Food Protein Analysis - Quantitative Effects on Processing (2002)

FOOD PROTEIN ANALYSIS Quantitative Effects on Processing

R. K. Owusu-Apenten The Pennsylvania State University University Park, Pennsylvania

Marcel Dekker, Inc.

New York • Basel

TM

Copyright © 2002 by Marcel Dekker, Inc. All Rights Reserved.

ISBN: 0-8247-0684-6 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-261-8482; fax: 41-61-261-8896 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright # 2002 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, micro®lming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

FOOD SCIENCE AND TECHNOLOGY A Series of Monographs, Textbooks, and Reference Books EDITORIAL BOARD

Senior Editors Owen R. Fennema University of Wisconsin–Madison Y.H. Hui Science Technology System Marcus Karel Rutgers University (emeritus) Pieter Walstra Wageningen University John R. Whitaker University of California–Davis

Additives P. Michael Davidson University of Tennessee–Knoxville Dairy science James L. Steele University of Wisconsin–Madison Flavor chemistry and sensory analysis John H. Thorngate III University of California–Davis Food engineering Daryl B. Lund University of Wisconsin–Madison

Food proteins/food chemistry

Rickey Y. Yada

University of Guelph

Health and disease Seppo Salminen University of Turku, Finland Nutrition and nutraceuticals Mark Dreher Mead Johnson Nutritionals Phase transition/food microstructure Richard W. Hartel University of Wisconsin– Madison Processing and preservation Gustavo V. Barbosa-Cánovas Washington State University–Pullman Safety and toxicology Sanford Miller University of Texas–Austin

1. Flavor Research: Principles and Techniques, R. Teranishi, I. Hornstein, P. Issenberg, and E. L. Wick 2. Principles of Enzymology for the Food Sciences, John R. Whitaker 3. Low-Temperature Preservation of Foods and Living Matter, Owen R. Fennema, William D. Powrie, and Elmer H. Marth 4. Principles of Food Science Part I: Food Chemistry, edited by Owen R. Fennema Part II: Physical Methods of Food Preservation, Marcus Karel, Owen R. Fennema, and Daryl B. Lund 5. Food Emulsions, edited by Stig E. Friberg 6. Nutritional and Safety Aspects of Food Processing, edited by Steven R. Tannenbaum 7. Flavor Research: Recent Advances, edited by R. Teranishi, Robert A. Flath, and Hiroshi Sugisawa 8. Computer-Aided Techniques in Food Technology, edited by Israel Saguy 9. Handbook of Tropical Foods, edited by Harvey T. Chan 10. Antimicrobials in Foods, edited by Alfred Larry Branen and P. Michael Davidson 11. Food Constituents and Food Residues: Their Chromatographic Determination, edited by James F. Lawrence

12. Aspartame: Physiology and Biochemistry, edited by Lewis D. Stegink and L. J. Filer, Jr. 13. Handbook of Vitamins: Nutritional, Biochemical, and Clinical Aspects, edited by Lawrence J. Machlin 14. Starch Conversion Technology, edited by G. M. A. van Beynum and J. A. Roels 15. Food Chemistry: Second Edition, Revised and Expanded, edited by Owen R. Fennema 16. Sensory Evaluation of Food: Statistical Methods and Procedures, Michael O'Mahony 17. Alternative Sweeteners, edited by Lyn O'Brien Nabors and Robert C. Gelardi 18. Citrus Fruits and Their Products: Analysis and Technology, S. V. Ting and Russell L. Rouseff 19. Engineering Properties of Foods, edited by M. A. Rao and S. S. H. Rizvi 20. Umami: A Basic Taste, edited by Yojiro Kawamura and Morley R. Kare 21. Food Biotechnology, edited by Dietrich Knorr 22. Food Texture: Instrumental and Sensory Measurement, edited by Howard R. Moskowitz 23. Seafoods and Fish Oils in Human Health and Disease, John E. Kinsella 24. Postharvest Physiology of Vegetables, edited by J. Weichmann 25. Handbook of Dietary Fiber: An Applied Approach, Mark L. Dreher 26. Food Toxicology, Parts A and B, Jose M. Concon 27. Modern Carbohydrate Chemistry, Roger W. Binkley 28. Trace Minerals in Foods, edited by Kenneth T. Smith 29. Protein Quality and the Effects of Processing, edited by R. Dixon Phillips and John W. Finley 30. Adulteration of Fruit Juice Beverages, edited by Steven Nagy, John A. Attaway, and Martha E. Rhodes 31. Foodborne Bacterial Pathogens, edited by Michael P. Doyle 32. Legumes: Chemistry, Technology, and Human Nutrition, edited by Ruth H. Matthews 33. Industrialization of Indigenous Fermented Foods, edited by Keith H. Steinkraus 34. International Food Regulation Handbook: Policy · Science · Law, edited by Roger D. Middlekauff and Philippe Shubik 35. Food Additives, edited by A. Larry Branen, P. Michael Davidson, and Seppo Salminen 36. Safety of Irradiated Foods, J. F. Diehl 37. Omega-3 Fatty Acids in Health and Disease, edited by Robert S. Lees and Marcus Karel 38. Food Emulsions: Second Edition, Revised and Expanded, edited by Kåre Larsson and Stig E. Friberg 39. Seafood: Effects of Technology on Nutrition, George M. Pigott and Barbee W. Tucker 40. Handbook of Vitamins: Second Edition, Revised and Expanded, edited by Lawrence J. Machlin 41. Handbook of Cereal Science and Technology, Klaus J. Lorenz and Karel Kulp 42. Food Processing Operations and Scale-Up, Kenneth J. Valentas, Leon Levine, and J. Peter Clark 43. Fish Quality Control by Computer Vision, edited by L. F. Pau and R. Olafsson 44. Volatile Compounds in Foods and Beverages, edited by Henk Maarse 45. Instrumental Methods for Quality Assurance in Foods, edited by Daniel Y. C. Fung and Richard F. Matthews 46. Listeria, Listeriosis, and Food Safety, Elliot T. Ryser and Elmer H. Marth 47. Acesulfame-K, edited by D. G. Mayer and F. H. Kemper 48. Alternative Sweeteners: Second Edition, Revised and Expanded, edited by Lyn O'Brien Nabors and Robert C. Gelardi

49. Food Extrusion Science and Technology, edited by Jozef L. Kokini, Chi-Tang Ho, and Mukund V. Karwe 50. Surimi Technology, edited by Tyre C. Lanier and Chong M. Lee 51. Handbook of Food Engineering, edited by Dennis R. Heldman and Daryl B. Lund 52. Food Analysis by HPLC, edited by Leo M. L. Nollet 53. Fatty Acids in Foods and Their Health Implications, edited by Ching Kuang Chow 54. Clostridium botulinum: Ecology and Control in Foods, edited by Andreas H. W. Hauschild and Karen L. Dodds 55. Cereals in Breadmaking: A Molecular Colloidal Approach, Ann-Charlotte Eliasson and Kåre Larsson 56. Low-Calorie Foods Handbook, edited by Aaron M. Altschul 57. Antimicrobials in Foods: Second Edition, Revised and Expanded, edited by P. Michael Davidson and Alfred Larry Branen 58. Lactic Acid Bacteria, edited by Seppo Salminen and Atte von Wright 59. Rice Science and Technology, edited by Wayne E. Marshall and James I. Wadsworth 60. Food Biosensor Analysis, edited by Gabriele Wagner and George G. Guilbault 61. Principles of Enzymology for the Food Sciences: Second Edition, John R. Whitaker 62. Carbohydrate Polyesters as Fat Substitutes, edited by Casimir C. Akoh and Barry G. Swanson 63. Engineering Properties of Foods: Second Edition, Revised and Expanded, edited by M. A. Rao and S. S. H. Rizvi 64. Handbook of Brewing, edited by William A. Hardwick 65. Analyzing Food for Nutrition Labeling and Hazardous Contaminants, edited by Ike J. Jeon and William G. Ikins 66. Ingredient Interactions: Effects on Food Quality, edited by Anilkumar G. Gaonkar 67. Food Polysaccharides and Their Applications, edited by Alistair M. Stephen 68. Safety of Irradiated Foods: Second Edition, Revised and Expanded, J. F. Diehl 69. Nutrition Labeling Handbook, edited by Ralph Shapiro 70. Handbook of Fruit Science and Technology: Production, Composition, Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam 71. Food Antioxidants: Technological, Toxicological, and Health Perspectives, edited by D. L. Madhavi, S. S. Deshpande, and D. K. Salunkhe 72. Freezing Effects on Food Quality, edited by Lester E. Jeremiah 73. Handbook of Indigenous Fermented Foods: Second Edition, Revised and Expanded, edited by Keith H. Steinkraus 74. Carbohydrates in Food, edited by Ann-Charlotte Eliasson 75. Baked Goods Freshness: Technology, Evaluation, and Inhibition of Staling, edited by Ronald E. Hebeda and Henry F. Zobel 76. Food Chemistry: Third Edition, edited by Owen R. Fennema 77. Handbook of Food Analysis: Volumes 1 and 2, edited by Leo M. L. Nollet 78. Computerized Control Systems in the Food Industry, edited by Gauri S. Mittal 79. Techniques for Analyzing Food Aroma, edited by Ray Marsili 80. Food Proteins and Their Applications, edited by Srinivasan Damodaran and Alain Paraf 81. Food Emulsions: Third Edition, Revised and Expanded, edited by Stig E. Friberg and Kåre Larsson 82. Nonthermal Preservation of Foods, Gustavo V. Barbosa-Cánovas, Usha R. Pothakamury, Enrique Palou, and Barry G. Swanson 83. Milk and Dairy Product Technology, Edgar Spreer 84. Applied Dairy Microbiology, edited by Elmer H. Marth and James L. Steele

85. Lactic Acid Bacteria: Microbiology and Functional Aspects, Second Edition, Revised and Expanded, edited by Seppo Salminen and Atte von Wright 86. Handbook of Vegetable Science and Technology: Production, Composition, Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam 87. Polysaccharide Association Structures in Food, edited by Reginald H. Walter 88. Food Lipids: Chemistry, Nutrition, and Biotechnology, edited by Casimir C. Akoh and David B. Min 89. Spice Science and Technology, Kenji Hirasa and Mitsuo Takemasa 90. Dairy Technology: Principles of Milk Properties and Processes, P. Walstra, T. J. Geurts, A. Noomen, A. Jellema, and M. A. J. S. van Boekel 91. Coloring of Food, Drugs, and Cosmetics, Gisbert Otterstätter 92. Listeria, Listeriosis, and Food Safety: Second Edition, Revised and Expanded, edited by Elliot T. Ryser and Elmer H. Marth 93. Complex Carbohydrates in Foods, edited by Susan Sungsoo Cho, Leon Prosky, and Mark Dreher 94. Handbook of Food Preservation, edited by M. Shafiur Rahman 95. International Food Safety Handbook: Science, International Regulation, and Control, edited by Kees van der Heijden, Maged Younes, Lawrence Fishbein, and Sanford Miller 96. Fatty Acids in Foods and Their Health Implications: Second Edition, Revised and Expanded, edited by Ching Kuang Chow 97. Seafood Enzymes: Utilization and Influence on Postharvest Seafood Quality, edited by Norman F. Haard and Benjamin K. Simpson 98. Safe Handling of Foods, edited by Jeffrey M. Farber and Ewen C. D. Todd 99. Handbook of Cereal Science and Technology: Second Edition, Revised and Expanded, edited by Karel Kulp and Joseph G. Ponte, Jr. 100. Food Analysis by HPLC: Second Edition, Revised and Expanded, edited by Leo M. L. Nollet 101. Surimi and Surimi Seafood, edited by Jae W. Park 102. Drug Residues in Foods: Pharmacology, Food Safety, and Analysis, Nickos A. Botsoglou and Dimitrios J. Fletouris 103. Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, edited by Luis M. Botana 104. Handbook of Nutrition and Diet, Babasaheb B. Desai 105. Nondestructive Food Evaluation: Techniques to Analyze Properties and Quality, edited by Sundaram Gunasekaran 106. Green Tea: Health Benefits and Applications, Yukihiko Hara 107. Food Processing Operations Modeling: Design and Analysis, edited by Joseph Irudayaraj 108. Wine Microbiology: Science and Technology, Claudio Delfini and Joseph V. Formica 109. Handbook of Microwave Technology for Food Applications, edited by Ashim K. Datta and Ramaswamy C. Anantheswaran 110. Applied Dairy Microbiology: Second Edition, Revised and Expanded, edited by Elmer H. Marth and James L. Steele 111. Transport Properties of Foods, George D. Saravacos and Zacharias B. Maroulis 112. Alternative Sweeteners: Third Edition, Revised and Expanded, edited by Lyn O’Brien Nabors 113. Handbook of Dietary Fiber, edited by Susan Sungsoo Cho and Mark L. Dreher 114. Control of Foodborne Microorganisms, edited by Vijay K. Juneja and John N. Sofos 115. Flavor, Fragrance, and Odor Analysis, edited by Ray Marsili 116. Food Additives: Second Edition, Revised and Expanded, edited by A. Larry Branen, P. Michael Davidson, Seppo Salminen, and John H. Thorngate, III

117. Food Lipids: Chemistry, Nutrition, and Biotechnology: Second Edition, Revised and Expanded, edited by Casimir C. Akoh and David B. Min 118. Food Protein Analysis: Quantitative Effects on Processing, R. K. OwusuApenten 119. Handbook of Food Toxicology, S. S. Deshpande 120. Food Plant Sanitation, edited by Y. H. Hui, Bernard L. Bruinsma, J. Richard Gorham, Wai-Kit Nip, Phillip S. Tong, and Phil Ventresca 121. Physical Chemistry of Foods, Pieter Walstra 122. Handbook of Food Enzymology, edited by John R. Whitaker, Alphons G. J. Voragen, and Dominic W. S. Wong 123. Postharvest Physiology and Pathology of Vegetables: Second Edition, Revised and Expanded, edited by Jerry A. Bartz and Jeffrey K. Brecht 124. Characterization of Cereals and Flours: Properties, Analysis, and Applications, edited by Gönül Kaletunç and Kenneth J. Breslauer 125. International Handbook of Foodborne Pathogens, edited by Marianne D. Miliotis and Jeffrey W. Bier

Additional Volumes in Preparation Handbook of Dough Fermentations, edited by Karel Kulp and Klaus Lorenz Extraction Optimization in Food Engineering, edited by Constantina Tzia and George Liadakis Physical Principles of Food Preservation: Second Edition, Revised and Expanded, Marcus Karel and Daryl B. Lund Handbook of Vegetable Preservation and Processing, edited by Y. H. Hui, Sue Ghazala, Dee M. Graham, K. D. Murrell, and Wai-Kit Nip Food Process Design, Zacharias B. Maroulis and George D. Saravacos

For Mum and Dad, Elizabeth, James, Richard, Candida, and A®a

Preface

There is no book dealing with food protein analysis exclusively, that is, with the analysis of proteins in the food system. This books attempts to ®ll this niche. Protein analysis comes in two forms: 1) Quantitative analysis, and 2) fractionation and characterization. The ®rst activity is described here. This publication provides a reference for planning, performing and interpreting assays for food proteins. Many approved methods derive from the late-19th century, but they have undergone rigorous testing and modernization. This book does not focus on reviewing the latest research methods for protein analysis. With the exceptions of Chapters 6 and 7, each of the 14 selfcontained chapters describes one protein assayÐprinciples, practices, and expected results. This book describes the effect of food processing on protein assay results with the emphasis on how to analyze proteins in real foods. A number of ``Methods'' sections provide instructions for speci®c tests. Sample pretreatment and clean-up procedures are described. General pretreatment strategies help in the avoidance of interference. More speci®c clean-up methods apply to particular protein assays and are described along with these. Example results, performance characteristics, case reports, and practical problems and solutions related to a wide range of foods are detailed in numerous ®gures, tables, and references. v

vi

Preface

Food protein analysis is a hugely important activity performed by thousands worldwide. The book should appeal to professionals interested in food proteins and anyone working in the food system formerly called the food chain. This includes researchers and workers in agricultural production, food processing, and wholesale and/or retail marketing. It provides information for the grain or dairy farmer, extension worker, agricultural scientist, food scientists and technologists, or college professor. Some techniques described in this book were ®rst used by clinicians, nutritionists, and veterinary scientists. The book may also be of interest to those in small businesses, private or government laboratories, research institutes, colleges, and universities. It will be useful to undergraduate, postgraduate, or postdoctoral students. Sections dealing with mechanisms assume graduate level chemistry and/or analytical biochemistry. Any shortcomings of this project are wholly my responsibility. I thank all those colleagues worldwide whose research is reported here. My thanks to Anna Dolezal, Mr. DeSouza and Professor Arthur Finch for teaching me to think for myself. I am grateful to my past students: Drs. Yetunde Folawiyo, Despina Galani, Michael Anaydiegwu, Kiattisak Duangmal, Pitaya Adulyatham, Kwanele Mdluli, Halima Omar and Sripaarna Banerjee for raising my awareness of protein assay issues and for reading parts of the manuscript. Thanks to Dr. Bob Roberts (The Pennsylvania State University) for his advice on combustion methods. I am grateful to Dr. S. Khokhar and Marcel Dekker, Inc., for their commitment. I am also grateful to my family for their support. R. K. Owusu-Apenten

Contents

Preface Part 1. Chapter 1.

Part 2. Chapter 2.

v Fundamental Techniques Kjeldahl Method, Quantitative Amino Acid Analysis and Combustion Analysis 1. Introduction to Food Protein Analyses 2. Kjeldahl Analysis 3. Colorimetric Analysis of Kjeldahl Nitrogen 4. Quantitative Amino Acid Analysis 5. Combustion Nitrogen Analyzers References

1 1 7 18 25 29 38

Copper Binding Methods The Alkaline Copper Reagent: Biuret Assay 1. Introduction 2. The Alkaline Copper Reagent Protein Assay 3. Chemistry of the Alkaline Copper Reagent Protein Assay 4. Interference Compounds

47 47 48 50 53 vii

viii

Chapter 3.

Chapter 4.

Part 3. Chapter 5.

Chapter 6.

Contents

5. 6. 7.

Sample Pretreatment and Avoiding Interferences The Micro-Biuret or Ultraviolet Biuret Protein Analysis Applications of the ACR Solution for Food Protein Analysis References

55 56

The Lowry Method 1. Introduction 2. The Lowry Protein Assay 3. Chemistry of the Lowry Assay 4. Calibration Features 5. Interference Compounds 6. Sample Pretreatment, Avoiding Interferences, and Ensuring Accuracy 7. Applications of Lowry Assays to Food Protein Analysis References

69 69 70 73 77 80

The Bicinchoninic Acid Protein Assay 1. Introduction 2. The BCA Protein Assay 3. Chemistry of the BCA Protein Assay 4. Calibration Features 5. Interference Compounds 6. Sample Pretreatment, Avoiding Interference, Ensuring Accuracy 7. Automated BCA Protein Assays 8. Applications of the BCA Assay to Food Protein Analysis References

57 64

86 87 93 99 99 103 105 109 110 112 113 116 121

Dye Binding Methods The Udy Method 1. Introduction 2. The Udy Method 3. Solid-Phase Dye-Binding Assays 4. The Chemistry of Dye-Binding Protein Assays 5. Interference Compounds and Their Avoidance 6. Applications of Dye-Binding Assays for Food Protein Analysis References

125 125 127 131 133 147

The Bradford MethodÐPrinciples 1. Introduction

169 169

147 160

Contents

ix

2. 3.

Theory of the Bradford Assay Effect of Protein-Dye Binding Parameters on the Bradford Assay 4. Linearization Plots for the Bradford Assays 5. Assay Sensitivity and the Maximum Number of Dye Binding Sites 6. Solid-Phase Dye-Binding Assays 7. Interference Compounds and Sample Pretreatment References Chapter 7.

Part 4. Chapter 8.

Chapter 9.

Bradford AssayÐApplications 1. Introduction 2. Coomassie Brilliant Blue Dye-Binding Assays 3. Performance Characteristics of CBBG Dye-Binding Assays 4. Applications to Food Protein Analysis References

171 183 184 184 185 186 191 195 195 195 201 204 218

Immunological Methods for Protein Speciation Immunological Assay: General Principles and the Agar Diffusion Assay 1. Introduction 2. Immunological Methods 3. Speciation of Proteins by Agar Gel Double Immunodiffusion Assay References Speciation of Meat Proteins by Enzyme-Linked Immunosorbent Assay 1. Introduction 2. Raw Meat Speciation by Indirect ELISA 3. Raw Meat Speciation by Sandwich ELISA 4. Muscle Protein Antigens for ELISA 5. Cooked Meat Analysis by ELISA 6. Monoclonal Antibodies for Meat Speciation 7. Fish and Seafood Identi®cation by ELISA 8. Performance Characteristics for Different ELISA Formats 9. Meat Testing for Transmissible Spongiform Encephalopathy Agents References

221 221 225 230 241 247 247 252 255 257 260 265 268 270 271 274

x

Contents

Chapter 10.

Chapter 11.

Part 5. Chapter 12.

Speciation of Soya Protein by Enzyme-Linked Immunoassay 1. Introduction 2. Sample Pretreatment and Analysis of Soy Protein 3. Structure, Denaturation, and Renaturation of Soybean Proteins 4. Solvent-Extractable Soybean Protein 5. Thermostable Antigens for Soybean Protein Analysis 6. Other Nonmeat Proteins References

285 289 289 292 292

Determination of Trace Protein Allergens in Foods 1. Introduction 2. Soya Bean Protein Allergens 3. Peanuts 4. Wheat and Related Cereals References

297 297 301 305 312 329

281 281 281

Protein Nutrient Value Biological and Chemical Tests for Protein Nutrient Value 1. Introduction 2. Human and Other In Vivo Assays for Protein Nutrient Value 3. Small Animal Bioassays for Protein Nutrient Value 4. In Vitro Methods for Assessing Protein Nutrient Value 5. Protein Digestibility References

341 341 346 348 354 366 374

Chapter 13.

Effect of Processing on Protein Nutrient Value 1. Introduction 2. Milk and Milk Powders 3. Infant Formulas 4. Feedstuffs and Concentrates for Livestock 5. Legumes and Oilseeds 6. Cereal and Cereal Products 7. Improving Cereal Protein Quality by Screening References

381 381 381 384 386 393 398 401 402

Chapter 14.

Protein Digestibility±Corrected Amino Acid Scores 1. Introduction 2. Protein Digestibility 3. Protein Denaturation 4. Chemical Deterioration of Protein Ingredients

411 411 411 414 416

Contents

xi

5.

Matrix Effects on the Rate of Deterioration of Protein Ingredients 420 6. Protein Digestibility±Corrected Amino Acid Scores (PDCAAS) 427 References 440 Index

447

1 Kjeldahl Method, Quantitative Amino Acid Analysis and Combustion Analysis

1. INTRODUCTION TO FOOD PROTEIN ANALYSES Protein analysis is a subject of enormous economic and social interest. The market value of the major agricultural commodities (cereal grains, legumes, ¯our, oilseeds, milk, livestock feeds) is determined partly by their protein content. Protein quantitative analysis is necessary for quality control and is a prerequisite for accurate food labeling. Proteins from different sources have varying aesthetic appeal to the consumer. Compliance with religious dietary restrictions means excluding certain protein (sources) from the diet. The variety of protein consumed is also extremely important in relation to food allergy. Detecting undeclared protein additives and substitutions is a growing problem. Proteins show differing nutritional quality or ability to support dietary needs. In summary, protein analysis has legal, nutritional, health, safety, and economic implications for the food industry (1). The estimated global food production total for 1988 was 4 billion metric tons. Allowing an average of 10% protein in foodstuffs yields 400 million metric tons of protein annually (2). Nonetheless, sensitivity is a major consideration for protein analysts. Some immunological methods can detect nanomole (10 9 mole) amounts of protein. Other important considerations when choosing a method for food protein analysis include 1

2

Chapter 1

TABLE 1 Approximate Chronology for Methods for Food Protein Analysis Date 1831 1843 1849 1859 1883 1927 1944 1951 1960 1960 1971 1975 1976 1985 a

Technique Dumasa Nessler's reagenta Biuret method Alkali-phenol reagent or Bethelot's methoda Kjeldahla Folin-Ciocalteau Dye bindinga Lowry Direct alkaline distillation Near-infrared re¯ectance (NIR)a Modi®ed Berthelot reaction Modi®ed Lowry method (Peterson) Bradford method (Coomassie Blue binding method) Bicinchoninic acid (BCA) method

Techniques for which semiautomated or fully automated apparatus has been manufactured.

high sample throughput, simplicity, and low capital costs. Some of the most signi®cant methods (Dumas, Kjeldahl, and biuret assays) date from the late 1800s (Table 1). Techniques for food protein analysis are described in this book. I will focus on the techniques that feature most often in the food science literature. Infrared analysis of food proteins is not discussed here. 1.1.

