaki et al. 1997). Magainins can then self-assemble into transmembrane pores that make the cell membrane leaky and can also lead to cell lysis, particularly in bacterial cells. The size of pores formed by magainins in lipid bilayers is estimated to be approximately 1 nm in diameter (Matsuzaki et al. 1994; Matsuzaki 1998). It was shown that magainin alone had no effect on skin permeability, probably because the relatively large magainin molecule had difficulty penetrating throughout the stratum corneum to make continuous transdermal pathways. However, when delivered from a formulation including an anionic surfactant, N-lauroyl sarcosine (NLS), in a 50% ethanol–phosphate buffer saline (PBS) solution, skin permeability was increased and thereby magainin penetration throughout the SC was facilitated (Kim et al. 2007). This enhancement was accompanied by increased SC lipid fluidity, as shown through differential scanning calorimetry (DSC), infrared spectroscopy, and x-ray diffraction measurements (Kim et al. 2007). To investigate the role of interactions between magainin and a drug as it diffuses through magaininmediated pathways in the SC, Kim et al. have used two model drugs—fluorescein and granisetron (Figure 24.12, up and down, respectively)—which are of similar molecular weight but carry opposite charge and changed the magainin charge state by changing pH. Measurements of skin permeability to fluorescein over a pH range of 7.4–11, where the charge of the skin, NLS surfactant, and fluorescein remain strongly negative, were carried out. However, magainin has an isoelectric point at pH 10.5, such that magainin changes from a + 2 positive charge at pH 7.4 to a neutral charge at pH 10.5, and a negative charge at pH 11 (Skoog and Wichman 1986; Kim et al. 2008). Transdermal permeation of fluorescein after treatment with magainin and NLS was increased by a factor of 35 (i.e., from an average of 0.037 to 1.302 μg of fluorescein) at pH 7.4, as shown in Figure 24.13 (Kim et al. 2008). However, raising the pH and thereby reducing magainin’s charge progressively removed magainin’s enhancement until

HO

OH

O

O

O Me N N S

H N N O

Me

R

Figure 24.12. Chemical structures of (up) fluorescein and (down) granisetron (Kim et al. 2008).

Fluorescein enhancement ratio

374

40 30 20 10 0

7.4

8

9 pH

10

11

Figure 24.13. Enhancement of transdermal fluorescein delivery as a function of pH. Skin was pretreated with NLS (䊏) or magainin + NLS (䊐) in 50% ethanol. Enhancement ratio represents the increase in transdermal fluorescein transported across skin over 5 hours at various pH values compared to delivery under identical conditions using a formulation of fluorescein in PBS (Kim et al. 2008).

pH 11. It was suggested that a positively charged magainin facilitated transdermal transport of negatively charged fluorescein due to electrostatic attraction at pH 7.4, but with increasing pH, as the

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

attraction decreased, the skin permeability enhancement decreased as well. Additional insight into the flux data presented in Figure 24.13 was obtained using multiphoton excitation microscopy, at various pH values, by imaging the amount of fluorescein delivered into the skin. Consistent with the flux data, optical sections through the epidermis show that the smallest amount of fluorescein was seen in the skin at pH 10, when the magainin charge was almost neutralized (Figure 24.14B); a greater amount was seen at pH 7.4, when magainin carried a + 2 charge that attracted nega-

375

tively charged fluorescein (Figure 24.14A). Z-stack images showing cross-sectional views of the epidermis in Figure 24.14C and D provide similar, complimentary results (Kim et al. 2008). In addition, CD measurements demonstrated that pH did not change the secondary structure of magainin in solution; microscopy indicated that pH did not affect the magainin content in the SC; and FTIR showed that pH did not affect lipid order, except at the lowest pH. These data are consistent with the proposed hypothesis that electrostatic forces between magainin

(A)

(B)

0 μm

5 μm

10 μm

15 μm

20 μm

26 μm

30 μm

35 μm

40 μm

Depth below skin surface (C)

(D)

Figure 24.14. Penetration of fluorescein into human epidermis imaged by multiphoton microscopy. Skin was treated with magainin + NLS. Fluorescein was delivered to skin for 5 hours at (A and C) pH 7.4, (B and D) pH 10. (A and B) Optical sections were taken at 5 μm increments starting at the stratum corneum surface on the left and proceeding deeper on the right. Scale bar is 100 μm. (C and D) Cross-sectional images were reconstructed as z-stacks with the stratum corneum surface on top and deeper tissue below. Scale bar is 20 μm (Kim et al. 2008).

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peptides and drugs mediate drug transport across the skin. The authors suggested that these electrostatic interactions occur between magainin loaded into the SC and drug molecules diffusing through transport pathways created by the magainin. However, they do not believe that magainin and drug molecules bind and diffuse together through the SC for several reasons. First, magainins were added as a pretreatment and then removed from the donor compartment of the diffusion cell before fluorescein or granisetron was added. Thus, there was no opportunity for magainin-drug binding in the donor compartment. Second, magainin was shown to be localized in the stratum corneum (i.e., the upper 10 μm of skin), whereas fluorescein was shown to penetrate deeply across the epidermis (i.e., > 40 μm). Furthermore, fluorescein and granisetron were revealed to be diffused across the full epidermis and into the receiver compartment of the diffusion cell. Finally, fluorescein has a molecular weight of 332 Da, and magainin has a molecular weight of 2,504 Da. Thus, a fluorescein-magainin complex would have a molecular weight of at least 2,836 Da. Magainin’s large size precludes it from diffusing across skin to an appreciable extent; it can only penetrate and remain within SC lipid bilayers due to its special physico-chemical properties as a pore-forming peptide. Given the large size of a fluorescein-magainin complex, it is unlikely that such a large complex would readily cross the skin. The transdermal studies conducted to date are still lacking information concerning the mode of absorption and distribution in the skin. A study by Bender et al. aimed to investigate distribution of a hydrophilic fluorescent model drug sulphorhodamine B (SRB; Figure 24.15) by visualizing its uptake in full-thickness human skin using four delivery systems (Bender et al. 2008). The delivery vehicles applied were two bicontinuous lipid cubic systems consisting of either GMO or phytantriol (PT) and water. Water and a commercial ointment have been used as reference vehicles for comparison formulations with cubic internal structures to an emulsion with discrete micrometer-sized aggregates (ointment) and lack the internal order (water). The formulations were applied on full-thickness human

SO 3H

–

Et 2N

O3 S

O+

NET2

Figure 24.15. The chemical structure of sulphorhodamine B (Bender et al. 2008).

skin (during 24 hours) and thereafter were investigated using two-photon microscopy (TPM). Twophoton excitation is an outcome of a nonlinear excitation process through simultaneous absorption of two photons, implying that near infrared light (NIR) can be used for excitation of fluorophores with fluorescence in the visible range of light (Denk et al. 1990). Since the NIR wavelengths lie in the so-called “optical window” of biological tissue, TPM enables imaging of fluorophores much deeper into highly light scattering and light absorbing tissue compared to confocal laser scanning microscopy, with minimal photo-bleaching and phototoxic effects (Cahalan et al. 2002; Zipfel et al. 2003). The in vitro distribution of SRB that has been visualized at different depths in human skin using TPM is illustrated in Figure 24.16. Variations in distribution patterns were observed and evaluated for each vehicle as seen in Table 24.2. In general, the observed SRB fluorescence was located in the intercellular spaces, creating a characteristic fluorescent pattern of polygonal structures surrounding the cells with a diameter of 30–50 μm. At the skin surface, nucleated keratinocytes in SC were observed as thin transparent sheets. The observed fluorescence within the keratinocytes implies that SRB had penetrated into the cytoplasm of the cells but not into

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

377

A

B

C

D

Figure 24.16. Two-photon fluorescence images showing the lateral distribution of sulphorhodamine B after 24 hours of passive diffusion in human skin using (A) a water vehicle, (B) commercial ointment, (C) GMO cubic phase, and (D) phytantriol cubic phase. The image depths (from left to right) are 0, 4, 8, and 12 μm. Each image plane corresponds to an area of 323 × 323 μm (Bender et al. 2008).

the nuclei. TPM revealed that the fluorescence distribution was different when comparing the traditional commercial ointment and water with the bicontinuous cubic phases. In the case of cubic phases, SRB was found to be located mainly in

microfissures and in threadlike structures. It should be noted that in a former study by the authors, the cubic vehicles were superior to commercial ointment for topical drug delivery of hydrophilic substances in vivo (Bender et al. 2005). Thus they assumed that

378

Table 24.2. Summary of fluorescent features observed in two-photon microscopy images at various tissue depths of human skin exposed to sulphorhodamine B using different vehicles. Vehicle: Depth (micrometer) Desquamated cells at skin surface Vehicle visible at skin surface

Water 0–5

6–10

Ointment

11–20 21–30 >30

6–10

11–20 21–30

>30

0–5

6–10

11–20 21–30

Phytantriol cubic phase >30

0–5

15/15

15/15

6/15

0

0

11/15

14/15

15/15

Polygonal cells

14/15 15/15

13/15

Cell nuclei

13/15 15/15

13/15

0

Micro-fissures

9/15

9/15

9/15

7/15

0

0

0

0

Threadlike structures

0–5

Monoolein cubic phase

Source: Bender et al. 2008.

3/15

0

15/15 15/15

0

13/15 13/15

3/15

0

4/15 14/15 14/15

14/15

13/15

0

0

0

5/15

5/15

3/15

0

0

6/15

11/15

0

5/15

11/15

5/15

0

7/15 14/15 14/15

14/15

13/15

9/15

0

0

12/15 12/15

6/15

0

6–10

0

14/15 14/15

0

5/15

11–20 21–30

4/15

>30

0

0

5/15

3/15

12/15 15/15 15/15

15/15

13/15

11/15

6/15

0

0

0

12/15 12/15

Chapter 24 Controlled Release and Delivery Technology of Biologically Active Proteins and Peptides

the intercluster penetration pathway was preferable for delivery of hydrophilic compounds. The commercial ointment and cubic phases were found to reside in skin wrinkles due to high viscosity and water insolubility of the formulations. Cubic phases, known to co-exist with excess water, can therefore resist washing with water or PBS. Moreover, their rheological characterization has revealed elastic rather than viscous behavior (Engström et al. 1992). The microfissures reported in the present study were approximately 5 μm wide and had an irregular and entangled structure. The intercellular pathway seems to be predominant when using the water vehicle and ointment, while the intercluster pathway seems to dominate the skin absorption from the cubic phases. The microfissures were not visible when performing TPM of skin autofluorescence (Paoli et al. 2008); however, these structures were expected to be present as a microscopic clustering of keratinocytes in normal skin. The elastic lipid cubic phases seemed to be able to penetrate deep into these preexisting microfissures, giving rise to the observed fluorescence pattern. Even if the microfissures become narrower further into the skin, SRB fluorescence was detectable deeper than 30 μm in about 50% of the GMO cubic and approximately 70% of the PT cubic phases. Thus, this route contributed to a more efficient delivery of hydrophilic substances using the bicontinuous cubic samples as drug delivery vehicles. Another obvious difference that was obtained by the cubic formulations, compared to water, was the thin threadlike structures that presented down to a depth of approximately 10 μm in the former. The authors interpreted this pattern as arising from lateral diffusion of the formulation into the lipid matrix between the cell layers, which can be explained by an interaction between the lipid bilayers of the formulation and the cellular lipid matrix. The GMO cubic phase, compared to the PT cubic phase, penetrated the lipid matrix in a finer and more homogenously distributed fluorescence pattern, implying different lipid bilayers—lipid matrix interactions between the cubic phase and the skin. This may be due to the different chain mobility of the oleoyl chains (GMO) and the phytanyl chains (PT).

379

4. Conclusion With the advancement in technology and formulation knowledge, a number of approaches have been used to overcome the limitations regarding proteins and peptides. Various liquid crystalline– based delivery systems have been developed for the delivery of proteins and peptides. These delivery systems were found to be efficient in incorporating large quantities of protein and peptides and protecting them from physical and enzymatic degradation. Additionally the liquid crystalline–based systems could contain permeation enhancers for achieving improved bioavailability and were able to sustain and control their delivery. It was demonstrated that the properties of the liquid crystalline structures can be tuned to regulate the rate of drug release. Additionally the additive size and shape had an impact on the transport rates (Daq was directly correlated to the size of the additive). A novel approach to increase skin permeability for transdermal delivery utilizing pore-forming peptides was introduced. Furthermore, their revealed transdermal-pH dependence can provide the opportunity to modulate or trigger transdermal delivery rates by altering the pH values. A recent investigation regarding distribution of hydrophilic fluorescent model drugs in fullthickness human skin was demonstrated.

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Index

A A beta protein precursor, 88t AB192, 318 ABriPP, 88t ABTS, radical-scavenging property of peptides and colorimetric binding assays for, 35 ACC phosphorylation, satiety, high-protein diets and, 143 ACE blood pressure regulation and, 207, 228, 256–257 first competitive inhibitors to, 170 forms of, 211 inhibition by peptides in vitro, derived from fish proteins, 208, 211 in vivo, derived from fish proteins, 212–214 structure-activity correlations among different peptide inhibitors of, 212 wheat peptide, prevention of hypertension and inhibition of, 298–299 ACE homologue (ACEH), 211 ACE inhibition mechanism, 211–212 ACE inhibitors, 6 blood pressure regulation and, 169–170 prototypes of, 207 soy proteins as, 80 synthetic, side effects with, 208

ACE inhibitory activity, bitterness and, 348–349 ACE inhibitory peptides blood pressure regulation and, 174, 175 design of, 44–46 in egg white albumin, 256 evaluating antihypertensive activity of, in spontaneously hypertensive rats, 173 from fish proteins, 207–215, 208 inhibition of renin-angiotensin system by, 46–47 from natural sources, potency of, 208 preparation of, 171–173 enzymatic hydrolysis, 171 fermentation products, 171–173 Acesulfame K, 351 Acid hydrolysis, fish gelatin and, 205 Actin, in myofibrillar tissue proteins, 204 Actinobacillus actinomycetemcomitans, lactoferrin and, 186 Actinomucor elegans peptidases, nonbitter protein hydrolysates and, 350 Activated carbon, debittering protein hydrolysates and, 349 Activator protein-1, 19

Acute hyperammonemia, GABA and prevention of, 125 Acyl-CoA oxidase, soy protein feeding in rats and, 73 ADanPP, 88t Adenovirus lactoferrin’s effects against, 185, 186 Lfcin’s antiviral activity against, 192 Adibi, S. A., 265 Adipocytes, inflammatory responses and, 71 Adipokines, 70 Adiponectin, 70, 71, 76 Adipose tissue, metabolic syndrome and, 70 Adiposity, soy protein action on, proposed molecular mechanism, 73 Adiposity reduction, soy protein’s effect on, 71, 72t, 73–74, 77–78t Advanced glycation end products, 227 Aerobic organisms, biochemical antioxidants and, 16 Affinity peptidomics, 308, 308–309 AGEs. See Advanced glycation end products Aggregation, protein and peptide drug inactivation with, 365 Aggregation profiles, of Humulin, Regular Iletin I, and Regular Iletin II, 365 383

384

Index

Aging process antioxidant-prooxidant balance and, 30 protein carbonyl compounds and, 29, 30 Agouti related peptide, 143 Akahane, Y., 213t Akpaffiong, M. J., 49 Alamethicin, liquid crystalline phases and, 363 Alanine, sweet taste of, 343 Alaska pollack ACE inhibitory peptides derived from, 208, 210t antioxidant peptides from, 217t antioxidative activity in gelatin extracted from, 218 branched amino acids present in antioxidant peptides from, 216 products of proteolytic digestion of gelatin extracts from, 211 Albumin, 247, 250 water-soluble, 290 wheat, 291 Alcalase, 154 Alcalase hydrolysates, 275 Alcalase-hydrolyzed zein, antioxidant activity of, 216 Alcohol consumption, reactive oxygen species and, 16 Aldehyde interactions between amino acids, flavor ingredients and, 354 orthonasal perception of aroma intensity and, 354 Aleurone layer bran, 233 rice kernel, 234 Alkali extraction, of rice bran proteins, 236 Alkaline hydrolysis, fish gelatin and, 205 Allergen epitope identification, 106–107 B-cell epitope mapping, 107 T-cell epitope mapping, 106–107

Allergens, hidden, 107 Allergic response clinical symptoms related to, 102 to food antigens: two-phase mechanism, 103 intestine mucosal barrier and, 102–103 T cells and, 105 Allergies. See also Food allergy; Wheat allergy defined, 295 lactoferrin’s anti-inflammatory properties and, 187 wheat, 295–298 Almeida, M. S., 282, 283 Alpha-amylase inhibitors, families of, in wheat, 298 Alpha blockers, 169 Alpha-chymotrypsin, nonbitter protein hydrolysates and, 350 Alpha-cyclodextrin, bitterness masked with, 350 Alpha-helical class of proteins and peptides, 361 Alpha-lactalbumin in bovine colostrum and milk, 153t putative biological functions of, 151 whey proteins and, 156–157 Alpha-synuclein, 92, 93t ALPMHIR, 162 Aluko, R. E., 62, 275, 280 Alzheimer’s disease, 89, 90 advanced glycation end products and, 227 amyloid beta peptide and, 32 amyloid beta protein and, 91 amyloid deposits and, 89, 95 amyloidosis and, 7 excessive levels of calmodulin and, 55 free radical attacks and, 29 nitric oxide levels and, 56 peptidomics and, 319 Amarowicz, R., 217t Amber wheat, 290 Ameal S, 163t, 170, 171t

Amino acids. See also Free amino acids antioxidant, endogenous, 29 antioxidant behavior of peptides and, 216 in eggs, 248, 249 in fish gelatins, 206 in fish protein, 204 flavor ingredients interacting with, 352 plasma, as central satiety signals, 142–143 plethora of research on, 354 potent antioxidant activity with, 17 residues of antioxidant peptides and, 35 aromatic, 45 design of ACE inhibitory peptides and, 44–45 flaxseed protein-derived peptides and, 58 as metal-ion binders, 37 in rice bran proteins, 240t serum essential amino acid levels in peripheral blood vessel after ingestion of test beverages, 266 sulfur-containing, in cereal grain proteins, 234 taste-active, development of, 355 taste-active properties of, 343–344 primary taste characteristics of, 343–344 synergistic interactions affecting taste and flavor, 344 umami taste and, 342 Aminopeptidases, reducing bitterness of protein hydrolysates and, 350 AMP-Activated Protein Kinase (AMPK), 143 Amylase, rice bran protein extraction and, 236 Amylin, 91 Amyloid beta peptide, 32 Amyloid beta protein, 88–89, 91, 93t

Index

Amyloid formation diseases associated with, 7 inhibitors, 95 preventing, 92, 94–95 Amyloidogenic proteins and peptides, 7, 87–95, 93t amyloidogenicity of small peptides, 92 natural occurrences of, 90–92 alpha-synuclein, 92 amylin, 91 amyloid beta protein, 91 beta2-microglobulin, 90 cystatin C, 90–91 huntingtin protein, 92 prion, 90 strategies for preventing amyloid formation, 92, 94–95 peptidic compounds, 94–95 phenolic compounds, 92, 94 treharose, 95 Amyloidosis, 88, 95 cutaneous, 87 defined, 89–90 diseases associated with, 7 localized, 90 systemic, 87, 90 understanding molecular basis of, 89 Amyloids defined, 89–90 precursor and names of, 88t Anaphylaxis, allergic response and, 102 Anderson, G. H., 138., 141 Anderson, J. W., 74, 77t Ando, Y., 88t Angelova, A., 363 Angiogenesis, bLf and hLf’s opposite effects on, 189–190 Angiotensin I, 207, 257 Angiotensin I-converting enzyme, inhibitory effects of, 280–281 Angiotensin II, 207, 257 Ang I, 44 Ang II, 44 Ang II-stimulation, effect of small peptides on, 47, 48

Aniline-Benzoic Acid Labeling (AniBAL), 316 Animal feeds lactoperoxidase in, 160 rice bran proteins in, 239 Animal muscle-based bioactive peptides, 225–229 biological activity and therapeutic applications, 226–228 antiaging effects, 228 antiglycation effects, 227 antihypertensive and cardiovascular effects, 227–228 anti-inflammatory and immune-modulating activity, 227 antioxidative activity, 226 neurological effects, 228 wound healing, 228 description of, 225–226 future studies on, 228–229 Animal proteins, bioactive proteins released from, 332 Animals, role of GABA in, 124–125 Animal sources of foods, 105 ANS (8-anilino-1-naphthalene sulfonic acid), defined, pea protein-derived peptides and, 63 Anserine, 225 in animal muscle, 225 antioxidative activity of, 30, 31t, 226 in chicken meat, 226 dose-dependent ACE-inhibitory activities of, 228 neurological effects of, 228 Anterior piriform cortex, detection of high-protein meals and, 145 Antiaging effects, of muscle-based bioactive peptides, 228 Antiamyloid agents, development of, 7 Anticancer peptides, in egg protein, 255 Antidepressant agents, GABA receptors and, 124

385

Antigen-presenting cells, 103 Antihypertensive effects, foods with specified health use and, 170–171 Antihypertensive food products, development of, 6 Antihypertensive peptides, 8, 43–52, 169 antihypertensive mechanisms of small peptides, 46–51 inhibition of renin-angiotensin system by ACE inhibitory peptides, 46–47 regulation of vascular events by dipeptides, 47–48 relaxation of vascular constrictive events by dipeptides, 48–51 derivation of, from caseins by proteolytic action, 172t design of ACE inhibitory peptides, 44–46 docking of, into ACE C-terminal and validation of computational modeling with experimental inhibition of peptides, 81 in egg protein, 256–258 in FOSHU products, 44t, 171t future prospects, 51–52 hypertension and renin-angiotensin system, 44 mode of action ACE inhibitory effects, 174 peptide absorption, 174 reported, from natural proteins in spontaneously hypertensive rats, 46t screening of vasorelaxant peptides of 50 mmol/L KCI-constricted aortic ring, 50t in vivo effect, 173–174 clinical trials, 173–174 effects on an animal model, 173 Antihypertensive pharmaceutical products, categories of, 169

386

Index

Anti-inflammatory proteins and peptides, 19, 20t, 21–23 egg proteins lysozyme, 19, 20t ovotransferrin, 19, 20t milk peptides, 19, 20t, 21–22 bovine casein, 19, 20t, 21 glycomacropeptide-κcaseinoglycopeptides, 20t, 21 lactoferrin, 20t, 21–22 proteose peptone-3, 20t, 22 plant proteins/herbal medicine, 23 soy, 22–23 Antimicrobial proteins and peptides, 169 alpha-helical motifs for, 184 from chickpea (Cicer arietinum), 283 from cowpea (Vigna unguiculata), 282–283 in eggs, 249t from garden pea (Pisum sativum), 281–282 from lysozyme, 251–252 from ovalbumin, 252–253 from pea, commercial utilization potential of, 283–284 Antioxidant proteins and peptides, 16–17, 29–39 action mechanisms of, 35–38 chemical reactions, 35–37 physical reactions, 37–38 activity of, in food systems, 34–35 antioxidant activity of in vitro protein digests, 32–34 chemical and physical mechanisms of: metal chelation, radical scavenging, and physical hindrance, 35 dietary, identifying fate of, 6 in eggs, 255–256 endogenous, 30–32 enzymatic oxidation, 214–215 from fish proteins, 214–215, 217t human health promoted with, 30 mechanism of lipid oxidation, 214 preparation of, 38–39 chemical synthesis, 39 enzymatic production, 38 microbial fermentation, 38–39

structure-function relationship of, 35–36 Antioxidants natural, 215 synthetic, 215 Antioxidant supplements, expanding market for, 30 Antioxidative peptides, with less bitterness, 349 Antioxidative stress food factors, 16 Antioxidative stress mechanisms, endogenous, 16 Antioxidative stress proteins and peptides, 16, 17–18, 17t egg yolk peptides, 17–18 milk protein, 18 plant proteins/herbal medicines, 18 Antitumor activities, lactoferrin and, 189–191 Aoyama, T., 268 AP-1. See Activator protein-1 APCs. See Antigen-presenting cells Apolipo-protein AI, 88t, 93t Apolipo-protein AII, 88t Apolipo-protein AIV, 88t Apoptosis in cancerous cells, lactoferrin’s promotion of, 190 Apo serum AA, 88t Aptamers, 313 Aqueous alcholol-soluble prolamin, 290 Aqueous channel size, drug release rate and, 373 Arachidonic acid production, soy protein and, 76 Arcan, I., 274 Ardo, Y., 349 Area postrema, detection of high-protein meals and, 145 Arginine ACE inhibitory activity, bitterness and, 349 blood pressure and, 80 hypocholesterolemic effects of pea proteins and levels of, 277 Arietin, 283 Ariyoshi, Y., 209t

Aroma perception, research on chemosensory stimuli and, 354 Aromatic amino acids, 274 Arthritis colostrum-based products with growth factors and, 161 lactoferrin’s anti-inflammatory properties and, 187 Artificial sweeteners, 342, 351 Ascorbic acid, 215 Ash, in bran, 233 Ashar, M. N., 162 Asia, rice production in, 233 Asparagines, in rice bran proteins, 238 Aspartame, 351, 355 Aspartic acid, 37 Aspergillus niger, pea protesase inhibitors expressed with, 279 Astawan, M., 209t, 210t, 213t Atherosclerosis advanced glycation end products and, 227 free radical attacks and, 29 reactive oxygen metabolites and, 226 Atlantic cod, serine collagenases isolated from, 205 Atlantic salmon, ACE inhibitory peptides derived from, 210t Atrial natriuretic factor, 88t Atwater factor, for protein, 142 Autistic spectrum disorders, carnosine supplementation and, 228 Avian eggs, 247 Avian eggshell, unique mechanical properties of, 249 Avidin anticancer properties of, 255 antimicrobial properties of, 254 Azadbakht, L., 68 Azeotropic extraction, debittering protein hydrolysates and, 349 Aziz, A., 141 Azzout-Marniche, D., 142

Index

B Baby foods, rice bran proteins in, 239 Bacillus cereus, ovotransferrin’s effect against, 252 Bacillus stearothermophilus lactoferrin’s antimicrobial activities and, 185 lysozyme effective against, 250 Bacillus subtilis, lactoferrin’s antimicrobial activities and, 185 Bacteria, LfcinB and LfcinH actions against, 191 Bacterial adhesions, lactoferrin binding to, 186 Bacterial GAD, 122 Bacterial infections, lactoferrin and, 187 Bacteriocins, applications for, 240 Bacteriorhodopsin, 361 Badii, F., 206 Baker’s asthma, 295, 296, 299 Bakery products, rice bran proteins in, 239 Balenine, antioxidant activity and, 30, 31t Balog, C. I., 318 Barley, rice protein net protein utilization compared to, 240 Baron, F., 249t Bar-Or, D., 37t Basmati rice, 237 Bassil, M. S., 143 BAT. See Brown adipose tissue Baumann, M., 88t Bay K 8644-stimulation, effect of small peptides on, 48, 48 BBIs. See Bowman-Birk protease inhibitors B-cell epitope-based immunotherapy (IgG-binding), food allergy and, 110 B-cell epitopes antigen, representation of, 106 food allergen, 105–106 mapping, 107

Beard, rice kernel, 234 Beef allergy model, 114 Beef gelatin, producing gelatin from fish to match gelling properties of, 219 Beer production, lysozyme used in, 251 Behavior, peptides and regulation of, 320 Bellamy, W., 183 Bender, J., 376 Benditt, E. P., 88t Beni-koji making, GABA content and, 126 Bensaid, A., 144 Benson, M. D., 88t Bergstrom, J., 88t Bertenshaw, E. J., 136 Berthoud, H. R., 144 Beta blockers, 169 Beta-carotene, 215 Beta cellulin, in bovine mammary secretions, 152, 160–161 Beta-conglycinin, 17 Beta-glucans, 233 Beta-hydroxyacyl-CoA dehydrogenase, soy protein feeding in rats and, 73 Beta-immunoglobulin peptide, health benefits with, 162 Beta-lactoglobulin antioxidant activity and, 34 in bovine colostrum and milk, 153t putative biological functions of, 151 whey proteins and, 157 Beta-lactorphin, health benefits with, 162 Beta-lactosin B peptide, antihypertensive activity with, 162 Beta-sheet class of proteins and peptides, 361 Beta2-microglobulin, 88t, 90, 93t BHA. See Butylated hydroxyanisole BHT. See Butylated hydroxytoluene Bicontinuous cubic phase medium, 363 Bicuculline, 125

387

Bigeye snapper skin, autolysis of, by indigenous serine proteinases, 205 Binding unit, 347 Bioactive fragments, release of, in silico by trypsin from milk proteins, 334, 336 Bioactive peptides classification of, 307 defined, 154 enzymatic release of, from precursor proteins in silico, 335–336t examples of commercial dairy products and ingredients based on, 163t future research areas about, 163 in health: overview, 5–11 health benefits with, 162 in silico analysis of, 10, 326–328, 331 milk proteins and, 8, 152 novel applications of, 161–162 plant proteins and, 332, 334 production of, 154–155 release of, from selected precursor proteins, 154 by enzymes–proteolysis in silico, 331–332, 334 strategy for research on, 327, 327 for therapeutic purposes, 326 Bioactive peptides and proteins, from lactoferrin, 8 Bioactive properties, potential correlation between bitterness of peptides and, 348–349 Bioactive whey proteins, occurrence and isolation of, 152–154 Biochemical enhancers, skin permeability for transdermal drug delivery and, 373 Biochemistry, bioinformatics methods applied in, 10 Biofilm development of bacteria, blocking, lactoferrin and, 158 Bioinformatics methods applications of, 10 increasing popularity of, 339

388

Index

Biomarkers defined, 317 peptides as, 317–318 BIOPEP database of proteins and bioactive peptides comprehensive analysis of proteins as source of bioactive peptides by, 331 development of, 10 proteolysis simulation example in, 333 proteolytic enzymes of gastrointestinal system in, 332 structure and function of, 326 Biopeptides, identification of selected, released by trypsin from milk proteins, 334, 336 Biopharmaceuticals, emergence of, 359 BioPURE-GMP, 163t Bioseparation techniques, immune milk preparations and, 156 Biotechnology, bioinformatics methods applied in, 10 BioZate, 163t Bitterness of peptides potential correlation between bioactive properties and, 348–349 predicting, 347–348 of protein hydrolysates and peptides, physico-chemical properties related to, 345, 347 Bitter peptides, of proteins and protein hydrolysates, isolated by different researchers, 346–347t Bitter substances, 342–343 Bitter taste human response to, 342 protein hydrolysis and, 345 Blackeyed peas (Vigna unguiculata), Bowman-Birk protease inhibitors and, 279 Black tea, GABA content in, 125 Blake, C., 89

bLf. See Bovine-milk derived lactoferrin Blom, W. A., 136 Blood glucose levels, insulin study in nonagitated and agitated cubic phase and effect on, 366 Blood pressure. See also Antihypertensive peptides; Hypertension antihypertensive peptides and, 8 regulation of ACE inhibitors and, 169–170, 174, 175, 207, 256–257 renin-angiotensin system and, 298 soy protein and, 79–80 Bloom gelatin, 205 Bloom value, hydrophobic amino acids and, 206 Blue whiting, ACE inhibitory peptides derived from, 210t Blundell, J. E., 143 BMI. See Body mass index Body composition, sustained high-protein diet and, 136 Body mass index, soy consumption and, 71 Body weight, gamma-aminobutyric acid and, 125 Boldyrev, A., 31t Bolton, D. C., 88t Bonito ACE inhibitory peptides derived from, 208, 210t hypotensive effects of, in spontaneously hypertensive rats, 213t Bonito bowels, ACE inhibitory peptides derived from, 209t Borderline hypertension, most beneficial improvement for, 43 Bothrops jararaca, bioactive peptides isolated from, 170 Bougatef, A., 217t Bovine κ-casein, anti-inflammatory properties of, 19, 20t, 21

Bovine caseins calmodulin-binding peptides isolated from peptic hydrolysate of, and potency against phosphodiesterase I, 57t functional peptides in, 171 Bovine CGP, anti-inflammatory properties of, 21 Bovine colostrum major bioactive whey proteins in, 153, 153t natural antibodies in, 156 Bovine lactalbumin alpha chromatograms of, 336, 338 mass spectrum of tryptic hydrolysate of, 338 profile of potential biological activity of, 337 Bovine lactoferrin, apoptosis-related gene expression changes and, 190 Bovine Lfcin (LfcinB) structure of, 184 structure of: LfcinB domain and LfcinB peptide, 184 Bovine mammary secretions, growth factors in, 152–153 Bovine-milk derived lactoferrin angiogenesis and, 189–190 anti-inflammatory activity associated with, 22 antimetastatic effects with, 189 sequences of Lfcin and Lfampin peptides from, 184t systemic host immunity promoted with, 189 Bovine spongiform encephalopathies, 89, 90 amyloid deposits and, 95 amyloidosis and, 7 demand for fish gelatin and, 205 Bowen, J., 140, 141 Bowm, A. W., 123 Bowman-Birk protease inhibitors anticancer effects due to, 278–280 inhibitory mechanism of, 229 Bowman-Birk trypsin inhibitor, 20t, 22, 23 BP. See Blood pressure

Index

Bradykinin, blood pressure regulation and, 207 Brain senescence, GABA and prevention of, 124–125 Bram, rice kernel, 234 Bran, components of, 233 Bray, T. M., 31t Brazzein, 351 Bread antioxidant caseinophosphopeptides in, 35 GABA used in, 129 Brockmann, A., 320 Brown adipose tissue, soy peptides and, 267–268 BTC. See Beta cellulin Buccal delivery, oleic acid and membrane permeation in, 370 Buee, L., 88t Buforin II, 192 Bull, rice kernel, 234 Burton-Freeman, B. M., 160 Butter fat, inhibiting oxidation of, 216 Butylated hydroxyanisole, 36, 215 Butylated hydroxytoluene, 214, 215 Butylhydroquinone, 215 Byun, H. G., 210t C Cabral, K. M. S., 283 Caffeine, 342 Caffrey, M., 361, 372, 373 Calbet, J. A., 141 Calcitonin, 93t, 319 Calcium binding, calmodulin activation and, 55 Calcium channel antagonists, 169 Calkins, E., 89 Calmodulin (CaM) effect of flaxseed protein-derived peptides on intrinsic fluorescence of, 61t effect of flaxseed protein-derived peptides on secondary structure fractions of, 61t functions of, 55 role of, 6

Calmodulin inhibitors flaxseed protein-derived peptides, 58–62 food protein-derived peptides, 55–64 milk protein-derived peptides, 56–58 pea protein-derived peptides, 62–64 Caloric intake, soy protein and reduction in, 73, 74 Calpis, 162, 163t CaM. See Calmodulin CaM-dependent peptides, current stage of knowledge about, 64 CAM-dependent protein kinase II (CAMKII), 56 cAMP. See Cyclic adenosine monophosphate Campbell, B., 156 CaM-PDE inhibition of, 56 milk protein-derived peptides and, 56–58 Cancer chronic inflammation and, 227 excessive levels of calmodulin and, 55 free radical attacks and, 29 lactoferrin’s anti-inflammatory properties and, 187 lactoferrin’s biological roles in, 189–191 peptidomics and, 319 prevalence of and mortality rates with, 255 rice bran proteins and prevention/ control of, 241 Candida albicans immune milk preparations and, 156 lactoferrin’s antimicrobial activity and, 158 lactoferrin’s effects against, 185, 193 LfambinB peptide’s activity against, 193 Lfcin and inhibiting growth of, 192

389

Candida glabrata, lactoferrin’s efficiency against, 193 Candida spp., lactoferrin’s effect on, 187 Capelin antioxidant peptides from, 217t antioxidative activity of protein hydrolysates from, 218 Captopril, 47, 174 antihypertensive effects of, in spontaneously hypertensive rat, 173 zinc deficiency effects of, 208 Carbohydrates, 105, 342 Carboxypeptidases, reducing bitterness of protein hydrolysates and, 350 Carcinoembryonic antigen, 319 Cardiac hypertrophy, excessive levels of calmodulin and, 55 Cardiovascular disease fat dysfunction and, 71 hypertension and, 169, 207 Carloric sweeteners, 351 Carnitine palmitoyltransferase, soy protein feeding in rats and, 73 Carnosine in animal meat, 225, 226 antiaging effects of, 228 antiglycation effects of, 227 anti-inflammatory and immune-modulating activity of, 227 antioxidant activity and, 30–31, 31t, 226 chemical structure of, 31 dose-dependent ACE-inhibitory activities of, 228 hypotensive effect of, 227–228 neurological effects of, 228 wound healing effects of, 228 Carnosine relaxation effect, in Sprague-Dawley (SD) rat aorta rings, 49 Caryopsis, 233 Casein hydrolysates ACE-inhibitory activity with, 154 bioactive peptides and, 162