Characteristics of Food Protein Assays

Techniques for food protein analysis need to be robust. This means one of several things. Foremost is compatibility with fresh produce (cereals, fruits, vegetables, meat, milk) and processed foods. Samples in various physical states (powders, slurries, dilute liquids, emulsions, gels, pastes) should be analyzable. A robust assay will also deal effectively with foods from either animal or plant sources. Such techniques are unaffected by the presence of dyes or pigments that absorb infrared, visible, or ultraviolet light. A robust protein assay needs mimimal sample pretreatment, which increases error and decrease analytical precision. Sample cleanup also increases the time per analysis (reduces sample throughput) and adds to costs. In the worst-case scenario, pretreatment can be too invasive, thereby invalidating results. In summary, a robust protein assay is simple, quick, sensitive, and reliable. It is also compatible with a diverse range of foods. The economic imperative

Kjeldahl Method

3

leads to a preference for techniques requiring low capital expenditure and minimum training. Laboratories handling more than 8000 analyses per year tend to select techniques on the basis of their speed and ease of operation. A high sample throughput is usually achieved by automation or continuous ¯ow analysis (CFA). A rough ``time line'' for some food protein assays is given in Table 1. Common descriptive terms for protein analysis are de®ned in Table 2. Kjeldahl analysis gives accurate protein readings no matter what the physical state of the sample. This technique has approved status and is the reference method adopted by many national and international organizations. However, the use of hazardous and potentially toxic chemicals in Kjeldahl analysis is creating concern. The Dumas combustion method is comparatively quicker, cheaper, easier to perform, safer, and more environment friendly; it is now considered on equal terms with Kjeldahl analysis in the United States, Canada, and Western Europe. Dye binding is another robust test for proteins (3,4). The biuret method is widely used,

TABLE 2 Some Important Calibration Indices and a Brief Explanation of Their Meaning Calibration feature Linear dynamic range Sensitivity

Accuracy Precision, repeatability, or reproducibility Speci®city Reliability Lower limit of detection (LLD) Sample throughput (time per analysis)

Explanation Range over which signal is proportional to analyte concentration Slope of the calibration graph; analytical response per unit change in protein concentration. cf. parameters a, a0 in Eqs. (1)±(4) Degree of agreement of results with a true value Agreement between repeated measurements taken with a single sample or with different paired samples Ability to discriminate between protein and interfering substance. Ratio of sensitivity for the analyte and interference A composite parameter combining speci®city, accuracy, precision, and sensitivity Minimal protein concentration detectable above background noise Numbers of samples analyzed per unit time, speed of analysis

4

Chapter 1

especially for cereal proteins (5). Procedures involving copper-based reagents (Lowry and bicinchoninic acid assays) continue to be important. Finally, a range of empirical (viscosity, refractive index, speci®c gravity) measurements are used for protein quantitation within industry.

1.2.

Calibration and Statistical Principles

The two common forms of calibration are (a) method calibration and (b) sample calibrations. With method calibration a set of food samples are analyzed using a new test method and a reference method that has been validated by a committee of the Association of Of®cial Analytical Chemists (AOAC). A calibration graph is then drawn by plotting results from the reference method (% Kjeldahl protein) on the Y-axis and the test results on the X-axis. The Xi and Yi observations are usually related by an equation for a straight line: Yi ˆ aXi ‡ b

…1†

where a is the gradient and b is the intercept for the calibration graph. For each Xi result we can determine the calculated % Kjeldahl protein value (Ycalc) via Eq. (2). Ycalc ˆ aXi ‡ b

…2†

Values for Yi and Ycalc can be compared in order to evaluate the test method (see later). Some investigators choose to plot the Kjeldahl results on the Xaxis. Therefore, rather than Eq. (1) we get Yi* ˆ a0 X * ‡ b0

…3†

where Xi* is % Kjeldahl protein and Yi* is the test result. To compare Eq. (1) and Eq. (3), notice that a0 ˆ 1/a and b0 ˆ Yi (Xi / a). For sample calibration, the assay technique is assumed to be valid. We analyze a set of (standard) samples containing known amounts of protein. In Eq. (1), Xi now represents a range of known protein concentrations and Yi are the corresponding instrument responses. Calibration factors (a, b, etc.) can be determined from simple algebra or statistical analysis of paired (Xi, Yi) results. From the principles of least-squares analysis, P …Xi Xm †= …Yi Ym † …4† aˆ P …X i X m †2

Kjeldahl Method

5

and b ˆ Ym

aXm

…5†

where Xm and Ym are the mean values for all Xi and Yi observations. Agreement between the reference and test results is measured by the correlation coef®cient (R); R&1 shows excellent agreement. When Yi and Ycalc observations are poorly correlated, R & 0. The squared correlation coef®cient (R2) can be calculated from Eq. (6). Most handheld calculators can perform this operation automatically. " # …Yi Ycalc †2 2 …6† R ˆ1 P …Yi Ym †2 Precision is another measure of the (dis)agreement between Yi and Ycalc values. This can be expressed as the standard deviation (SD) or coef®cient of variation (CV). High-precision methods produce low values for the SD and CV. P …Yi Ycalc †2 2 …7† …SD† ˆ n 2 CV ˆ …SD=Ym †6100

…8†

We can also measure precision (commonly called error) from n-replicate (Yi) measurements on a single test sample. Thereafter, the numerator in Eq. (7) becomes (Yi Ym)2, which is the square of the differences between individual observations and the mean for all observations. A low CV implies good agreement between successive test results.

1.3.

Assay Performance

Calibration parameters can provide a great deal of other information about assay performance (Table 2). The linear dynamic range is the concentration range over which a linear relationship exists between the instrumental response and protein concentration. Sensitivity is the slope of the calibration graph, and the lower limit of detection (LLD) is smallest quantity of sample that triggers an instrumental response above the background noise. The LLD is dependent on the instrument baseline quality and assay sensitivity. It is common to refer to ``sensitivity'' when we mean the LLD.We differentiate between sensitivity and LLD via the following exercise. Measure the instrument baseline noise by recording the output (Yo) and

6

Chapter 1

the standard deviation (SDo) using a sample blank. The smallest instrumental response that can be distinguished from ``random noise'' in 95% of all cases is Yo +2:326SDo . Now substitute for Yi (ˆYo ‡ 2.326 SDo) and Xi (ˆ LLD) in Eq. (1), leading to the following expression: LLD ˆ

…Yo ‡ 2:326SDo † a

b

…9†

Usually Yo and b are both set to zero when the analyst sets the instrument baseline response to zero. Consequently, Eq. (9) becomes LLD ˆ 2:326SDo =a

…10†

This relation shows that LLD decreases with increasing assay sensitivity and with increasing baseline quality (see decrease in the value for SDo). In order to ensure high sensitivity, it is important to obtain a stable instrumental baseline.

1.4.

Calibrating Protein Assays

The Kjeldahl method is used for calibrating other protein assays. Duda and Szot (6) evaluated six methods for analyzing porcine plasma protein during its manufacture. The techniques are simple and therefore of wider interest (Table 3). The protein content of porcine plasma was 5.58% (w/v). All techniques showed a good correlation with Kjeldahl results (R ˆ 0.905± 0.952). The precision for density and Kjeldahl assays was the same (CV ˆ 10.8%). The sensitivity of the former method was better. With appropriate calibration, density or viscosity measurements could be suitable for the routine analysis during the manufacture of plasma proteins.

TABLE 3 Some Simple Methods for Evaluating Porcine Plasma Protein Method Densitometry Refractometry Modi®ed refractometry UV absorbance (215/225 nm) UV absorbance (241 nm) UV absorbance (280 nm)

Instrument Standard picnometer Laboratory refractometer Laboratory refractometer UV spectrophotometer UV spectrophotometer UV spectrophotometer

Kjeldahl Method

7

Williams et al. (7) calibrated beer protein analyses using quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis (QSDS-PAGE). A range of test methods were investigated including biuret, bicinchoninic acid (BCA), Bradford, Kjeldahl, Lowry, and pyrogallol-red molybdate (PRM) assays. QSDS-PAGE revealed that beer has between 0.5 and 1 mg mL 1 protein. Only the Bradford and PRM assays gave accurate results (Fig. 1). The main sources of error were low-molecular-weight interferences. Beer contains plant pigments, starch, sugars, alcohol, and natural dyes of barley origin. Both Kjeldahl and combustion analyses were subject to interferences by nonprotein nitrogenous (NPN) compounds. Dialysis did not improve accuracy for BCA, Lowry, and biuret assays, which were affected by high-molecular-weight Cu- reducing agents such as pectin and starch. Calibration issues are discussed in two articles by Pomeranz and coworkers (8,9). They considered the reliability of several test methods (biuret, dye binding, infrared re¯ectance, alkaline distillation method) for analyzing proteins in hard red winter wheat varieties from the American Great Plains. The test methods were highly correlated with the Kjeldahl assay (R ˆ 0.976± 0.992). The order of precision was Kjeldahl > biuret > dye binding > infrared analysis. Pomeranz and More (9) also considered the reliability of four ``rapid'' methods for barley or malt protein analysis.* A summary of assay performance statistics is given in Table 4. For barley samples, the precision and sensitivity of analysis were highest for the Kjeldahl and infrared analyses. The use of Kjeldhal analysis to calibrate protein assays for dairy products was discussed by Luithi-Pent and Puhan (10) and also Lynch and Barbano (11). 2. KJELDAHL ANALYSIS Johan Kjeldahl was born on August 16, 1849 in the town of Jaegerpris in Denmark. In 1876 he was employed by the Carlsberg brewery to develop an improved assay for grain protein. The Kjeldahl method was published in 1883. The original technique has been extensively modi®ed. Key steps for the assay are (a) sample digestion, (b) neutralization, (c) distillation and trapping of ammonia, and (d) titration with standard acid. An exhaustive * For the purposes of calibration, 44 samples of barley and 49 samples of malt were analyzed with biuret, dye binding, infrared, alkaline distillation, and Kjeldahl tests. Such results were the basis for deriving calibration relations between Kjeldahl and each test method. Then a further 76 samples of barley and 72 samples of malt were analyzed using only the rapid test methods. Each Xi result gave rise to a corresponding Kjeldahl protein (Ycalc) value.

8

Chapter 1

FIGURE 1 Apparent protein concentrations in stout beer as determined by seven methods. (Data from Ref. 7.)

TABLE 4 Barley Protein Analysis Using a Range of Techniques

Test method

Analysis time (min)

Biuret

10

Dye bindingc Infrared

15 0.5±1.0

Alkaline distillation

90

a

Regression line and correlation coef®cienta Y ˆ 0.857 cP ‡ 1.942 R ˆ 0.972 Ð Y ˆ 1.060 cP 1.03 R ˆ 0.96 Y ˆ 1.070 cP 0.670

Standard error of analysisb 0.336 and 0.2336 Ð 0.838±1.980 2.383 and 1.540

cP, crude protein determined by Kjeldhal method (N 6 6.25); Y, response from the test method. P b Assay standard error calculated from …Ycalc Yi †2 =…n†. c No information given. Source: Ref. 8.

Kjeldahl Method

9

account of the Kjeldahl method can be found in the monograph by Bradstreet (12). The book is divided into ®ve chapters: 1, introduction to the Kjeldahl method; 2, the Kjeldahl digestion; 3, digestion procedure (for fertilizers, leather, cereals, foods and proteins, coal and fuels); 4, the distillation and detection of ammonia. Chapter 5 is an extensive bibliography. A standardized Kjeldahl procedure appears in the International Standard ISO-1871 (13). Further descriptions are given by Gaspar (14) and Osborne (15). Initially, only sulfuric acid was used for sample digestion. Then solid potassium permanganate was added to facilitate sample oxidation. Mercuric oxide was introduced as a catalyst in 1885. During the acid digestion phase, the food sample is heated with concentrated sulfuric acid, which causes dehydration and charring. Above a sample decomposition temperature, carbon, sulfur, hydrogen, and nitrogen are converted to carbon dioxide, sulfur dioxide, water, and ammonium sulfate [Eq. (11)]. NH2 …CH2 †p COOH ‡ …q ‡ 1†H2 SO4 ?…p ‡ 1†CO2 ‡ q…SO2 † ‡ 4p…H2 O† ‡ NH4 HSO4

…11†

Digestion is complete when the mixture turns clear (light green color), usually after 20±30 minutes of heating. A further (after-boiling) period of heating is necessary to ensure quantitative recovery of nitrogen. Data from McKenzie and Wallace (cited in Ref. 14) show that adding X (mg) of potassium sulfate per mL of sulfuric acid increases its boiling point according to the relation Y (8C) ˆ 55.8X ‡ 331.2. A maximum boiling point elevation of 1308C is achivable by adding 2 mg (potassium sulfate) per mL acid. A high boiling point reduces the sample digestion time. Sample digestion can also be facilitated by using a catalyst; the order of effectiveness for metal oxide catalysts is Hg > Se > Te > Ti > Mo > Fe > Cu > V >W > Ag (16). A proprietary brand of Kjeldahl catalyst (Kjeltabs from Foss Electric Ltd.) comes as tablets. Each tablet contains 0.25 g of mercuric oxide and 5 g of potassium sulfate. A working selenium catalyst can be formulated with potassium sulfate (32 g), mercuric sulfate (5 g), and selenium powder (1 g). Chemical oxidants (hydrogen peroxide, perchloric acid, or chromic acid) can be added to the sulfuric acid to speed up sample digestion. Ammonium sulfate is ®rst neutralized with alkali to form ammonia. This is then distilled and trapped using 4% boric acid. Ammonium borate is then titrated with standard acid in the presence of a suitable indicator. Lowcost Quick-®t glassware is readily available for distillation and titration. Sophisticated semiautomatic distillation systems are also available. The

10

Chapter 1

processes of neutralization, distillation, and titrimetric analyses are summarized as follows. distill

NH4 HSO4 ‡ 2OH ? NH3 ‡ 2H2 O ‡ SO24

…12†

NH3 ‡ H3 BO3 …excess†?NH4 H2 BO3 ?NH‡ 4 ‡ H2 BO3

…13†

H2 BO3 ‡ HCl?…titration†?H3 BO3

…14†

Suitable titration indicators include methyl orange, methyl red, Congo red, and Tashiro indicator (a 1:1 mixture of 0.2% methyl red solution and 0.1% methylene blue). 2.1.

Nitrogen-to-Protein Conversion Factors

The Kjeldahl technique measures sample nitrogen (SN) as ammonia. The value for SN is later converted to crude protein (cP) by multiplying by a Kjeldahl factor, FK. cP…%† ˆ S N F K

…15†

The units for SN are g-N 100 g 1 (g-nitrogen released per 100 g of sample). The Fk (g-protein g 1 N) is the amount of protein that produces a gram of nitrogen. Fk is also called the nitrogen-to-protein conversion factor. AOACrecommended values for FK for meat and other food are summarized by Benedict (17). Frequently, FK is given a default value of 6.25 or 5.7 for animal and plant proteins, which are assumed to have an average N content of 16% and 17.5%, respectively. In fact, most proteins deviate signi®cantly from these averages (18). FK is also affected by the presence of NPN (e.g., adenine, ammonia, choline, betaine, guanidine, nucleic acid, urea, free amino acids). Soya beans have 3±10% NPN, which increases to about 30% for immature seeds. The amount of NPN also changes with growth conditions as well as with geographic factors. There is generally no correlation between NPN and protein content (19). No single FK value applies to all food types. Ideally, FK should be determined for each individual food type (Table 5). FK can be calculated from amino acid data (18,20±24). Table 5 lists the 20 naturally occurring amino acids along with their formula weight, number of nitrogen atoms, percent nitrogen, and the value for FK. For arginine, FK is 3.11 …ˆ 100=32:15†. An idealized protein having all 20 amino acids in equal numbers has a nitrogen content of 14.73%. The FK value is therefore 6.79 (100 g/14.73). Evaluating FK from amino acid data (for

32.15 27.06 21.20 19.16 19.15 18.64 15.71 13.71 13.32 12.16 11.96 11.75 11.56 10.67 10.67 10.52 9.52 9.38 8.47 7.73 14.73 6.79

Average ˆ FK(1) ˆ

4 %N

4 3 2 2 2 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1

N atoms

3

FK ˆ

3.11 3.70 4.72 5.22 5.22 5.36 6.36 7.29 7.51 8.22 8.36 8.51 8.65 9.37 9.37 9.51 10.51 10.66 11.80 12.94 Sum ˆ

5 Amino Acid FK

6.01

234.0 188.0 292 783 487.0 128.0 219.0 90.0 338.0 571.0 428.0 278.0 47.0 290.0 600.0 214.0 574.0 148.0 341.0 297.0 5669.7

6 AA composition (mg/g N) 75.2 50.9 61.9 150.1 93.3 23.9 34.4 12.3 45.0 69.5 51.2 32.7 5.4 30.9 64.0 22.5 54.6 13.9 28.9 22.9 943.5

7 AA-N (mg/g N) 0.001343 0.001211 0.00221 0.005359 0.003331 0.001704 0.002458 0.000441 0.003216 0.004961 0.003655 0.002334 0.000388 0.00221 0.004573 0.001608 0.003902 0.000992 0.002064 0.001639 0.04960

8 AA (moles/g N) 0.027082 0.024422 0.044564 0.108048 0.067157 0.034362 0.049553 0.008886 0.064837 0.100016 0.073687 0.047059 0.007825 0.044563 0.092199 0.032415 0.078669 0.019999 0.041615 0.033045 1.0

9 Mole fraction (Xi)

4.71761 3.79022 5.88694 15.7859 9.81828 2.58058 4.4152 1.81447 6.81433 11.5118 8.6288 5.60469 0.94756 5.84662 12.0964 4.3144 11.5723 2.98379 6.87481 5.98774 132.0

biX

10

a FK(1) is determined from the average nitrogen content for all amino acids, i.e., 14.73%. FK(2) ˆ value of whole protein or sum of amino acid nitrogen divided by sum of weight of nitrogen. FK ˆ 5669.7/943.5. Columns 8±10 contain data for calculating total protein content from quantitative amino acid analysis (Sec. 4).

174.2 155.2 132.1 146.1 146.2 75.1 89.1 204.2 105.1 115.1 117.1 119.1 121.1 131.2 131.2 133.1 147.1 149.2 165.2 181.2

2 Formula weight (bi)

Determination of the Kjeldahl Factor for Milk Protein Using Amino Acid Composition Dataa

Arginine Histidine Asparagine Glutamine Lysine Glycine Alanine Tryptophan Serine Proline Valine Threonine Cystine Isoleucine Leucine Aspartic acid Glutamic acid Methionine Phenylalanine Tyrosine

1 Amino Acid

TABLE 5

Kjeldahl Method 11

12

Chapter 1

skimmed milk) can be achieved in the following steps: (a) express each amino acid as mg per gram of total nitrogen (see column 6 of Table 5) and (b) calculate the mass of nitrogen derived from each amino acid (column 7 in Table 5). FK is the weight of amino acids divided by the weight of amino acid nitrogen (AA-N). FK ˆ

total weight of AA 5696:7 ˆ ˆ 6:01 total weight of AA N 943:5

…16†

Some typical values for FK are listed in Table 6 for a range of foods. The use of FK values for quantitative amino acid analysis is discussed in Sec. 4.

TABLE 6 Nitrogen-Protein Conversion (Fk) Factors for Selected Food Protein Sources Food product Dairy products and egg Casein Milk Cheese Egg Egg white solids Meat and ®sh products Beef Chicken Fish Leafy vegetables Lettuce Cabbage Cereals and legumes Wheat Rice Corn Sorghum Field pea Dry bean Soya bean Source: Data from Refs. 18 and 24.

Fk 6.15 6.02 6.13 5.73 5.96 5.72 5.82 5.82 5.14 5.30 5.71±5.75 5.61±5.64 5.72 5.93 5.40 5.44 5.69

Food product Roots and tuber Carrot Beet Potato Potato protein

Fk 5.80 5.27 5.18 5.94

Fruit Tomato Banana Apple Microbial and fungal Yeast Mushrooms

5.78 5.61

Buckwheat Oats Millet Mustard seed Rapeseed meal Sun¯ower meal Flax meal

5.53 5.50 5.68 5.40 5.53 5.36 5.41

6.26 5.32 5.72

Kjeldahl Method

2.2.

13

Macro- and Micro-Kjeldahl Analysis

Kjeldahl digestion methods are discussed in this section. Illustrative examples are given to establish a pattern of work. Individual results are described in later parts of the chapter.

A.

Grain and Cereals

Kaul and Sharma (25) analyzed a range of legume and cereal grains by micro-Kjeldahl analysis. About 200 mg of each sample was weighed into several 75-mL Kjeldahl digestion tubes. Concentrated sulfuric acid (3 mL), hydrogen peroxide (1.5 mL), and one Kjeltab tablet were added. The tubes were heated using a Tectator digestion block at 3748C for exactly 25 minutes and then allowed to cool. The contents of each tube were diluted to 75 mL and any ammonia produced quanti®ed by colorimetric analysis (Sec. 3).

B.

Potatoes

Mohyuddin and Mazza (26) analyzed proteins from 14 potato cultivars. Potato tubers were peeled, sliced, diced, and dried in a vacuum oven at 708C and 48.8 mm Hg pressure. Each sample was milled and sieved through a 40mesh sieve. Potato ¯our (100 mg) was added to each 100-mL Kjeldahl ¯ask, followed by concentrated sulfuric acid (3 mL), hydrogen peroxide (30% solution; 1.5 mL), and commercial catalyst (500 mg; 10:0.7 w/w ratio of potassium sulfate and mercuric oxide). Heating for 45 minutes digested the samples. After cooling to room temperature, the contents of Kjeldahl ¯asks were diluted to 75 mL and then subjected to colorimetric analysis to determine ammonia.

C.

Dried Milk Powder

Venter et al. (27) described a semimicro-Kjeldahl analysis for low-fat, medium-fat, and high-fat dried milk. About 200±250 mg of sample was mixed with 2.1 g of selenium catalyst and digested by heating with 10 mL of concentrated sulfuric acid. The digest was cooled and diluted to 100 mL with distilled water. Then 60 mL of 45% (w/v) NaOH solution was added and the liberated ammonia was distilled into 20 mL of 4% (w/v) boric acid solution. Titration was with 0.02 M HCl to an end point of pH 4.8. The results agreed well with the macro-Kjeldahl method [International IDF Standard (1962) No. 20].

14

D.

Chapter 1

Beer Protein

Concentrated sulfuric acid (2 mL) was added to 50 mL of beer (bitter, lager, or stout) and the mixture was heated until nearly dry (7). Kjeldahl catalyst (10 g) and more sulfuric acid (20 mL) were added, followed by further heating for 25 minutes. After cooling for 2 hours, water (250 mL) was added and the Kjeldahl ¯ask was connected to a condenser with one end immersed in a 2% boric acid solution (200 mL). Bromocresol green was used as indicator. Sodium hydroxide (10 M, 70 mL) was added, followed by heating until the distillate tested neutral. The borate solution was later titrated with 0.1 N HCl. The nitrogen content in beer was calculated from the relation %N ˆ 14Va =Wd

…17†

where Va (mL) is the volume of HCl required to neutralize ammonia and Wd (g) is the dry weight of beer. Table 7 summarizes characteristics of the Kjeldahl method used in the brewing and allied industries (28).

TABLE 7 Macro-Kjeldahl Procedures Currently in Use in the Brewing and Allied Industries Parameter Instrumentationb Sample weight Catalyst (weight range)c Volume of conc. H2SO4 Digestion temperatured Digestion timee End-point detection a

Commenta Kjel-Tec 1030 (Auto), Kjel-Tec 1007/ 1002*; Kjel-Foss 16120y 0:92+0:23 g (barley or malt) range 0.5±1.5 g, 14+8:4 mL (beer) range 5±25 mL K2SO4 ‡ CuSO4 ‡ TiO2; 3.5±15.8 g 16+4:3 mL, range 10±25 mL 411148C, range 380±4308C 75 min or 9.6 min Mainly colorimetric

Average values unless otherwise stated. Local suppliers *Tectator Ltd. and Perstop Ltd., both of Bristol, UK, { Foss Electric (UK) Ltd., The Chantry, Bishopthorpe, York, UK. c A large amount of a single catalyst or a smaller quantity of a combination catalysts was used. HgO was used in 2 of the 25 laboratories. d Digestion temperatures were not reported for seven laboratories using a manual Kjeldahl technique. e Digestion time plus after-boiling time. The digestion time is 9.6 min when H2O2 is used as a prooxidant. Source: Summarized from Ref. 28. b

Kjeldahl Method

2.3.

15

Automated Kjeldahl Analysis

Kjeldahl analysis has undergone three forms of automation. The KjelFoss1 instrument mechanizes the entire micro-Kjeldahl procedure (digestion, neutralization, distillation, and titration). The Kjel-Tec1 technique uses a digestion block in conjunction with apparatus for automated distillation and titrimetric analysis. The ®nal form of automation is the Technicon AutoAnalyzer Instrument1, which uses continuous ¯ow analysis (CFA). A.