390

Index

Casein peptides, importance of helix structure configuration of, and effective binding to CaM, 58 Caseins antihypertensive peptides derived from, by proteolytic action, 172t antioxidant activity of peptides in, 34 functional properties of rice bran proteins and, 237 increase in GLP-1 concentrations in whey proteins vs., 141 Caspase-3, lactoferrin and, 190 Catalase (CAT), aerobic organisms and, 16 Cataracts, advanced glycation end products and, 227 Cathepsin G, 23 CCK. See Cholecystokinin CCK1 receptor, 140 CD4 antigen, lactoferrin and, 188 CD4+ T cells, food allergy and, 103 CD spectra. See Circular dichroism spectra CEA. See Carcinoembryonic antigen Cefaclor, 140 Celiac disease, 10, 114, 295, 297, 299 Cell migration, lactoferrin and, 188 Cell-permeable peptides, 192 Cell proliferation, lactoferrin’s biological roles in, 189–191 Cell signaling mechanisms, inflammation and, 19 Cellular homeostasis, histidyl dipeptides and, 225 Cellulose, 233 Central nervous system, mammalian, GABA and, 121 Central neuronal pathways, protein-induced satiety and, 143–144 Centripetal obesity, metabolic syndrome and, 67 Cereals antioxidant caseinophosphopeptides in, 35 natural antioxidants in, 215

proteins in, 234 world wheat production and, 9 worldwide production of, 233 Cerebral ischemia, oxidative stress and, 31–32 Ceruloplasmin, lactoferrin and, 183 Cetyltrimethylammonium bromide, hydrophobicity of insoluble glutelins and, 235 Chand, R., 162 Chaotropic solutes, properties of liquid crystalline phases and, 362, 363 Chapatti (flat bread), rice bran proteins in, 239 Charter, E. A., 249t Chatterton, D. E. W., 157 Cheese, bioactive peptides and, 161 Cheese ripening, GABA levels in, 126 Cheese starter bacteria, bioactive peptide production and, 155 Cheese whey beta cellulin in, 152 large-scale preparation of bLf from, 193 Cheison, S. C., 38 Chelikani, P. K., 140 Chemically Linked Peptides on Scaffolds, 107 Chen, H. M., 36, 37t Chen, K. M., 216 Chewing gum, bLf added to, 193 Chickpea (Cicer arietinum), 9, 273, 274 antimicrobial peptides/proteins and, 283 antioxidant effects of, 274–275 effects of proteolytic treatments on physicochemical and bitterness properties of, 275 future prospects for biological activities related to, 284 hypocholesterolemic effects and, 276 Chickpea protein hydrolysates effect of, on serum antioxidant activity of aged mice, 275t fractions from (Fra. I, Fra. II, Fra. III, Fra. IV), 274

China, wheat production and consumption in, 9, 289 Chocolate antioxidant caseinophosphopeptides in, 35 GABA in, 129 Cholecystokinin dose-dependence of, in protein-induced satiety, 140 food intake regulation and, 139, 140 protein and, 74 protein-induced satiety and, 138, 145 putative satiety mechanisms diagram, 139 soy protein and, 76 Cholesterol-rich diets, hyper-hypo-response phenomenon and, 277 Chondroitin sulphate, 186 Chromatograms, of bovine lactalbumin alpha, 336, 338 Chromatographic separation technique debittering protein hydrolysates and, 349 for fractionation and isolation of bioactive milk proteins, 153 Chronic disease bioactive peptides and proteins and, 5 CaM-dependent peptides and, 64 oxidative stress and inflammation and, 23 Chu, K. T., 283 Chuang, W. L., 217t Chum salmon, antioxidant peptides from, 217t Chun, H., 266 Chymase, 23 Chymotrypsin, 21, 154, 171 in BIOPEP database, 332 Bowman-Birk inhibitors and, 278, 278t Cicerarian, 283 Cicerin, 283 CIR. See Cosmetic Ingredient Review

Index

Circadian rhythm regulation, N-acetyl-5methoxytryptamine and, 31 Circular dichroic (CD) spectroscopy, of insulin in liquid crystalline system, 366, 367 Circular dichroism spectra, 361 CLA. See Conjugated linoleic acid Clark, A., 88t Class intervals, 328 Clemente, A., 279 CLIPS. See Chemically Linked Peptides on Scaffolds Clogston, J., 372, 373 Closed-interface domains, phenolic compounds and, 92–93, 93 Clostridium difficile enterotoxins, immune milk preparations and, 156 Clostridium perfringens, lactoferrin’s antimicrobial activity and, 158 Clostridium thermosaccharolyticum, lysozyme effective against, 250 Clostridium tyrobutyricum, lysozyme effective against, 250, 251 Clostripaine, 332 Codex Alimentarius Committee, 159 Coenzyme Q10, in animal muscle, 225 Cohen, A. S., 89 Cohen, D. H., 88t Coho salmon, ACE inhibitory peptides derived from, 210t Cola, GABA used in, 129 Colitis, anti-inflammatory effect of GMP and TNBS models of, in rats, 21 Collagen in connective tissue proteins, 204 hydroxyproline in, 204 Collagenase, 205, 332 Collagen triple helix structure, fish gelatin and, 205, 206 Colon carcinomas, Lfcin’s antitumor effects against, 192–193 Colostral immunoglobulins, biological function of, 156

Colostrum beta cellulin in, 152 growth factors in, 160–161 health-promoting proteins and peptides in, 7–8 immunoglobulins in, 152 natural antibodies in, 156 Combinatorial peptidomics, 308, 309 Conditioned taste aversion, 144 Conjugated linoleic acid, in animal muscle, 225 Connective tissue proteins, in fish proteins, 204 Controlled release of biologically active proteins and peptides, molecular and transport characteristics in bulk aqueous medium of additive molecules, 373t Convulsions, nitric oxide levels and, 56 COOH-terminal dipeptide residue, substrate specificity of ACE and, 212 Cookies, rice bran proteins in, 239 Cope, W. B., 74 Copper, 215 Coprinus comatus, sativin’s antifungal activity against, 282 Corn, rice protein net protein utilization compared to, 240 Cornish, J., 158 Corn oil, inhibiting oxidation of, 216 Corn protein, antioxidant acitivty of peptides in, 34 Cos, M. L., 74 Cosmetic Ingredient Review, 242 Cosmetics, bLf added to, 193 Costa, P. P., 88t Cota, D., 143 Cowpea (Vigna unguiculata), 9, 273, 274 antimicrobial peptides/proteins and, 282–283 future prospects for biological activities related to, 284 hypocholesterolemic effects and, 276

391

Cow’s milk allergy model, 112–113 COX-2. See Cyclo-oxygenase-2 CPK levels. See Creatine phosphokinase levels Cp-thionin II, bactericidal activity of, 283 Craft, I. L., 265 Creatine, in animal muscle, 225 Creatine phosphokinase changes in, after beverage ingestion: comparison of placebo, soy protein, and soy peptide on GH and CPK level, 268 soy peptides in sports and levels of, 267 Creutzfeldt-Jakob disease, 90 Crohn’s disease colostrum-based products with growth factors and, 161 fecal lactoferrin and, 194 “Cross-beta” conformation, 89 Cryptosporidium parvum, immune milk preparations and, 156 CS. See Chondroitin sulphate CTA. See Conditioned taste aversion C-terminal glutamate residue, for some inhibitory peptides, 212 CTLs. See Cytotoxic T lymphocytes Cubic phase delivery of proteins and peptides in, 360, 363 drug release rate and, 372 GMO, two-photon fluorescence images showing lateral distribution of sulphorhodamine B, after 24 hours of passive diffusion in skin and, 377, 377, 379 gramicidin D ‘s destinations within, 361 insulin and optical density of, 366 microfissures, SRB fluorescence and, 379 time course of in vitro skin penetration and percutaneous delivery of cyclosporin A incorporated in, 369–370, 370

392

Index

Cubic phase (continued) in vivo skin penetration of CysA incorporated into, compared to olive oil formulation, 371, 371 Cubic phase domains, drug release rate and, 373 Cubosome particles, 363 Cui, M., 351 Curculin. See Neoculin Curcumin, 16, 92 CVD. See Cardiovascular disease Cyclamate, 351 Cyclic adenosine monophosphate, 56 Cyclo (His-Pro), antioxidant activity and, 31 Cyclo-oxygenase-2, 18 Cyclosporin A (CysA) penetration experiments with liposomes with ethanol and, 371 time course of in vitro skin penetration and percutaneous delivery of, incorporated in cubic and hexagonal liquid crystalline phases, with olive oil control, 369–370, 370 in vitro penetration of, in stratum corneum and [E + D] at 6 and 12 hours following its topical application using hexagonal phase nanodispersion or olive oil control, 372 in vivo skin penetration of, incorporated in cubic and hexagonal liquid crystalline phases, compared to olive oil control formulation, 371, 371 Cystatin anticancer property of, 255 in egg, antimicrobial properties of, 254 as serine protease inhibitor, 258 Cystatin C, 88t, 90–91, 92, 93t Cysteine, 29, 274, 343 in cereal grain proteins, 234 radical quenching activity of peptides and, 35

Cystine, in cereal grain proteins, 234 Cytochrome C, liquid crystalline phases and, 363 Cytokine environment, food allergy and, 103 Cytokines, 307 Cytomegalovirus, 192 lactoferrin’s antimicrobial activity and, 158 lactoferrin’s effects against, 185 Cytotoxic T lymphocytes, food allergy and, 103 D Daddaoua, A., 20t Dahi, bioactive peptides and, 161, 162 Dairy products, novel applications of bioactive peptides and, 161–162 DC-SIGN receptor, lactoferrin binding and, 186 Debitrase, 350 Debittering approaches, for protein hydrolysates, 349–351 Decker, E. M., 216 Defatted (DF) bran, protein extraction from, 236 De Freitas, S. M., 229 Degree of milling, 233, 234 Dehydrin proteins, functional properties of, 237 Deibert, P., 77t Delacourte, A., 88t Delivery technology for biologically active proteins and peptides, 359–379 barriers and mechanism of bioavailability of proteins and peptides, 366–3709 entrapment and stability of proteins and peptides by liquid crystalline systems, 360–366 overview of difficulties related to, 359–360 prerequisites for success in, 360 Denatonium, 342 Denaturation, protein and peptide drug inactivation with, 365

Deng, S. G., 350 Dental health care products, lactoperoxidase in, 160 Dermatophytes, Lfcin and inhibiting growth of, 192 “Desensitization” induction, food allergy and, 108 Dextran sodium sulfate, 18 DF. See Diafiltration Dia, V. P., 20t, 23 Diabetes advanced glycation end products and, 227 chronic inflammation and, 227 cyclo (His-Pro) and amelioration of, 31 flavor-active components and novel therapeutic approaches to, 355 metabolic syndrome and, 67 overconsumption of salty and sweet foods and, 342 reactive oxygen metabolites and, 226 sodium substitutes and, 345 sweet proteins and, 351 wheat albumin and prevention of, 298 Diabetes mellitus, defined, 298 Diafiltration, manufacture of whey powder and whey protein concentrates and, 153–154 Dialysis-related amyloidosis, beta2-microglobulin and, 90 Diepvens, K., 139 Diet high-protein, satiety and food intake inhibition in, 137–138 high-protein meals, satiety and, 136–137 improvement in hypertension and, 43 therapeutic, metabolic syndrome and, 67, 68 Dietary antioxidant supplements, expanding market for, 30 Dietary Approaches to Stop Hypertension (DASH) diet, 68

Index

Diet-disease associations, peptides and, 319–320 Digestion process, antioxidant peptides and, 32–34 Di-glycerine peptides, absorption of, 265 Dilute acid- or alkali-soluble glutelin, 290 Dimeric domain-swapped L68Q human cystatin C, open-interface and closed-interface, 92, 94 Dioleoyl phosphatidylcholine vesicles, 361 Dipeptide ACE inhibitors, 212 Dipeptides development of, from soy, 266 intestinal adsorption of, 174 relaxation of vascular constrictive events by, 48–51 release of, from milk proteins, 332 salty taste and, 345 Disaccharides, polyglutamine aggregate formation and, 95 Disease lipid oxidation and, 214 oxidation and, 29 peptide biomarkers in, 319 Diuretic agents, 169 Doan, F. J., 30 DOM. See Degree of milling Domains of cubic structures, bicontinuous cubic phase medium and, 363 DOPC vesicles. See Dioleoyl phosphatidylcholine vesicles Douchi, antioxidant peptides in, 38 DPPH antioxidant effects from peas and, 274 radical-scavenging property of peptides and colorimetric binding assays for, 35 Drug penetration enhancement of, 368 hydrophilic and lipophilic pathways of, and mode of action of penetration enhancers, 368, 369

routes across the stratum corneum, 368 Drug release rate, properties of liquid crystalline structures and, 372, 379 Dry extrusion, rice bran stabilization and, 235 DSS. See Dextran sodium sulfate D3 protease antihypertensive effect of, 350–351 less bitter protein hydrolysates and, 350 Dunshea, F. R., 74 Dyslipidemia, 67, 71 Dziuba, J., 332, 339 E EAI. See Emulsion activity index Edible films, lactoferrin and, 194 “Edible vaccines,” 115 EDTA. See Ethylene di-amine tetra acetic acid EGF. See Epidermal growth factor Egg allergy model, 113–114 Egg proteins, 215 antioxidant activity of peptides in, 34 bioactive, recent advances in, 9 digestibility of, 248 Eggs anticancer proteins and peptides in, 255 antihypertensive peptides in, 256–258 antimicrobial proteins and peptides from, 249–254 avidin, 254 cystatin, 254 lysozyme, 250–252 ovalbumin, 252–253 ovomacroglobulin, 253 ovomucin, 253 ovotransferrin, 252 phosvitin, 253–254 antimicrobial proteins in, 249t antioxidant proteins and peptides in, 17, 17t, 255–256 components of, 247 fried vs. boiled, ACE inhibitory activities and, 257

393

immunomodulating proteins and peptides in, 254–255 major chemical compositions of different parts of, 248t as “nature’s perfect food,” 258 protease inhibitors in, 258 proteins in, 258 Eggshell, 249 Egg white, 247 biological functions of proteins in, 247–248 protease inhibitors in, 258 Egg yolk peptides, phosvitin phosphopeptides, 17–18, 17t Eimeria stiedai lactoferrin’s effects against, 186 Lfcin’s effects on, 192 Elastase, 23, 332 Electron spin resonance microscopy, radicals detected with, 35 Elicitation phase, in allergic response to food antigens, 103 Elimination diets, food allergy and, limits with, 107–108 Embryo, 233 Emulsion activity index, 236 Emulsion stability index, 236 Enalapril, 208 Enari, H., 210t Endocytosis, 102 Endogenous antioxidant peptides, 30–32 anserine, 30, 31t balenine, 30, 31t carnosine, 30–31, 31t cyclo (His-Pro), 31 glutathione, 30, 31t melatonin, 31, 31t SS31 peptide, 31, 31t Endogenous antioxidative stress mechanisms, 16 Endopeptidases, 317 Endosperm, 233 Endothelial isozyme, of NOS (eNOS), 56 Endothelial NOS protein molecule, CaM-dependent, effect of flaxseed protein-derived peptides on, 59–60

394

Index

Endothelin antagonist, 169 Energy balance, proposed model for role of mTOR signaling in hypothalamic regulation of, 144 Energy expenditure, as metabolic signal in protein-induced satiety, 142 Energy intake, regulation of, 7, 135 Enrichment bioactive milk proteins and, 153 immune milk preparations and, 156 Entamoeba histolytica, lactoferrin’s effects against, 185 Enterococcus spp., lactoferrin’s antimicrobial activities and, 185 Environmental factors, antioxidant-prooxidant balance and, 30 Enzymatic digestion, antioxidant peptides and, 38 Enzymatic extraction, of rice bran proteins, 236 Enzymatic hydrolysis, 171 Enzymatic oxidation, 214–215 Enzymatic treatment, for reducing bitterness of protein hydrolysates, 350 Enzymes, in bran, 233 Epidermal growth factor, in bovine mammary secretions, 152, 161 Epidermis, penetration of fluorescein into, as imaged by multiphoton microscopy, 375 Epitalon, 31 Epithalamin, 31 Ericsson, B., 366 Escherichia coli GABA biosynthesis with, 126 immune milk preparations and, 156 lactoferrin’s antimicrobial activities and, 158, 185, 186 lysozyme and, 250 Escherichia coli K-12, ovotransferrin’s effect against, 252

ESI. See Emulsion stability index ESR microscopy. See Electron spin resonance microscopy ESR spectrum, gel filtration of whey protein and, for OH signals of samples containing different peptide fractions, 36 Essential hypertension, most beneficial improvement for, 43 Ethanol-phosphate buffer saline solution, skin permeability study and, 374 Ethoxyquin, 214 Ethylene di-amine tetra acetic acid, 250 EU. See European Union Eulitz, M., 88t European Union, wheat production and consumption in countries of, 9, 289 Evolus, 162, 163t Exercise. See also Wheat-dependent exercise-induced anaphylaxis efficacy of soy peptides in, vs. with soy protein, 267 improvement in hypertension and, 43 overexertion during, reactive oxygen species and, 16 Exogeneous protein/peptide antioxidants, purpose of, 16 Exopeptidases debittering and, 354 reducing bitterness of protein hydrolysates and, 350 Expander cooker, rice bran stabilization and, 235 Extended class of proteins and peptides, 361 Extendin-4, 141 F Fahmi, A., 210t, 211, 213t Fahr, A., 371 Faipoux, R., 144 Familial amyloidosis, 90 Farina, 289 Farm animals, colostral Ig preparations for, 156

FAS. See Fatty acid synthase Fatal familial insomnia, 90 Fat dysfunction, 71 Fat replacers, rice bran as, 239–240 Fats, removing from bran, advantages with, 235–236 Fat substitutes, rice-based, 243 Fatty acids, unsaturated, oxidation of, 214 Fatty acid synthase, 71 FDA. See U.S. Food and Drug Administration FDEIA. See Food-dependent exercise-induced anaphylaxis FD-RBPs. See Freeze-dried rice bran proteins Fecal lactoferrin, use of, as diagnostic tool, 194 Feline calcivirus, Lfcin’s antiviral activity against, 192 Fermentation products, 171–173 Fermented dairy products, novel applications of bioactive peptides and, 161–162 Fermented food, bioactive peptides and, 326 Fermented milks, bLf added to, 193 Ferreira, S. H., 170 Ferritin, 37 Ferulic acid, 92, 126 FFAs. See Free fatty acids FGF1, in bovine mammary secretions, 152, 161 FGF2, in bovine mammary secretions, 152, 161 Fibrinogen alpha-chain, 88t Fibroblast growth factor, in bovine mammary secretions, 152, 161 Fibrosarcomas, Lfcin’s antitumor effects against, 192 Fickian diffusion model, drug release measured by, 372 Filamentous fungi, Lfcin and inhibiting growth of, 192 Findeis, M. A., 94 Fish collagen, sensitivity of, 204 Fish gelatin, 204–207 Bloom value and, 206–207 chemical composition of, 205

Index

demand for, 205 physical and chemical properties of, 205 properties and production processes for, 206 Fish muscle and collagen, bioactive peptides and protein from, 8 Fish peptides, antioxidant, 215–218 Fish processing, utilizing and upgrading waste from, 203 Fish products, bioactive peptides isolated from, 171 Fish proteins, 215 ACE inhibitory peptides derived from, 207–214, 209–210t ACE and blood pressure, 207 ACE inhibition mechanism, 211–212 ACE inhibitors, 207–208 ACE inhibitory peptides isolated from fish proteins, 208 in vitro ACE inhibition by peptides derived from fish proteins, 208, 211 in vivo ACE inhibition by peptides derived from fish proteins, 212–214 antioxidant peptides derived from, 214–215, 217t enzymatic oxidation, 214–215 mechanism of lipid oxidation, 214 overview, 214 food antioxidants and, 215–218 antioxidant fish peptides, 215–218 natural antioxidants, 215 synthetic antioxidants, 215 health benefits of, 204 hypotensive effects of fish-derived peptides in spontaneously hypertensive rats, 213t Fish sauce ACE inhibitory peptides isolated from, 208 inhibitory activities in, 172 Fish skin, as source of gelatin, 205 Fish supplies, threats to, 203

Flavonoids, 16, 215 Flavor, synergistic interactions with effect on, 344 Flavor-active components consumer acceptance and, 341 novel therapeutic approaches to chronic diseases and, 355 Flavor enhancers, rice bran proteins as, 238–239 Flavor ingredients amino acids interacting with, 352 peptides and protein hydrolysates interacting with, 352–353 proteins interacting with, 353–354 Flavourzyme, 238, 350 Flavourzyme hydrolysate, 275, 280 Flaxseed, omega-3 fatty acids in, 58 Flaxseed protein-derived cationic peptide fractions effect of, on intrinsic fluorescence of Ca2+/CaM-dependent nitric oxide synthases, 59–60, 60t kinetics of inhibition of neuronal nitric oxide synthase by, 59 Flaxseed protein-derived peptides as calmodulin inhibitors, 58–62 effect of on intrinsic fluorescence of calmodulin, 61, 61t on secondary structure fractions of calmodulin, 61, 61t Fluorescein chemical structure of, 374 penetration of, into human epidermis after treatment with magainin + NLS, 375 Fluorescein delivery, transdermal, enhancement of as function of pH, 374, 374 Fluorophores, two-photon microscopy and imaging of, 376 Folic acid, from animal muscle, 225 Foltz, M., 140 FONDAPARINUX, 318 Food allergens B- and T-cell epitopes, 105–106 “big eight,” 111, 114 molecular properties of, 105

395

Food allergy, 112–113 B-cell epitope mapping and, 107 current management of, 107–108 defined, 7, 101–102 egg allergy model, 113–114 IgE-mediated, potential immunotherapeutic approaches for, 109t novel immunotherapeutic strategies for, 108 peanut allergy model, 113 PIT investigations and murine models of, 116 sequence of events leading to type 1 hypersensitivity, 104 T-cell epitope mapping and, 106–107 T lymphocytes and role in, 103–105 wheat and beef allergy model, 114 Food antigens, allergic response to: two-phase mechanism, 103 Food antioxidants, 215–217 antioxidant fish peptides, 215–218 natural antioxidants, 215 synthetic antioxidants, 215 Food-based hydrolyzates, food allergy and, 109–110 Food choices, improvement in hypertension and, 43 Food-color carriers, rice bran proteins as, 239 Food-dependent exercise-induced anaphylaxis, 296 Food industry, lysozyme used in, 251 Food intake inhibition high-protein preload and, 136–137 satiety and, in high-protein diets, 137–138 Food intake in humans, physiological and psychological factors related to, 135 Food preservation and safety, lactoferrin and, 194 Food products, lipid oxidation in, 214

396

Index

Food protein-derived calmodulin-binding peptides, typical protocol during production of, 57, 57 Food protein-derived peptides as calmodulin inhibitors, 55–64 flaxseed protein-derived peptides, 58–62 milk protein-derived peptides, 56–58 pea protein-derived peptides, 62–64 Foods for specified health use, 6, 8 antihypertensive peptides in, 171t antihypertensive products, 44t design of ACE inhibitory peptides and, 45 in Japan, 43 in the market, 170–171 Food systems, antioxidant activity of peptides in, 34–35 FOSHU. See Foods for specified health use Fourier transform-ion cyclotron resonance, 310 Fourier transform-Raman spectroscopy, predicting bitterness of peptides and, 348 FOXp3 molecules, food allergy and, 116 Fractionation of bioactive milk proteins and peptides, 153, 162 immune milk preparations and, 156 Franco, O. L., 283 Free amino acids benefits related to protein hydrolysates consisting of, 344 reducing bitterness of protein hydrolysates and, 350 taste of foods and, 344 Free fatty acids, 70 Free radical attacks, disease, pathogenesis and, 29 Free radical oxidative reactions, termination of, 214 Free radicals, defined, 15

Freeze-dried rice bran proteins, 237 Fricker, L. D., 316 Friend virus complex, lactoferrin’s effects against, 185 Froetschel, M. A., 142 Fruits, natural antioxidants in, 215 FT-ICR. See Fourier transform-ion cyclotron resonance Fujita, H., 209t, 210t, 213t Full-fat-stabilized (FFS) bran, protein extraction from, 236 Full-fat-unstabilized (FFU) bran, protein extraction from, 236 Fumio, T., 123 Funa-sushi, GABA synthesis and, 126 Functional foods. See also Soy peptides as functional food system with antihypertensive effects, 169–175 global interest in promotion of, 162 in the market, 170–171 mode of action ACE inhibitory effects, 174 peptide absorption, 174 preparation of ACE inhibitory peptides, 171–173 enzymatic hydrolysis, 171 fermentation products, 171–173 in vivo effect, 173–174 clinical trials, 173–174 effects on an animal model, 173 worldwide interest in, 326 Functional proteins, within nutraceutical food sector, 5 Fungi filamentous, Lfcin and inhibiting growth of, 192 lactoferrin and, 187 Fusarium oxysporum, sativin’s antifungal activity against, 282 Fuzeon, 115 G GABA. See Gamma-aminobutyric acid

GABA-A receptors, mood regulation in animals and, 124 GABA-B receptors, mood regulation in animals and, 124 GABA shunt, 127 GABA-T, 127, 128, 129 GABA tea, 125, 129 GAD. See L-glutamic acid decarboxylase GAD65 molecule, 128 GAD67 molecule, 128 GAGs. See Glycoamino-glycans Gamma-aminobutyric acid, 121–130 animals and roles of, 124–125 curing neurological disease, 124 hypotensive effects, 125 inhibiting acute hyperaammonemia, 125 mood regulation, 124 preventing brain senescence, 124–125 sleep regulation, 125 weight loss, 125 biological functions of, 121 biosynthesis of, 127t biosynthetic pathway and metabolism, 130 conversion of intermediate to, 128 discovery of, 7, 121 formation of Schiff-base by substrate and PLP, 128 functions of, 122–125 human application of, 129–130 metabolic pathway of, 127–129 neutral and zwitterionic structures, 122 physical properties of, 122 preparation of, 125–127 other foodstuffs, 125–126 synthesis by microbes, 126–127 in tea, 125 properties of, from lactic acid bacteria, 123t proposed transformation of L-Glu to, by GAD, 129

Index

reaction mechanism of, 127 roles of synthesis, in plants, 122–124 nitrogen storage, 123 pH regulation, 123 plant defense, 123 plant development, 123–124 potential modulator of ion transport, 124 Garden/green pea (Pisum sativum), 9, 273, 274 anticancer effects due to Bowman-Birk inhibitors and, 278 antimicrobial peptides from, 281–282 antioxidant effects of, 274–275 future prospects for biological activities related to, 284 hypocholesterolemic effects and, 276 Gardenia jasminoides Ellis, 18 Gastric inhibitory peptide, 76 Gauthier, S. F., 161 GBR. See Germinated brown rice Gejyo, F., 88t Gelatin antioxidant activity of peptides in, 34 antioxidant proteins/peptides in, 17 bitterness masked with, 350 fish, 204–207 Gel filtration, whey protein isolates and, 154 Gelsolin, 88t “Generally Recognized as Safe” designation, 159 Genistein, 92 Gerber, S. A., 315 Germ, 233 rice kernel, 234 German-Straussler-Scheinker syndrome, 90 Germinated brown rice, GABA used in, 126, 129 GH. See Growth hormone Ghrelin food intake and, 141–142 protein and, 74

GHRH. See Gonadotrophichormone-releasing hormone Giant squid, antioxidant peptides from, 217t Giardia lamblia, Lfcin’s effects on, 192 GI enzymes, antioxidant peptides and, 32 Giméneze, B., 217t GIP. See Gastric inhibitory peptide Giroux, I., 276 GJE. See Gardenia jasminoides Ellis Glenner, G. G., 88t Gliadin, 10, 290, 291–293, 295 Globulin, 247 Glucagon-like peptide-1 (GLP-1), 139 anorexigenic actions of, 141 effect of 3-hour intravenous infusions of, on cumulative food intake in rats, 141 protein and, 74 protein-induced satiety and, 138 short-term, 145 Glucagon secretion, dietary proteins and, 76 Glucofructans, 233 Gluconeogenesis, protein-induced satiety and, 145 Glucose, as metabolic signal in protein-induced satiety, 142 Glucose 6-phosphatase (G6Pase), protein-induced satiety and, 142 GLUT-2, protein and, 75 Glutamate, taste of, 344 Glutamate decarboxylase, 122 Glutamic acid, 37, 238 Glutamines, in rice bran proteins, 238 Glutathione aerobic organisms and, 16 in animal muscle, 225 antioxidant activity and, 30, 31t chemical structure of, 31 redox homeostasis and, 16 Glutathione peroxidase, aerobic organisms and, 16 Glutathione reductase, aerobic organisms and, 16

397

Glutathione synthetase, 16 Glutelin, 290, 293, 295 Gluten, 295 viscoelastic property of, 9–10, 295, 299 Glutenin, 290, 293, 295 Glutenin subunits, repeat motifs in structure of, 293, 295 Gly, in gelatin, 205 Glycemic control, soy protein and, 74–76 Glycerol monooleate, 361 Glycine bitterness masked with, 350 sweet taste of, 343 Glycinin, 17 Glycoamino-glycans, lactoferrin binding to, 186 Glycomacropeptide, 19, 20t anti-inflammatory properties of, 20t, 21 health benefits with, 160 purification of, from bovine colostral or cheese whey, 154 whey proteins and, 160 Glycosylated peptides, enrichment of, 314 Gly-Thr-Trp, 155 Gly-Val-Trp, 155 GM-CSF. See Granulocytemacrophage colony stimulating factor GMO. See Glycerol monooleate GMO cubic phase, PT cubic phase vs., in hydrophilic fluorescent model drugs investigation, 379 GMO/water cubic phase, insulin protected from agitation-induced aggregation and, 365 GMO/water systems diffraction patterns of, with incorporated insulin, 364 small-angle x-ray scattering patterns and lattice constants a of lamellar and cubic phases of, loaded with 4% insulin, 364

398

Index

GMP. See Glycomacropeptide Gonadotrophic-hormone-releasing hormone, angiotensin II and, 207 G protein-coupled receptors (GPCRs), 342 GPR93 receptor, 140 GPx. See Glutathione peroxidase GR. See Glutathione reductase Gramicidin, liquid crystalline phases and, 363 Gramicidin A, space-filing model of channel form of, 362 Gramicidin D, 361 Gram positive/gram negative bacteria LfambinB peptide’s activity against, 193 LfcinB and LfcinH actions against, 191, 192 lysozyme and, 250, 251 Gram positive/negative pathogens, lactoferrin and, 185, 186 Granisetron, chemical structure of, 374 Granulocyte-macrophage colony stimulating factor, lactoferrin and production of, 189 Grape seed extracts, 30 GRAS. See “Generally Recognized as Safe” designation Grass carp, antioxidant peptides from, 217t Green tea extracts, 30 GABA content in, 125 Grosman, M. V., 236 Growth factors, 7–8, 151, 307 in bovine colostrum and milk, 153t in mammary secretions, 152–153, 160–161 Growth hormone, changes in, after beverage ingestion: comparison of placebo, soy protein, and soy peptide on GH and CPK level, 268 GS. See Glutathione synthetase GSH. See Glutathione

GSSG, 16 Guérin-Dubiard, C., 249t H Habener, J. F., 140 Haemophilus influenzae, lactoferrin’s antimicrobial activities and, 158, 185, 186 Haggqvist, B., 88t Hall, W. L., 138, 141 Halorhodopsin, 361 Hardeland, R., 31t Hartog, A., 20t Hata, I., 20t Haversen, L., 22 Hayashida, K., 20t Health promotion, bioactive peptides and proteins and, 5 Heart disease, chronic inflammation and, 227 Heat stabilized rice bran, 242 HEL. See Hen egg lysozyme Helicobacter pylori immune milk preparations and, 156 lactoferrin’s antimicrobial activity and, 158 lactoperoxidase system and, 160 Helix-loop-helix pattern, lysozyme and, 251 Hemicelluloses, 233 Hemodialysis-associated disease, 89, 90 amyloid deposits and, 95 amyloidosis and, 7 Hen egg lysozyme, anti-inflammatory activity with, 19 Hen eggs as important food worldwide, 258 lysozyme in, 250 Hen egg yolk phosvitin, 17, 18 Heparan sulphate, 186 Hepatic lipogenesis, soy protein feeding in rats and, 71 Hepatitis B, lactoferrin’s effects against, 185 Hepatitis C bLf, IFN therapy and eradication of, 189

lactoferrin’s effects against, 185, 186 Hepatitis viruses, lactoferrin’s antimicrobial activity and, 158, 159 Herbal medicine anti-inflammatory mechanisms and, 23 antioxidative stress proteins and peptides in, 18 Herbs, natural antioxidants in, 215 Hernandez-Ledesma, B., 37t Herpes simplex virus lactoferrin’s antimicrobial activity and, 158 lactoferrin’s effects against, 185 Herring, antioxidant peptides from, 217t Herring fish protein hydrolysate, antioxidative activity of, 218 Herring processing wastes, enhancing gelatin from, 205 Hexagonal phase time course of in vitro skin penetration and percutaneous delivery of cyclosporin A incorporated in, 369–370, 370 in vivo skin penetration of CysA incorporated into, compared to olive oil formulation, 371, 371 Hexane, 214 “Hidden allergens,” 107 High blood pressure, cardiovascular disease and, 207 High-density lipoprotein cholesterol, metabolic syndrome and, 67, 68t High-density lipoproteins in egg yolk, 248 soy protein and, 76 High fructose corn syrup, 351 High-molecular-weight glutenin, 293 High-protein diet, defined, 137 High-protein meal, high-protein diet vs., 137

Index

High-protein preload, high-protein meal-induced satiety and food intake inhibition and, 136–137 Himeno, K., 126 HINUTE-AM launching of, 266 typical analysis of, 267t Hipkiss, A. R., 227 His amino acid, potent antioxidant activity with, 17 Histadyl dipeptides carnosine and anserine, 225, 226 health benefits with, 226 Histidine, 29 antioxidant activity of peptide derived from hoki skin, 218 antioxidant behavior in peptides, 216 food intake and, 142–143 radical quenching activity of peptides and, 35 HIV-1, lactoferrin’s antimicrobial activity and, 158 HLA. See Human leukocyte antigen HLA-DQ2, celiac disease and, 297 HLA-DQ8, celiac disease and, 297 HLA-DR, expression of seven alleles of, in Caucasian population, 115 hLf effect of, on angiogenesis, 189–190 sequences of Lfcin and Lfampin peptides from, 184t Hockerman, G. H., 50 Hoie, L. H., 77t Hoki, antioxidant peptides from, 217t Hoki skin branched amino acids present in antioxidant peptides from, 216 histidine and antioxidant behavior of peptide derived from, 218 Holst, J. J., 141 Holton, J. L., 88t Hormone replacement therapy, transdermal route of administration and, 360

Host cells, adsorption of lactoferrin in, 187 Howell, N. K., 206 HS. See Heparan sulphate HSRB. See Heat stabilized rice bran HSV, Lfcin’s antiviral activity against, 192 Human immunodeficiency virus lactoferrin binding to co-receptors of, 186–187 lactoferrin’s effects against, 185, 186 Lfcin’s antiviral activity against, 192 Human leukocyte antigen, 115 Human Lfcin (LfcinH), structure of, 185 Humiski, L. M., 275, 280 Humulin, aggregation profiles of, 365, 365 Huntington protein, 92 Huntington’s disease, 90, 92 amyloid deposits and, 89 treharose and, 95 HVP. See Hydrolyzed vegetable protein Hydrolysis of proteins, desirable sensory attributes with, 344–345 Hydrolyzed vegetable protein, 352 Hydrophilic fluorescent model drugs, distribution of, in full-thickness human skin, 376–377, 378t, 379 Hydrophilic pathways, of drug penetration, 368, 369 Hydrophobicity, bitterness and, 345, 347 Hydroxyl radicals, 29 Hydroxyproline in collagen, 204 in fish gelatins, 206 Hyperammonemia, acute, GABA and prevention of, 125 Hyper-hypo-response phenomenon, cholesterol-rich diets and, 277 Hyperinsulinemia metabolic syndrome and, 67 soy protein consumption and, 75, 76

399

Hypertension, 71. See also Antihypertensive peptides cardiovascular disease and, 169, 207 improving treatment of, 6 metabolic syndrome and, 67, 68t morbidity and mortality related to, 256 overconsumption of salty and sweet foods and, 342 renin-angiotensin system and, 44 sodium substitutes and, 345 soy protein and, 79–80 wheat peptide and prevention of, 298–299 Hypoallergenicity, tolerogenicity vs., 112–113 Hypotension, gamma-aminobutyric acid and, 125 Hypothalamus food intake, energy homeostasis and, 144, 144 food intake regulation and, 145 I IAPP. See Islet amyloid polypeptide Ibrahim, H. R., 249t ICAT. See Isotope-Coded Affinity Tag IC50 value, potency of ACE inhibitory peptide and, 208 IgE. See Immunoglobulin E IgE-mediated food allergy etiology of: two-phase phenomenon, 103 potential immunotherapeutic approaches for, 109t IGF-I in bovine colostrum vs. in human colostrum, 153 in bovine mammary secretions, 152, 161 IGF-II in bovine colostrum and milk, 153 in bovine mammary secretions, 152, 161 Ikemoto, F. IL-6. See Interleukin-6 IL-8. See Interleukin-8