The Kjel-Foss Instrument

The Kjel-Foss instrument (N. Foss Electric Ltd., Hillerùd, Denmark) performs the entire Kjeldahl procedure automatically (29±31). Automation reduces the analysis time from 3 hours to 6 minutes. The ®rst analysis is completed in 12 minutes and succeeding analyses every 3 minutes. The sample throughput is 120±160 analyses per day. The Kjel-Foss instrument requires a reliable supply of electricity and tap water for installation and adequate drains and ventilation. A fume cupboard is not essential. Accuracy, precision, and economics of the Kjel-Foss method were compared with those of the manual Kjeldahl method, neutron activation analysis, proton activation analysis, combustion analysis, and the Kjel-Tech method (32). Results of the Kjel-Foss and manual Kjeldahl methods were highly correlated. Fish meal was analyzed using the Kjel-Foss instrument by Bjarno (33). Seven collaborators compared the ef®ciency of antimony versus mercuric oxide as catalyst. After modifying the Kjel-Foss procedure slightly with higher acid settings, the differences in recovery and repeatability of the two procedures were < +1%. Using mercuric oxide catalyst poses environmental concerns if the ef¯uent from the Kjel-Foss instrument is to be disposed of through the sewers. McGill (34) compared the Kjel-Foss method with an improved AOAC Kjeldahl method for meat and meat products. Over 80 analyses were performed with low (25%) fat, high (40%) fat, and dry sausage (50% fat). As shown in Fig. 2 and Table 8, the two techniques were highly correlated (Y ˆ 0.9904X ‡ 0.1797; R2 ˆ 0.9896). There was no systematic error in the Kjel-Foss technique over the range of protein concentrations examined by McGill. This work validated the Kjel-Foss instrument for meat product analysis. Suhre et al. (35) also evaluated the Kjel-Foss instrument for meat analysis using the AOAC Kjeldahl method as reference. Twenty-three different laboratories analyzed six meat products having 10±30% crude protein. Eight laboratories used the automated Kjel-Foss instrument, ®ve used the of®cial AOAC method, and eleven used a block digester with steam

16

Chapter 1

FIGURE 2 Calibration graph for the Kjel-Foss automated method for protein determinations. (Data from Table 1 of Ref. 34.)

distillation. Recommendations from this work led to the block digester± steam distillation method being awarded ``®rst action'' status. B.

Automated Kjeldahl Continuous Flow Analysis

The Technicon AutoAnalyzer has two reaction modules (36). The ®rst module digests water-dispersible samples. The digest is then pumped to a second module (AutoAnalyzer Sampler II). Colorigenic reagents are added in quick succession before the ¯ow stream passes to a delay coil to allow color formation. Ammonia is detected using Berthelot's reaction or the ninhydrin assay (Sec. 3). The AutoAnalyzer was applied for protein determinations in plant material (37), feedstuffs (38), grain ¯our (39), instant breakfasts, meat analogues (40), meat products (41), and over 40 assorted canned and processed foods (42,43). In general AutoAnalyzer results agreed with micro-Kjeldahl analysis. The AutoAnalyzer digestion unit is heated in two stages at 380±4008C and 300±3208C. To achieve ef®cient digestion, the ratio of acid to sample is

Kjeldahl Method

17

TABLE 8 A Comparison of the Automated Kjel-Foss and Approved Kjeldahl Method for Protein Analysis in Sausage Samples Sausage type Low-fat sausages Bologna Polish Krakow Liver Hungarian Medium-fat sausages Breakfast Italian Pizza Pork Dry sausages Pepperoni Summer

Kjel-Foss (% protein)

Kjeldahl (% protein)

14.64 14.42 16.76 16.64 16.01

14.54 14.43 16.60 16.74 16.02

14.40 14.77 17.42 14.09

14.52 14.80 17.33 14.21

14.33 18.87

14.41 18.86

Source: Ref. 34.

higher than normal for batch digestion. A superheated layer of acid forms, which facilitates sample digestion (44). Later tests showed that the recovery of nitrogen from refractory materials (arginine, creatine, or nicotinic acid) was only 70%. Davidson, et al. (45) concluded that the AutoAnalyzer digestion module was not reliable if an accuracy of 1% was desired for Kjeldahl analysis. Over 70 different animal feeds (corn grain, wheat, barley, rice, alfalfa, mixed feeds, feed concentrates) were analyzed using the AutoAnalyzer digestion module. The recovery of nitrogen was 88±90% (46). In contrast, using a Technicon block digestor followed by AutoAnalyzer Sampler II led to 100% recovery of nitrogen from cattle supplement, swine ration, pig starter, and poultry ration (47). Suitable catalysts include copper sulfate and oxides of mercury, selenium, or titanium. Ammonia was detected using alkaline phenol reagent (Sec. 3.1). Quantitative recovery of nitrogen was also demonstrated by Kaul and Sharma (25), who used a Tectator1 heating block to digest 23 assorted strains of rice and 15 other cereal-legume mixtures. The electrically heated block digests 40 samples per hour under controlled temperature conditions. Samples were then transferred to the AutoAnalyzer Sampler II for ammonia detection using the alkaline phenol reagent.

18

3.

Chapter 1

COLORIMETRIC ANALYSIS OF KJELDAHL NITROGEN

Colorimetric analysis simpli®es Kjeldahl analysis and increases the sample throughput. Other bene®ts include increased sensitivity and a greater potential for automation. Reagents for colorimetric Kjeldahl-N analyses include (a) alkali-phenol reagent (APR), also called indophenol reagent; (b) ninhydrin (indanetrione hydrate) reagent; (c) Nessler's reagent; and (d) acetylacetone formaldehyde reagent. These colorimetric techniques are reviewed next. 3.1.

Alkali-Phenol Reagent (Indophenol) Method

The alkali-phenol reagent is frequently used for the Technicon AutoAnalyzer. Under alkaline conditions, ammonia, sodium hypochlorite, and phenol react to form a blue product. Berthelot ®rst reported this reaction in 1859. The principles of the APR assay have been reviewed (48±51) although the underlying reactions remain uncertain. Ammonia probably reacts with hypochlorite to form chloramine (NH2Cl). This reacts with phenol to form N-chloro-p-hydroxybenzoquinone monoimine or quinochloroamine (I). NH3 ‡ OCl ?NH2 Cl PhO ‡ NH2 Cl?…I† PhO ‡ …I†?…II†

(I) Quinochloroamine and (II) Indophenol

Alternatively, ammonia may ®rst react with phenol to form paminophenol, which is then oxidized by hypochlorite to form (I). Irrespective of how (I) forms, it reacts with 1 mole of phenol to form indophenol (II). The yellow compound ionizes under strongly alkaline conditions (pKa 13.4) giving a blue anion …De ˆ 1 26104 L mol 1 cm 1 †. Substituted phenols (o-chlorophenol, m-cresol, and guaiacol) undergo the indophenol reaction. However, para-substituted phenols and some metaderivatives do not react. Color intensity and the rate of color formation increase in the presence of manganese (‡2) ions or acetone. Sodium nitroprusside (10±40 mg L 1) is another catalyst. A simple APR assay suitable for detecting 3 ppm ammonia is described in Ref. 48 (Fig. 3). Indophenol formation is pH and temperature dependent. The linear dynamic range for ammonia was 0.3±3 ppm with a sensitivity of 0.3284 (absorbance units/1 ppm NH3). The assay precision (for 1 ppm NH3) was +3%. Although performed with boric acid as the background medium, the simple APR assay is probably not suitable for

Kjeldahl Method

FIGURE 3

19

Calibration graph for ammonia determination using the alkali-phenol reagent assay. (Drawn from results in Ref. 48.)

Kjeldahl-N determination. Copper, zinc, and iron salts were found to act as interferences. Tetlow and Wilson (49) added ethylenediaminetetraacetic acid (EDTA) to APR to reduce metal ion interference. Temperature control was also improved using a thermostated water bath. An outline protocol is described below. Method 1 Analysis of ammonia using the APR assay (48,49). Reagents 1. Phenol (crystalline,  85% pure) 2. Sodium hydroxide solution (5 N) 3. Sodium hypochlorite solution (or commercial bleach) 4. Ammonium chloride solid 5. EDTA (6% w/v) 6. Acetone

20

Chapter 1

Procedure Preparation of alkali-phenol reagent. Place 62.5 g of solid phenol in a 500-mL beaker and add 135 mL of sodium hydroxide (5 N) slowly with stirring. Caution: Use an ice bath to avoid excessive heat buildup. Add 12 mL of acetone and make up the volume to 500 mL with deionized water. Sodium hypochlorite (1% w/v available chloride). Prepare by diluting commercially available bleach. Ammonium chloride standards (1000 ppm NH3 and 100 ppm NH3). Dissolve 314.1 mg of solid NH4Cl in 100 mL of water and then dilute 10-fold. Prepare a working standard solution (0.5 ppm NH3) daily. The APR assay sequence. Place 1 mL of sample (or standard) in a test tube. Add EDTA solution (100 mL) with gentle shaking. Next, add APR (1 mL) and hypochlorite (0.5 mL) in quick succession, mixing after each addition. Finally, add 2.5 mL of water and incubate at 258C for 60 minutes. Take A625 readings for samples. Prepare a reagent blank as described next. Reagent blank (reverse addition method). First, mix hypochlorite (0.5 mL) and APR (1 mL) solutions and allow to react for 5±10 minutes. Next add EDTA (100 mL) followed by 3.4 mL of water (or the designated blank solution). When reverse addition is used, hypochlorite reacts with phenol ®rst. Traces of NH3 present in the blank are not detected (48,49). Reverse addition is useful where ammonia-free water is not available for sample preparation. After optimization, the linear dynamic range for ammonia analysis was 50± 500 ppb. Assay sensitivity was 200% greater than the results shown in Fig. 2. Color formation with 50±800 ppb NH3 was virtually complete after 15 minutes at 14±308C. Temperature variations had little effect on the reaction. Thermostating at 258C for 60 minutes improved the precision. Addition of acetone to APR increased the response to ammonia by 10fold. The color yield with 500 ppb ammonia declined by 2.65%, 4.8%, and 6.8% for 4.5-hour-, 1-day- or 5-day-old APR. Addition of EDTA prevented interference from 100 ppb copper. The intervals between addition of various reagents must not exceed 1 minute to ensure optimum precision. Comparing results for normal and reverse addition provides a means for detecting very small amounts of NH3 in samplesÐsuch as water. Otherwise, ammonia-free water is needed for preparing reagents and blanks. The calibration curve for the APR assay is described by the equation A625 ˆ 0.7120X, where X is the concentration of nitrogen (ppm). The analytical sensitivity was 0.7120 (absorbance units) for 1 ppm ammonia. The SD for the reagent blank was +0:0005 ppm. These values lead to an expected LLD for ammonia of 1.6

Kjeldahl Method

21

TABLE 9 The Comparative Costs of Manual APR Assay and Other Techniquesa Technique Manual APR method Micro-Kjeldahl AutoAnalyzer APR test

Capital costb

Running cost per yearc

Analysis per year

Cost per analysisd

6000 (1)

5200 (1)

8000

0.72 (1)

12000 (2) 81,000 (13.5)

7000 (1.3) 17,000 (3.3)

2000 32,000

4.1 (5.4) 0.78 (1)

a

Costs are given in deutsche marks. At current exchange rate 2.8 DM ˆ $1.4 ˆ £1. All methods employ a digestion unit costing DM6000. b Capital costs for the micro-Kjeldahl method include the cost of a distillation unit and an autotitrator. c The running cost includes DM5000 for miscellaneous chemicals. d Calculated for a 10-year period as capital cost/10 ‡ running cost)/no. of samples. Ratios of costs are given in parentheses for each column.

ppb. Assuming a default FK of 6.25, the LLD for protein is 10 ppb. The APR assay is widely used in conjunction with the AutoAnalyzer.The composition of the APR used in CFA is pretty much the same as described earlier (45,52). Kaul and Sharma (25) describe a rare attempt to deploy a manual Kjeldahl-APR assay for protein analysis. They used a Tectator heating block for micro-Kjeldahl digestion of grain. Sample nitrogen was then analyzed by the APR assay. The analytical performance was similar to results obtained with the AutoAnalyzer-APR assay or the conventional micro-Kjeldahl analysis. From Table 9, the capital cost for the manual Kjeldahl-APR assay was 2 times lower than for the micro-Kjeldahl and 13.5 times lower than for the AutoAnalyzer method. Running costs were also lowest for the manual APR assay. For laboratories handling 40 or more analyses per day, it may be worth investing in an automated technique. The manual Kjeldahl-APR analysis was advantageous for small laboratories lacking the wherewithal to purchase an AutoAnalyzer. Mohyuddin and Mazza (53) used the manual Kjeldahl-APR assay to analyze potatoes (see Sec. II.B.2). The mean protein content for 14 potato cultivars was 10.65 (+1.23)% by the manual APR assay and 10.53 (+1.13)% using the AutoAnalyzer. 3.2.

Nessler's Reagent

Ammonia reacts with alkaline potassium iodomercurate II (Nessler's reagent) to form a colloidal complex (lmax ˆ 430±460). The linear range

22

Chapter 1

for analysis extends to 75 mg (ammonia) ml 1. A possible reaction scheme for Nesslerization is 2K2 HgI4 ‡ 3KOH ‡ NH3 ?OHg2 NH2 I ‡ 2H2 O ‡ 7KI

…18†

Hach et al. (54) developed a commercial Nesslerization reagent for use in Kjeldahl analysis. A sulfuric acid±digested sample (0.4 mL) is diluted with 24.6 mL of 0.01% (w/w) polyvinyl alcohol (PVA) solution. One ml of Nessler's reagent is added and the sample is agitated mechanically before absorbance measurements are recorded at 430 nm. As the product of Nesslerization is colloidal in nature, spectrophotometric analysis is sensitive to the degree of sample agitation. PVA acts as a colloidal stabilizer and improves the precision of the Nessler method.

3.3.

Acetylacetone-Formaldehyde Reagent

The acetylacetone-formaldehyde assay is based on the Hantzsch reaction for the synthesis of pyridine (55). Prediluted digest is reacted with a mixture of acetyltacetone and formaldehyde in the presence of sodium acetate. The yellow product (3,5-diacetyl-1,4-dihydrolutidine) is measured at 410 nm …De ˆ 1:46103 Lmol 1 cm 1 †. The color-forming reaction is shown in Eq. (19).

…19†

Acetylacetone-formaldehyde reagent was used for the analysis of medicinal agents such as paracetamol, sulfanilamide, and chloropramide. The potential for colorimetric Kjeldahl analysis is obvious.

Kjeldahl Method

23

Method 2 Determination of ammonia using acetylacetone-formaldehyde reagent (55). Reagents Acetylacetone-formaldehyde reagent. Place 15 mL of formaldehyde (37% w/v) and acetylacetone (7.8 mL) into a 100-mL ¯ask. Make up to 100 mL with distilled water. Sodium acetate (2M). Dissolve sodium acetate (82 g) in 1 L of distilled water. Procedure Add prediluted Kjeldahl digest (< 2 mL; 25±100 mg N) to a 25-mL conical ¯ask followed by sodium acetate solution (3 mL) and acetylacetone-formaldehyde reagent (3 mL). Incubate the mixture at 97.88C for 15 minutes and cool to room temperature. Bring the total volume to 25 mL and record A412 values using a 1-cm cuvette. The linear dynamic range for the preceding assay was 0.5±6.0 mg N (per ®nal reaction mixture). The calibration graph was described by A412 ˆ 9:8610 2 X ‡ 4:2610 3 …R2 ˆ 0:9999†, where X is the amount of nitrogen present in the ®nal (25-mL) reaction mixture. The new method shows levels of accuracy and precision equal to those of the micro-Kjeldahl method. 3.4.

Ninhydrin (Indanetrione Hydrate) Assay

Ninhydrin* reacts with amino acids (Fig. 4) in two stages: (a) the amino acid is oxidized to aldehyde and ammonia while ninhydrin is converted to hydrindantin and (b) hydrindantin and 1 mole of ninhydrin react with ammonia to form Ruhemann's purple (56,57). For ammonia determination an added reducing agent is necessary to convert ninhydrin to hydrindantin. Ninhydrin solution is available commercially. Results from the ninhydrinKjeldahl assay agree closely with those from Kjeldahl analysis (56±58). The linear dynamic range for colorimetric Kjeldahl assay depends on the extent of sample dilution just before ninhydrin analysis. A 2-mL standard ammonium sulfate solution containing 5.6 mg ( or 2.8 ppm) reacted with 2 mL of ninhydrin solution yields an A570 reading of 0.805. Interference was noted for concentrations of selenium above 86 mg mL 1 (prediluted digest). No interferences were observed with Fe, Zn, Pb, Cu, Ca, Ba, Al, Mg, Co, or * Ninhydrin is often encountered in detective novels as a reagent for ®ngerprint analysis.

24

Chapter 1

(a)

(b) FIGURE 4

Reaction scheme between amino acids and ninhydrin reagent.

Kjeldahl Method

FIGURE 5

25

Colorimetric ninhydrin analysis of Kjeldahl nitrogen calibrated against the conventional macro-Kjedahl analysis. (Drawn from data in Ref. 58.)

Ni at concentrations of 50 mg mL 1 or from Hg at 30 mg mL 1. The overall impression is that the ninhydrin assay is resistant to metal ions. Quinn et al. (58) analyzed rapeseed ¯our, rapeseed concentrate, soybean concentrate, and bovine serum albumin using the ninhydrin assay in conjunction with an AutoAnalyzer (Fig. 5). The precision of analysis was 1.40±1.76%. A manual Kjeldahl-ninhydrin assay has not been reported recently. This seems a pity. Compared with other colorimetric methods, the ninhydrin assay is more resistant to interferences from metal catalysts. The color is also formed at a more easily buffered pH between 4.9 and 5.4.

4. QUANTITATIVE AMINO ACID ANALYSIS The following steps are involved in quantitative amino acid analysis: (a) hydrolyze a sample of food using concentrated hydrochloric acid, (b) determine the amino acid pro®le, (c) calculate the concentration of each amino acid in the sample, and (d) calculate the weight of each amino acid. Quantitative amino acid analysis is reportedly one of the most reliable methods for protein quantitation (59±61).

26

4.1.

Chapter 1

Principles of Quantitative Amino Acid Analysis

Crude protein (cP) is expressed by Eq. (20), where Ci (mole) is the amount of each amino acid in the sample and bi (g mole 1), is the formula weight for each amino acid. cP ˆ

20 X

Ci bi

…20†

iˆ1

However, amino acid pro®les are reported in terms of mole fraction of each amino acid (Xi): Xi ˆ Ci =Cnet

…21†

where Cnet is the net concentration of amino acids found in the sample. Substituting Ci ˆ Cnet Xi in Eq. (20), cP ˆ Cnet

20 X

…bi Xi †

…22†

iˆ1

P The term (biXi) is called the mean residue weight, W ( g mole 1). This is the average formula weight for all amino acids in the sample adjusted for their frequency.* X W… g mole 1 † ˆ …bi Xi † …23† and cP ˆ WCnet

or

cP ˆ FCnet

…24†

In Eq. (24), F is the mean residue weight. Usually, W (g mole 1) is adjusted to take into account two routine errors in amino acid analysis: (a) many colorimetric reagents for amino acids do not react with proline and (b) tryptophan is destroyed during acid hydrolysis of proteins. Proline and tryptophan are usually determined by separate experiments. After correcting for such errors, one gets the conversion factor, F (g mole 1): 18 P

Fˆ

Cnet

iˆ1

18 P

…bi Ci †

CPro

CTrp

or

Fˆ

1

iˆ1

…bi Xi †

XPro

XTrp

…25†

where XPro and XTrp are the mole fractions of proline and tryptophan. For a * Horstmann called this parameter the weight equivalent (WE).

Kjeldahl Method

27

range of meat products, F (g mole 1) was 10±20% larger than W [Eq. (23)]. Calculations of F (g mole 1) appear in last three columns of Table 5. Typical values for F (g mole 1) are given in Table 10. Values for F range from 100 to 125 g mole 1 for most proteins.Most protein sources are now routinely analyzed for amino acid scores during nutritional evaluations (Chapter 14). This yields all the information necessary for total protein estimation. Zarkadas and co-workers in Canada are strong exponents of quantitative amino acid analysis (Table 11).

4.2.

Quantitation of Speci®c Proteins

Meat collagen was determined by measuring 5-hydroxylysine (5OH-Lys). This amino acid is found in collagen and no other meat protein. The concentration of collagen (Pj) was calculated from a modi®ed Eq. (24) (64,65); Pj ˆ Ci

1000Wj nj M i

…26†

TABLE 10 Corrected Mean Residue Weights (F) for Selected Food Protein Sources Protein source Barley ¯our Soya bean ¯our Pea ¯our Fish meal Beef sausage Pig skin (rind) Bone meal Soya bean protein Flour Isolate Concentrate Wheat Flour Gluten Egg white solids Potato protein Milk (nonfat) powder

F (g mole 1) 129.84 119.82 118.85 112.70 107.06±109.01 94.02 104.21 114.43 114.48 115.69 113.13±116.00 108.4±108.52 118.43 108.52 112.98

Source: Data calculated or taken from references in Table 11.

28

Chapter 1

TABLE 11

Protein Determination by Quantitative Amino Acid Analysis

Sample/comments NASAÐSkylab meals Mushroom total protein Porcine muscle and connective tissue proteins (myosin, actin, elastin, and collagen Actin, myosin, and collagen in composite meat products; mixed meat sausages, bologna, frankfurters, sausages, hamburgers Additives and ingredients for meat products including soybean, wheat products, potato protein Porcine skin (rind) Chicken meat and connective tissue Apple ¯ower buds Bone isolates New soybean cultivars

Reference Heidelbaugh et al. (59) Weaver et al. (62), Braaksma and Schaap (63) Zarkadas et al. (64), Zarkadas et al. (65) Karatzas and Zarkadas (66)

Zarkadas et al. (67) Nguyen et al. (68) Karatzas and Zarkadas (69) Khanizadeh et al. (70) Zarkadas et al. (71) Zarkadas et al. (72)

where Ci is the concentration of 5OH-Lys in the meat hydrolysate, Wj is mean residue weight from the amino acid pro®le for collagen (averaged for the different collagen types), nj is the number of 5OH-Lys residues per 1000 residues in collagen, and Mi is the formula weight for 5OH-Lys. Typically, Wj ˆ 91:01 g mole 1 , Ni ˆ 10 residues, Mj ˆ 145:18, and consequently Amount of collagen ˆ 62:75‰5OH-LysŠ

…27†

Similarly, the concentration of Nt-methylhistidine and 4-hydroxyproline was the basis for assessing the amount of myo®brillar protein and connective tissue (collagen and elastin) in meat. These methods are satisfactory for meat and meat products. They may have questionable validity for composite foods. Plant foods may contain 4-hydroxyproline± rich glycoproteins (extensins, lectins, salt-extractable glycoproteins). For example, alfalfa protein and potato protein contain signi®cant levels of 4OH-Pro.

Kjeldahl Method

4.3.

29

Examples and Relation to Kjeldahl Method

Quantitative amino acid analysis is arguably one of the most accurate methods for food protein quantitation. One source of error is the high concentration of free amino acids in some foods. The protein content in nine strains of Agaricus was 28% (+3.4)% by quantitative amino acid analysis (62). The results were poorly correlated …R ˆ 0:4† with Kjeldahl results (22.4% protein per dry weight basis). A more recent analysis of freeze-dried mushroom powder led to estimates of 7.0% protein by dry weight (63). In the later study, mushroom powder was extracted with 0.5 M NaOH and precipitating with trichloroacetic acid (TCA) before analysis. This removed large amounts of TCA-soluble NPN associated with mushrooms. Of the total NaOH-soluble nitrogen extracted from mushrooms, 20% was protein, 60% was urea or ammonia, and 20% was free amino acids. Food samples are now routinely extracted with organic solvents to remove NPN before quantitative amino acid analysis (Table 11). Advocates for quantitative amino acid analysis point to its compatibility with plant proteins. There is no interference from phenols, tannins, and lignin. By contrast, the Kjeldahl method is unsuitable for plant tissues regardless of the conversion factor used (73).

5. COMBUSTION NITROGEN ANALYZERS The Dumas assay predates Kjeldahl analysis by 50 years (Table 1). The former technique was invented by Jean Baptiste Dumas. Early applications include the analysis of plant materials (74,75), meat (76), casein, whole powdered milk, soybeans, and maize ¯our (77). The ®rst-generation instruments for the Dumas method were not user friendly. The volume of nitrogen gas produced by combustion was determined with a manometer. The advent of easy-to-use and highly accurate combustion nitrogen analyzers (CNAs) rekindled interest in the Dumas method. CNAs from various manufacturers work on the same principle. The sample is dropped into a 950±10508C furnace, purged free of atmospheric gas, and ®lled with pure (99‡%) oxygen. Complete sample combustion leads to CO2, water, SO2, NO2, and N2. The product gases are cooled and a portion is passed through tubing packed with hot lead chromate, copper, sodium hydroxide (solid), or phosphorus pentoxide to remove SO2, O2, CO2, and water, respectively. The NO2 is then reduced to N2 and measured with a thermal conductivity detector (TCD). Sample protein content is calculated by taking into account the mass of sample injected, the

30

Chapter 1

proportion of the combustion gases analyzed, and the nitrogen-protein conversion factor (FK). The calculations are now automated. 5.1.