400

Index

IL-10. See Interleukin-10 IL-beta. See Interleukin-beta Ile-Ala-Pro, hypertension prevention and, 299 Ile-Pro-Pro, 162, 172 absorption of, 174 antihypertensive effect of milk tested in hypertensive patients and, 173 casein hydrolyzate with, 170 hypotensive capacity of, 155 Ile-Tyr, vascular relaxation effect of, in 18-week-old SHR thoracic aorta rings constricted by 30 mmol/L KCI, 49 Ile-Val-Tyr, hypertension prevention and, 299 Immobilized metal affinity capture (IMAC), 314 Immune-enhancing nutraceuticals, bLf added to, 193 Immune milk, history behind concept of, 156 Immunoglobulin E, common form of food allergy mediated by, 102 Immunoglobulin heavy chain, 88t Immunoglobulin light chain, 88t Immunoglobulins (Igs), 7, 105–106, 151 in bovine colostrum and milk, 153t in colostrum, 152 whey proteins and, 156 Immunomodulating proteins and peptides, 254–255, 326 Immunostimulating peptides, 169 Incretins, 76 India, wheat production and consumption in, 9, 289 Indonesian dried-salted skipjack tuna, ACE inhibitory activity exhibited by, 211 Inducible isozyme, of NOS (iNOS), 56 Inducible Tregs, food allergy and, 104 Infant formulas, bLf and, 193

Infections, lactoferrin’s efficiency against, 193 Inflammation cell signaling mechanisms associated with, 19 chronic disease and, 6, 23 reactive oxygen metabolites and, 226 reactive oxygen molecules and, 18 reactive oxygen species and, 16 at root of chronic diseases, 227 Inflammatory bowel disease fecal lactoferrin and, 194 free radical attacks and, 29 lactoferrin’s anti-inflammatory role in, 187 Inflammatory diseases lactoferrin as clinical marker of, 194 lactoferrin’s efficiency against, 193 Inhalant allergens, PIT investigations carried out with, 114 in meso crystallization process, description of, 361 Inoue, K., 125 In silico analysis, of proteins and bioactive peptides, 326–328, 331 Insulin, 88t changes in circular dichroic spectra of, in solution and cubic liquid crystal compared with control insulin, 366, 367 GMO/water system loaded with, 363–365, 364 monomer as biologically active form of, 363 protection of, from agitationinduced aggregation and GMO/water cubic phase, 365, 366 Insulin A chain, 93t Insulin B chain, 93t Insulin/glucagon (I/G) ratio, soy protein consumption and, 76

Insulin-like growth factor, in bovine mammary secretions, 152, 161 Insulin molecule, dimeric, schematic representation of, 363 Insulin resistance metabolic syndrome and, 67, 69 soy protein and, 74–76, 75 Intercellular route of administration, across stratum corneum, 368, 368 Interleukin-6, 18 Interleukin-8, 18, 19 Interleukin-10 food allergy and, 104, 116 lactoferrin and, 187, 188 Interleukin-beta, 18–19 Intestinal inflammation, fecal lactoferrin and, 194 Intestine mucosal barrier, allergic response and, 102–103 Intramuscular route of administration, for biopharmaceuticals, 360 Intravenous injections, of biopharmaceuticals, difficulties related to, 360 In vitro protein digests, antioxidant activity of, 32–34 Ion transport, gamma-aminobutyric acid and modulation of, 124 IR. See Insulin resistance Iron, from animal muscle, 225 Iron-binding properties, of lactoferrin, 181–182 Ishikado, A., 20t Islam, fish gelatin acceptable for, 205 Islet amyloid polypeptide, 88t, 91, 93t Isoflavones, 30 Isoleucine ACE inhibitory activity, bitterness and, 348, 349 bitterness associated with, 343 Isotope-Coded Affinity Tag, 316 Isotope Tags for Relative and Absolute Quantification, 316 Itou, K., 213t

Index

J Jao, C. L., 217t Japan fish consumption in, 171 foods for specified health use in, 43 number of hypertensives and high-normal hypertensives in, 43 wheat production and consumption in, 289 Japanese fermented foods, ACEI peptides analysis in, 172–173 Japanese Minstry of Health, Labour and Welfare, 43 Je, J. Y., 210t, 217t Jean, C., 144 Johansson, B., 88t Johnson, J., 141 Johnson, P., 31t Johnstone, A. M., 137 Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure criteria, 80 Judaism, fish gelatin acceptable for, 205 Juices, antioxidant caseinophosphopeptides in, 35 Julka, S., 315 Jumbo flying squid, antioxidant peptides derived from, 217t, 218 Jun, S. Y., 37t, 217t Jung, W. K., 210t, 213t, 217t

Kawashima, K., 216 Kazuhito, A., 123 KDR/Flk-1, lactoferrin and, 190 Kerato-epithelin, 88t Kernel, 233 Kieffer, T. J., 140 Kim, H-O, 345, 348 Kim, K. S., 31t Kim, S. K., 210t, 217t Kim, Y-C, 373, 374 Kingman, S. M., 277 Kinin-nitric oxide system, blood pressure regulation and, 207 Kininogen, 207 Kinnersley, A. M., 124 Kizawa, K., 58 Klompong, V., 217t KNOS. See Kinin-nitric oxide system Ko, W. C., 217t Kobayashi, H., 20t Kodera, T., 350 Kohama, Y., 209t Kohno, M., 77t Komatsu, T., 269 Komatuszaki, N., 126 Konig, D., 78t Kono, I., 126 Korpela, J., 249t Korvatska, E., 88t Kosmotropes, 362 Kosmotropic solutes, properties of liquid crystalline phases and, 362 Kovacs-Nolan, J., 249t Kraineva, J., 363 Kruzel, M. L., 159 Kunitz trypsin inhibitor (KTI), soyderived, anti-inflammatory mechanisms and, 20t, 22–23 Kuru disease, 90

K Kallikrein, 207 Karagiannis, E. D., 318 Karelin, A. A., 325 Kasaoka, S., 143 Katayama, S., 256 Katsuobushi (dried bonito) oligopeptide, 43, 44t

L LAB. See Lactic acid bacteria Lactadherin, 88t Lactalbumin alpha Lactalbumin alpha, bovine chromatograms of, 336, 338 mass spectrum of tryptic hydrolysate of, 338

iTRAQ. See Isotope Tags for Relative and Absolute Quantification

401

profile of potential biological activity of, 337 Lacteal secretions, in vivo activities of lactoferrin in, 157 Lactic acid bacteria, 240 antioxidant activity of, 38–39 fermented products and, 172 GAD properties from, 123t Lactic acid bacteria (LAB)-based starter cultures, proteolytic properties of, 155 Lactobacillus, GABA biosynthesis with, 126 Lactobacillus acidophilus, high radical-scavenging activity in, 39 Lactobacillus brevis, GABA content and, 126–127 Lactobacillus casei, 21 Lactobacillus helveticus ACE inhibitory peptides isolated from, 172 antihypertensive peptides and, 155 effect of, in spontaneously hypertensive rat, 173 Lactobacillus helveticus-fermented milk (sour milk), 44, 44t Lactobacillus jensenii, high radical-scavenging activity in, 39 Lactobacillus paracasei NFRI, GABA synthesis and, 126 Lactobacillus plantarum, lysozyme and, 251 Lactobacillus rhamnosus GG-degraded bovine casein, 21 Lactococcus lactis subsp. cremoris FT4, ACEI peptides produced in fermented milk started by, 172 Lactoferrampin, 180 Lactoferricin, 180 Lactoferrin (Lf), 7, 20t, 88t, 151, 250 anti-inflammatory activity associated with, 20t, 21–22 antitumor activity of, 158 applications of, and related peptides, 193–194

402

Index

Lactoferrin (continued) beneficial health effects with, 158 biological roles of, in cancer and cell proliferation, 189–191 biological roles of, in host defense, 185–189 anti-inflammatory properties of, 187–188 antimicrobial activities of, 185–187 pro-inflammatory properties of, 188–189 in bovine colostrum and milk, 153t characteristics of, 180 iron-binding properties and dynamics of, 181–182 localization of Lfcin domain and Lfampin sequence on, 183 mechanisms related to antimicrobial activity of, 157–158 overall structure of, 181 protective effects of, 8, 179 secreted, origin of, 180 structure of, 180–181 gene structure and amino acid sequence, 180 glycosylation of, 181 in 3D, 181 structure of biologically active peptides of, 182–185 biologically active peptide sequences of, 182–184 structure of Lfampin, 185 structure of Lfcin, 184–185 whey proteins and, 157–159 Lactoferrin-related peptides biogical properties and mechanisms of, 191–193 of Lfampin, 193 of Lfcin, 191–193 Lactoglobulin beta, 334 Lactoperoxidase, 151 in bovine colostrum and milk, 153t in colostrum, 152 health benefits with, 159 whey proteins and, 159–160 Lactotransferrin, 180

Lagarde, G., 249t Lam, 281, S. S. L. Lamellar phase, delivery of proteins and peptides in, 360, 363 L-amino acids bitterness associated with, 343 taste, detection threshold, and taste-enhancing effects of, 343t Lang, V., 138. Lasekan, J. B., 277 Late embroygenesis-abundant protein group, 237 Lateral hypothalamus with orexin-containing neurons, detection of high-protein meals and, 145 satiety and, 143 Latham, C. J., 143 Laying hens, feeding formula for, 239 L-carnitine in animal muscle, 225 antihypertensive and cardiovascular effects of, 227 health benefits with, 226 hypotensive effect of, 227–228 LEA protein group. See Late embroygenesis-abundant protein group Lectins, rice bran, 241–242 Lee, M., 19, 20t Lee, S. J., 20t Lee, Y. J., 320 Legionella pneumophila, lactoferrin’s antimicrobial activities and, 185 Legnin, 233 Legumes health promotion and, 9 natural antioxidants in, 215 as sources of dietary protein, 273 Legumin, in chickpea seeds, 280 Lejeune, M. P., 141, 142 Leptin, 70, 71 Leucine, 248 ACE inhibitory activity, bitterness and, 349 bitterness associated with, 343 food intake and, 143

Leuconostoc mesenteroides ssp., high radical-scavenging activity in, 39 Leu-Lys-Pro, antihypertensive effects of, in spontaneously hypertensive rat, 173 Leu-Lys-Pro-Asn-Met antihypertensive effects of, in spontaneously hypertensive rat, 173 preparation of, 171 Leu-Val-Tyr, preparation of, 171 Leventhal, A. G., 125 Lf. See Lactoferrin Lfampin biological properties and mechanisms of, 193 sequences of, from hLf and bLf, 184t structure of, 185 LfampinB peptide, 193 LfampinH, 193 Lfampin sequence, localization of, on lactoferrin, 183, 183 Lfcin, 191 antimicrobial activities of, 194 biological properties and mechanisms of, 191–193 sequences of, from hLf and bLf, 184t structure of, 184–185 LfcinB, actions of, against bacteria, 191 Lfcin domain, localization of, on lactoferrin, 183, 183 LfcinH, actions of, against bacteria, 191 Lf gene, transcription of, 180 L-glutamic acid decarboxylase biosynthetic pathway and GABA metabolism and, 130 proposed transformation of L-Glu to GABA by, 129 L’Heureux-Bouron, D., 142 Li, B. F., 217t Li, H., 62 Liao, F-H, 77t Li-Chan, E. C. Y., 345, 348 Lichtenstein, G. R., 20t

Index

Lifestyle modifications, hypertension management and, 43 Lim, K. T., 20t Lin, F., 124 Lin, L., 217t Linke, R. P., 88t Linoleic acid, 216 Lipases, isolated from rice bran, 238 Lipid metabolism, efficacy of soy peptides on, vs. with soy protein, 267 Lipid oxidation inhibiting, 216 mechanism of, 214 Lipids, 105 free radical attacks on, 29 functions of, 214 Lipid transfer proteins, 283 Lipophilic pathways, of drug penetration, 368, 369 Lipopolysaccharides inflammation and, 18 lactoferrin and, 187, 188 Lipoprotein Receptor-related Protein, 182 Liposomes with ethanol, penetration experiments of CysA and, 371 Lipoxygenase, 214, 215 Liquid chromatography, investigating bioactive peptide proteins with BIOPEP database and, 336 Liquid crystalline phases cubic and hexagonal time course of in vitro skin penetration and percutaneous delivery of CysA in, compared to olive oil control formulation, 369–370, 370, 371 in vivo skin penetration of CysA in, compared to olive oil as control formulation, 371, 371 delivery of proteins and peptides and, 360, 379 Liquid crystalline systems, entrapment and stability of proteins and peptides by, 360–366

Liquid crystal mesophases (“in meso” method), 361 Lisinopril, 208 Listeria monocytogenes, lactoferrin’s antimicrobial activities and, 158, 185 Liu, W., 361 Livetin, in egg yolk, 248 Localized amyloidosis, 90 Loop class of proteins and peptides, 361 Lopes, L. B., 369, 371 Lovati, M. R., 79 Low-density lipoproteins, in egg yolk, 248 Low-molecular-weight glutenin, 293 LOX. See Lipoxygenase LOX-1, 215 LOX-2, 215 LP. See Lactoperoxidase LPS. See Lipopolysaccharides LPS-binding protein (LBP), 187 LRP. See Lipoprotein Receptor-related Protein L68Q cystatin C, 90, 91, 91, 92 LTPs. See Lipid transfer proteins Lukaszuk, J. M., 78t Lung tumors, in mice, Bowman-Birk inhibitors and, 278 Luppi, P. H., 125 Lutein, 30 LXR alpha, soy protein and, 79 Lycopene, 30 Lys amino acid, potent antioxidant activity with, 17 Lysine ACE inhibitory activity, bitterness and, 349 in cereal grain proteins, 234 sweet and bitter taste of, 343 Lysozyme (LZM), 19, 20t, 88t, 247, 258 antimicrobial activity of, 250 antimicrobial peptides from, 251–252 antioxidant activity of, 256 in bovine colostrum and milk, 153t in cow’s colostrum, 152 in egg white and shell membranes, 249–251

403

hen, 93t human, 93t immunomodulating properties of, 254–255 sweetness threshold value of, 351 LzP, antimicrobial activity of, 251–252 M Mackerel antioxidant peptides from, 217t hypotensive effects of, in spontaneously hypertensive rats, 213t Macromolecules, increasing permeability of, new technologies for, 360 Maebuchi, M., 266 Magainin, penetration of fluorescein into epidermis after treatment with N-lauroyl sarcosinen and, 375, 375 Magainin peptides, skin permeability and, 373–376 Maize zein, antioxidant activity of, 216 Makin, O. S., 89 MALDI, 310 MALDI-Q-TOF, 310 MALDI-TOF-MS, 310 Mammalian Target of Rapamycin model for role of, in hypothalamic regulation of energy balance, 144 satiety, high-protein diets and, 143 Mammals, metabolic pathway in, 127 Manganese, 215 MAPKs. See Mitogen activated protein kinase Marine peptide, 171t Mass spectrometry investigating bioactive peptide proteins with BIOPEP database and, 336 peptidomics and, 307, 308, 309–311 Mastitis, lactoferrin and prevention of, 194 Masuda, K., 267

404

Index

Matsufuji, H., 209t, 213t Matsui, T., 174, 209t Matsumura, N., 209t Matthews, D. M., 265 Maury, C. P., 88t Mayonnaise, antioxidant caseinophosphopeptides in, 35 MBP. See Milk basic protein M cells, 102 MCP-1. See Monocyte chemoattractant protein 1 MDA-MB-231 breast cancer line, lactoferrin and, 190 Meat products lysozyme and bacteria control in, 251 rice bran proteins in, 239 Medium-chain acyl-CoA dehydrogenase, soy protein feeding in rats and, 73 Melanomas, Lfcin’s antitumor effects against, 192 Melatonin antioxidant activity and, 31, 31t chemical structure of, 31 Melittin, liquid crystalline phases and, 363 Membrane proteins, as important diagnostic and prognostic markers, 313 Membrane separation technique, for fractionation and isolation of bioactive milk proteins, 153 Mendis, E., 217t Mero, A., 161 Metabolic disorder, flavor-active components and novel therapeutic approaches to, 355 Metabolic parameters, soy protein diets and effect on, 77–78t Metabolic syndrome. See also Diabetes; Obesity; Soy protein for metabolic syndrome chronic inflammation and, 227 criteria for definitions and diagnosis of risk factors in, 68t

incidence of, 67 milk-derived components and reducing risk of, 155 pathogenesis of, 69–71, 70 quintet of factors in, 67 soy protein action on insulin resistance in, 75 soy protein and, 6 sweet proteins and, 351 whey proteins and, 152 Metal-ion binders, 37 Metal-ion catalysts, sequestering, peptides inhibiting oxidative processes and, 37 Metallo-collagenase MMP-13, 205 Metal prooxidants, sequestering and stabilization of, 37 Met amino acid, potent antioxidant activity with, 17 Metastasis, lactoferrin as defense against, 189 Methionine, 29 in cereal grain proteins, 234 hypocholesterolemic effects of pea proteins and levels of, 276–277, 277 radical quenching activity of peptides and, 35 MHC polymorphism, food allergy and, 115 Microalbuminuria, metabolic syndrome and, 68t Microbe fermentation, 172 Microbes, lactoferrin binding to, 186 Microbial fermentation, antioxidant peptide preparation with, 38–39 Microbial growth, rice bran as substrate for, 240 Microencapsulation, bitter taste reduction and, 354 Microorganisms, GABA synthesis by, 126–127, 127t Migraine headaches, nitric oxide levels and, 56 Mikkelsen, T. L., 20t, 21 Milk immune, history behind, 156 major bioactive whey proteins in, 153, 153t

Milk basic protein, bone-strengthening effects of, 161 Milk growth factors biological functions of, 161 health benefits with, 161 Milk lysozyme, anti-inflammatory activity with, 19 Milk peptides nutritional benefits with, 154 protein-derived, as calmodulin inhibitors, 56–58 Milk proteins, 215 antioxidative stress proteins and peptides in, 18 bioactive peptides and, 8 high nutritional value of, 7–8, 151 release of bioactive proteins from, 332 selected biopeptides released by trypsin from, 334, 336 Milk whey proteins, health benefits with, 155 Miller, D. J., 227 Miller, G. M., 30 Millet, rice protein net protein utilization compared to, 240 Mine, Y., 251 Minerals, 215 salty taste and, 342 soluble peptides, 169 Miraculin, 351 Mitogen activated protein kinase, 19 Miyagawa, S., 249t, 253 Molla, A., 249t Monascus, GABA biosynthesis with, 126 Monascus-fermented rice, 126, 129 Monascus purpureus CCRC 31615, GABA synthesis and, 126 Monellin, 351 Monnai, M., 20t Monoacylglycerols aqueous channel size, drug release rate and, 373 hydrated, effect of drug radius of gyration on transport from cubic phase of, 372 Monocolonal antibody-based products, 359

Index

Monocyte chemoattractant protein 1, 71 Monocytes, 19 Monoolein aqueous channel size, drug release rate and, 373 as penetration enhancer, 370 Monoolein cubic phase, summary of fluorescent features observed in two-photon microscopy images at various tissue depths of skin exposed to sulphorhodamine B in, 378t Monopalmitolein, aqueous channel size, drug release rate and, 373 Monosodium glutamate bitterness masked with, 350 protein hydrolysates used with, 238 Monovaccenin, aqueous channel size, drug release rate and, 373 Mood regulation, in animals, GABA and, 124 Moran, L. J., 142 Morrison, C. D., 143 MRM. See Multireaction monitoring MS. See Mass spectrometry; Metabolic syndrome MSG. See Monosodium glutamate mTOR. See Mammalian Target of Rapamycin Multidimensional protein identification technology (MudPIT), 313 Multifunctional peptides, in BIOPEP, characteristic activity of, 332 Multireaction monitoring, 310 Murai, T., 125 Murphy, C. L., 88t, 94 Muscle-based bioactive peptides, 8 Muscle protein, antioxidant acitivty of peptides in, 34 Mycoleptodonoide aitchisonii, aqueous extract from, 43–44, 44t Myofibrillar proteins, in fish proteins, 204

Myoglobin, 89, 93t Myosin, in myofibrillar tissue proteins, 204 Mytilus coruscus muscle protein, peptide with potent antioxidative activity from, 218 N N-acetyl-5-methoxytryptamine, 31 N-acetylglucosamine, 250 N-acetylmuramic acid, 250 N-acetylneuraminic residues, anti-inflammatory activity and, 21 NAG. See N-acetylglucosamine Nagai, T., 217t Nagasawa, T., 31t Nakajima, K., 210t, 352 NAM. See N-acetylmuramic acid Nanofiltration techniques, fractionation of bioactive peptides and, 155 National Cancer Institute Best Practices of Biospecimen Resources, 311 National Cholesterol Education Program, 67 Natto antioxidant peptides in, 38 inhibitory activities of, 172 Natural antioxidants, 215 Natural killer cells, food allergy and, 103, 105 Navab, M., 37t NCEP. See National Cholesterol Education Program NDGA. See Nordihydroguaiaretic acid Near infrared light, 376 Neoculin, 351 Neotame, 351, 355 NEPS. See Neutral endopeptidase system Net protein utilization, 240 Neuraminidase, 21 Neurodegenerative diseases lactoferrin and, 188 lactoferrin’s anti-inflammatory role in, 187

405

reactive oxygen metabolites and, 226 Neurological diseases, curing in animals, GABA and, 124 Neurological effects, muscle-based dipeptides and, 228 Neuronal isozyme, of NOS (nNOS), 56 Neuronal nitric oxide synthase, flaxseed protein-derived cationic peptide fractions and kinitecs of inhibition of, 59 Neuropeptides, 307, 319 Neuropeptide Y, satiety, high-protein diets and, 143, 144 Neutral endopeptidase system, blood pressure regulation and, 207 Neutrophils, 19 Ney, K. H., 345 Ng, T. B., 282, 283 Nguyen, S. D., 37t Nicholls, W. C., 88t Nicin, 250 Nicotinamide adenine dinucleotide phosphate oxidase complex, 16 Nielson, K. L., 249t Nilsson, M. R., 88t NIR. See Near infrared light Nitric oxide, excessive levels of, 56 Nitric oxide synthases, main isozymes of, 56 Nitrogen solubility index, 236 Nitrogen storage, of gamma-aminobutyric acid, 123 NK cell cytotoxicity, lactoferrin and promotion of, 189 N-lauroyl sarcosine (NLS) penetration of fluorescein into epidermis after treatment with magainin and, 375, 375 skin permeability study and, 374 NMBzA-induced esophageal tumors, Bowman-Birk inhibitors and, 278 Nomura, A., 210t Nonheat stabilized rice bran, 242

406

Index

Noninfectious pathologies, lactoferrin and, 188 Nonsteroidal anti-inflammatory drugs, colostrum-based products with growth factors and side effects of, 161 Noodles, instant, GABA used in, 129 Nordentoft, I., 76 Nordihydroguaiaretic acid, 92 Noriega-Lopez, L., 75 NOS. See Nitric oxide synthases Novel peptides, peptidomics and, 320 NPU. See Net protein utilization NPY. See Neuropeptide Y NSI. See Nitrogen solubility index NSRB. See Nonheat stabilized rice bran N-terminal helix, lysozyme and, 251 Nuclear factor-kappa B, inflammation and activation of, 18–19 Nuclear layer, bran, 233 Nucleic acid-based medicinal products, 359 Nucleic acids, free radical attacks on, 29 Nutraceuticals, growing demand for, 354. See also Functional foods Nuts, natural antioxidants in, 215 O OBBR. See Office of Biorepositories and Biospecimen Research Obese rat, blood lipid level of, 269 Obesity. See also Metabolic syndrome; Protein-induced satiety and food intake inhibitions; Satiation/satiety; Weight loss bioactive milk peptides and, 163 centripetal, metabolic syndrome and, 67 flavor-active components and novel therapeutic approaches to, 355 insulin resistance promoted by, 70 metabolic syndrome and, 68t, 71

overconsumption of salty and sweet foods and, 342 soy protein and, 6 sweet proteins and, 351 type II diabetes and, 67–68, 298 OCX-36. See Ovocalyxin-36 Odashima, M., 20t Odontogenic ameloblast-associated protein, 88t O’Dowd, A., 227 “Off-flavor” problems, interactions with proteins and, 353 Office of Biorepositories and Biospecimen Research, 311 Ogundele, M. O., 20t Oh, P. S., 20t Ohtsubo, K., 126 Oilseed proteins, antioxidant activity of, 216 Oilseeds, natural antioxidants in, 215 Ointment, commercial summary of fluorescent features observed in two-photon microscopy images at various tissue depths of skin exposed to sulphorhodamine B and, 378t two-photon fluorescence images showing lateral distribution of sulphorhodamine B, after 24 hours of passive diffusion in skin and, 377, 377, 379 Okamoto, A., 210t Okara, 266 Oligopeptidases, reducing bitterness of protein hydrolysates and, 350 Oligophosphopeptides, 17 Olive oil biophenols, 16 Omega-3 fatty acids, in flaxseed, 58 Ono, S., 210t Oolong tea, GABA content in, 125 Opioid peptides, 169 Oral administration of biopharmaceuticals, difficulties related to, 360 Oral health care products, lysozyme used in, 251 Oram, J. D., 159

Ornithyl-beta-alanine hydrochloride, salty taste attributed to, 345 Ornithyltaurine hydrochloride, salty taste attributed to, 345 Orthonasal contributions, to aroma perception, 354 Osajima, K., 208, 209t Osborne classification, cereal proteins and, 235 Osteoblast proliferation, lactoferrin and, 191 Osteoporosis, lactoferrin and, 191 Otani, H., 20t OTAP-92, 252 Ovalbumin, 247 antihypertensive peptides in, 257 antimicrobial peptides in, 252–253 in egg white, 254 in serpin family, 258 Ovocalyxin-36, 250 Ovoinhibitor, as serine protease inhibitor, 258 Ovokinin, 257 Ovomucin, 247 anticancer property of, 255 antimicrobial activity of, 253 immunomodulating properties of, 254 Ovomucoid, 247 Ovotransferrin, 247, 250 anti-inflammatory activity with, 19, 20t antimicrobial activity of, 252 antioxidant activity of, 256 immunomodulating properties of, 254 Ovo-vegetarians, egg proteins and, 249 Ovovmacroglobulin, antimicrobial activity of, 253 Oxidation, disease and pathogenesis associated with, 29 Oxidative stress, 15–16 antioxidative stress food factors, 16 chronic disease and, 23 defined, 5, 15 endogenous antioxidative stress mechanisms, 16 exogenous protein/peptide antioxidants, 16

Index

Oyster, antioxidant peptides and GI digests of, 33 P Pain, nitric oxide levels and, 56 Pain management, transdermal route of administration and, 360 Pan, A., 68 Pan, Y., 158 Pancreatic elastase, in BIOPEP database, 332 Papain, 171 Papain hydrolysates, high molecular weight peptides in, 275 Parasites, lactoferrin and, 187 Parenteral route of administration for biopharmaceuticals, difficulties related to, 360 Parkinson’s disease, 90 alpha-synuclein and, 92 amyloid deposits and, 89 gamma-aminobutyric acid and, 124 nitric oxide levels and, 56 peptidomics and, 319 Parotha (oily flat bread), rice bran proteins in, 239 Pastries, antioxidant caseinophosphopeptides in, 35 Pasupuleti, V. K., 74 Pathogenesis, oxidation and, 29 Pattern-recognition receptors, 18 PBPCs. See Rice bran protein concentrates PCA. See Principal component analysis PCR. See Polymerasae chain reaction PCT-SPS. See Pressure Cycling Technology Sample Preparation System PDGF. See Platelet-derived growth factor Peanut allergy model, 113 Pea protein-derived cationic peptide fractions effect of, on intrinsic fluorescence of Ca2+/CaM-dependent protein kinase, 63, 63t

inhibition of CaM-dependent protein kinase II activity by, at varying CaM levels, 62, 62t Pea protein-derived peptides as calmodulin inhibitors, 62–64 increases in Fmax/Fo values for CaMKII interactions and, 60 Pea protein hydrolysates, enzymatic, free radical (DPPH)-scavenging activity of, 275 Peas angiotensin-I converting enzyme inhibitory effects of, 280–281 anticancer effects due to Bowman-Birk protease inhibitors, 278–280 antimicrobial effects due to peptides, 281–284 antimicrobial peptides from garden pea, 281–282 chickpeas, 283 commercial utilization potential of, 283–284 cowpeas, 282–283 antioxidant effects with, 274–275 bioactivity of proteins and peptides from, 9 hypocholesterolemic effects of, 275–278 Pedroche, J., 280 Pelagic thresher, ACE inhibitory peptides isolated from, 208 Pellegrini,A., 251 Pellet cooker, rice bran stabilization and, 235 Pellet mill, rice bran stabilization and, 235 Penetratin, 192 Peng, X., 35 Pentadecapeptide (gramicidin D), 361 Pentadin, 351 Pentane, 214 Pentosans, 233 PEPCK. See Phosphoenolpyruvate carboxylase kinase

407

Pepsin, 154, 171 in BIOPEP database, 332 PepT1, 140 Peptide antioxidants, exogenous, 16 PeptideAtlas, 311 Peptide-based immunotherapy advantages of, 110–111 aim and rationale for, 108 B-cell epitope-based immunotherapy, 110 current experimental models of, in food allergy, 111–114 cow’s milk allergy model, 112–113 egg allergy model, 113–114 inhalant allergens, 114 peanut allergy model, 113 wheat and beef allergy model, 114 food allergies and, 7, 102 food-based hydrolyzates, 109–110 strategies for, 108 suggested mechanisms underlying, 111 T-cell epitope-based immunotherapy, 110 translation of, into human clinical applications, 114–115 large-scale production of peptides, 115 MHC polymorphism, 115 use of overlapping synthetic peptides, 109 Peptide-based vaccines, practical issues relative to, 115 Peptide hormones, 307 Peptide libraries, 107 Peptide mass fingerprinting, 336 Peptide precursors, 328 Peptides. See also Amyloidogenic proteins and peptides; Antihypertensive peptides; Anti-inflammatory proteins and peptides; Antioxidant peptides; Antioxidative stress proteins and peptides antimicrobial proteins and, from eggs, 249–254 avidin, 254 egg cystatin, 254

408

Index

Peptides (continued) lysozyme, 250–251 ovalbumin, 252–253 ovomacroglobulin, 253 ovomucin, 253 ovotransferrin, 252 phosvitin, 253–254 antioxidant, 16–17 assessing radical-scavenging property of, 35 barriers and mechanism of bioavailability of, 366–379 binding of metal ions by, 37 bioactive, production of, 154–155 bitter taste and spatial arrangement of, 347 classes of, 361 controlled delivery of, 360 designed, 93t enzymatic release of, with antithrombotic and antiamnestic activity and control of gastric mucosal function, 332, 334t exogenous, examples of those exhibiting antioxidant activity in vitro and in model systems: sources and sequences, 37t flavor ingredients interacting with, 352–353 identification of, 314–315 immunomodulating, 254–255 large-scale production of, 115 liquid crystalline systems and entrapment and stability of, 360–366 novel, peptidomics and, 320 within nutraceutical food sector, 5 physico-chemical properties related to bitterness of, 345, 347 plethora of research on, 354 potential correlation between bitterness and bioactive properties of, 348–349 predicting bitterness of, 347–348 production of, from food sources, 344 from proteins, benefits with, 325

quantification of, 315–316 release of, from milk proteins, 332 separation, 313–314 size and bitterness of, 345, 347 synthetic, food allergy and use of overlapping, 109 taste-activating properties of, 344–345, 347–351 taste-active, development of, 355 therapeutic, 359 Peptide sequences, for calmodulin-binding peptides isolated from peptic hydrolysate of bovine casein and potency against phosphodiesterase I, 57t Peptide soup, 171t Peptide synthesis, automated, 115 Peptide therapeutics, improving delivery of, 10 Peptide transporters, 140 Peptide transport system, 265 amino acids transport system and, on small intestine cell, 266 Peptidic compounds, preventing amyloid formation and, 94–95 Peptidomics (or “peptide proteomics”) applications of, 317–320 peptide biomarkers in disease, 318t, 319 peptides and diet-disease associations, 318t, 319–320 peptides as biomarkers, 317–319, 318t peptides in regulation of behavior, 318t, 320 peptidomics and novel peptides, 318t, 320 for bioactive peptide analysis, 307–320 defined, 10, 307 methods in, 308–311 affinity peptidomics, 308–309 combinatorial peptidomics, 309 mass spectrometry, 309–311 methods of, 308

sample preparation for, 311–317 enrichment and detection of proteins useful to peptidomic studies, 312–313 method validation, 317 peptide identification, 314–315 peptide quantification, 315–316 peptide separation, 313–314 posttranslational modifications, 316–317 sample collection and preanalytical treatment, 311–312 steps in sample preparation for, 312 Peptidyl antioxidants, as multifunctional compounds, 30 PeptoPro, 163t Pepys, M. B., 88t Pericarp, 233 Permeability of macromolecules, increasing, technologies for, 360 Peroxyl radicals, 29 Peroxynitrite, 29 Pessi, T., 20t Pet care supplements, bLf added to, 193 Petersen, W. E., 156 PHA. See Phytohaemagglutinin Phagocytes, lactoferrin and activation of, 188 Pharmaceutical Research and Manufacturers of America, 359 Pharmacological applications, lysozyme used in, 251 Phe, 45 Phenolic antioxidants, 215 Phenolic compounds in flaxseed, 58 preventing amyloid formation and, 9294 Phenylalanine, 274 bitterness associated with, 343 Phifer, C. B., 144 Phormones, 326

Index

Phosphatidylcholine, hypocholesteremic effect of legume protein and, 276 Phosphatidylethanol-amine (PE) ratio, of liver microsomes, hypocholesteremic effect of legume protein and, 276 Phosphodiesterase I, 56 calmodulin-binding peptides isolated from peptic hydrolysate of bovine casein and potency against, 57t Phosphoenolpyruvate carboxylase kinase, 142 Phosphopeptides, 326 Phosvitin antimicrobial properties of, 253–254 antioxidant activity of, 256 in egg yolk, 248 Phosvitin phosphopeptides, 17, 17t, 18 Physical instability processes, protein and peptide drug inactivation with, 365 Phytantriol, two-photon fluorescence images showing lateral distribution of sulphorhodamine B, after 24 hours of passive diffusion in skin and, 377 Phytantriol cubic phase, summary of fluorescent features observed in two-photon microscopy images at various tissue depths of skin exposed to sulphorhodamine B in, 378t Phytic acid, in germinated brown rice, 126 Phytochemicals, 30 Phytohaemagglutinin, 21 Phytonutrients, bitter taste in, 342 Pichon, L., 137 Picrotin, 342 Piglet development, Lfcin and Lfampin chimeras and, 194 Pig skin gelatin, preparing, 205 Pisavin, 282 Pisumin, antifungal activity of, 282

PIT. See Peptide-based immunotherapy Pittaway, J. K., 276 Pituitary hormones, angiotensin II and, 207 Plant development, gamma-aminobutyric acid and, 123–124 Plant food allergens, structural superfamilies of, 105 Plant phenols, 215 Plant proteins anti-inflammatory mechanisms and, 23 antioxidant activity of, 216 antioxidative stress proteins and peptides in, 18 bioactive peptides and, 332, 334 Plants metabolic pathway in, 127 roles of GABA synthesis in, 122–124 Plant serine protease inhibitors, structurally distinct families of, 278 Plant sources of foods, 105 Plasma amino acids, as central satiety signals, 142–143 Plasmin, 171 Plasmodium falciparum, lactoferrin’s effects against, 186 Platelet-derived growth factor, in bovine mammary secretions, 152, 161 Playford, R. J., 161 Pleurotus ostreatus, sativin’s antifungal activity against, 282 PLP-dependent enzymes, reaction mechanism of GAD and, 127 PMF. See Peptide mass fingerprinting Pneumonia, lactoferrin and, 188 Pokeweed (PW) mitogen, 21 Polaprezinc anti-inflammatory activity of, 227 ulcer healing and, 228

409

Polar headgroup interaction, hydrophilic and lipophilic pathways of drug interaction and, 368, 369 Poliovirus, lactoferrin’s antimicrobial activity and, 158 Polisb, rice kernel, 234 Poly-enzymatic method, for reducing bitterness of protein hydrolysates, 350 Polymerase chain reaction, 311 Polymeric glutenins, elastic property of, 9–10 Polymorphonuclear cells, 19 Polyomvirus, lactoferrin’s effects against, 185 Polyphenol compounds, inhibitory mechanism of amyloid fibril formation by, 94 Polyphenols, bitter taste in, 342 Polyphosphates, bitterness masked with, 350 POMC. See Pro-opiomelanocortin POMC neurons, food intake and activation of, 144 Popel, A. S., 318 Pore-forming peptides, increasing skin permeability for transdermal delivery and, 373, 379 Pork gelatin, producing gelatin from fish to match gelling properties of, 219 Port protein, 216 Posttranslational modifications, 316–317 Potatoes antioxidant proteins/peptides in, 17 “wound-induced” inhibitors I and II in, 278 Potato protein, antioxidant activity of peptides in, 34 PPPs. See Phosvitin phosphopeptides Praventin, 163t Precipitation, protein and peptide drug inactivation with, 365