Collaborative Studies and Approved Status for CNAs

CNAs were calibrated with the Kjeldahl method. Interlaboratory studies appearing after 1987 are listed in Table 12. Such trials led to CNAs receiving approved status from the AOAC (Association of Of®cial Analytical Chemists), AOCS (American Oil Chemists' Society), ASBC (American Society of Brewing Chemists), AFI (American Feed Industry), BRFInternational (Brewing Research Foundation-International), IOB (Institute of Brewing), and EBC (European Brewing Convention). The Canadian Grain Commission and U.S. Department of Agriculture (USDA) Federal Grain Inspection Services (FGIS) approved CNAs in TABLE 12

Food Protein Analysis Using the Dumas Combustion Method

Samplea Animal feedstuffs, fertilizers Beer Brewing grainsÐbarley, malt, rice Cereal grainsÐwheat, barley, corn, sorghum Dairy productsÐskimmed, powdered milk etc. chocolate milkshake, cheeses, etc. FruitÐguava, peaches, plum Infant food Meat and meat products, ®sh (raw, ®sh in oil, tuna) Oilseeds (soybean, canola, sun¯ower, corn) Potatoes VegetablesÐcabbages, broccoli, ketchup, tomato a

Reference Sweeney and Rexroad (78), Schmitter and Rhihs (79), Sweeney (80), Sachen and Thiex (81), Tate (82) ASBC (83), Johnson and Johansson (84,85) ASBC (86), Buckee (28,87), Krotz et al. (88), Johansson (89), Angelino et al. (90) Bicsak (91), Bicsak (92), Williams et al. (93) Wiles and Gray (94), Wiles et al. (95), Simonne et al. (96,97) Simonne et al. (96,97), Huang et al. (98) Bellemonte et al. (99) King-Brink and Sebranek (100), Simonne et al. (96,97), Buschmann and Westphal (101) Bicsak (91), Duan and DeClercq (102), Berner and Brown (103) Young et al. (104) Simonne et al. (96,97)

Approximate sample classi®cation; classes contain the other foodstuffs.

Kjeldahl Method TABLE 13 1. 2. 3. 4. 5. 6. 7. 8.

31

Advantages of the Dumas Method

Greater ease of operation Higher operator safety owing to the nonrequirement for harzadous chemicals The absence of wetchemistry Reduced time of analysis Higher performance characteristics (greatar accuracy, repeatablility) Absence of waste disposal concerns (Table 14) Simple instrument installation without a requirement for specialized ventilation Low cost per analysis

1994 and 1996, respectively (91±93). Trials for the combustion method usually follow guidelines described by Youden and Sleiner (105): 1. The number of laboratories ranges from 7 to 12. Studies involving as few as three laboratories have been reported. 2. All studies compare CNAs with Kjeldahl analysis. 3. Interlaboratory studies focus on a single food group. Therefore, CNAs tend to receive approval for one food group at a time (Table 12). 4. Trials usually test a ``generic combustion method'' and are independent of the choice of instruments. Minimum performance guidelines for CNA instruments include (a) a furnace temperature of 9508C, (b) a separation system for trapping CO2 and water, (c) a thermal conductivity detector for nitrogen, (d) suf®cient accuracy to produce results within ‡ 0.15% of the mean (% nitrogen) results for 10 successive measurements using a standard compound, and (e) suf®cient precision to produce a relative standard deviation of 0.01%.The LECO FP-428 analyzer was used by about 80% of the laboratories involved in collaborative trials. The Foss-Heraue Macro-N analyzer, Carlo Erba NA-5000, and Perkin Elmer PE2410 also feature. The LECO FP-2000 combustion analyzer appears in the latest trials. 5.2.

Advantages of the Combustion Method

The modern CNA has advantages over the Kjeldahl method (Table 13). There is greater speed of analysis and greater operator safety stemming from the nonuse of aggressive chemicals. The estimated cost for analysis is $0.37± $0.50 per sample with the LECO FP-2000 protein analyzer (LECO Corporation, Saint Joseph, MI) compared with $1.0 per test for the Kjeldahl method (106±109).

32

Chapter 1

TABLE 14 A Comparison of Materials Reqirement for the Kjeldhal and Dumas Methods (74,84) Requirement

Kjeldahl

Dumas

Chemical requirements

Conc. H2SO4, 40% NaOH, K2SO4, TiO2/CuSO4 (or HgO), H3BO3 KH, phthalate, methyl red, phenolphthalein, pumice, water Kjeldahl and Erlenmeyer ¯asks, burettes, acid, alkali and water dispensers, stirring equipment, large containers for acid, etc. Ductwork for corrosive fumes, acid-resistant fans, fume washer, fans, etc. Collected, professionally disposed 120 min (24 samples) 6 70±98 1.2

Air, oxygen, helium, copper, turnings, EDTA, nitrogen catalyst, Mg perchlorate, sodium hydroxide, alumina oxide pellets Tinfoil squares, brushes, tin capsules, combustion, reduction and absorption tubes, cotton wool, steel wool, particle ®lters, tubing

Other suppliesa

Ancillary equipment Disposal of chemical Time per analysis Degree of hazardc Accuracy Precision (CV %)

Ductwork for warm airb Nontoxic, wastebin or sink disposal 3 min 2 100 0.7

a Does not include main equipment (Kjeldahl digester and distillation apparatus, or CNA instrument b Optional, but advisable for large-scale testing. c Arbitrary scale of 1±10, with 10 being extremely hazardous and 1 completely safe. There may be a risk of burns when maintaining the combustion instrument.

A more detailed discussion of the relative costs of protein analysis by Kjeldahl or combustion analysis has to consider factors such as number of analyses per year, capital costs for instrumentation, depreciation, maintenance costs, and savings of labor, chemicals, and other consumables costs (106). It has been suggested that the combustion method provides cost savings of about 30% with a payback period within 2 years. For research institutes, universities, and small-scale laboratories, the safety of modern CNAs probably outweighs cost considerations. Further comparisons of the CNAs and Kjeldahl analysis are summarized in Table 14.

Kjeldahl Method

5.3.

33

Combustion Analysis of Feeds, Cereal Grains, and Oilseeds

Combustion analysis ®rst received AOAC approval for feeds in 1968. The classical instruments (Coleman model 29A nitrogen analyzer) used a manometer for the volumetric assay of nitrogen (110). Comprehensive testing using modern TCD-based CNAs appeared in 1987 (78). A ninelaboratory collaborative trial to determine nitrogen in feeds was successfully completed in 1989 (80). The AOAC approved CNAs for animal feed testing in 1990. The small sample sizes (150±500 mg) used with modern CNAs raised concerns about sampling. Extensive grinding and mixing are essential to ensure sample homogeneity and representative sampling. Sweeney and Rexroad (78) analyzed 14 different animal feeds using the LECO FP-228 instrument with a prescribed sample size of 25%) and the triple-helix structure of collagen, which is apparently stable in alkaline media. Komsa-Penkova et al. (77) described two procedures for improving the Lowry assay sensitivity for collagen. In the ``standard modi®cation'' protocol, samples of collagen (200 mL) are heated with 180 mL of Lowry reagent A (see Method 1) at 508C for 20 minutes. Reagent B is added and the mixture is allowed to react at room temperature for 10 minutes. Finally, Folin-Ciocalteu reagent (600 mL) is added and the mixture heated at 508C for another 10 minutes before taking absorbance readings at 562 nm. With the ``enhanced protocol,'' collagen is reacted with Lowry reagent C at 508C for 20 minutes, cooled to room temperature, and then Folin-Ciocalteu reagent is added. Absorbance readings are taken 10 minutes later. Table 7 shows the performance characteristics of the proposed Lowry methods for collagen. The relative response to type I, type II, and type IV collagen was the same and 50% lower than the response obtained with type V collagen. The modi®ed methods were 10- to 20-fold more sensitivity than Method 1 for collagen analysis. The sensitivity increase is due to the denaturation of collagen by thermal treatment. Gelatin can be analyzed with the normal Lowry method at room temperature.

REFERENCES 1.

OH Lowry, NJ Rosebrough, AL Farr, RJ Randall. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265±273, 1951.

94

Chapter 3

2.

GL Peterson. A simpli®cation of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83:346±356, 1977. H Wu. Contribution to the chemistry of phosphomolybdic acid, phosphotungstic acids and allied substances. J Biol Chem 43:189±220, 1920. H Wu. A new colorimetric method for the determination of plasma proteins. J Biol Chem 51:33±39, 1922. O Folin, V Ciocalteu. On tyrosine and tryptophan determination in proteins. J Biol Chem 73:627±650, 1927. RM Herriot. Reactions of Folin [phenol] reagent with proteins and biuret compounds in the presence of cupric ion. Proc Soc Expt Biol Med 46:642±644, 1941. E Lane. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol 13:447±453, 1957. GL Peterson. Review of the Folin phenol method of Lowry, Rosebrough, Farr and Randall. Anal Biochem 100:201±220, 1979. GL Peterson. Determination of total protein. Methods Enzymol 91:95±119, 1983. A Bensadoun, D Weinstein. Assay of proteins in the presence of interfering materials. Anal Biochem 70:241±250, 1976. S-C Chou, A Goldstein. Chromogenic groupings in the Lowry assay protein determination. Biochem J 75:109±115, 1960. A Livitski, M Anbar, A Berger. Speci®c oxidation of peptides via their copper complexes. Biochemistry 6:3757±3767, 1967. JL Kurtz, GL Burce, DW Margerum. Trivalent copper catalysis of the autooxidation of copper (II) tetraglycine. Inorgan Chem 17:2455±2460, 1978. G Legler, CM Muller-Plantz, M Mentges-Hettkamp, G P¯ieger, E Julich. On the chemical basis of the Lowry protein determination. Anal Biochem 150:278± 287, 1985. ME Anderson, RT Marshall. An automated continuous protein analyzer: modi®cation of Lowry method. J Food Sci 40:728±731, 1975. YW Huang, RT Marshall, ME Anderson, C Charoen. An automated modi®ed Lowry method for protein analysis of milk. J Food Sci 41:1219±1221, 1976. CE Stauffer. A linear standard curve for the Folin Lowry determination of protein. Anal Biochem 69:646±648, 1975. WT Coakley, CJ James. A simple linear transform for the Folin-Lowry protein calibration curve to 1.0 mg/ml. Anal Biochem 85:90±97, 1978. MA Peters, JR Fouts. Interference by buffers and other chemicals with the Lowry protein determination. Anal Biochem 30:299±301, 1969. TH Ji. Interference by detergents, chelating agents and buffers with the Lowry protein determination. Anal Biochem 52:517±521, 1973. J Bonitati, WB Elliott, PG Miles. Interference by carbohydrates and other substances in the estimation of protein with the Folin-Ciocalteu reagent. Anal Biochem 31:399±404, 1969.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

The Lowry Method

95

22. J O'Sullivan, GE Mathieson. Interference by monosaccharides with the estimation of tyrosine and proteins using the Folin-Ciocalteu phenol reagent. Anal Biochem 42:540±543, 1970. 23. F Toldra. Effect of glucose and maltose on the Lowry assay. Nahrung 33:795± 796, 1989. 24. I Alkorta, MJ Llama, JL Serra. Interference by pectin in protein determination. Lebensm Wiss Technol 27(1):39±41, 1994. 25. DH Berg. Hexoseamine interference with the determination of protein by the Lowry procedure. Anal Biochem 42:505±508, 1971. 26. JMO Eze, ED Dumbroff. A comparison of the Bradford and Lowry methods for analysis of protein in chlorophyllous tissue. Can J Bot 60:1046±1049, 1982. 27. GW Pace, MC Archer, SR Tannenbaum. The effect of cryoprotective agents on the Lowry protein assay. Anal Biochem 60:649±652, 1974. 28. MK Zishka, JS Nishimura. Effect of glycerol on Lowry and biuret methods of protein determination. Anal Biochem 34:291±297, 1970. 29. B Gerhadt, H Beevers. In¯uence of sucrose on protein determination by the Lowry procedure. Anal Biochem 24:337±352, 1968. 30. H Schuel, R Schuel. Automated determination of protein in the presence of sucrose. Anal Biochem 20:86±93, 1967. 31. HL Rosenthal, WA Sobieszczanska. In¯uence of reducing sugars on protein determination by the Lowry procedure. Anal Biochem 34:591±598, 1970. 32. C-H Lo, H Stelson. Interference by polysucrose in protein determination by the Lowry method. Anal Biochem 45:331±336, 1972. 33. J Eichberg, LC Mokrasch. Interference by oxidized lipids in the determination of protein by the Lowry procedure. Anal Biochem 30:386±390, 1969. 34. MB Lees, S Paxman. Modi®cation of the Lowry procedure for the analysis of proteolipid protein. Anal Biochem 47:184±192, 1972. 35. AAK Markwell, SM Hass, LL Bieber, NE Tolbert. A modi®cation of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206±210, 1978. 36. LP Kirazov, LG Venkov, EP Kirazov. Comparison of the Lowry and the Bradford protein assays for protein estimation of membrane-containing fractions. Anal Biochem 208:44±48, 1993. 37. CG Vallejo, R Lagunas. Interferences by sulfhydryl, disul®de reagents and potassium ions on protein determination by Lowry's method. Anal Biochem 36:207±212, 1970. 38. PJ Geiger, SP Bessman. Protein determination by the Lowry method in the presence of SH reagents. Anal Biochem 49:467±473, 1972. 39. E Ross, G Schatz. Assay of protein in the presence of high concentrations of sulfhydryl compounds. Anal Biochem 54:304±306, 1973. 40. M Higuchi, F Yoshida. Lowry determination of protein in the presence of sulfhydryl compounds or other reducing agents. Anal Biochem 77:542±547, 1977. 41. M Higuchi, F Yoshida. An improved Lowry procedure by using chloramine-T under the neutral or alkaline conditions. Agric Biol Chem 42:75±77, 1978.

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42. V Hii, WC Herwig. Determination of high molecular weight proteins in beer using Coomassie Blue. J Am Soc Brew Chem 40(2):46±50, 1982. 43. KM Williams, P Fox, T Marshall. A comparison of protein assays for the determination of the protein concentration of beer. J Inst Brew 101:365±369, 1995. 44. VW Padhye, DK Salunke. Biochemical studies on black gram (Phaseolus mungo): I. Solubilization and electrophoretic characterization of the proteins. J Food Biochem 1:111±129, 1977. 45. SK Sathe, DK Salunkhe. Solubilization and electrophoretic characterization of the Great Northern bean (Phaseolus vulgaris L.) proteins. J Food Sci 46(1):82± 87, 1981. 46. B Sebecic. A new possibility of wheat protein content determination. Nahrung 31:817±823, 1987. 47. O Paredes-Lopez, LF Gueverra, ML Schevenim-Pinedo, R Montes-Rivera. Comparison of procedures to determine protein content of developing bean seeds (Phaseolus vulgaris). Plant Foods Hum Nutr 39(2):137±148, 1989. 48. M Seguchi. Study of wheat starch granule surface proteins from chlorinated wheat ¯ours. Cereal Chem 67:258±260, 1990. 49. M Seguchi. Effect of wheat ¯our aging on starch-granule surface proteins. Cereal Chem 70:362±364, 1993. 50. Y W Huang, RT Mashall, ME Anderson, C Charoen. Automated modi®ed Lowry method for protein analysis of milk. J Food Sci 41:1219±1221, 1976. 51. TA Kroening, P Mukerji, RG Hards. Analysis of beta-casein and its phosphoforms in human milk. Nutr Res 18:1175±1186, 1998. 52. EM Ogunbunmi, O Bassir. Proteins and amino acid contents of some Nigerian food condiments. Nutr Rep Int 22:497±502, 1980. 53. JE Hoff. A simple method for the approximate determination of soluble protein in potato tubers. Potato Res 18:428±432, 1975. 54. J Vigue, PH Li. Correlation between methods to determine the protein content of potato tubers. HortScience 10:625±627, 1995. 55. SJ Latlief, D Knorr. Tomato seed protein concentrates: effects of methods of recovery upon yield and compositional characteristics. J Food Sci 48:1583± 1586, 1983. 56. P Vananuvat, JE Kinsella. Production of yeast protein from crude lactose by Saccharomyces fragilis. Batch culture studies. J Food Sci 40:336±341, 1975. 57. DL Gierhart, NN Potter. Effects of ribonucleic acid removal methods on composition and functional properties of Candida utilis. J Food Sci 43:1705± 1713, 1978. 58. ShA Abramov, DA Efendieva, STS Kotenko. Effect of the growth medium on the protein content of the yeast Saccharomyces cerevisiae. Appl Biochem Microbiol 30:225±227, 1994. 59. L Kovar, V Benda, B Hodrova, M Marounek. Fermentation of glucose, xylose, cellulose and waste paper by the rumen anaerobic fungus Orpinomyces joyonii. A J. Anim Feed Sci 9:727±735, 2000.

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60. MA Oliveira, C Rodrigues, EM dos Reis, J Nozaki. Production of fungal protein by solid substrate fermentation of cactus Cereus peruvianus and Opuntia ®cus indica. Quim Nova 24:307±310, 2001. 61. SJ Galluzzo, JM Regenstein. Emulsion capacity and timed emulsi®cation of chicken breast muscle myosin. J Food Sci 43:1757±1760, 1978. 62. KM Tran, MA Einerson. A rapid method for the evaluation of emulsion stability of non-dairy creamers. J Food Sci 52:1109±1110, 1987. 63. HS Juffs. Proteolysis detection in milk. I. Interpretation of tyrosine value data for raw milk supplies in relation to natural variation, bacterial counts and other factors. J Dairy Res 40:371±381, 1973. 64. RL Ory, AA Sekul. Spectrophotometric assay curves as anomalous indicators of proteolysis of oilseed proteins. J Food Biochem 1:67±74, 1977. 65. KKH Kwan, S Nakai, BJ Skura. Comparison of four methods for determining protease activity in milk. J Food Sci 48:1418±1421, 32, 1983. 66. SF Siddiqui, MK Pasha, F Ahmad, M Ahmad. Digestibility of some nonconventional seed proteins. J Oil Technol Assoc India 26(2):49±51, 1994. 67. MV Ramana-Murthy, Sriram-Padmanabhan, M Ramakrishna, BK Lonsane. Comparison of nine different caseinolytic assays for estimation of proteinase activity and further improvement of the best method. Food Biotechnol 11:1±23, 1997. 68. RK Robinson, DF Toerien. The algae: a source of protein. In BJF Hudson, ed. Developments of Food Proteins, Vol 1. Barking-Essex NJ: Applied Science Publishers, 1984, pp 289±325. 69. ML Anson. Estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 22:79±89, 1938. 70. ME Hull. Colorimetric determination of partial hydrolysis of the proteins in milk. J Dairy Sci 30:881, 1947. 71. BMK Gmeiner, CC Seelos. Measurement of phosphotyrosine phosphatase activity using the Folin-Ciocalteu phenol reaction. Biochem Mol Biol Int 36:943±948, 1995. 72. DJ Crossman, KD Clements, GJS Cooper. Determination of protein for studies of marine herbivory: a comparison of methods. J Exp Mar Biol Ecol 244:45±65, 2000. 73. EA Magee, CJ Richardson, R Hughes, JH Cummings. Contribution of dietary protein to sul®de production in the large intestine: an in vitro and a controlled feeding study in humans. Am J Clin Nutr 72:1488±1494, 2000. 74. CL Cooney, CK Rha, SR Tannembaum. Single-cell protein: engineering, economics and utilization in foods. In CO Chichester, ed. Advances in Food Research 26. New York: Academic Press, 1980, pp 1±52. 75. M Guzman-Juarez. Yeast protein. In BJF Hudson, ed. Developments in Food ProteinsÐ3. London: Elsevier Applied Science, 1982, pp 263±291. 76. E Orban, GB Qualia, I Casini, M Moresi. Effect of temperature and yeast concentration on the autolysis of Kluyveromyces fragilis grown on lactose based media. J Food Eng 21:245±261, 1994.

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77. R Komsa-Penkova, R Spirova, B Bechev. Modi®cation of Lowry's method for collagen concentration measurement. J Biochem Biophys Methods 32:33±43, 1996.

4 The Bicinchoninic Acid Protein Assay

1. INTRODUCTION The bicinchoninic acid (BCA) protein assay was developed by Smith et al. (1). The plan was to substitute BCA for the Folin-Ciocalteu reagent in the Lowry assay. The advantages of the BCA method include decreased sensitivity to interferences, a need for one working reagent, and color stability. The BCA protein assay has the same sensitivity as the Lowry method. Reagents for the BCA assay are available commercially. As yet, the BCA assay does not feature greatly in the food science literature. As of June 2001, there were 16 references to bicinchoninic acid or bicinchoninate in the Food Science and Technology abstracts. General citations in the Science Citation Index number over 8500. The BCA assay is without a doubt more popular than indicated by the low number of formal citations. The characteristics of the BCA protein assay are reviewed in this chapter. Section 1 is an account of the history of BCA reagent and its use for chemical analysis. In Sec. 2, the basic BCA procedure is presented, followed, in Sec. 3, by a discussion of the chemistry underlying color formation. In Secs. 4±6 are descriptions of calibration features, interfering compounds, and sample pretreatment strategies for avoiding error. After a discussion of 99

100

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automated formats (Sec. 7), we turn to application of the BCA assay to food protein analysis in Sec. 8. 1.1.

Determination of Copper Using BCA

BCA is the trivial name for 2,20 -diquinolyl-4,40 -dicarboxylic acid. Early applications include the analysis of Cu2‡ in metal alloys and blood sugars. Hoste (2) evaluated 2,20 -diquinolyl (2,20 DQ) and nine related heterocyclic compounds for Cu2‡ analysis after reducing Cu2‡ to Cu1‡ with hydroxylamine hydrochloride. He found that 2,20 DQ forms a 2:1 complex with Cu1‡ (Fig. 1) having maximum absorbance (lmax) at 540 nm and a molar extinction coef®cient (De540) of 5490 M 1 cm 1. To determine Cu1‡, the working solution of 2,20 DQ (0.02% w/v in ethanol, 10 mL) was added to 3 mL of sample. The mixture was diluted with 25 mL of ethanol and A540 measurements were recorded. The Cu1‡ was quantitatively determined in the presence of Cu2‡, with which there is no reaction. KerteÂsz (3) employed 2,20 DQ (0.05% w/v in glacial acetic acid) for the analysis of copper at the active site of the enzyme tyrosinase. To calibrate the assay, 2 mL of 2,20 DQ was added to enzyme-free samples containing

FIGURE 1 Diagram of the 2:1 complex formed between 2,20 -diquinolyl (R ˆ H) or 2,20 -bicinchoninic acid (R ˆ COOH) and Cu1‡

The Bicinchoninic Acid Protein Assay

101

< 11.5 mg of Cu1‡ in 2 mL of phosphate buffer (0.05 M, pH 6.8). First, Cu2‡ was reacted with hydroxylamine hydrochloride to produce Cu1‡. Upon the addition of 2,20 DQ, a purple complex formed in 5±10 minutes at room temperature. There was no reaction with Cu2‡, although preincubating Cu2‡ with BSA or conalbumin led to reactivity with 2,20 -DQ. Initial studies using 2,20 DQ suggested that tyrosinase had a Cu1‡ atom at the active site. Fesenfeld (4) also investigated the reaction of tyrosinase with 2,20 DQ. He proposed that in the absence of substrate mushroom tyrosinase had an active site Cu2‡ species and that this might easily be reduced to Cu1‡ by a protein SH group. Colorimetric analysis of Cu1‡using BCA was studied by Gershuns et al. (5). They showed that Cu1‡ forms a complex with BCA and that this was insoluble at pH < 4. At high pH the red-violet complex had a lmax value of 560 nm. The linear range for Cu1‡ analysis was 1±100 mg mL 1. The analytical precision was 1%. There were no interferences from common metal ions when present at 6±12 6 103-fold mole excess. The Cu1‡ (BCA)2 complex was reportedly stable for a few hours at pH 4±12, although the optimal pH for Cu1‡ analysis was pH 6. As with previous investigations, Cu2‡ was analyzed after reduction using hydroxylamine hydrochloride. Nakano (6) also employed BCA for the analysis of Cu2‡. In a longrunning study, the characteristics of several 4,40 -substituted DQ derivatives were investigated. As shown in Table 1, BCA was the second most colorigenic derivative. Values in Table 1 are with water or isoamyl alcohol as solvent. The ratio of BCA to Cu1‡ found in the complex was con®rmed as 2:1. The Cu1‡ (BCA)2 complex had a lmax value of 565 nm and De565 equal

TABLE 1 Properties of Cu1‡ Complexes with 2,20 DQ derivativesa 4,40 Substituent (R) 2 2COOHb 2 2CONHEt 2 2CONHCHMe2 2 2CONMe2 2 2CONEt2 2 2CON (CHMe2)2 2 2CONBt2 2 2COOEt 2 2COOBt a

max (nm) 560±565 565 566 560 561 556 561 576 576

Values are with water or isoamyl alcohol as solvent. R ˆ COOH for bicinchoninic acid (BCA). Source: Compiled from Ref. 6. b


" (M

1

cm 1)

7920±8000 3100 7100 6130 6200 28100 5150 6600 6020

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to 8000 M 1 cm 1. The color produced from BCA and Cu1‡ is reportedly stable for 48 hours at temperatures < 658C and at pH 3 to 13. The linear range for Cu1‡ analysis was 0.02±20 mg mL 1. Reports of the analysis of Cu2‡ using BCA were produced by Musta®n et al. (7) and Tikhonov (8). Noskova (9) used BCA for the photometric determination of copper in blood serum. The dissociation constant (Kd) for the Cu1‡ (BCA)2 complex was estimated as 1 6 10 11 M at 208C by Buhl et al. (10). Capitan et al. (11) developed a solid-phase analysis of copper in natural water. The microdetermination of copper involved the reduction of Cu2‡ to Cu1‡, reaction with BCA, and adsorption of the resulting complex with a dextran cation exchange resin. Absorbance measurements were recorded directly using the resin phase. Copper was determined at concentrations of 1±20 6 10 9 g L 1. The relative standard deviation for analysis was 1.7%. The typical sample size was 2 L. Brenner and Harris (12) determined serum copper levels using BCA. Blood plasma (0.75 mL) was deproteinized by treating with 0.25 mL of TCA (30% w/w). After microcentrifugation, 0.5 mL of deproteinized serum was added to 0.1 mL of ascorbic acid (35±2 mg% w/w) to reduce Cu2‡ to Cu1‡. Then 0.4 mL of buffered BCA reagent was added and absorbance readings were measured. For calibration, standard amounts of copper were analyzed using 0.1 M sodium phosphate buffer as solvent. A second lmax value was reported for the Cu1‡ (BCA)2 complex at 354.5 nm (13). At this wavelength the sensitivity toward copper is six to seven times greater (De354.5 ˆ 4.6 6 104 M 1 cm 1) than the sensitivity 562 nm (De562 ˆ 7.7 6 103 M 1 cm 1). For a system containing 50 mM BCA, the linear dynamic range for copper detection was 2±25 mM. The upper limit of detection is consistent with the 2:1 stoicheometry for BCA binding to Cu1‡. The pH stability characteristics of the Cu1‡ (BCA)2 complex can be seen from Table 2. These results show that Cu1‡ (BCA)2 is

TABLE 2 The pH Stability of the Cu1‡ (BCA)2 Complex Solution pH 4.5 7.0 9.5 10.5 12.5 Source: Adapted from Ref. 12.