410

Index

Precursor proteins number of fragments with antithrombotic, antiamnestic, and regulating stomach mucosal membrane activity released from, 334t in silico, enzymes releasing bioactive peptides from, 335–336t PREDICT 7 application, 326 Pregerminated brown rice, GABA used in, 129 PREMIER clinical trial, on soy protein and blood pressure, 80 Pressure Cycling Technology Sample Preparation System, 311 Preventative medicinal chemistry, 6 Primary amyloidosis, 90 Principal component analysis, predicting bitterness of peptides and, 348 Prion diseases, 89, 90 amyloid deposits and, 89 identifying, 90 Prion elk, 93t Prion human peptide, 93t Prion mouse, 93t Prion protein, 88t, 90 Prion Syrian hamster, 93t Pripp, A. H., 349 Pro, 45 Probiotic bacteria, commercial, bioactive peptide production and, 155 Pro calcitonin, 88t Procaspase-3 activation, lactoferrin and, 190 ProDiet F200/Lactium, 163t ProDom, 326 “Prodrug” peptides, 354 Prolactin, 88t Prolamin, 290, 291–293, 295 Proline ACE inhibitory activity, bitterness and, 348 radical quenching activity of peptides and, 35 sweet and bitter taste of, 343

Proline endopeptidase, 332 Proline oligopeptidase, in BIOPEP database, 332 Pronase, 332 Pro-opiomelanocortin, 143 Propanol, 214 Prophylactic effect, 113 Propyl gallate, 214, 215 Prorenin, 207 PROSITE, 326 Protease/amylase mixture, rice bran protein extraction and, 236 Protease inhibitors, in eggs, 258 Protein animal muscle as valuable source of, 225 Atwater factor for, 142 cereal, 234 Protein antioxidants, exogenous, 16 Proteinase K, 171, 332 Protein-based therapeutics, physico-chemical and biological properties and delivery challenges with, 359–360 Protein-derived antioxidants in food systems, evaluation of, 34–35 Protein diet, weight loss and, 136 Protein hydrolysates antioxidant efficacy of, 38 assessing antioxidant activity of peptides in, 34 bitter peptides of, isolated by different researchers, 346–347t bitter taste in, 342 debittering approaches for, 349–351 flavor ingredients interacting with, 352–353 peripheral, gut-derived satiety hormones and, 139–140 physico-chemical properties related to bitterness of, 345, 347 plethora of research on, 354 production of, from food sources, 344

satiety triggering and, 136, 144–145 taste-activating properties of, 344–345, 347–351 Protein hydrolysis, bitter taste and, 345 Protein in diet, metabolic syndrome and, 68 Protein-induced satiety and food intake inhibition, 7, 136–144 central neuronal pathways and, 143–144 energy expenditure and glucose as metabolic signals in, 142 high-protein diets and, 137–138 high-protein preload and highprotein meal-induced satiety and food intake inhibition, 136–137 peripheral, gut-derived satiety hormones, 139–142 plasma amino acids as central satiety signals, 142–143 role of protein source and type in, 138–139 Proteins, 105. See also Amyloidogenic proteins and peptides; Anti-inflammatory proteins and peptides; Antioxidative stress proteins and peptides antioxidant, 16–17 antioxidant peptides in, 32 barriers and mechanism of bioavailability of, 366–379 bitter peptides of, isolated by different researchers, 346–347t in bran, 233 classification of, 290, 361 into families and subfamilies, 328, 336 controlled delivery of, 360 in eggs, 247, 249, 258 in egg yolk, 248 factors related to nutritional quality of, 203 fish, 204 flavor ingredients interacting with, 353–354

Index

free radical attacks on, 29 immunomodulating, 254–255 in silico analysis of, 326–328, 331 interaction between sweet-taste receptors and, 351–352 in large eggs, 249 liquid crystalline systems and entrapment and stability of, 360–366 plethora of research on, 354 profiles of potential biological activity of, 337 richest subfamilies of–precursors of peptides with selected activities, 329–331t strategy for research on, 327, 327 sweet, 351 taste-active, development of, 355 taste-active properties of, 351–352 interaction between proteins and sweet-taste receptors, 351–352 sweet proteins, 351 taste-modifying property of sweet proteins, 352 therapeutic, 359 Protein sequences, BIOPEP database and motifs with 23 types of activity in, 328 Protein solubility, role of, in processed foods, 237 Protein therapeutics, improving delivery of, 10 Proteolysis in silico, release of bioactive peptides from selected precursor proteins by enzymes or enzyme conjugates and, 331–332, 334 Proteolysis simulation example, in BIOPEP database, 333 Proteolytic enzymes bioactive peptides and, 162 BIOPEP data on, 331 Proteomic techniques, use of, 154 Proteose peptone-3, 20t anti-inflammatory mechanisms and, 20t, 22

Protozoans, Lfcin’s effects on, 192 PrP. See Prion protein PRRs. See Pattern-recognition receptors Psd1, 282 Psd2, 282 Pseudomonas aeruginosa, lactoferrin’s antimicrobial activity and, 158 Pseudomonas spp., ovotransferrin’s effect against, 252 PT cubic phase, GMO cubic phase vs., in hydrophilic fluorescent model drugs investigation, 379 PTMs. See Posttranslational modifications p21 protein, lactoferrin-induced overexpression of, 190 Pungent foods, 342 Purification, of bioactive milk proteins, 153 Puroindolines, 291 Q Q-ion-trap (Q-IT), 310 QSAR. See Quantitative structure and activity relationship QSAR modeling, ACE inhibitory activity, bitterness and, 349 Q-TOF, 310 Quantitative structure and activity relationship, 257, 258 development of functional ingredients and, 354 predicting peptide bitterness and, 347–348 Quark, bioactive peptides and, 161 Quercetin, 92 Q values, bitterness of peptides and, 345 R Rainbow trout fibroblasts, metallo-collagenase MMP-13 isolated from, 205 Rajapakse, N., 37t, 217t Ramipril, 174, 208 Ramputh, A. R., 123

411

RAS. See Rennin-angiotensin system RB. See Rice bran RBDPs. See Rice bran dehydrin proteins RBEE. See Rice bran enzymatic extract RBL. See Rice bran lectin RBPIs. See Rice bran protein isolates Rb promoters, lactoferrin and, 190, 191 RBPs. See Rice bran proteins RDA. See Recommended daily allowance RDI. See Recommended daily intake Reactive oxygen species, 5, 29, 163 defined, 15 GI diseases and, 32 lactoferrin and, 188 production of, 16 Reaven, G. M., 67 Recombinant hLf, antimetastatic effects with, 189 Recommended daily allowance, 248 Recommended daily intake, 249 Red wheat, 290 Regnier, F., 315 Regular Iletin I, aggregation profiles of, 365, 365 Regular Iletin II, aggregation profiles of, 365, 365 Regulatory T cells, food allergy and, 103 Rehault, S., 249t Reidelberger, R. D., 139 Reiter, B., 159 Ren, J., 217t Renin, 207, 298 Renin-angiotensin, circulatory, updated, 45 Renin-angiotensin system, 45 blood pressure regulation and, 207, 298 hypertension and, 44 inhibition of, by ACE inhibitory peptides, 46–47 Reperfusion injury, oxidative stress and, 31–32

412

Index

Residual proteins, in cereal, 235 Resistin, 70, 71 Retino-blastoma protein-mediated growth arrest, lactoferrin and, 190 Retinoid signaling pathways, lactoferrin and, 190 Retronasal contributions, to aroma perception, 354 Reverse hexagonal phase, delivery of proteins and peptides and, 360 Reverse osmosis, manufacture of whey powder and whey protein concentrates and, 153–154 Reverse phase strong cation exchange cartridge system, 318–319 Rheumatoid arthritis, lactoferrin’s anti-inflammatory role in, 187 rhLf. See Recombinant hLf Rhodobacter sphaeroides, 361 Rhodopsin/transducer complex, 361 Rhus verniciflua Stokes glycoprotein, anti-inflammatory mechanisms and, 20t, 23 Ribosome inactivating proteins, 282 Rice, production and milling of, 233 Rice-based vaccine, efficiency of, 115 Rice bran composition of, 233, 234t defatted, proximate composition and caloric content of, 234t enzymes isolated from, 238 future trends with, 242–243 protein provided by, 234 stabilization of, 234–235 Rice bran dehydrin proteins, functional properties of, 237 Rice bran enzymatic extract, 239 Rice bran lectin, 241–242 Rice bran protein concentrates, functional properties of, 237 Rice bran protein isolates, functional properties of, 237

Rice bran proteins, 234 amino acid composition of, 240t antinutritional behavior of, 242 extraction and isolation of, 235–237 alkali extraction, 236 enzymatic extraction, 236 miscellaneous methods, 236–237 functional properties of, 237–238 health benefits of, 240–242 as antioxidants, 242 lectins, 241–242 in prevention and control of cancers, 241 human health promotion and, 8–9 in product development, 238–242 in animal feeds, 239 in baby foods, 239 in bakery products, 239 as fat replacers, 239–240 as flavor enhancers, 238–239 as food-color carriers, 239 in meat products, 239 miscellaneous applications, 240 as substrate for microbial growth, 240 Rice bran proteolysate, 237 Rice cultivators, proteins consecutively solubilized with water, salt, alcohol, acetic acid, and sodium hydroxide from ether-defatted brans of, 235 Rice flour, composition of, 234t Rice kernel, parts of, 234 Righetti, P. G., 313 RIPs. See Ribosome inactivating proteins Ririe, D. G., 227 Riticum aestivum amino acid sequence of low-molecular-weight glutenin subunit in, 295 amino acid sequence of x-type high-molecular-weight glutenin subunit in, 294 amino acid sequence of y-type high-molecular-weight glutenin subunit in, 294

Rival, S. G., 37t RNase, 93t RO. See Reverse osmosis Rolls, R. J., 136 Ropelle, E. R., 143 ROS. See Reactive oxygen species Rosmarinic acid, 92 Rotaviruses immune milk preparations and, 156 lactoferrin’s antimicrobial activity and, 158 lactoferrin’s effects against, 185, 186 Roufik, S., 162 Round scad antioxidant peptides from, 217t antioxidative activity of hydrolysates from, 218 RP-SCX cartridge system. See Reverse phase strong cation exchange cartridge system rTI1B, 278, 278t rTI2B, 278, 278t Rukimini, C., 241 RVS glycoprotein. See Rhus verniciflua glycoprotein S S. enteriditis, lactoferrin’s antimicrobial activity and, 158 S. flexneri, lactoferrin and, 186 Saccharin, 351 Saccharomyces cerevisiae, ACE inhibitory peptides isolated from, 172 S-adenosyl methionine, hypocholesterolemic effects of pea proteins and, 277 Sadhale, Y., 365, 366 Saito, K., 37 Saito, M., 267 Saito, Y., 173 Sake, ACEI peptides in, 173 Sake lee, ACEI peptides in, 173 Salicin, 342 Salmon ACE inhibitory peptides derived from, 210t

Index

ACI inhibitory peptides derived from, 208 hypotensive effects of, in spontaneously hypertensive rats, 213t Salmonella enteritidis, ovotransferrin’s effect against, 252 Salmonella typhimurium, lactoferrin’s antimicrobial activities and, 158, 185 “Salting-out” effect, liquid crystalline phases and, 362 Salt-soluble globulin, 290 Salty taste, 10, 342 dipeptides and, 345 triggering of, 342 SAM. See S-adenosyl methionine Sarcoplasmic proteins, in fish proteins, 204 Sardine, ACE inhibitory peptides derived from, 209t, 210t Sardine muscle, hypotensive effects of, in spontaneously hypertensive rats, 213t Sardine peptide, 44, 44t Sathivel, S., 217t, 218 Satiation/satiety, 135 brain’s response to retronasal aroma release and, 354 complex and complementary pathways and mediation of, 145 defined, 135 egg breakfasts vs. bagel breakfasts and, 248 food intake inhibition in high-protein diets and, 137–138 high-protein meal-induced, high-protein preload and, 136–137 luminal events within GI tract and, 138 plasma amino acids as central signals in, 142–143 protein-induced, 7 central neuronal pathways and, 143–144

energy expenditure and glucose as metabolic signals in, 142 role of protein source and type in, 138–139 Satiety hormones, peripheral, gut-derived, 139 Sativin, antifungal activity of, 282 Savory peptides, in foods, 344 Savory taste, 343 Sawai, Y., 125 SAXS patterns. See Small angle x-ray scattering patterns SBEAMs architecture, 311 SBP. See Systolic blood pressure SCD-1. See Stearoyl-CoA desaturase Schiff-base, formation of, by substrate and PLP, 128 Schiff-base exchange reaction, PLP enzymes and, 127 Schulz-Knappe, P., 314 Scrutton, H., 157 SD-RBPs. See Spray-dried rice bran proteins SDS. See Sodiumdodecyl sulfate SDS-PAGE, 237 Sea bream ACE inhibitory peptides derived from, 208, 210t hypotensive effects of, in spontaneously hypertensive rats, 213t “Search for active fragments” icon, in BIOPEP, 332 Secondary amyloidosis, 90 Selenium, 215, 225 Semenogelin I, 88t Semolina, 289 Sensitization phase, in allergic response to food antigens, 103 Sensory profile of food, amino acids and, 344 Sensory rhodopsin, 361 Septicemia, 194 Septic shock, lactoferrin and prevention of, 187 Serine, 37 collagenases, 205 in fish gelatin, 206 sweet taste of, 343

413

Serpell, L. C., 89 Serpins, 291 Serum lipids, soy protein and, 76, 79 Sesame peptides, 44 Sesamine tea, 171t Severe Acute Respiratory Syndrome, 194 Shah, J. C., 365, 366 Shahidi, F., 217t Shanghai Women’s Health Study, 79 Shell eggs, 247 Shellfish antioxidant peptide production in GI system and, 33 antioxidant peptides from, 217t branched amino acids present in antioxidant peptides from, 216 Shigella dysenteriae, lactoferrin’s antimicrobial activities and, 185 Shigella flexneri, immune milk preparations and, 156 Shin, K., 20t Short-term preload paradigm, 136 SHRs. See Spontaneously hypertensive rats Sick fat, 71 SISCAPA. See Stable Isotope Standards and Capture by Anti-Peptide Antibodies SIT. See Specific immunotherapy Sites, C. K., 78t Skarra, L., 353 Skeletal muscle, oxidative deterioration of, 225 Skim milk, large-scale preparation of bLf from, 193 Skin allergy, lactoferrin and, 187, 188 Skin barrier, pathways of drug penetration across, 368, 368 Skin penetration, time course of percutaneous delivery of CysA incorporated in cubic and hexagonal liquid crystalline phases, with olive oil control, 369–370, 370

414

Index

Skin permeability biochemical enhancers and increase in, for transdermal drug delivery, 373 fluorescein and measurements of, 374, 374 magainin peptides and, 373–376 Skip-jack tuna ACE inhibitory peptides derived from, 209t Indonesian dried-salted, ACE inhibitory activity exhibited by, 211 Skp1 promoters, lactoferrin and, 190, 191 Sleep regulation, gamma-aminobutyric acid and, 125 Sletten, K., 88t Small angle x-ray scattering patterns, 363, 364 Small peptides amyloidogenicity of, 92 antihypertensive mechanism of, 46–51 inhibition of renin-angiotensin system by ACE inhibitory peptides and, 46–47 relaxation of vascular constrictive events by dipeptides and, 48–51 effect of, on Ang II stimulation or Bay K 8644 stimulation, 48, 48 Smeets, A. J., 136, 141 Smoking cessation, transdermal route of administration and, 360 Smooth hound (shark), antioxidant peptides from, 217t SOD. See Superoxide dismutase Sodiumdodecyl sulfate, hydrophobicity of insoluble glutelins and, 25 Sodium sterate, hydrophobicity of insoluble glutelins and, 235 Sodium substitutes, 345 Sole, antioxidant peptides from, 217t Soleimanpour, M. R., 265

Solubilization, of rice bran proteins, 235 Somatic and testicular ACE, 211 Son, D. O., 227 Sour milk, bioactive peptides and, 161 Sour taste, triggering of, 343 Soy anti-inflammatory mechanisms and, 22–23 di- and tri-enriched peptides developed from, 266 Soybean paste, ACEI peptides in, 173 Soybean protein, antioxidant activity of, 216 Soybeans, typical industrial processing of, 69 Soybean seedlings, GABA used in, 129 Soymilk, GABA content and, 126 Soy peptide ingestion, comparison of placebo, soy protein, and soy peptide on GH and CPK levels measured at 30 minutes and 18 hours after, 268 Soy peptides antiobesity effect of, 268–269 antioxidant activities related to, 17 di- and tri-enriched peptides from soy and, 266 efficacy of on lipid metabolism compared with soy protein, 267–269 in sports, soy protein vs., 267 as functional food system, 265–269 health promotion and, 9 manufacturing process for, 266 peptide transport system, 265 production of, 267 Soy proteins antioxidant acitivty of peptides in, 34 effects of animal protein and, on body fat ratio of obese rat and genetically obese mice, 269

efficacy of soy peptides in sports, soy proteins vs., 267 flavor ingredients interacting with, 353 health benefits with, 68, 81 high demand for, 239 lipid metabolism and efficacy of soy peptides vs., 267 for metabolic syndrome, 6, 67–81 blood pressure and, 79–80 glycemic control and insulin resistance, 74–76 reduction of caloric intake, 74 serum lipids and, 76, 79 weight loss and adiposity reduction, 71, 72t, 73–74 soy peptides prepared from, 9 Soy sauce, inhibitory activities in, 172 Specific immunotherapy, food allergy and, 108 Spellman, D., 350 Spices, natural antioxidants in, 215 Spielmann, J., 276 Spontaneously hypertensive rats, 8, 155, 170 ACE-inhibitory fish peptides and, 212–214 evaluating antihypertensive activity of ACEI peptides in, 173 hypotensive effects of fish-derived peptides in, 213t ovokinin’s effects in, 257 stroke-prone, ACE activity in aorta of, 174 Sports, soy peptides vs. soy protein and, 267 Sprague-Dawley (SD) rat aorta rings, carnosine relaxation effect in, 49 Spray-dried rice bran proteins, 237 Spring wheat, 290 SPS. See Stiff-person syndrome SP-sepharose, 58 Squid, ACE inhibitory peptides derived from, 210t SRB. See Sulphorhodamine B SREBP-1. See Sterol regulatory element-binding protein-1

Index

SREBP-2. See Sterol regulatory element-binding protein-2 SREBPs. See Sterol regulatory element binding proteins SSADH. See Succinic semialdehyde dehydrogenase SS31 peptide, antioxidant activity and, 31, 31t Stable Isotope Standards and Capture by Anti-Peptide Antibodies, 314 Staphylococcus aureus lactoferrin’s antimicrobial activity and, 158 ovotransferrin’s effect against, 252 Staphylococcus spp., lactoferrin’s antimicrobial activities and, 185 Starchy endosperm, rice kernel, 234 STC-1 cells, 140 Stearoyl-CoA desaturase, soy protein and, 79 Stemplot analysis, 328, 336 Sterol regulatory element-binding protein-1, soy protein and, 79 Sterol regulatory element-binding protein-2, 276 Sterol regulatory element binding proteins, soy protein and, 71 Stiff-person syndrome, 124 Stimulating site, 347 St-Onge, M. P., 78t Stratum corneum permeation of, by drug molecules, 367–368 in vitro penetration of CysA in, at 6 and 12 hours following topical application using hexagonal phase nanodispersion or control olive oil formulation, 372 water content in, 368 Streptococcus mutans immune milk preparations and, 156 lactoferrin’s antimicrobial activity and, 158

lysozyme effect against, 251 ovotransferrin’s effect against, 252 Streptococcus spp. GABA biosynthesis with, 126 lactoferrin’s antimicrobial activities and, 185 Streptococcus thermophilus, lactoferrin and, 194 Streptokinase, 360 Stress food factors, antioxidative, 16 Stress relief, alpha-lactalbumin and, 156–157 Stroke, nitric oxide levels and, 56 Subcutaneous route of administration, for biopharmaceuticals, 360 Subtilisin, 154, 171 Succinic semialdehyde dehydrogenase, 127, 128, 129 Succinyl-1-proline, 207 Sucralose, 351 Sucrose, 351 Suetsuna, K., 208, 209t Sugiyama, K., 276 Sulphorhodamine B chemical structure of, 376 summary of fluorescent features observed in two-photon microscopy images at various tissue depths of skin exposed to using different vehicles, 378t transdermal study of, using four delivery systems, 376–377 two-photon fluorescence images showing lateral distribution of, after 24 hours of passive diffusion in skin using four delivery systems, 377 Sunde, M., 89 Sunflower oil, amino acids with antioxidant activity in, 216 Supercritical water, rice bran protein extraction and, 237 Superoxide anion, 29 Superoxide dismutase, 16, 256

415

Suppressor T cells. See Regulatory T cells Surface adsorption, protein and peptide drug inactivation with, 365 Surface nucleolin, lactoferrin binding and, 186 Sweet proteins, 351, 352 Sweet taste, 10, 342 peptides with, 344 triggering of, 342 Sweet-taste receptors, interaction between proteins and, 351–352 SWISS-PROT, 326 Syndrome X. See Metabolic syndrome Synthetic antioxidants, 215 Synthetic peptides, overlapping, food allergy and, 109 Synthetic vaccines, 359 Systemic amyloidosis, 90 Systolic blood pressure, ovokinin and, 257 T T. gondii, lactoferrin’s effects against, 186 Tagging-via-substrate approach, for global identification of O-glycosyl enrichment, 314 Tamaru, S., 268 Tammen, H., 318 Tanaka, M., 95 Tancredi, T., 351 TAS approach. See Tagging-viasubstrate approach Taste, synergistic interactions with effect on, 344 Taste-active properties of peptides and protein hydrolysates approaches for debittering or production of less bitter protein hydrolysates, 349–351 physico-chemical properties related to bitterness of, 345, 347

416

Index

Taste-active properties (continued) potential correlation between bitterness and bioactive properties of peptides, 348–349 prediction of bitterness in peptides, 347–348 of proteins, 351–352 interaction between proteins and sweet-taste receptors, 351–352 sweet proteins, 351 taste-modifying property of sweet proteins, 352 Taste characteristics, of amino acids, 343–344 Taste modalities, 10, 342 Taste receptor cells, 342–343 Tau, 88t Tau, 93t Taurine, in animal muscle, 225 Taylor, A. A., 49 Taylor, C. G., 31t TBARS. See Thiobarbituric acid reactive substances T-cell epitope-based immunotherapy food allergy and, 110 potential mechanisms underlying, 112 T-cell epitopes antigen, representation of, 106 food allergen, 105–106 mapping, 106–107 T-cell receptors, 106 T-cell regulation, currently accepted view on, 105 T cells, allergic responses and, 105 TCRs. See T-cell receptors Tea antioxidant caseinophosphopeptides in, 35 GABA and, 125 natural antioxidants in, 215 Telomere shortening, carnosine protective against, 228 Tempeh, antioxidant peptides in, 38 Testa, 233 Textural profile analysis, 239

TGF-beta, food allergy and, 104, 116 TGF-beta1, in bovine mammary secretions, 152, 153, 161 TGF-beta2, in bovine mammary secretions, 152, 153, 161 THAA. See Total hydrophobic amino acids content Thaumatin, 351 T helper cell (ThO), food allergy and, 103 Therapeutic effect, 113 Therapeutic macaromolecules, oral administration of, difficulties related to, 360 Therapeutic proteins and peptides, 359 Thermal-processing, rice bran stabilization and, 235 Thermogenesis, diet-induced by protein, 142 Thermolysin, 154, 171 Thiansilakul, Y., 217t Thiobarbituric acid reactive substances, 34 THM. See Tsukuba-Hypertensive Mouse Thomas, D. A., 31t Th1 response, lactoferrin and, 188–189 Th1/Th2 cytokine balance, lactoferrin and, 188 Th1/Th2 model, food allergy and, 103 Threonine, 37, 343 Th17 cells, 105 Thymus-derived Tregs, food allergy and, 104 Tissue array technology, 311 Tityus serrulatus venom peptides, identifying, 320 TLR4. See Toll-like receptor 4 TLRs. See Toll like receptors T-lymphocytes food allergy and roles of, 103–105 lactoferrin and, 188 TMAB. See Trimethylammoniumbutyrate TNBS. See Trinitrobenzenesulfonic acid

Tobacco smoke, reactive oxygen species and, 16 Tofu, fermented, inhibitory activities in, 172 Togawa, J., 20t “Tolerance” induction, food allergy and, 108 Tolerogenicity, hypoallergenicity vs., 112–113 “Tolerogenic” peptides, 108 Toll-like receptor 4, lactoferrin and, 187 Toll like receptors, 18 Tomato, “wound-induced” inhibitors I and II of, 278 T1R2-T1R3 receptors, proteins and, 351–352 Total hydrophobic amino acids content, antioxidant effects from peas and, 274 Toxoplasma gondii, Lfcin’s effects on, 192 TPA. See Textural profile analysis TPM. See Two-photon microscopy Transcellular route of administration, across stratum corneum, 368, 368 Transdermal route of administration, for biopharmaceuticals, 360 Transepidermal route of administration across stratum corneum, 368 divisions of, 368 for permeation of stratum corneum by drug molecules, 367–368 Transfollicular route of administration, across stratum corneum, 368 Transforming growth factor, in bovine mammary secretions, 152, 161 Transglandular route of administration, across stratum corneum, 368 Transthyretin, 88t TRCs. See Taste receptor cells Tregs, food allergy and, 103, 104–105 Treharose, 95

Index

TrEMBL, 326 Tremblay, F., 75 Tricitum sphaerococcum, 290t Triglycerides metabolic syndrome and levels of, 68t soy protein and, 76 Tri-glycine peptides, absorption of, 265 Trimethylammoniumbutyrate, 316 Trinitrobenzenesulfonic acid, in rats, anti-inflammatory effect of GMP and, 21 Tripeptide ACE inhibitors, 212 Tripeptides antioxidant properties and, 37 development of, from soy, 266 intestinal adsorption of, 174 release of, from milk proteins, 332 Triplet repeat disease, 92 Triticum aegilopoides, 290t Triticum aestivum, 289, 290, 290t amino acid sequence of alpha-amylase inhibitor in, 291 amino acid sequence of alphabeta-gliadin in, 292 amino acid sequence of y-gliadins in, 292 Triticum compactum, 289, 290, 290t Triticum dicoccoides, 290t Triticum dicoccum, 290t Triticum durum, 289, 290, 290t Triticum macha, 290t Triticum monococcum, 290t Triticum orientale, 290t Triticum polonicum, 290t Triticum spelta, 290t Triticum turgidum, 290t Triticum urartu, 290t Triticum vavilovii, 290t Tritrichomonas foetus, lactoferrin’s effects against, 185 Tropomyosin, in myofibrillar tissue proteins, 204 Troponin, in myofibrillar tissue proteins, 204 Trp, 17, 45

Trp-His schematic representation of binding site to voltage-gated L-type Ca2 channel in VSMC, 52 vascular relaxation profiles of, in endothelium-intact (+) or endothelium-denuded Sprague-Dawley rat aorta rings, 51 Trypanosoma brucei, lactoferrin’s effects against, 185 Trypanosoma cruzi, lactoferrin’s effects against, 185 Trypsin, 154, 171, 250 in BIOPEP database, 332 Bowman-Birk inhibitors and, 278t identification of selected biopeptides released by, from milk proteins, 334, 336 Trypsin hydrolysates, 275 Tryptic hydrolysates, of casein, 43, 44t Tryptophan, 274 ACE inhibitory activity, bitterness and, 348 radical quenching activity of peptides and, 35 Tsukuba-Hypertensive Mouse, 46 Tsushida, T., 125 TR family, of bitter receptors, 343 Tumorigenesis, lactoferrin as defense against, 189, 190 Tumor necrosis factor-alpha, 18, 19, 187, 188 Tumor suppressor proteins, lactoferrin acting as, 191 Tuna ACE inhibitory peptides derived from, 208, 209t antioxidant peptides from, 217t hypotensive effects of, in spontaneously hypertensive rats, 213t Twin-screw extruder, rice bran stabilization and, 235 Two-dimensional electrophoresis, investigating bioactive peptide proteins with BIOPEP database and, 336

417

2-D-methylsuccinyl-1-proline, 207 Two-photon microscopy, transdermal studies and use of, 376, 377, 377, 379 Type I diabetes, 298 Type II diabetes, 71, 75, 298 Tyr, 17, 45 Tyrosine, 29, 274 antioxidant activity of peptides and, 216 bitterness associated with, 343 food intake and, 142, 143 radical quenching activity of peptides and, 35 Tyr-Val, vascular relaxation effect of, in 18-week-old SHR thoracic aorta rings constricted by 30 mmol/L KCI, 49 U U. S. Department of Agriculture Foreign Agriculture Service, 9 UCPs. See Uncoupling proteins UDN. See Ulmus davidiana Nakai UF. See Ultrafiltration Ulcers, carnosine and healing of, 228 Ulmus davidiana Nakai, anti-inflammatory mechanisms and, 20t, 23 Ultrafiltration fractionation of bioactive peptides and, 155 manufacture of whey powder and whey protein concentrates and, 153–154 Umami taste, 10, 342, 343 glutamate and, 344 peptides with, 344 triggering of, 342 Uncoupling proteins, soy protein and, 74 United States, rice bran production in, 233 U.S. Department of Agriculture, Foreign Agricultural Service, 289 U.S. Food and Drug Administration, 115, 129, 159

418

Index

USDA. See U. S. Department of Agriculture UV radiation, reactive oxygen species and, 16 V Vaccines, synthetic, 359 Vagal afferent pathways, protein sensing and signalling to brain and, 144 Valenti, P., 249t Valine ACE inhibitory activity, bitterness and, 348, 349 bitterness associated with, 343 Valio (Finland), 170 Val-Pro, antihypertensive effects of, in spontaneously hypertensive rat, 173 Val-Pro-Pro, 162, 172 absorption of, 174 antihypertensive effect of milk tested in hypertensive patients, 173 casein hydrolyzate with, 170 hypotensive capacity of, 155 Val-Tyr with ACE inhibitory activity, 171 antihypertensive effects of, in spontaneously hypertensive rat, 173 change in systolic blood pressure and ACE activities of 18-week-old spontaneously hypertensive rat after administration of, 47 inhibition of renin-angiotensin system and, 46–47 regulation of vascular events by dipeptides and, 47–48 vascular relaxation effect of, in 18-week-old SHR thoracic aorta rings constricted by 30 mmol/L KCI, 49 Vascular constrictive events, dipeptides and, 47–51 Vascular smooth muscle cell, 47 Trp-His binding site to volatge-gated L-type Ca2 channel in, 52

“Vectors of Hydrophobic, Steric, and Electronic,” predicting bitterness of peptides and, 348 Vegetable oils, amino acids with antioxidant activity in, 216 Vegetable proteins, hypocholesterolemic effects of, 275–278 Vegetables, natural antioxidants in, 215 Vegetable sources of foods, 105 Ventromedial nucleus, satiety and, 143 Verbeek, M. M., 88t Verma, D. D., 371 Vermeirssen, V., 281 Very-low-density lipoproteins, 71, 276 VHSE. See “Vectors of Hydrophobic, Steric, and Electronic” Vibrio cholerae, lactoferrin’s antimicrobial activities and, 158, 185 Vibrio parahaemolyticus, halophilic, Lfcin and Lfampin chimeras and, 194 Vickers, Z., 141 Vidal, R., 88t “View the report with the results” option, in BIOPEP, 332 Virchow, Rudolf, 89 Virtanen, T., 39 Viscozyme, 237–238 Visfatin, 70 Vitamin C, 30 Vitamin E, 30 Vitamins, 215 from animal muscle, 225 in bran, 233 Vivinal Alpha, 163t VLDL. See Very-low-density lipoproteins VMN. See Ventromedial nucleus VSMC. See Vascular smooth muscle cell W Wagner, J. D., 74 Wako, Y., 210t

Walleye pollack antioxidant peptides from, 217t antioxidative activities of enzymatic hydrolysates from, 218 Wang, D., 38 Wang, H. F., 125 Wang, H. X., 282 Wang, L., 320 Wang, Y. F., 80 Ware, J. H., 20t Watanabe, K., 249t Water, as penetration enhancer, 368 Water channels, drug rate of transfer and size of, 373 Water-soluble albumin, 290 Water-soluble oryzanol enzyme extract, antioxidative function of, 242 Water-soluble proteins and peptides, properties of liquid crystalline phases and, 362 Water vehicle summary of fluorescent features observed in two-photon microscopy images at various tissue depths of skin exposed to sulphorhodamine B and, 378t two-photon fluorescence images showing lateral distribution of sulphorhodamine B, after 24 hours of passive diffusion in skin and, 377 WDEIA. See Wheat-dependent exercise-induced anaphylaxis Wei, C., 37t Weighted holistic invariant molecular index descriptors, predicting bitterness of peptides and, 348 Weight loss eggs and, 248 gamma-aminobutyric acid and, 125 high-protein meals and diets and, 145 soy protein’s effect on, 71, 72t, 73–74, 77–78t sustained high-protein diet and, 136

Index

Weight management, whey proteins and, 152 Weight reduction, high-protein diets and, 68 Westermark, P., 88t, 91 Westerterp-Plantenga, M. S., 137 Wheat classification of, 289–290 families of alpha-amylase inhibitors in, 298 grain color of, 290 major cultivated species of, 289, 290t rice protein net protein utilization compared to, 240 sowing seasons for, 290 world-wide production and consumption of, 289, 290 Wheat albumin, 291, 298 Wheat allergy, 295–298 baker’s asthma, 295, 296, 299 celiac disease, 295, 297, 299 reduction of, 297–298 symptoms of, 295–296 types of, 295 wheat-dependent exercise-induced anaphylaxis, 295, 296–297, 299 Wheat allergy model, 114 Wheat-dependent exercise-induced anaphylaxis, 10, 295, 296–297, 299 Wheat gliadin, antioxidant activity of, 216 Wheat peptides, hypertension prevention and, 298–299 Wheat production, cereal production and, 9 Wheat protein antioxidant acitivty of peptides in, 34 classification of, 290–295 albumin, 291 glutelin (glutenin), 293, 295 gluten, 295 prolamin (gliadin), 291–293 epitope structure of, 297 Whey, health-promoting proteins and peptides in, 7–8

Whey powder, membrane separation processes and, 153–154 Whey protein hydrolysate, gel filtration of OH signals for samples containing different peptide fractions, 36 Whey protein isolates, gel filtration and, 154 Whey proteins antioxidant acitivty of peptides in, 34 beneficial health effects with, 155–156 bioactive in bovine colostrum and milk, 153t occurrence and isolation of, 152–154 biological functions and applications of, 155–160 alpha-lactalbumin, 156–157 beta-lactoglobulin, 157 glycomacropeptide, 160 immunoglobulins, 156 lactoferrin, 157–159 lactoperoxidase, 159–160 concentrates of, membrane separation processes and, 153–154 functional peptides in, 171 glutathione concentrations in, 18 health benefits in, 151–152 increase in GLP-1 concentrations in casein vs., 141 membrane separation processes and, 153–154 membrane system to produce and separate antioxidant peptides from, 38 WHIM descriptors. See Weighted holistic invariant molecular index descriptors White kidney bean (Phaseolus vulgaris), antioxidant effects of, 274 White wheat, 290 Wine production, lysozyme used in, 251 Winter wheat, 290

419

Wolosiak, R., 274 World Health Organization, 255, 298 Worobiej, E., 274 Wound healing, muscle-based dipeptides and, 228 WPI. See Whey protein isolates WSOEE. See Water-soluble oryzanol enzyme extract Wu, H. C., 217t Wu, J., 249t. Y Yamashita, H., 296 Yang, H., 320 Yang, Y., 276 Ye, X. Y., 282, 283 Yeast prion sup35, 93t Yeasts, Lfcin and inhibition of, 192 Yellowfin sole ACE inhibitory peptides derived from, 208, 210t antioxidant peptides from, 217t Yellowfin sole frame protein, 211 Yellowfin sole frame protein hydrolysates, 216 Yellowfin sole protein, branched amino acids present in antioxidant peptides from, 216 Yellowsole, hypotensive effects of, in spontaneously hypertensive rats, 213t Yellow stripe trevally antioxidant peptides from, 217t antioxidative activity of protein hydrolysates from, 218 Yemenicioglu, A., 274 YFPHs. See Yellowfin sole frame protein hydrolysates Yogurt bioactive peptides and, 155, 161 GABA concentration in, 126 Yokoyama, K., 209t Yolk of egg, major proteins in, 248 Yoshikawa, M., 209t, 213t Yust, M. M., 280

420

Index

Z Zein, 290 antioxidant peptides and in vitro digests of, 32–34

reducing, metal chelation, and ABTS scavenging activity of, in vitro digests, 33 Zimecki, M., 20t, 159

Zinc, 215 Zinc ion, active site of ACE and, 212 Zittel, T. T., 144

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