"354.5 /104 (M 3.95 4.58 4.43 4.73 3.88

1

(0.1) (0.1) (0.2) (0.2) (0.2)

cm 1)

The Bicinchoninic Acid Protein Assay

103

stable at pH 3±13; a similar conclusion is supported by absorbance measurements at 560±565 nm. At the time of writing, no protein assays have been conducted at 354.5 nm. 1.2.

Analysis of Sugars Using BCA

Between 1971 and 1973, researchers af®liated with Pierce-Warner Chemical Company used BCA for sugar analysis. An early iteration of the BCA reagent developed by Grindler (14) and Mopper and Grindler (15) was prepared from two stock solutions. Reagent A comprised BCA (170 g) and sodium carbonate (27 g) dissolved in 500 mL of distilled water. Reagent B contained copper sulfate (1 g) and aspartic acid (2.55 g) dissolved in 500 mL of distilled water. A working BCA solution was prepared by mixing equal volumes of reagents A and B. The analysis of sugars was performed in a ¯ow system using a reaction coil at 808C. The reaction time was 25 minutes and absorbance readings were recorded at 562 nm. McFeeters (13) described a manual BCA assay for sugars. Reagent A comprised 0.085% (w/v) BCA in 1.0 M phosphate buffer (pH 8.5). Reagent B contained 25 g of aspartic acid and 33.4 g of sodium carbonate predissolved in 500 mL of water to which copper sulfate (1.34%, 500 mL) was then added. A working BCA reagent was prepared from 23 parts of reagent A and 1 part of reagent B. In a typical analysis, 3 mL of working BCA reagent was added to a 1-mL solution of sugar. The mixture was heated in a boiling-water bath for 10 minutes. After allowing the samples to cool, absorbance readings were taken at 560 nm. The following sugars were assayed: galacturonic acid, glucuronic acid, glucose, galactose, fructose, mannose, xylose, maltose, melibiose, and cellobiose. The average color yield was 1.9 (+1.0) DA560 per mmole. The utmost color yield was obtained with fructose (10.76 DA560 per mmole). For a within-cuvette fructose concentration of 120 6 10 9 mole, DA560 was 1.29.

2. THE BCA PROTEIN ASSAY As described earlier, the BCA protein assay was developed by a 10-member team (1) from the Biochemical Research Division, Pierce Chemical Company, Rockford, Illinois.* Their objective was to ®nd an alternative to the Folin-Ciocalteu reagent for detecting Cu1‡, which is formed when Cu2‡ is reduced by proteins. Actually, the BCA assay may be a totally new * In Europe, Pierce and its sister company HyClone trade under the name Perbio Science.

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concept in protein analysis. The underlying reactions are signi®cantly different from those for the Lowry assay. The distinctiveness of the BCAprotein reaction probably accounts for the greater resistance to certain interferences. BCA reagent is prepared from two stock reagents. Reagent A and B are then mixed in a ratio of 50:1 to produce the working BCA solution. Method 1 The bicinchoninic acid protein assay (1). Reagents 1. Reagent A (alkaline BCA reagent). Add BCA (10 g), sodium carbonate (20 g), sodium tartrate (1.6 g), sodium hydroxide (4 g), and sodium hydrogen carbonate (9.5 g) to 500 mL of distilled water. Adjust to pH 11.25 using sodium hydroxide or solid sodium carbonate. Make up to a volume of 1 L. Reagent A is apparently stable inde®nitely at room temperature. 2. Reagent B (4% copper sulfate). Dissolve 4 g of copper sulfate …CuSO4
5H2 O† in 100 mL of deionized water. Reagent B is, reportedly, stable inde®nitely at room temperature. 3. Working BCA solution. Mix 50 volumes of reagent A with 1 volume of reagent B. Prepare the working BCA solution daily.* Procedure Add 100 mL of sample (20±120 mg protein) to exactly 2 mL of BCA working reagent. As long as the 1:20 volume ratio is maintained, other sample and BCA reagent volumes can be used. Prepare a reagent blank by replacing 100 mL of sample with the same volume of distilled water. Incubate the mixture at 378C (30 minutes) or at room temperature (2 hours). The assay time may be reduced to 10±15 minutes by performing the reaction at 608C. Record A562 against a reagent blank. The BCA assay was characterized with respect to the linear dynamic range, assay temperature, reaction pH, effect of interferences, and reagent stability. Performance characteristics were also compared with those from the Lowry assay. At room temperature, DA562 readings increased slowly over 21 hours. Heating the reaction mixture at 378C for 60 minutes * This is a precautionary measure. The working BCA reagent was stable for over 7 days. A calibration graph produced using the 7-day-old BCA working reagent was virtually identical to a graph produced with a freshly prepared reagent.

The Bicinchoninic Acid Protein Assay

105

produced a similar amount of color. Performing the assay at 608C increased the assay sensitivity four- and ®vefold (1). The effect of pH on the BCA assay was evaluated by altering the pH of reagent A with sodium hydroxide or solid sodium carbonate. The DA562 readings showed a bell-shaped response over the pH range 10.2±12.0. The optimal color yield occurred at pH 11.25. The working BCA reagent had suf®cient buffer capacity to provide accurate results for protein samples containing 0.1 N sodium hydroxide or 0.1 N hydrochloric acid. Because Cu1‡(BCA)2 is stable at pH 3±13 (Table 2), the pH range of Smith's assay is possibly dictated by the effect of pH on the protein-copper reaction. Protein-protein variations in the color yield follow a pattern similar to those for the Lowry assay (Chapter 3). The order of increasing color formation is gelatin < avidin < BSA < immunoglobin G < chymotrypsin < insulin < ribonuclease. The color yield from gelatin was only 50% of the value for avidin. A high concentration of (hydroxyl) proline residues probably reduces Cu2‡ binding to collagen. There is a further loss of color yield assay owing to the low concentrations of tyrosine and tryptophan in the gelatin chain (see later). Protein-protein variations in assay results are lower at 608C. For samples incubated at room temperature or 378C, DA562 readings increased slightly (0.25% per minute) after the prescribed assay time. For increased precision, the absorbance changes for all samples should be taken within a short time of each other. 3. CHEMISTRY OF THE BCA PROTEIN ASSAY Cu2‡ reacts with the protein to produce to Cu1‡, which then binds to BCA. The reaction is different from those taking place for the Lowry assay (Chapter 3). In the latter case, the concentration of Cu1‡ remains very low due to the action of Mo6‡/W6‡. This strong oxidant converts Cu1‡ to Cu2‡ and Cu3‡. 3.1.

Reactions of the BCA Protein Assay

Two reactions occur between proteins and Cu2‡ during the BCA assay. The ®rst is a temperature-insensitive reaction between Cu2‡ and oxidizable protein side chains (tyrosine, tryptophan, cysteine). The second reaction is a temperature-sensitive process involving the binding of Cu2‡ to the peptide backbone. Once bound, Cu2‡ undergoes reduction to Cu1‡. Assays performed at high temperatures assist the peptide pathway. Because the peptide backbone is the same for different proteins, a high-temperature BCA assay lessens protein-protein variations in results. Wiechelman et al.

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TABLE 3 Some Features of the BCA Protein Interactions 1. Four amino acids (cysteine, tryptophan, tyrosine, and phenylalanine) react with BCA working reagent. 2. Biuret and dipeptides (unable to form a tetradentate Cu2‡ complex) do not react with the BCA reagent. 3. Di- or tripeptides containing tyrosine or tryptophan react with the BCA reagent. 4. There is no correlation between the redox potential and the color yield from the BCA assay. 5. The colors from amino acids and peptide backbone are not additive. 6. Oxidizable side chains do not react completely in the BCA assay at 378C. 7. The BCA assay is susceptible to interfering compounds that reduce Cu2‡ to Cu1‡.

(16) assessed the reactivity of various model compounds with BCA and Cu2‡. They also determined the redox potential for some of these compounds. Their ®ndings are summarized in Table 3. Clearly, the twotier reaction scheme proposed by Smith et al. (1) is correct. However, oxidizable side chains do not react easily at either 37 or 608C. The following half-reactions probably lead to the reduction of 2 moles of Cu2‡. 2Cys?Cys

Cys ‡ 2e ‡ 2H‡

…1† dopa ‡ 2e ‡ H‡

Tyr?semiquinone radical?L

TABLE 4

Determination of Reducing Compounds Using BCA

Compound Fructose Glucose Indole Tryptophan Tyrosine Cysteine Acetol Ascorbic acid 2,4-Dinitrophenyl hydrazine Hydroxylamine a

…2†


" (M

1

cm 1)a

Moles per Cu1‡b

43,000 6,120 3,621 1,589 1,172 1,357 10,000 3,030 16,400

0.2 0.8 2.2 5.0 6.8 5.8 0.8 0.26 0.5

3,230

2.5

Values for the apparent extinction coef®cient are from Ref. 16 and 22. Moles of each compound necessary to reduce 1 mole of Cu2‡ to Cu1‡. Value is calculated as De (M 1 cm 1) divided by 8000 (M 1 cm 1). b

The Bicinchoninic Acid Protein Assay

107

The majority of the half-reactions leading to the reduction of Cu2‡ are unknown. The
" values for reactions with BCA are given in Table 4. Dividing
" by 8000 (M 1 cm 1) yields the probable number of moles of each compound needed to form 1 mole of Cu1‡(BCA)2 complex. Clearly, acetol undergoes a one-electron reaction with Cu2‡. One mole of ascorbic acid apparently yields four electrons. 2,4-Dinitrophenylhydrazine contributes two electrons per mole. The reactions with protein side chains are dif®cult to describe in quantitative terms. Between 5 and 6.8 moles of tyrosine, cysteine, or tryptophan appear to be involved in the reduction of one Cu2‡. 3.2.

Metal Ion±Catalyzed Oxidation of Proteins and Polypeptides

Metal ion±catalyzed oxidation (MCO) reactions require a transition metal ion (Cu2‡, Zn2‡, Fe3‡, or Ni2‡), hydrogen peroxide, and a reducing agent (cysteine, glutathione, or 2-mecaptoethanol, ascorbic acid). The reducing agent transform Cu2‡ to Cu1‡, which then reduces hydrogen peroxide to form the highly reactive hydroxyl radical … OH†. The literature concerning MCO of protein was reviewed by Levine et al. (17) and also Paci®ci and Davies (18). Possible initiation reactions for MCO involving ascorbic acid are shown next. 2Cu2‡ ‡ ascorbic acid?2Cu1‡ ‡ DHA*

…3†

H2 O2 ‡ Cu1‡ ? OH ‡ OH ‡ Cu2‡

…4†

Hydrogen peroxide can also be produced from a two-electron reduction of oxygen. O2 ? O2 ?H2 O2

…5†

Attack by .O2 or .OH can initiate a free radical reaction leading to covalent modi®cations of amino acid side chains. Depending on the reaction conditions, MCO can result in subtle changes in protein tertiary and quaternary structure, aggregation, or fragmentation into small polypeptides. Madurawe et al. (19) and also Bush and Lumpkin (20) showed that lactate dehydrogenase (LDH) is inactivated by MCO in the presence of added ascorbic acid, Cu2‡, and hydrogen peroxide. Inactivation of LDH by MCO also occurred without added ascorbic acid. Under such * DHA=dehydro ascorbic acid.

108

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circumstances, the reducing power is probably derived from oxidizable protein side chains (tyrosine, tryptophan, cysteine). The MCO process is site speci®c. Protein structural modi®cations seem to occur close to the site for Cu2‡/Cu1‡ binding. The degradation of hydrogen peroxide via Fenton-type reactions leads to a protein-bound .OH that attacks adjacent groups. BCA inhibits MCO by forming a complex with Cu1‡. Other MCO inhibitors include catalase, thiourea, and ethylenediaminetetraacetate. These inhibitors remove hydrogen peroxide, quench free radicals, or chelate Cu2‡, respectively. Inhibitors did not wholly eliminate MCO when LDH was exposed to Cu2‡ alone in the absence of reducing compound. Superoxide dismutase, mannitol, isopropanol, and sodium formate were also ineffective MCO inhibitors under such circumstances. The failure of radical scavenges to eliminate MCO may be partly explained by the localized nature of these reactions. Protein-bound free radicals may be inaccessible to dissolved inhibitors. Lactate dehydrogenase modi®ed by MCO showed increased susceptibility to proteolysis due to changes in the enzyme quaternary and tertiary structure. The structural changes induced by MCO are subtle. Protease susceptibility was increased even when the residual LDH activity after MCO was > 95%. There was no fragmentation and cross-linking after LDH activity was reduced to < 50% by MCO. One view is that MCO alters the charge on histidine residues. The consequent changes in enzyme charge generate conformational changes including subunit dissociation. Due to the inhibitory effect of BCA on the progress of MCO reactions, we expect that proteins will not be degraded extensively during the BCA protein assay.

3.3.

A Note on Protein-BCA Interactions with Copper

1‡

Cu -protein binding (Kd *10 6 M) is signi®cantly weaker than Cu1‡ binding to BCA (Kd ˆ 10 11 M). Consequently, Cu1‡ will be sequestered as the Cu1‡ (BCA)2 complex. On the other hand, Cu2‡ will mainly complex with tartrate and protein. As BCA does not bind Cu2‡, the formation of a Cu1‡ (BCA)2 complex will alter the Cu2‡/Cu1‡ redox equilibrium in favor of Cu1‡. Cu2‡ ‡ e?Cu1‡ ;

E ˆ ‡ 0:153

…6†

In the presence of BCA, the apparent redox potential (E8*) for the Cu2‡/Cu1‡ redox couple can be estimated from Eq. (7) (21). E * ˆ E  ‡ 0:059 log…1=Kd †

…7†

The Bicinchoninic Acid Protein Assay

109

From the Kd value given for Cu1‡ (BCA)2 the E8* calculated from Eq. (7) is approximately ‡ 0.802. The BCA increases the oxidizing power of the Cu2‡/ Cu1‡ system. By comparison, Cu2‡ is a relatively mild oxidizing agent (E8 ˆ ‡ 0.153). This suggests that BCA is not a passive ligand for Cu2‡ but also plays an integral role in the protein detection mechanism.

4. CALIBRATION FEATURES The linear dynamic range for the BCA protein assay extends to  40 mg of protein. The standard graph for 20±120 mg of BSA is curvilinear. The linear range is approximately the same for gelatin, avidin, immunoglobulin G, chymotrypsinogen, insulin, ribonuclease, and soybean trypsin inhibitor (1). The limit of linearity is probably an intrinsic feature of the BCA assay. Depending on the protein, the maximum DA560 ˆ 1±2 units 120 mg 1 of protein. The calibration graph for the BCA protein assay is nonlinear just like the standard curve for the Lowry assay (Chapter 3). The two assays have roughly equal sensitivities. Protein-protein variations in color yield are also similar. The Lowry assay for inorganic reductants leads to straightline graphs. Nonlinearity was ascribed to a slow reaction with proteins that allows time for the degradation of Mo6‡/W6‡ at high pH. With the BCA assay, the instability explanation for nonlinearty is less tenable. In contrast to Mo6‡/W6‡, BCA is stable in alkaline media. What is clear is that the reduction of Cu2‡ to Cu1‡ becomes less quantitative at high protein concentrations. Some possible causes for nonlinearity are listed in Table 5. A hyperbolic standard curve can be readily explained in terms of saturation phenomenon. Protein binding to a ®xed concentration of Cu2‡ is according to Eq. (8) P ‡ Cu2‡ ?PCu2‡

…8†

Therefore, Kd ˆ ‰PŠ‰Cu2‡ Š=‰PCu2‡ Š and total number of sites ‰Cu2‡ Š0 ˆ ‰Cu2‡ Š ‡ ‰PCu2‡ Š. Therefore, the fraction of Cu2‡ bound is   PCu2‡ b  ˆ  2‡   …9† ‡ PCu2‡ Cu ‰Cu2‡ Š0 where b is the concentration of the PCu2‡ complex. Substitution for [PCu2‡] (ˆ [P][Cu2‡]/Kd) and a brief rearrangement of Eq. (9) give the hyperbolic

110

Chapter 4

TABLE 5 Plausible Explanations for Nonlinear BCA Protein Assay and Their Rebuttal Explanations 1. Incomplete reduction of Cu2‡ by proteins. 2. Cu1‡ is bound to the protein and unavailable to BCA, 3. Cu1‡ is lost as the insoluble hydroxide, 4. Cu2‡ concentration is limiting in the BCA assay. Rebuttals for points 1±3 1. High temperature increases sensitivity or extent of reaction. There is no effect on linear range for analysis. 2. The binding af®nity of BCA for Cu1‡ is 10,000 times greater than the af®nity of proteins for Cu1‡. 3. Formation of insoluble copper precipitates is curtailed by tartrate or BCA.

function. bˆ

‰Cu2‡ Š0 ‰PŠ ‰PŠ ‡ Kd

…10†

The absorbance change from the BCA reaction is proportional to the amount of bound copper ions; therefore, A562 nm ˆ eb1

…11†

As with the Lowry assay (19), the calibration graph for the BSA-protein assay can be linearized using a range of transformations (Chapter 3).

5.

INTERFERENCE COMPOUNDS

Smith et al. (1) evaluated 41 potential interferences for the BCA assay. These are either chelators or reducing agents. A further group of miscellaneous compounds disrupt the assay by virtue of their strong color. Glucose (100 mM) caused a large increase in the reagent blank. Sucrose (10%) had little effect on the BCA assay. EDTA, guanidine hydrochloride, sorbitol, and ammonium sulfate produced signi®cant color losses compared with control samples. A list of additional interfering compounds includes biogenic amines, pharmaceutical agents, and Benedict-positive compounds (Table 6). The Benedict test is a method for determining easily oxidizable compounds in biological samples. Upon heating with the test sample, Cu2‡

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111

TABLE 6 List of Potential Interferences for the BCA Protein Assaya Interference Biogenic amines. Dopamine, norepinephrine, epinephrine, tyrosine, serotonin (5-HT), tryptophan Buffers ( ) Ada, Ampso, Bes, Bicine, Bistris, Caps, Epps, Hepes, Hepps, Mes, Mops, Pipes, Tes, Tricine Benedict-positive compounds Acetol, aminophenol, ascorbic acid, 2,3butanedione, glucose, glyoxal, 2,4dinitrophenylhydrazine, pyruvic aldehyde Drugs Chlorpromazine, caffeine ( ), carbachol ( ), chloramphenicol ( ), codeine phosphate ( ), lidocaine ( ), penicillin G, paracetamol Lipids Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, sphingomyelin Phenolsb Gallic acid, tannic acid, pyrogallic acid, pyrocatechol a

Reference Slocum and Duepree (23)

Kaushal and Barnes (24), Lleu and Rebel (25)

Chen et al. (22,26)

Marshall and Williams (27)

Kesller and Fenestil (28)

Kamath and Pattabiraman (29)

( ) With the exception of Tricine, buffers showed no response with BCA reagent. Effects have been carefully documented in food systems (see Sec. 5.2).

b

is reduced, forming a brown precipitate of Cu1‡ (hydr)oxide. Chen et al. (22) used BCA to detect Cu1‡ formed in the Benedict procedure. Biogenic amines interfered with the BCA assay of proteins from brain or adrenal medulla (23). Catecholamines were highly reactive with the BCA reagent. In protein-free samples, there was a linear response for 1± 100 6 10 9 mole of biogenic amine. Very small quantities (1 6 10 9 mole) of biogenic amine affected protein results. Indeed BCA was suggested as a highly sensitive reagent for assaying biogenic amines.

112

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Zhang and Halling (30) showed that samples containing high concentrations of NaOH (1 M) gave unexpectedly low readings with the BCA assay. The interference was higher for the microassay format, where sample volume constitutes up to 50% of the total assay volume. The normal BCA response was restored by neutralizing samples. To analyze highly alkaline samples, the BCA reagent was adjusted to pH 10.27 in order to improve its buffer capacity. 6.

SAMPLE PRETREATMENT, AVOIDING INTERFERENCE, ENSURING ACCURACY

The TCA-DOC precipitation method (31) was adapted for the BCA assay by Brown et al. (32). Protein pellets were prepared as described in Method 3 of Chapter 3 and resuspended in 50 mL of alkaline SDS solution (5% w/v SDS dissolved in 0.1 N NaOH). Then 1 mL of a standard BCA working solution was added, followed by sample incubation at 378C for 30 minutes. Interferences by glucose, DTT, 2ME, and ammonium sulfate were eliminated after the TCA-DOC procedure. Protein was recovered quantitatively from media containing various proprietary ampholytes or polybuffer. Shahabi and Dyers (33) proposed a two-point kinetic assay for dealing with interferences. Their approach exploits differences in the kinetics of the BCA reaction with proteins and interfering compounds. Many interferences (ascorbic acid, cysteine, uric acid ) react with BCA according to ®rst-order kinetics. By contrast, the reaction with proteins is usually zero order. This means that interfering compounds produce a ®xed amount of background color rapidly. In contrast, the absorbance change in the presence of proteins increases linearly with time. Using the rate of color formation as the index of protein concentration eliminated the effect of interferences. Solid-phase assays are another means for avoiding interferences. Gates (34) adsorbed protein samples (50 mL) on 1 6 2-cm strips of cationized nylon membrane (Zeta-Probe, Biorad Ltd.). The bound protein was rinsed free of interferences and then air dried. Each membrane was placed in a 1.2 6 10-cm test tube and 2 mL of BCA working solution was added. The reaction was incubated at 608C for 30 minutes and the concentration of nylon-immobilized protein determined from DA562 measurements. The sensitivity of the solid-phase protein assay was 78 (+5)±93 (+3.4)% of the sensitivity obtained with the conventional solution assay. The limits of detection (*10 mg protein) and the precision of analysis were the same as for the conventional BCA assay. Accurate results were obtained in the presence of glucose (20 mM), Tris-HCl buffer (200 mM), or DTT (0.1% w/v). Drying the protein solution facilitated binding to the nylon support.

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113

7. AUTOMATED BCA PROTEIN ASSAYS Automated BCA protein assays were developed using microwell plate readers or ¯ow injection analysis. Microwell plates are widely used for enzyme-linked immunoassay (EIA) and other colorimetric assays. The advantages of microwell plates assays include (a) a requirement for a small sample size (5±10 mL), (b) reduced reagent requirements (50±200 mL), (c) compatibility with multichannel pipettes and dispensers, (d) automated measurement using microwell plate spectrophotometers, and (e) the ability to download results to a computer for enhanced data processing. Flow injection analysis and continuous ¯ow analyzers allow rapid and more precise sample analysis compared with conventional batch analysis. 7.1.

Microwell Plate Assays

A microwell plate BCA assay was applied for the rapid analysis of protein fractions from sucrose gradient centrifugation by Redinbaugh and Turley (35). The assay involved a commercial BCA reagent kit from Pierce Chemical Company with BSA as the standard protein. Protein samples (10 mL) were placed in the 96-well microwell plate, to which BCA working reagent (200 mL) was added. The samples were incubated at 228C for about 14 hours (i.e., overnight) or at 608C for 2 hours. Absorbance readings were recorded at 570 nm. Lane et al. (36) also described a BCA microwell plate protein assay, as have Sorensen and Brodbeck (37). Typically, color formation took place at 378C for 30 minutes. Absorbance readings for 96 samples could be completed in 5 minutes. The performance of microwell plate-based BCA protein assays depends on a range of factors including sample size, volume of protein to BCA working reagent, incubation temperature, and time. With the standard assay (1:20 ratio of protein to BCA reagent), the linear dynamic range for analysis was between 1 and 12 mg of protein (compare with a linear range of 10±120 mg for the conventional assay). The calibration graph remains curvilinear (1). The proposal that microwell plate-based BCA assays are 10fold more sensitive than the conventional assay is not supported by the available evidence when results are normalized for differences in the assay temperature or for protein±protein variations in the response. The maximum absorbance expected for a given amount of protein can be estimated for the BCA assay. Consider 10 mg of BSA in a 210-mL assay volume. First, six peptide bonds bind each Cu2‡ ion. Taking the molecular weights of BSA and amino acids as 66,000 and 110, then 100 moles of Cu2‡ ions will bind to 1 mole of BSA (see Chapter 2, Table 2). Consequently, 10 mg of protein (1.51 6 10 10 mole BSA) will bind with 15.1 6 10 9 mole of

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Cu2‡, which is equivalent to 72 6 10 6 M of Cu2‡ in the 210-mL reaction volume. Assuming that the bound Cu2‡ is fully reduced to Cu1‡, the maximum absorbance change expected (A ˆ ecl) is 0.58. In practice, the maximum absorbance change obtained for 10 mg of BSA during a microwell plate assay was between 0.29 and 0.93 (Table 7). To account for the higher than expected absorbance change, note that the preceding calculation did not consider the role of oxidizable protein side chains. Groups such as tyrosine, tryptophan, and cysteine react directly with Cu2‡. It was also assumed that each sextet of peptide groups undergoes a one-electron redox reaction with bound Cu2‡. Table 4 shows that this assumption is probably unwarranted.

7.2.

Flow Injection Analysis and Continuous Flow Analysis Using BCA Reagent

The principles of ¯ow injection analysis (FIA) were described in the monograph by Ruzicka and Hansen (38) and also by Valcarcel and Luque de Castro (39). In FIA, a pump delivers reagents and analyte to a mixer (chamber) and then to a reaction coil. The products of the reaction then ¯ow to a detector and from there to a waste receptacle. Electrochemical and colorimetric detectors are popular. Davis and Radke (40) illustrated the use of BCA in a simple FIA system for proteins. They employed a peristaltic pump to impel BCA reagent (containing 0.5±2% BCA) through a ¯owline of Te¯on or polyethylene tubing [inside diameter (ID) 0.5 mm]. Protein samples were injected into the ¯ow stream via a septum using a (25- or 50-mL) Hamilton syringe. The mixture then passed through a 3-m length of Te¯on tubing incubated in a constant-temperature water bath. The color formed was monitored with a spectrophotometer ®tted with a ¯ow-through cuvette. Absorbance (peak height) measurements were recorded using a strip chart recorder. The BCA-FIA system's performance depends on variables such as sample volume (10±50 mL), length of the reaction coil, ¯ow rate (0.5 or 1 mL min 1), and reaction temperature (72±1008C). Note that the BCA assay is a kinetic method. The color-forming reactions do not go to completion. This is especially true for FIA when the sample residence time in the reaction coil is short. Precision can be ensured only by keeping the sample ¯ow rate (and hence residence time) constant. To improve the color yield (peak height) during FIA, sample ¯ow rate may be reduced, thereby increasing the sample residence time in the reaction coil. Another strategy is to use a longer reaction coil. Assay sensitivity can also be increased by using higher reaction temperatures and/or concentrations of BCA.

0.113 (0.003) 0.093 (0.002) 0.0547 0.0245

10:50B 10:200C

0.5±2.5 2±12

Sensitivity
A/g (BSA)

10:200A 10:200A

BSA/BCA volume (L)

0.2±2 1±10

Linear range (g)

0.9940 0.9967

R

3.3 3.5

Ð Ð

% Error

Results with bovine serum albumin as standard. Samples were incubated at (A) 228C overnight, (B) 608C for 30 minutes, or (C) 378C for 30 minutes.

a

Redingbaugh and Turley (35) Microassay Standard assay Lane et al. (36) Microassay Standard assay

Assay format

TABLE 7 Microwell Plate-Based BCA Protein Assaysa

The Bicinchoninic Acid Protein Assay 115

116

Chapter 4

The FIA devised by Davis and Radke (40) employed a sample ¯ow rate of 0.5 mL per minute, a sample size of 10 mL and a reaction temperature of 808C. The reaction coil was 3 m in length. With such design features, the sample residence time was 4.7 minutes. The throughput achieved with this system was 60 samples per hour. The linear range for analysis was 1±10 mg of protein. The precision of analysis was surprisingly good (1%) in view of the crude system for sample injection. With BSA, ovalbumin, hemoglobin, betalactoglobulin, conalbumin, and myoglobin as standard proteins, the average sensitivity for analysis (DA560/mg) was 0.024 (+0.0013). Therefore, the sensitivity for FIA is comparable to that obtained for batch analysis (Table 7). At reaction temperatures above 808C, the ¯ow tubing became blocked by an unidenti®ed precipitate. A further example of protein FIA using BCA reagent was described by Wolfe and co-workers (41,42). Their setup was essentially as described by Davis and Radke (40) with the following modi®cations: (a) sample injection was via a Rheodyne2 sample valve or an autosampler device with a 20-mL sample loop, (b) samples were thoroughly degassed before use, (c) the carrier stream was phosphate buffer (0.1 M, pH 7.4) with 1% Triton X-100, and (d) protein analysis was performed at 808C with a ¯ow rate of 0.9 mL min 1. The linear range for analysis was 0±260 mg mL 1 (0±5.6 mg). The Technicon AutoAnalyzer was adapted for the BCA assay by Hawkes and Craig (43). The reagent, air, and sample ¯ow lines were assembled from 2.4-mm (ID) glass tubing. The sample stream was segmented with air bubbles before mixing with BCA reagent. It then passed to a reaction coil incubated at 558C at a ¯ow rate designed to produce a residence time of 4.2 minutes. Color formation (peak heights) was monitored at 570 nm. Calibration graphs for standard proteins were curvilinear and ®tted a polynomial equation. To save on the reagent cost, commercial BCA reagent was diluted 1:4 before use. The sensitivity of analysis decreased with decreasing concentrations of BCA reagent. Proteinprotein variations in assay results were similar to those observed with the batch assay: gelatin < BSA < chymotrypsinogen < RNA < insulin. The within-day precision of analysis was 1.04±2.99% and day-to-day precision was 1.62±14%, depending on the nature and concentration of the protein analyzed. 8.

APPLICATIONS OF THE BCA ASSAY TO FOOD PROTEIN ANALYSIS

There are only a small number of reports describing the use of the BCA assay for food protein analysis. No doubt, the numbers of applications will

The Bicinchoninic Acid Protein Assay

117

increase in the near future.* Applications reported in the published literature are discussed next. 8.1.

Solid-Phase Analysis of Cereal Proteins

Chan and Wasserman (44) determined protein in corn meal ¯our. Commercial corn meal samples and/or zein (2±7 mg) were placed in microcentrifuge tubes. The BCA working reagent (1 mL) was added and the mixture was incubated with intermittent shaking at 378C for 30 minutes. Samples were cooled over an ice bath for 5 minutes and particulate material removed by centrifuging (13,000g; 10 minutes). Thereafter, 0.2 mL of supernatant was diluted in 1 mL of BCA reagent A (see Method 1 for the reagent composition) and A562 readings were recorded. The BSA (50± 400 mg) was used as the standard protein while method calibration involved Kjeldahl analysis. Fig. 2 shows generally good agreement between Kjeldahl and BCA results. However, the former technique gave higher values for protein than the BCA assay. The discrepancy between Kjeldahl and BCA assays was

FIGURE 2 Correlation between Kjeldahl protein and BCA assay of corn meal protein and samples of zein. (Drawn from Ref. 44.)

* From personal experience, use of the commercially available reagent is underreported.

118

Chapter 4

ascribed to the presence of NPN. It is also likely that BSA is not a satisfactory standard for corn protein analysis. 8.2.

Analysis of Forage Plant Leaf Proteins

Some 23 forage plant (leaf) samples were analyzed using the BCA assay by Messman and Weiss (45). These included alfalfa (fresh, wilted, hay, silage, leaves), crown vetch (fresh, wilted, silage), spinach (fresh), perennial ryegrass (fresh), orchard grass (fresh, wilted), corn plant (silage), and peal millet (fresh). For sample pretreatment, leaves were lyophilized and ground using a 1-mm Wiley mill. The resulting powders were extracted with boratephosphate buffer (0.1 M ionic strength) containing SDS (1% w/w). Sonicating the suspension of leaf powder for up to 2 minutes facilitated protein extraction. In most cases, protein recovery was 85%. The protein extracts were analyzed with the BCA and Kjeldahl methods (N 6 6.25). The BCA method gave unreliable estimates of leaf proteins. There was poor agreement between BCA and Kjeldhal results. Leaf samples contain numerous interfering compounds (plant pigments, peptides, sulfhydryl compounds, and phenol derivatives) that can interfere with both the BCA and Kjeldahl methods. Attempts to circumvent interferences using the DOC-TCA procedure were not successful. The yield of leaf protein recovered by precipitation with DOC-TCA ranged from 40 to 80%. Protein recovery by cold-acetone precipitation was not signi®cantly higher. 8.3.

Analysis of Seed Proteins in the Presence of Phenolic Compounds

Salt-soluble proteins from soybean, tamarind, ragi, jack fruit, mango kernel, and sorghum were analyzed by Kamath and Pattabiraman (29). Whole meals were extracted with 0.3 M NaCl (buffered with 20 mM phosphate buffer, pH 6.9). Protein extracts were then analyzed using the BCA, Bradford, and Lowry assays. A range of pure proteins (BSA, casein, chymotrypsinogen, lysozyme, myoglobin, trypsin, zein) were also analyzed. The BCA, Bradford, and Lowry assays showed differences in proteinprotein variations in color yield. Apparently, endogenous seed compounds affected the results. High responses were obtained with sorghum, mango kernel, and other samples known to have high concentrations of total phenol. The BCA reagent was more sensitive to phenolic substances than to protein. On a scale of 1.0 for BSA, the color yields from a range of phenolic compounds were pyrogallic acid (86), gallic acid (2.1), pyrocatechol (106.0), tannic acid (9.3), and phenol (0.8). The BCA response to protein and phenolic substances was additive. There was a linear response between A567

The Bicinchoninic Acid Protein Assay

119

values and the concentration of phenol. Most seed contained comparable amounts of protein and phenolic compounds. Therefore, the BCA response to these systems is likely to be due to the phenol. The BCA analysis of soybean protein was error free owing to the low concentrations of phenolic compounds in this seed. 8.4.

Identi®cation of High-Lysine Cereals Using the BCA Assay

The A562 readings for ribonuclease, chymotrypsinogen, insulin, and BSA apparently showed a high degree of correlation with the number of lysine residues in each protein (R ˆ 0.99; P > Bradford. The performance of the BCA assay may be better than indicated from results in Table 8. First, the BCA assay was not adversely affected by SDS-NaOH or GnHCl when serum albumin was

The Bicinchoninic Acid Protein Assay

121

employed as the standard protein. Inadequate explanation was given for the ability of the BCA assay to detect only 75% of the carcass protein. Calibration graphs for the BCA are nonlinear. However, a hyperbolic function was not applied by the authors although such an equation was ®tted to results from the Bradford assay. There are good prospects that the BCA method can be adapted for animal protein analysis, perhaps with SDSNaOH as the extraction solvent. 8.7.

Analysis of Proteins from Freshwater Algae

There were several sources of error during attempts to analyze proteins from freshwater algae. Meijer and Wijffels (51) noted that the ef®ciency of protein extraction from cells was variable. Attempts to facilitate extraction using chemical means led to interferences with the Bradford and BCA protein assays. Proteins could also undergo severe damage under harsh extraction conditions such as boiling with alkali. Such harsh treatments could lead to standard proteins being less representative of the sample. Boiling Chlorella cells with 1 M NaOH for 30 minutes led to a recovery of between 3% (Bradford assay) and 14% (BCA assay) of the crude protein. By comparison, extracting yeast cells under similar conditions produced protein recoveries between 76% (Bradford method) and 85% (BCA method). When BSA standard protein was exposed to boiling 1 M NaOH for 30 minutes, there was 32% (Bradford method) or 85% (BCA assay) of the response recorded for the untreated proteins. Apparently, chemical damage due to heating at high pH is only partly responsible for the poor assay results. Chlorella protein was ef®ciently extracted by sonicating 50-mL samples of fresh algae suspended in sodium phosphate buffer (25 mM) containing 1% SDS. After sonication for 0.5, 1, and 3 minutes, there was 36, 80, and 104% recovery of crude protein as determined by the BCA assay. To avoid foaming and a rise in sample temperature, the period of sonication was divided into six 30-second intervals. In the presence of SDS, the Bradford assay could not be used.

REFERENCES 1. 2. 3.

PK Smith, RI Krohn, GT Hermanson, AK Mallia, FH Gartner, MD Provenzano, EK Fujimoto, NM Goeke, BJ Olson, DC Klenk. Measurement of protein using bicinchoninic acid. Anal Biochem 150:76±85, 1985. J Hoste. On a new copper speci®c group. Anal Chim Acta 4:23±37, 1950. D KerteÂsz. State of copper in phenoloxidase (tyrosinase). Nature 180:506±507, 1957.

122 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Chapter 4 G Felsenfeld. The determination of cuprous ion in copper proteins. Arch Biochem Biophys 87:247±251, 1960. AL Gershuns, AA Verezubova, ZhA Tolstykh. Photocolorimetric determination of copper with 2,20 -bicinchoninic acid. Izv Vyssh Uchebn Zaved Khim Khim Technol 4 (1):25±27, 1961. S Nakano. 2,20 -Biquinoline derivatives VI. Copper (I) chelates of the 4,40 substituted 2,20 -biquinoline derivatives and the determination of copper by 2,20 bicinchoninic acid. Yakugaku Zasshi 82:486±491, 1962. IS Musta®n, NS Frumina, VS Kovashova. Determination of copper in various samples with 2,20 -bicinchoninic acid. Zavod Lab 29:782±785, 1963. VN Tikhonov. Highly selective titration method for determining copper with 2,20 -bicinchoninic acid. Zh Anal Khim 27:673±677, 1972. NN Noskova. Photometric determination of copper in blood using 2,20 bicinchoninic acid. Mikroelem Sib 9:159±161, 1974. F Buhl, Z Gregorowicz, B Piwowarska. Complex compound of 2,20 bicinchoninic acid with copper (I) ions. Pr Nauk Uniw Slask Katowicach 91:27±33, 1975. F Capitan, JMR Navarro, LF Capitanvallvey, Solid-phase spectrophotometric microdetermination of copper. Anal Lett 24:201±1217, 1991. AJ Brenner, ED Harris. A quantitative test for copper using bicinchoninic acid. Anal Biochem 226:80±84, 1995 [see Anal Biochem 230:360, 1995 for erratum]. RF McFeeters. A manual method for reducing sugar determination with 2,20 bicinchoninic acid reagent. Anal Biochem 103:302±306, 1980. EM Grindler. Automated determination of glucose via reductive formation of lavender Cu(I)-2,20 -bicinchoninate chelate. Clin Chem 16:519, 536, 1970. K Mopper, EM Grindler. A new noncorrosive dye reagent for automatic chromatography. Anal Biochem 56:440±442, 1973. KJ Wiechelman, RD Braun, JD Fitzpatrick. Investigation of the bicinchoninic acid protein assay: identi®cation of the groups responsible for color formation. Anal Biochem 175:231±237, 1988. RL Levine, D Garland, CN Oliver, A Amici, I Climent, A-G Lenz, B-W Ahn, S Shaltiel, ER Stadtman. Determination of carbonyl content in oxidatively modi®ed proteins. Methods Enzymol 186:464±478, 1990. RE Paci®ci, KJA Davies. Protein degradation as an index of oxidative stress. Methods Enzymol 186:485±502, 1990. RD Madurawe, Z Lin, PK Dryden, JA Lumpkin. Stability of lactate dehydrogenase in metal-catalyzed oxidation solutions containing chelated metals. Biotechnol Prog 13:179±184, 1997. KD Bush, JA Lumpkin. Structural damage to lactate dehydrogenase during copper iminodiacetic acid metal af®nity chromatography. Biotechnol Prog 14:943±950, 1998. DA Skoog, DM West. Fundamentals of Analytical Chemistry. 3rd ed. New York: Holt, Rinehart and Winston, 1976, p 305. Q Chen, N Klemm, I Jeng. Quantitative Benedict test using bicinchoninic acid. Anal Biochem 182:54±57, 1989.

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23. TL Slocum, JD Duepree. Interference of biogenic amines with the measurement of proteins using bicinchoninic acid. Anal Biochem 195:14±17, 1991. 24. V Kaushal, LD Barnes. Effect of zwitterionic buffers on measurement of small masses of protein with bicinchoninic acid. Anal Biochem 157:291±294, 1986. 25. PL Lleu, G Rebel. Interference of Good's buffers and other biological buffers with protein determination. Anal Biochem 192:215±218, 1991. 26. Q Chen, N Klemm, G Duncan, K Jeng. Sensitive Benedict test. Analyst 115:109±110, 1990. 27. T Marshall, KM Williams. Drug interference in the Bradford and 2,20 bicinchoninic acid protein assay. Anal Biochem 198:352±354, 1991. 28. RJ Kesller, DD Fanestil. Interference by lipids in the determination of protein using bicinchoninic acid. Anal Biochem 159:138±142, 1986. 29. P Kamath, N Pattabiraman. Phenols interfere in protein estimation by the bicinchoninic acid assay method. Biochem Arch 4:17±23, 1988. 30. J-X Zhang, PJ Halling. pH and buffering in the bicinchoninic acid (4,40 dicarboxy-2.20 -biquinoline) protein assay. Anal Biochem 188:9±10, 1990. 31. A Bensadoun, D Weinstein. Assay of proteins in the presence of interfering materials. Anal Biochem 70:241±250, 1976. 32. RE Brown, KL Jarvis, KJ Hyland. Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem 180:136±139, 1989. 33. ZK Shahabi, MS Dyers. Protein analysis with bicinchoninic acid. Ann Clin Lab Sci 18:235±239, 1988. 34. RE Gates. Elimination of interfering substances in the presence of detergent in the bicinchoninic acid protein assay. Anal Biochem 196:290±295, 1991. 35. MG Redinbaugh, RB Turley. Adaptation of the bicinchoninic acid assay for use with microtiter plates and sucrose gradient fractions. Anal Biochem 153:267±271, 1986. 36. RD Lane, D Federman, JL Flora, BL Beck. Computer-assisted determination of protein concentrations from dye-binding and bicinchoninic acid protein assays performed in microtiter plates. J Immunol Methods 92:261±270, 1986. 37. K Sorensen, U Brodbeck. A sensitive protein assay method using microtiter plates. Experientia 42:161±162, 1986. 38. J Ruzicka, EH Hansen. Flow Injection Analysis. 2nd ed. New York: Wiley, 1988. 39. M Valcarcel, MD Luque de Castro. Flow Injection Analysis: Principles and Applications. Chichester, England: Ellis Horwood, 1987. 40. LC Davis, GA Radke. Measurement of protein using ¯ow injection analysis with bicinchoninic acid. Anal Biochem 161:152±156, 1987. 41. AC Wolfe, DS Hage Automated determination of antibody oxidation using ¯ow injection analysis. Anal Biochem 219:26±31, 1994. 42. CAC Wolfe, MR Oates, DS Hage. Automated protein assay using ¯ow injection analysis. J Chem Educ 75:1025±1028, 1998. 43. WC Hawkes, KA Craig. Adaptation of the bicinchoninic acid protein assay to a continuous ¯ow analyzer. Lab Robot Autom 3:13±17, 1990.

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44. K-Y Chan, BP Wasserman. Rapid solid-phase determination of cereal proteins using bicinchoninic acid. Cereal Chem 70:27±28, 1993. 45. MA Messman, BP Weiss. Extraction of protein from forages and comparison of two methods to determine its concentration. J Agric Food Chem 41:1085± 1089, 1993. 46. H Ahokas, L Naskali. Bicinchoninic acid protein assay reveals high-lysine contents in barley seed protein extracts. J Cereal Sci 7:43±48, 1988. 47. ML Kakade, E Liener. Determination of available lysine in proteins. Anal Biochem 27:273±280, 1969. 48. A Orr, BA Wagnaer, CT Howard, OA Schwartz. Assay of plant proteins with bicinchoninic acid for high-resolution two-dimensional polyacrylamide gel electrophoresis. Plant Cell Rep 7:598±601, 1988. 49. SPJ Brooks, JJ Lampi, G Sarwar, HG Botting. A comparison of methods for determining total body protein. Anal Biochem 226:26±30, 1995. 50. J Torten, JR Whitaker. Evaluation of the biuret and dye-binding methods for protein determination in meats. J Food Sci 29:168±1174, 1964. 51. EA Meijer, RH Wijffels. Development of a fast, reproducible and effective method for the extraction and quanti®cation of protein of micro-algae. Biotechnol Tech 12:353±358, 1998.

5 The Udy Method

1. INTRODUCTION Proteins react with certain organic dyes to produce insoluble complexes. The quantity of dye bound is proportional to (a) the dye-binding capacity (DBC)* and (b) the protein concentration. Farm-gate prices for milk (in Australia, Denmark, France, Netherlands, New Zealand, United States) is partly determined by its protein content. Dye-binding assays are widely used for milk protein determination. Amido Black 10B (C.I. 20470), Acid Orange 12 (C.I. 15970), and Orange-G (C.I. 16230) are the three main dyes used. Dye-binding assays are presented in Sections 1±3 of this chapter. Section 4 covers the chemistry of protein binding with azo dyes. Section 5 is a review of interference compounds and other sources of error. Section 6 covers applications of dye-binding assays in food protein analysis. Protein-dye interactions are of interest in relation to (a) dyeing of natural ®bers and textiles, (b) dyeing of tissue sections for microscopic observation, (c) the use of dyes to evaluate renal function, (d) application of dyes as pH indicators in protein-rich media, and (e) uptake of dyes by photographic materials. In 1925 Grollman (1) described phenol red

* DBC is the amount of dye bound by a gram of protein.

125

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Chapter 5

(phenolsulfonphthalein) binding using the Freundlich adsorption isotherm: 1=n

…Db =cP† ˆ KDf

…1†

where Db (moles) is the dye bound by cP (grams) of crude protein and Df is the equilibrium concentration of free dye. The empirical constants K and n were identical for serum albumin samples from healthy and diseased persons (2). Eq. (1) applied to solid adsorbents (charcoal, casein, granular gelatin) or soluble proteins (serum albumin). The Df was inversely related to serum albumin concentration. Robinson and Hogden (3) also showed that Df (measured as a decreases in A565) was inversely related to protein concentration (0±3 mg mL 1). Schmidt and co-workers (4±6) found that several proteins (gelatin, casein, ®brin, edestin) bound with acidic acid dyes (Biebrich Scarlet, Naphthylamine Brown,* Metanil Yellow, Tropaeolin O) at pH < 4.0 with maximum binding at pH 1.56±2.5. Contrary to earlier ®ndings, binding did not conform to the Fruendlich isotherm.{ The DBC was proportional to the concentration of basic amino acids in different proteins. Binding with basic dyes (Methylene Blue, Safranine-Y, and Induline Scarlet) depended on the number of acidic groups in the protein. The DBC was the same for soluble or granular gelatin. Fraenkel-Conrat and Cooper (7) con®rmed that Orange-G binds to basic amino acids. The basic dye Safranine-O bound with acidic amino acid side chains (carboxylic, phenolic, sulfhydryl groups). This study is recognizably the ®rst protein dye-binding assay.{ The number of basic (guanidyl, imidazole, amino) groups in several proteins is the same whether determined by titration or by Orange-G binding (Fig. 1).} The application of dye-binding assays to food proteins was developed and later commercialized by Udy (8,9). This subject has been reviewed by McGann (10), Lakin (11,12), Hartley (13), McGann (14), Lowe (15), and * Naphthylamine Brown is also called Amido Black 10B (C.I. 20470), Amidoschwarz 10B, Naphthylamine Black 10BR, Aniline Blue Black, Naphthol Blue Black, Acid Black 1, or Pontacyl Blue Black SX. { From the Freundlich equation, log(Db/cP) ˆ log K ‡ (1/n) log Df. Thus, a plot of log(Db/cP) versus log Df should give a straight line graph with a slope (1/n) and an intercept ˆ log K. { About 5 mg of protein (egg albumin, beta-lactoglobulin, casein, ®brin, and zein) was placed in a 15-mL test tube, wetted with 1 mL of buffer followed by the addition 1±5 mL of dye solution (e.g. 0.1% w/v Orange-G in 0.2 M citrate±0.1 M phosphate buffer, pH 2.2). After shaking for 20±24 hours, the suspension was centrifuged to remove the insoluble protein-dye complex. The supernatant was diluted 100-fold with buffer and absorbance readings were taken. The free dye concentration was determined from a standard curve of absorbance readings plotted against known dye concentration. } Both collagen and casein appear to have a typical dye-binding characteristics.

The Udy Method

127

McGann (16). The literature through to 1967 was discussed by Cole (17). Sherbon (18) reviewed dye-binding assays for milk proteins. 2. THE UDY METHOD The Udy assay using Acid Orange 12 is described here (Method 1). Information in the public domain deals with milk and dairy products. Other applications developed by Udy Corporation during the 1960s were probably commercially sensitive and not published (Table 1). The Udy method is the basis of Udy Protein Systems2. 2.1.

Protein Determination Using Acid Orange 12

Method 1 is equivalent to AOAC Method 967.12 for milk protein analysis (19). Instrument requirements include a spectrophotometer, short-pathlength ¯ow cuvette, and automatic pipettes. For small-scale analysis, a degree of improvisation is possible. For large numbers of samples the required accessories include the Udy calorimeter, a 40-mL dye regent

FIGURE 1

Basic amino acid concentration in a range of proteins as measured by Orange G binding and by titration. Units of the Y-axis are 6 10 4 moles per gram protein. (Drawn using data from Ref. 7.)

128

Chapter 5

TABLE 1

A Range of Commodities Analyzed by the Udy Method

Alfafa Barley Beans Bermuda grass Caseinate Cheese (hard) Chickpeas Corn silage Cottonseed meal Cowpeas Fish meal

Gaines burger Gram Grass peas Lentils Linseed meal Malted barley Meat MilkЯuid MilkÐpowders Mung beans Mustard meal

Oats Oat groats Peanut meal Peas Pigeon peas Rapeseed meal Rice Rye Saf¯ower meal Sesame meal Sorghum (milo)

Soybean Soybean hulls Soybean meals Sun¯ower meal Triticale Urd beans Wheat Wheat germ WheatÐgluten WheyÐdelactose WheyÐfresh WheyÐpowdered

Source: Adapted from Udy Corporation advertising literature.

dispenser, and a React-R-Shaker* for highly ef®cient mixing of powdered samples with dye solution. Method 1 Acid Orange 12 dye binding (20±22). Reagents{ 1. Acetic acid (glacial) 2. Acid Orange 12 3. Oxalic acid 4. Potassium dihydrogen phosphate Procedure Puri®cation of Acid Orange 12. Dissolve 400 g of dye in 400 mL of boiling water. Add 400 mL of reagent-grade ethyl alcohol. Cool to room temperature and refrigerate at 0±58C overnight. Vacuum ®lter dye solids using a Buchner ¯ask-®ltration unit ®tted with a polypropylene ®lter. Wash with cold ethyl alcohol and dry the resulting solid in an oven at 1258C. * Udy and React-R-Shaker are trademark terms for the Udy Corporation, 201 Rome Court, Fort Collins, CO 80524. Fax: 1-970-482-2067. Telephone: 1-970-482-2060. Internet address: http://www.udycorp.thomasregister.com { Other requirements include 2-oz polyethylene bottles or 125-mL conical ¯asks, automatic pipettes for dispersion of 40 mL of reagent, syringe pipettes (2±5 mL), sample mill, and ®ltration equipment or a low-speed centrifuge.

The Udy Method

129

Phosphate buffer (0.05 M, pH 1.8±1.9). Dissolve potassium dihydrogen phosphate (3.4 g) and oxalic acid (2 g) in 100 mL of warm water. Add to 800 mL of water containing phosphoric acid (3.4 mL), acetic acid (60 mL), and propionic acid (1 mL) and dilute the mixture to 1 L. Working Acid Orange 12 dye reagent (0.13% w/v). Dissolve 1.3 g of Acid Orange 12 in 100 mL of warm phosphate buffer. Allow to cool and dilute to 1 L with phosphate buffer. Reference dye solution (0.06% w/v). Dilute the working dye reagent with phosphate buffer. Prepare further dilutions and produce a calibration curve of free dye concentration versus A480 readings. Performing a dye-binding assay. Place 1.5±2.4 mL of liquid sample (or 0.25±0.5 g of solid) in a 2-oz plastic polyethylene bottle. Add 40.44 g (40 mL) of working dye solution and shake vigorously for 30 seconds. Solid samples may be shaken for 5 minutes. Centrifuge at 3500 rpm for 30 minutes or ®lter to remove insoluble dye-protein complex. Dilute* the supernatant 100-fold with phosphate buffer and record A480 readings versus an appropriate buffer blank. Determine the amount of free dye from the calibration graph. Calibration for protein determination. Determine dye binding for samples with known amounts of crude protein (%N 6 6.25). Establish the regression equation relating Db versus crude protein. Analyses of 73 whole milk or 34 spray-dried milk samples by Orange G binding led to a highly signi®cant correlation between crude protein (%N 6 6.38) and the amount of free dye (23); cP ˆ 100

…V1 D V2 Df † kEm

…2†

where k (226 g mole 1) is the dye equivalent weight,{ E (moles g 1) is the DBC expressed as equivalents of dye bound, and m is the weight of sample in grams. For a solid sample V1 & V2 (i.e., volume of sample & volume of

* Colorimetric measurements are possible without dilution when using a very short (0.3 mm) path-length ¯ow-through cuvette. { The equivalent weight (k) for an ionic species (g mole 1) is the molecular weight divided by the number of charges per molecule; k ˆ 226 (g mole 1) assuming that this dye has two positive charges. Note that the number of equivalents of dye bound is DBC/k.

130

Chapter 5

sample ‡ dye) and hence cP ˆ 100

V1 …D Df † kEm

…3†

Rearranging Equation (2) leads to Eq. (4) or Eq. (5) for liquid or solid samples, respectively. Df ˆ

V1 D V2

Df ˆ D

cPEkm V2

cPEkm V1

…4†

…5†

Therefore a graph of Df versus cP yields a straight line with a slope of mkE. The parameter E was 0.792 (mEq g 1) for spray-dried milk and 0.805 (mEq g 1) for fresh milk. See Section 6 for further discussions of milk protein analysis.

2.2.

Protein Determination Using Orange-G

Fraenkel-Conrat and Cooper (7) employed Orange-G in their seminal study of 1944. Udy (8) also used Orange-G for protein determination and later changed to Acid Orange 12 in 1963 (22). The color change for Acid Orange 12 was apparently 100% greater than that obtained with Orange-G.* The former dye is also less hygroscopic and more easily puri®ed. These days, high-grade samples of Orange-G are readily available. The use of Orange-G is described further in Refs. 8,9,24,25, and 26.

2.3.

Protein Determination Using Amido Black 10B

Milk protein analysis using Amido Black 10B is important in both North America and Europe (27±30). Commercial instruments such as the ProMilk Mark II or ProMilk PMA (manufactured by Foss Food Technology Corp.) use Amido Black 10B. Several investigators reported dif®culties with Amido Black 10B staining of plant proteins; some Amido Black 10B samples may contain impurities with different af®nities for plant proteins (31). [Method 2 is adapted from Sherbon (32,33).] * This view is incorrect (Section 3.2). Proteins bind equal amounts of Orange-G and Acid Orange 12. The molar extinction coef®cients for Orange-G and Acid Orange 12 are also similar.

The Udy Method

131

Method 2 Protein analysis using Amido Black 10B dye binding* (32,33). Reagents 1. Amido Black 10B 2. Citric acid 3. Disodium hydrogen phosphate 4. Thymol blue (optional preservative) Procedure 0.05M Citrate±0.01 M phosphate buffer. Dissolve citric acid (52.6 g), sodium dihydrogen phosphate (3.3 g), and thymol blue (1 g) in 660 mL of water. Working dye solution (0.075% w/v). Dissolve Amido Black 10B (3 g) in 1 L of water by heating to 708C. Mix with citrate-phosphate buffer and add 3.33 kg of water. Reference dye solution. Dilute the working dye solution (1 volume) with 2.5 volumes of distilled water. Add 1 mL of sample to 20 mL of dye solution. Mix for 0.5±3 minutes. Filter to remove insoluble dye-protein complex. Dilute the supernatant 100-fold with phosphate buffer. Record A620 readings. Determine the amount of free dye from a calibration graph of A620 plotted against reference dye solutions.{ For calibration analyze at least 10 samples of known protein concentration in duplicate. Methods 1 and 2 were readily scaled down by mixing 50±100 mL of sample with 1 mL of dye reagent in a polyethylene microcentrifuge tube. The protein-dye precipitate was then removed by microcentrifugation (13,000 rpm; 5 minutes). Absorbance readings were recorded after diluting the dyesupernatant solution 100-fold. For strongly colored samples, the dry weight for the protein-dye complex was recorded. These micro-dye-binding assays led to signi®cant savings of reagent and improved convenience (34). 3. SOLID-PHASE DYE-BINDING ASSAYS Protein is adsorbed on a ®lter support such as nitrocellulose, ®lter paper, or a glass ®ber ®lter. Sometimes, the adsorbed protein is ``®xed'' by treating * Amido Black 10B dye binding is also called the ProMilk method. { Absorbance measurements can be taken without dilution if using the purpose-made 0.3 mm path length cuvette. Speci®c instructions are given for use with commercial instruments.

132

Chapter 5

with dilute TCA. Exposure to dye solution is followed by a destaining solution to remove excess dye. The dyed protein spot is then excised and placed in a test tube with elution solvent, which dissolves the protein-dye complex. Absorbance measurements are then recorded as before. The advantages of the solid-phase assay include (a) increased sensitivity and (b) improved resistance to interfering compounds. In the best cases, an LLD of 0.25 mg is attainable with a linear range extending to 200 mg of protein. 3.1.

Nitrocellulose and Cellulose Acetate Membranes

Kuno and Kihara (35) employed nitrocellulose membranes for solid-phase dye-binding assays.* The linear range for analysis was 10±50 mg protein. The ef®ciency of protein binding was >97%. Compared with the Lowry assay, there was increased resistance to interference from tyrosine or Tris. Heil and Zillig (36) analyzed protein (0.5±2.5 mg) using cellulose acetate membranes. Staining was with Amido Black 10B (0.24% w/w) dissolved in (10:45:45) acetic acid±methanol±water solvent. The same solvent was used for destaining. Protein spots were air dried, excised, and eluted with 0.5 mL of solvent (glacial acetic acid, formic acid, water, TCA). Schaffner and Weissmann (37) treated protein samples with 10% (w/w) TCA and recovered the resulting precipitate with a Millipore (HAWP 025CO) membrane ®lter. The rest of their methodology is essentially as already described. The linear dynamic range was 5±30 mg with a sensitivity of 0.027 DA630 mg 1 BSA. The LLD was 1.5 mg + 5% for a sample volume of 2 mL. The following compounds did not interfere: dextran (100 mg mL 1), polyethylene glycol (0.5 mg mL 1), glycogen (0.5 mg mL 1), RNA or DNA (30 mg mL 1), NaCl (>1 M), ammonium sulfate (2.5 M), magnesium chloride (>0.1 M), EDTA (>100 mM), 2-mecaptoethanol (>100 mM), SDS (1%), and sucrose (20%). The Schaffner-Weissmann method was modi®ed for protein analysis in the presence of 1000-fold excess lipid (38).BSA (20 mg) was accurately determined in the presence of 20 mg phospholipid (43% phosphatidylcholine, 30% phosphatidylethanolamine, 27% unidenti®ed lipids). The linear dynamic range (2±24 mg BSA) was unaffected by added phospholipid. Assay sensitivity was also unaffected by the presence of lipid. The A630 was 0.744 (+ 0.054) and 0.775 (+ 0.048) for 20 mg of BSA without and with 20 mg of * Protein (5±50 mg) dissolved in 0.2 M magnesium chloride was ®ltered with a nitrocellulose membrane under suction. Each membrane was stained with 2 mL of Amido Black 10B (4 mg mL 1 in acetic acid±methanol±water (1:5:4) solvent). After destaining with 5 mL of acetic acid (1% w/w), membranes were eluted with 3.5 mL of 10 mM NaOH. The released dye was measured (A620).

The Udy Method

133

lipid, respectively. There was compatibility with a great many buffer salts (200 mM): NaCl, KCl, Na phosphate, K phosphate, HEPES, Tris-HCl, MES, MOPS. However, protein-protein variations in the dye response were evident. The proceding technique was apparently more accurate than the modi®ed Lowry assay or biuret method. The speed for solid-phase analysis was increased by Nakamura and co-workers (39). They used micro®ltration apparatus* to apply multiple samples to nitrocellulose membranes. Stained protein spots were measured directly via a densitometer. The linear dynamic range for analysis was 1±10 mg. Sensitivity using Amido Black 10B staining was twofold higher than with Ponceau Red. For both dyes the order of increasing sensitivity was trypsin < lysozyme < cytochrome c < bovine serum albumin < human serum albumin < concanavalin A < histone II < human g-globulin. Assay results were unaffected at pH 3.6±9. There was no interference from a range of salts, sugars, amino acids, nucleotides, polyols or EDTA. Detergents (SDS, Triton X-100, Tweens) or high concentrations of denaturants reduced protein binding to the nitrocellulose membrane. 3.2.

Whatman Paper and Glass Membrane Filters

Protein samples (5±200 mg) were dried on to Whatman No. 42 paper followed by treatment with 7.5% (w/v) TCA (40). McKnight (41) spotted 100 mL of protein (0.5±5 mg) on glass ®ber ®lters (Whatman GF/C), dried the liquid using hot air, and then ®xed the protein with 20% (w/v) TCA. Esen (42) and Almand and Saleemuddin (43) dried protein solutions (5 mL; 1±4 mg mL 1) on Whatman No. 1 with no TCA ®xation step. The ®lter paper±bound protein was stained with Amido Black 10B or Coomassie Brilliant Blue G250 (see Chapter 7).

4. THE CHEMISTRY OF DYE-BINDING PROTEIN ASSAYS 4.1.

Characteristics of Azo Dyes

Azo dyes represent 60% of all known dye structures (44). The ®rst azo compounds were synthesized by Peter Gries in 1858 using building units designated A, D, E, M, and Z (Fig. 2). Mono-azo dyes were formed via electrophilic attack of a diazotized species (A) on a sulfonated amino* BIO-DOT apparatus; Bio-Rad Laboratories, Richmond, CA. Membrane-bound protein was stained using Amido Black 10B or Ponceau Red (0.1% w/v) dissolved in 7% acetic acid and destained with 7% (w/v) acetic acid.

134

FIGURE 2

Chapter 5

Building blocks for synthesis of azo dyes with a brief explanation of A, D, E, M, and Z notation. (Top) Diazotized (group A) compounds (left) or (right) a tetrazotized (group D) compound. (Bottom) Coupling agents with a capacity to react with one equivalent or two equivalents of a group A compound. M is an aromatic amine that can react with A. The product may be diazotized for a second round of coupling.

naphthol nucleus (E or Z). By altering the reaction pH, temperature, and concentration of reagents, diazo, triazo, or tetrakisazo dyes may be formed. The structure of azo dyes can written in shorthand: A?Z refers to a monoazo dye produced by reacting a diazotized aromatic compound (A ˆ benzenediazonium chloride) with Z. Examples of A?Z dyes are Acid Orange 12, T-azo-R, and Acid Orange 1. A typical diazo dye is Amido Black 10B (1-amino-2p-nitrophenylazo-7-phenylazo-8-naphthol-3,6-disulfonic acid) with the formula A1?Z/A2. The A1 and A2 units are attached to a central (Z) unit, 1-amino-2-naphthol-3,6-disulfonic acid. These structures are shown in Figs 3±5. All azo dyes possess one or more azo (22N55N22) groups. The nitrogen-nitrogen double bond allows cis-trans isomerism. The naphthalene 2-hydroxyl group hydrogen bonds to the azo-group nitrogen, thereby stabilizing the trans isomer. The characteristics of some azo dyes are listed in Table 2. The absorptivity for Acid Orange 12 (w/v) is 26% higher than for Orange-G. The molar extinction coef®cients for the two dyes differ by only about 6%. The sulfonic acid group of azo dyes remains ionized at most

The Udy Method

135

FIGURE 3 The structure of T-azo-R and Acid Red 1.

accessible pH values. However, the exact acidity of benzenesulfonate or naphthylenesulfonate groups is uncertain (pKa ˆ * 0.7±1.5).

4.2.

Protein Dye Binding

Azo dyes bind with the guanidino, imidazole, and the e-NH2 side chain of arginine, histidine, and lysine, respectively (4,5,7,45±48). Interactions with wool (composed of the protein keratin) occur via ionic bonding. Further

136

FIGURE 4 The structure of Acid Orange 12 and Orange G.

Chapter 5

The Udy Method

137

FIGURE 5 The structure of Amido Black 10B.

bonding is by van der Waals and hydrophobic interactions. These increase with the area of contact between the dye and protein (44). Nonionic interactions become more important at high dye/protein ratios. Formerly, the order of sensitivity for dye-binding assays was given as Amido Black 10B > Acid Orange 12 > Orange-G (21,22,26). Protein assays using Orange G were thought to be 100% less sensitive than assays using Acid Orange 12 because the two dyes bound 2 or 1 mole of arginine per mole of dye, respectively. However, more recent data (47) show that both Orange G and Acid Orange 12 form 1:1 mole complexes with protein basic amino acid residues. For Amido Black 10B the ratio of dye bound to basic amino acids is 1:0.5 (Table 3) (49,50). The small distance of separation between the sulfonate groups of Orange G may exclude binding to two sites.

TABLE 2 Characteristics of Some Acid Azo Dyes Used for Protein Assay Dye C.I. No. Molecular weight Net charge lmax e (Abs mL/mg)a

AB 10B

AO 12

OG

20470 616.50 1 620 81.5 (43,684)

15790 350 1 482 59.0 (22,066)

16230 452.38 2 480 46.9 (20,683)

AB10, Amido Black 10B; AO12, Acid Orange 12; OG, Orange G. a Extinction coef®cient, absorbance per unit concentration of dye bound (mg mL 1). Value in parentheses is molar extinction coef®cient. Source: Refs. 21 and 26.

138

Chapter 5

TABLE 3 Dye-Binding Capacity for a Range of Samples for Orange G (OG), Acid Orange 12 (AO 12), and Amido Black 10B (AB 10B) DBC (mmoles g Sample BSA HSA HGG k-Casein Meat meal Fish meal Milk protein Soybean Average Ratiob

1

cP)a

OG

AO12

AB 10B

1365.0 1466.8 1028.8 692.5 630.5 708.0 800.9 984.5 959.6 0.8

1775.7 1780.0 1354.3 857.1 905.7 920.0 1094.3 1345.7 1254.1 1.0

743.5 834.4 582.8 274.4 339.3 336.0 490.3 555.2 519.5 0.54

a

Dye-binding capacity (mmoles of dye bound per gram protein) from Ref. 50. Average dye: BAA ratio. BSA, bovine serum albumin; HSA, human serum albimin; HGG, human gamma globulin. b

In summary, careful perusal of information in Table 3 shows that the sensitivities of dye-binding assays using Orange-G, Acid Orange 12, and Amido Black 10B are the same.

4.3.

Soluble Protein Dye Complexes

Protein-dye complexes can be studied by spectrophotometry or equilibrium dialysis (51). To avoid precipitate formation, the concentration of dye used (1±10 mM) is 350±1000 times below those used for the Udy assay. BSA binding with azosulfathiozole, Orange I, Orange II, methyl orange, and tetrazine yellow was investigated by Klotz et al. (52), Klotz (53), and Sheppard et al. (54). Pesavento and Profumo (55) examined T-azo-R binding to BSA. Other interesting reports describe protein binding to phenol red (3), bromophenol blue (56,57), thymol blue (58), and the reactive dye cibracron blue (59±61). Protein dye binding shifts the equilibrium between nonionized and ionized dye forms. The extinction coef®cient for the bound dye (eb) increases while the wavelength for maximum absorption (lmax) shifts to lower values. The hyperchromic effect is explained by reference to the conjugation theory. The lmax for dye molecules is determined by the energy required for p?p* electron transition. Protein binding alters the degree of conjugation

The Udy Method

139

involving the p orbital and lowers the energy of the p* state. Lysine, arginine, or histidyl (auxochromic) groups donate electrons to the dye molecule, thereby increasing its conjugation extent. A further explanation centers on the transfer of free dye molecules from a polar low-viscosity solvent phase to a relatively nonpolar or restricted protein phase. Dye transfer to more nonpolar solvents and micelles leads to spectral changes resembling those observed during protein binding (62±64). Usually, a ®xed concentration of dye is exposed to increasing amounts of protein. Absorbance readings are recorded with a reference cuvette containing a dye solution of the same concentration as the sample cuvette (Table 4). Measuring the ``difference absorption'' (DA) is useful where a dye solution has a high background. The absorbance change for dye reagent depends on the total dye concentration (D), extinction coef®cient (ef), and the cuvette path length (1 cm) as described in Equation (6). A1 ˆ ef D

…6†

Df, protein, and the protein-dye complex are in equilibrium. Db has its own extinction coef®cient (eb). The net absorption change is described by

TABLE 4 A Summary of Symbols Used in Describing Protein-Dye Binding Symbol A1 and A2 DA ( ˆ A2 A1) a D Db Df ef eb De ( ˆ ef eb) eapp P, Pf Kd limax liso n, ns

De®nition Absorbance for dye and dye ‡ protein Difference absorbance Fraction of dye bound Total concentration of dye Concentration of bound dye Concentration of free dye Extinction coef®cient for free dye Extinction coef®cient for bound dye Extinction coef®cient difference for the free and bound dye Apparent molar extinction change when a fraction of dye is bound Added, free concentration of protein Conditional dissociation constant Wavelength for maximum absorbance Isobestic wavelength where De ˆ 0 Number of dye molecules bound per molecule protein; ns ˆ strong sites

140

Chapter 5

Equation (7). A2 ˆ ef …D

Db † ‡ eb Db

…7†

From Eqs (6) and (7) it can be seen that A2±A1 ˆ DA ˆ Db(eb A2

A1 D

ˆ

Db …eb ef † D

or

eapp ˆ a…eb

ef † ‡ ef

ef) and also …8†

and therefore aˆ

…eapp ef † …eb ef †

…9†

where a is the fraction of dye bound and eapp ( ˆ A2/D) is the apparent extinction coef®cient change when dye is bound. The isobestic point (liso) is the wavelength at which bound and free dye molecules have equal absorptivity (ef ˆ eb). By running absorbance spectra with increasing dye or protein concentration, liso can be identi®ed as the wavelength at which there is no absorbance change (DA ˆ 0). The existence of an isobestic point is indication that the dye exists as two interconvertible forms (e.g., bound and free). No isobestic point will appear if ef = eb over the wavelength range examined. The corollary is that proteindye binding will not generate an absorbance change if De ˆ 0.

4.4.

Analysis of Protein Dye-Binding Reactions

The protein dye-binding reaction is summarized by the following equation Df ‡ nPf „ Db

…10†

Replacing Db with DA/De, we can de®ne the dissociation constant (Kd) as Kd ˆ

…D

DA=De†…nP DA=De

DA=De†

…11†

The concentration of dye species changes with pH and ionic strength. Therefore, Kd is a conditional constant with a value that depends on the pH and ionic strength (52). Depending on the protein/dye ratio Eq. (11) takes on the two forms described in Cases 1 and 2.

The Udy Method

141

Case 1, Low dye/protein ratio. With excess protein we have nP (DA/De) & nP in Eq. (11). This approximation is also justi®ed if the number of binding sites is large; hence, …D

Kd ˆ

DA=De†nP DA=De

and DA ˆ

nDePD Kd ‡ nP

…12†

For high protein concentrations (nP >10Kd) DA reaches a maximum (DAmax) where De ˆ

DAmax D

…13†

Equation (13) is the chief means by which De and also eb may be determined (65). First, invert all terms in Eq. (12). The resulting double-reciprocal relation [Eq. (14)]* allows the determination of DAmax by graphical means (see the following). 1 Kd 1 ˆ ‡ DA DeDnP DeD

…14†

Multiplying the former relation by DADeD gives Eq. (15). Other linearized forms result from multiplying Eq. (15) by 1/(DeKd) or 1/(DeKdD). DA ˆ DeD

DAKd nP

…15†

DA D ˆ nPDe Kd

DA DeKd

…16†

DA n ˆ PDDe Kd

nDA DeDKd

…17†

* The transformation is analogous to linearization of the Michaelis-Menten equation to give the Lineweaver-Burke double reciprocal plot, Eadie plot, Hanes plot, etc.

142

Chapter 5

Finally, Equation (17) may be restated as Gˆ

nD Kd

nGP Kd

…18†

where G ˆ Db/P. To evaluate DAmax, De, and Kd/n, proceed as follows: 1. Add varying concentrations of protein to a ®xed concentration of dye (D). 2. For each sample measure DA. 3. Using Equation (14), plot a graph of 1/DA (Y-axis) versus 1/P (X-axis). From the X ˆ 0 intercept ®nd 1/DAmax ( ˆ 1/DeD). 4. Use the estimate for DAmax and ®nd De from Eq. (13). 5. From the slope and known values for De and D calculate Kd/n. It is not possible to determine Kd independently using Eqs (14)±(17). An alternative stratagem is to translate DA values to Db (e.g., Db ˆ DA/De) and Df ( ˆ D DA/De). Thereafter, use Eq. (18) to evaluate all binding parameters. Note that Eqs (12)±(18) are valid only at high protein/dye ratios. Under these circumstances, only high-af®nity protein sites (strong sites) are occupied. Binding parameters therefore relate to strong sites. The number of strong binding sites (ns) is distinct from the total number of sites (n). Case 2. High dye/protein ratio. Eq. (11); therefore, DA ˆ

With excess dye D

(DA/De) & D in

DenPD Kd ‡ D

…19†

Eq. (19) describes protein ligand binding when a small ®xed concentration of protein is exposed to varying concentrations of dye. As the concentration of dye increases (e.g., D > 10Kd), DA increases to a maximum value (DAmax) and Eq. (19) becomes* DAmax ˆ nDeP

…20†

Using the De value determined before (see Case 1), ®nd the total number of binding sites (n) as follows: 1. Add varying concentrations of dye to a ®xed concentration of protein. * A high dye concentration is de®ned in relation to Kd and not protein concentration.

The Udy Method

143

2. For each mixture measure DA. 3. Plot a graph of 1/DA (Y-axis) versus 1/D (X-axis). The X ˆ 0 intercept yields 1/(nDeP) and the slope is Kd/nDeP. 4. Calculate the number of binding sites from Eq. (21). n ˆ DAmax =…DeP†

…21†

Dividing the graph slope by the intercept gives Kd under conditions such that both strong and weak binding sites are ®lled. In summary, two different experimental designs and analyses for protein dye binding are possible. Case 1 employs a ®xed (low) concentration of dye and varying amounts of protein. The estimates of De, Kd, and n obtained are related to high-af®nity sites. Case 2 employs a ®xed (low) concentration of protein and varying (high) concentrations of dye. This study is useful mainly for determining the total number of binding sites. Values of Kd are average parameters for both weak and strong dye-binding sites (57,58,65). The application of these relations to a study of BSA binding with T-azo-R is described next. A ®xed concentration of dye (D ˆ 10.8 mM in 5 6 10 3 M HCl solvent, pH 2.3) was titrated with increasing concentrations of BSA (see Case 1). Fig. 6 shows the pattern of binding of T-azo-R to BSA (55). At concentrations of dye above 100 mM, an insoluble protein-dye complex formed. Apparently T-azo-R binding to BSA could not be analyzed using the Scatchard plot (55). I have reanalyzed such data and others from Refs. 57, 58, and 65 using Eqs (14), (15), and (18) (Figs 6 and 7). Parameters for BSA binding with T-azo-R, bromophenol blue, and thymol blue are shown in Table 5. The original studies were not designed to measure the total number of (low- and high-af®nity) dye-binding sites. The proportion of basic amino acids (*110 per mole BSA) functioning as high-af®nity binding sites for T-azo-R did not exceed 50%. With bromophenol blue there was dye binding to a very small proportion of basic amino acids.

4.5.

Solubility Relations for Protein Dye Complexes*

The reaction between a charged dye molecule (D ) and protein (P ‡ ) produces a soluble, complex [PD]AQ that later forms an insoluble complex * There is no strict adherence to the use of squared brackets to indicate concentration. Brackets are included only where their presence renders equations more readable.

144

FIGURE 6

Chapter 5

Analysis of protein-dye binding. A ®xed concentration T-azo-R dye (D ˆ 10.8 mM) was titrated with increasing concentrations of bovine serum albumin (P ˆ 0±6 mM). (Top graph) Difference absorbance changes monitored at 510 nm (DA510) plotted versus total added protein concentration (P). (Lower graph) Determination of binding parameters using a double reciprocal plot of 1/P versus 1/DA. The Y-intercept is 1/DAmax. Furthermore, DAmax/D ˆ De [see Eq. (13)]. The slope ˆ Kd/(nDeD).

The Udy Method

145

FIGURE 7 Analysis of T-azo-R binding with bovine serum albumin. Same data as shown in Fig. 6. (Top graph) Determination of binding parameters in accordance with Eq. (15). DA is plotted versus DA/P. The slope is Kd and intercept is DAmax. (Bottom graph) Determination of binding parameters according to Eq. (18). As P ? 0, then Db ? nP and Kd * D. Under such conditions it follows that G ˆ n (see Refs. 58 and 59).

146

Chapter 5

TABLE 5 Binding Parameters for Soluble Serum Albumin±Dye Complexes Kd n

Dye, equation T-azo-R Eq. (14) Eq. (15) Eq. (18) Bromophenol blue Eq. (14) Eq. (15) Eq. (18) Thymol blue Eq. (18) a

…mM 1 †

De (M

1

cm 1)

0.162 0.177 0.181

4709 5024 Ð

3.11 2.03 2.23

87787 69250

69.0

1430

n Ð Ð 62.a

6.0 26

The intercept from Equation (18) ˆ n. See Refs. 57 and 58.

[PQ]S. P‡ ‡ D „ PDAQ ; PDAQ „ PDS ;

Kd ˆ ‰P‡ Š‰D Š=‰PDŠAQ

K2 ˆ ‰PDŠAQ =‰PDŠS

…22† …23†

The net reaction is P‡ ‡ D „ ‰PSŠS

…24†

The overall process is comparable to isoelectric precipitation (4). Precipitation occurs when suf®cient numbers of dye molecules bind to neutralize all protein charges. In contrast, excess protein produces a ``colloidal protective effect'' that maintains the solubility of protein-dye complexes. From the de®nition of solubility product (KS) we have KS ˆ Kd K2 ˆ ‰P‡ Š‰D Š

…25†

The activity for [PQ]S is given a value of 1. To describe the effect of pH on protein-dye interactions, consider the ionization of dye-binding sites (pKa *12.5); P ‡ H‡ „ P ‡ ;

Ka ˆ ‰Pf Š‰H‡ Š=‰P‡ Š

…26†

The total protein concentration (P) ˆ [P ‡ ) ‡ [Pf] and after substituting for Pf, Ka ˆ …‰PŠ

‰P‡ Š†H‡ =P‡

…27†

The Udy Method

147

and P‡ ˆ

P …Ka =‰H‡ Š† ‡ 1

…28†

The concentration of P ‡ changes with the concentration of H ‡ in accordance with Equation (28) and consequently KS is given by Equation (29). KS ˆ

P‰D Š …Ka =‰H‡ Š† ‡ 1

…29†

To attain very low concentrations of soluble protein (in equilibrium with the protein-dye precipitate) requires a high dye concentrations at a low pH. Strong protein-dye binding (small Kd) will also facilitate quantitative precipitation of protein from solution. At pH 4.84 the gelatin complex with Amido Black 10B yields KS & 4 6 10 12. Under higher acidity conditions the value KS was too low to determine (4). Refer to Skoog and West (66) for more information on KS-solubility relations. 5. INTERFERENCE COMPOUNDS AND THEIR AVOIDANCE There was no interference from low-molecular-weight compounds, including amino acids and peptides. Lipids do not affect protein-dye binding. Ionic surfactants and benzoic acid derivatives (e.g., p-aminobenzoic acid) might interfere if present in high concentrations. Chaotropic agents such as urea are also likely to affect dye-binding results. Dye binding with nonprotein food components is possible. Calibration graphs for wheat ¯our protein had a nonzero intercept owing to dye binding with wheat bglucan (67). Mass transfer or diffusion limitations may be important for solid or powdered food materials. Physical effects can be overcome by achieving high sample agitation, increasing the mixing time, and reducing sample particle size. 6. APPLICATIONS OF DYE-BINDING ASSAYS FOR FOOD PROTEIN ANALYSIS 6.1.

Milk, Ice Cream, and Dairy Products

Udy (9) analyzed whole milk and spray-dried milk samples by Orange-G binding. The milk samples and Kjeldahl protein values were supplied by Ashworth and co-workers at the Department of Dairy Science, Washington State University (Pullman, WA). Dye-binding studies at Ashworth's

148

Chapter 5

laboratory led to one of the ®rst Ph.D. dissertations in this area (68). Literature covering milk or dairy protein analysis using Orange-G and Acid Orange 12 is summarized in Table 6. Ashworth et al. (24) analyzed 354 milk samples from six breeds of cows. Milk powders were also analyzed. The average protein content for milks was 3.49 (+ 0.273)%. Some 95% of protein determinations were within + 0.67% of the crude protein content. NPN, proteose peptone, milk fat, and lactose caused little or no interference. Sample preservatives (hydrogen peroxide, formaldehyde, or mercuric hydrochloride) also did not affect the results. Adding mercuric chloride (1.35 mg %) to milk samples allowed room temperature storage before analysis. Antibiotics were not effective preservatives.

TABLE 6 Dye-Binding Assay of Milk and Dairy Protein Dye, application Orange-G Milk (fresh, powder) Milk (fresh, evaporated, powdered), buttermilk, cheese, sherbet, cream, ice cream Ice cream, frozen desert Acid Orange 12 Milk (fresh, evaporated, powdered), buttermilk, cheese, sherbet, cream, ice cream Milk Chocolate milk drink, buttermilk Nonfat dry milk powder, ice cream, half-and-half Various dairy products Milk Cheese Various dairy products, NFDM Ice cream, ice milk, diet ice cream, dietetic ice cream Other dyes Milk powder (delactosed)ÐRamazol Blue R

Reference Udy (9), Ashworth et al. (24), Dolby (25), Ashworth and Chaudry (26), and Conetta et al. (69), Park and King (70) Ashworth (20) Kroger et al. (71) Ashworth (20) Sherbon (21) Sherbon and Luke (72) Sherbon and Luke (73) Sherbon (74) Conetta et al. (69), Lakin (75,76), Wilkinson and Richardson (77), Kristoffersen (78) Sherbon and Fleming (79), Bruhn et al. (80) Rawson and Mahoney (81,82)

The Udy Method

149

The DBC for milk protein fractions was assessed by Ashworth and Chaudry (26). Milk protein fractions should have similar DBC values; otherwise, assays may be affected by variations in milk composition. Compared with whey proteins, caseins had a lower DBC (Table 7). Presumably the proportions of casein and whey protein remain fairly constant in different milk samples. The quantity of protein in several brands of milk drink (chocolate milk, two-ten, half-and-half, vitamin D milk, etc.) were determined by Ashworth (20). The Orange-G binding capacity for milk proteins was remarkably constant, notwithstanding processing into products such as ice cream and evaporated milk (Table 8). Fresh milk had a protein content of 3.5%, whereas evaporated milk contained 7% protein. Cheese manufacture had a signi®cant lowering effect on DBC, probably because of proteolysis. Notice that the values for the DBC are 50% lower than those reported in Table 3 for reasons discussed earlier. 6.2.

Of®cial Approval of Dye-Binding Assays

Collaborative studies led to dye-binding assays being granted AOAC approval for milk protein determination (21,72,83). The studies involved laboratories attached to the of®ces of the U.S. Federal Milk Market TABLE 7 Apparent DBC for Different Milk Protein Fractionsa Milk protein (fraction) Whole milk Skim NFDP Whole casein Paracasein a-Casein k-casein b-casein Whey protein b-Lactoglobulin Proteose peptone Average SD

AB 10B (mmoles g 566.6 561.7 595.8 589.3 556.8 577.9 592.5 504.9 730.5 763.0 584.4 602.1 76.1

1

cP)

Orange G (mmoles g

1

cP)

389.4 396.0 407.1 446.9 415.9 398.2 420.4 376.1 539.8 557.5 278.8 420.6 76.2

a DBC was calculated from Ref. 26 using dye molecular weights from Table 2. Values are between 50 and 100% lower than expected from the basic amino acid content in milk proteins (see Table 3).

150

Chapter 5

TABLE 8 Dye-Binding Capacity and Protein Content for Different Milk Productsa Product Milk Fresh Evaporated Powdered, nonfat Powdered, whole Buttermilk Cottage cheeses Creamed Dry curd Cheddar cheese Mild Sharp Cream 20±50% fat Ice cream Vanilla Chocolate

OG (mmoles g 1)

AO12 (mmoles g 1)

Protein (%)

393.5 386.8 394.6 366.9 381.1

1000.0 932.8 1000.0 963.6 902.0

3.5 7.0 36.0 26.0 4.0

394.8 394.8

966.4 966.4

15.0 18.0

306.8 245.4

801.1 700.3

24.0 24.0

394.8

1000.06

2.5

394.8 362.3

1000.0 963.6

4.0 4.0

a A comparison with data from Table 3 shows that the values for OG are lower than expected. Source: DBC values calucalated from Ref. 21.

Administrators.* The extent of interlaboratory differences was considered practically insigni®cant. The agreement between dye-binding results and Kjeldahl results was excellent. Dye binding was granted of®cial ®rst action status for protein quantitation in fresh milk, dairy chocolate milk drink, cultured buttermilk, and half-and-half cream. A collaborative study to examine dye-binding assays for ice cream mix and nonfat dry milk powder is described by Sherbon and Luke (73). Five commercial samples of vanilla ice cream mix and 10 samples of nonfat dry milk were analyzed in powder form or after reconstitution. Seven laboratories performed dye-binding assays while three did Kjeldahl analysis. Tests using nonfat dry milk powder gave the same results as for the reconstituted material. The protein content for nonfat dry milk was 34.946% as determined by Kjeldahl analysis and 35.141% by dye binding. The average difference between Kjeldahl and dye-binding results was ‡ 0.195%.

* One lot of sterile, canned milk was analyzed by ®ve laboratories. Three laboratories carried out crude protein determination by the Kjeldahl method. A further 25 fresh milk samples were analyzed via 140 replicate determinations.

The Udy Method

151

Ice cream mix had 3.852% protein or 3.968% protein by the Kjeldhal and dye-binding analysis, respectively. Consequently, dye binding received AOAC approval as a method for protein determination in ice cream and nonfat dry milk Analysis of ice cream mix and ice cream proteins using dye binding is further described by Kleyn (84) and by Bruhn (85). Further examples of these studies are listed in Table 9. Sherbon (32) compared the Pro-Milk and Udy methods for the analysis of milk proteins. The two techniques gave comparable results. Greater care was needed in calibrating the Pro-Milk instrument. A recommendation to grant of®cial status to the Pro-Milk method was deferred pending further work. A year later, a six-laboratory study of the Pro-Milk method led to AOAC approval (33). In addition to reports cited in Table 9 the Pro-Milk Analyzer is discussed in Refs. 86±91.

6.3.

Wheat and Other Cereal Proteins

Udy (8) found that wheat albumin, gluten, albumin ‡ gluten, and residue proteins bound constant amounts of Orange-G regardless of the variety of seed. These observations paved the way for a quantitative analysis of wheat TABLE 9 Application of Amido Black 10B for Milk and Dairy Protein Analysisa Amido Black 10B Cheese Condensed milk Ice cream, frozen dessert Milk

Whey protein, casein Milk (goat) Milk (mastitis) Skimmed milk powder a

Reference Kroger and Weaver (92) Lueck (93) Kroger et al. (71) Dolby (25), Ashworth and Chaudry (26), Radcliffe (94), O'Connell (86), Conetta et al. (69), Sherbon (95), Kroger (96), Uzonyi (97), Patel et al. (98), Ng-Kwai-Hang and Hayes (99), van Reusel and Klijn (100) Roper and Dolby (101), McGann et al. (102), Renner and Ando (103), Reimerdes and Flegel (104) Grappin et al. (105), Mabon and Brechany (106) Waite and Smith (107) Sanderson (108), O'Connell and McGann (109)

The majority of investigators used the Pro-Milk Analyzer

152

Chapter 5

protein using dye binding (9). In all, 128 samples of whole wheat ¯our and 218 samples of re®ned wheat ¯our (from 58 known and 34 unknown wheat varieties) were examined for Orange-G binding capacity at pH 2.2. The samples were also analyzed for crude protein content by the Kjeldahl method. The correlation between amounts of dye bound and crude protein (%N 6 6.25) is described by Equation (30) (re®ned wheat ¯our) or Equation (31) (whole wheat ¯our). cP ˆ 1:092Db

4:62

…30†

cP ˆ 1:000Db

5:53

…31†

The nonzero intercept indicates dye binding to nonprotein components, probably starch and/or wheat bran. Greenaway (110) at the U.S. Department of Agriculture (USDA; Beltsville, MD) reported a positive correlation between dye binding and Kjeldahl results for soft wheat (< 10% protein), hard winter wheat, hard spring wheat, and durum wheat; cP ˆ 0:8842X ‡ 1:7938

…R ˆ 0:988†

…32†

where X (% protein) is wheat protein content determined from dye binding and cP ˆ Kjeldahl protein (%N 6 6.25). Over 220 protein assays were performed. Methods were compared with respect to average protein content, correlation coef®cients, and standard error of estimates. The dyebinding assay gave reliable estimates for protein content for hard red spring wheat. For other classes of wheat, protein results were slightly lower than expected from Kjeldahl analysis. The mean difference between Kjeldahl and dye-binding tests was  0.5%. For wheat samples having less than 10% protein, dye-binding and Kjeldahl results differed by *1%. The reliability of the Udy method was compared with that of ®ve other techniques (Kjeldahl, alkaline distillation, biuret, Dumas, and infrared re¯ectance) for wheat protein determination by Pomeranz and More (111). Reliability encompasses speci®city, accuracy, precision, sensitivity, and the LLD (Chapter 1). Dye binding was the least precise method tested. Interestingly, no strong case is made for preferring any one method. Forty®ve varieties of rice produced in the 1966 season in the Philippines by the International Rice Research Institute (IRRI) were analyzed for protein (112). A typical set of results highlighting the performance of the dyebinding assay for rice protein analysis is given in Table 10. Barley and malt proteins were analyzed by Pomeranz et al. (113) using ®ve methods including the Udy method. About 120 samples each of barley and malt from all over the United States were analyzed. No details of the dye-binding assay were given other than a reference to Udy's 1971 paper

The Udy Method TABLE 10 Assay

153

Determination of Rice Protein Using Acid Orange 12 Dye-Binding

Parameter

Milled rice

Regression linea Protein (%) SY.X (%) R

Y ˆ 14.67 13.60A485 5.55±11.65 + 0.48 0.961

Brown rice Y ˆ 14.78 14.12A485 6.00±11.95 + 0.26 0.984

a Y ˆ crude protein content (%). Protein (%) is range for 45 samples. SY.X (%) ˆ standard deviation from the regression line. Data are from experiments using a laboratory shaker (112).

(22). The correlation coef®cient for dye-binding and Kjeldhal results was 0.974 (barley) or 0.984 (malt). The average mean squared error for analysis (with the Kjeldahl method as reference) was 0.897%. Using commercial apparatus, 200 protein determinations were completed daily. Baker and Hunt (114) evaluated the Pro-meter instrument (Foss America Inc) for dye-binding analysis of wheat protein. About 107 wheat samples were ground to pass 20-mesh screen and then analyzed according the instrument manufacturer's instructions. The graph of instrument response versus protein content was curvilinear for 50 samples of red wheat (hard red spring, hard red winter, durum wheat). By contrast, a linear calibration graph was obtained for 57 white wheat samples. The Pro-meter instrument was judged satisfactory despite some mechanical dif®culties. The ®lter system was periodically clogged, necessitating dismantling and cleaning of the measuring unit. 6.4.

Legumes and Other Seed Proteins

The Udy method is applicable to range of legumes including, chickpeas, cowpeas, gram, mungbeans, peas, and soybean (Table 1). Pomeranz (115) analyzed 24 soy ¯our samples using Orange-G and commercial apparatus from the Udy Corporation. The results were compared with the biuret and Kjeldahl methods. A highly signi®cant correlation was found between protein content assessed by dye binding (X, %) and crude protein (N 6 6.25%). For samples containing up to 80% protein, the regression equation was cP ˆ 1:003X

4:559

…R ˆ 0:989†

…33†

The standard error of analysis was 1.8213%. Flour particle size had negligible effects on protein results. Mild heat did not affect dye-binding

154

Chapter 5

results although other studies show that severe heating reduces the DBC of soy proteins (116±120). Romo et al. (121) assessed seed protein extractability using the Udy method. The following DA482 changes were noted for the different seed protein solutions (10 mg mL 1): 1.363 (®eld bean), 1.197 (cowpea), 2.976 (rapeseed), 2.2454 (sesame seed), and 1.203 (cotton seed). Clearly, the assay sensitivity is different for different seed proteins. Medina et al. (122) proposed that a single calibration graph might be used for cereal, legume, and oilseed protein analysis. A composite graph would save time. Sesame ¯our, rapeseed meal, and rapeseed ¯our were analyzed by the standard Udy (shaker mixing) method. Fig. 8 shows a composite calibration graph for Acid Orange 12 binding to cereal, legume, and oilseed proteins. The leastsquares equation for the composite graph is* cP ˆ 0:2152Db ‡ 4:7333

…R ˆ 0:981†

…34†

FIGURE 8 A composite calibration graph relating dye binding (X-axis) and crude protein content for *28 samples of legumes, cereals, and oilseeds. (Drawn from Table IV in Ref. 123.) * Actually, the regression equation reported in the literature was Y ˆ 0.245X ‡ 2.532 (R ˆ 0.995). In contrast, Fig. 1 was drawn using only 50% of the experimental data.

The Udy Method

155

Apparently the average DBC for Acid Orange 12 is 465 (+ 17.11) mg g 1 (cP) or 1328.6 mmole g 1. The Udy method was further optimized for seed protein determination (122). Vacuum drying (558C) or atmospheric drying (1008C) had no effect on dye binding. Improving the degree of mixing, extending the shaking time from 30 to 150 minutes, and/or reducing the particle size to 40 mesh increased the perceived sample protein content. Table 11 summarizes results for sesame ¯our, rapeseed ¯our, and rapeseed meal. For all cases, a good correlation was obtained between dye binding and Kjeldahl results. Rapeseed protein was also determined by Goh and Clandinin (123). Twelve commercial meals and two laboratory samples were analyzed using the Udy method with Orange G dye reagent. The investigators also examined Acid Orange 12 as a dye reagent. Rapeseed meal had 30.1±44.8% (w/w) protein. For Orange-G the least-squares line relating dye binding and Kjeldahl results was cP ˆ 0:49Db ‡ 3:91

…R ˆ 0:93†

…35†

For Acid Orange 12 dyes the corresponding equation is cP ˆ 0:28Db ‡ 0:36

…R ˆ 0:98†

…36†

From such data it may be shown that the apparent DBC for Orange-G is 204 mg (dye) g 1 (cP) or 490 mmoles (dye) g 1 (cP). With Acid Orange 12 the DBC is 357 mg g 1 (cP) or 580 mmoles (dye) g 1 (cP). The calibration data were not affected by ¯our particle size (40 or 60 mesh). However, DBC was higher for a protein/dye ratio of 2:1 as compared with a ratio of 4:1. Values for the DBC were proportional to the net concentration of arginine, histidine, and lysine. The standard deviation for analysis was 1.3% (Orange G) or 0.80% (Acid Orange). From the higher DBC (per weight), precision, TABLE 11 Protein Content in Sesame and Rapeseed Products Determined from Acid Orange 12 Dye Binding and Kjeldahl Analysis Protein (% w/w)a

Sample

Sesame ¯our Rapeseed ¯our Rapeseed meal

Dye binding

Kjeldahl

59.4 (+ 0.524) 59.4 (+ 1.743) 36.1 (+ 0.595)

58.9 (+ 1.093) 60 (+ 2.91) 36 (+ 0.338)

a Values are mean (+ SD). Source: Summarized from Ref. 122.

156

Chapter 5

and sensitivity of analysis, Goh and Clandinin (123) concluded that Acid Orange 12 was a more suitable dye reagent for rapeseed protein determination.

6.5. A.

Fish, Meat, and Egg Products Animal Feedstuffs

Bunyan (124) determined the protein content of feedstuffs containing animal protein. The procedure using Orange-G was essentially as given in Method 2. The dye-binding response was dependent on sample particle size. The extent of dye binding also increased with mixing time. With care, values of Db could be correlated with the protein content (Table 12). However, animal feeds were found to be highly heterogeneous owing to their different manufacturing and thermal histories. A number of meat meal samples had an unusually high content of gelatin. In one case, meat meal was positively identi®ed as feather meal (techniques for establishing protein authenticity are described in Chapters 9±11). It was concluded that dye-binding assays were not suited for animal feedstuffs. Differences in processing history, protein quality, and possible adulteration led to large variations in results.

TABLE 12 Binding

Analysis of Protein Content of Animal Feeds Using Orange G Dye

Sample (na) Meat meal (21) Whale meat meal (12) Fish meal (8) Soy bean (8) or Groundnut meals (6) Miscellaneous foodsb a

Regression line

DBC (mmole g 1 cP)

% Error (CV)

cP ˆ 0.278Db ‡ 30

796±925

6.4

cP ˆ 0.216Db ‡ 30

842±770

2.0

cP ˆ 0.325Db ‡ 24 cP ˆ 0.217Db ‡ 28

675 1020

2.3 2.0

cP ˆ 0.414Db ‡ 12

Ð

n ˆ number of different feed samples. Including casein, dried blood protein, egg, brewer's yeast, roller dried milk, and grass meal. Source: Summarized from Ref. 124. b

The Udy Method TABLE 13

157

Protein Determination in Raw Meat Using Orange G Dye Binding Regression linea

R

cP ˆ 0.301Db ‡ 8.18 cP ˆ 0.602Db 2.50 cP ˆ 0.367Db ‡ 5.45 cP ˆ 0.632Db ‡ 3.00

0.90 0.94 0.80 0.95

Sample Beef Chicken breast Pork loin Cod ®llet

DBC (mg g

1

cP)

DBC ˆ 209.2 1.135cP DBC ˆ 265.5 3.721cP DBC ˆ 271.2 4.040cP DBC ˆ 246.9 3.397cP

a Symbols cP and Db are as de®ned previously. Source: Summarized from Ref. 125.

B.

Meat Proteins

Raw beef, chicken, pork (loin), and cod ®llets were analyzed using OrangeG or Amido Black 10B* dye binding by Torten and Whitaker (125). Their procedure was described in Chapter 4. A signi®cant correlation was observed between crude protein values (Kjeldhal-N 6 6.25) and Db (Table 13). The DBC for raw meat proteins decreased linearly with increasing sample protein (see last column of Table 13). The amount of dye bound depended on the dye/protein ratio. In general, inadequate amounts of Orange-G were used in many early studies. Dye limitations and inadvertent changes in protein/dye ratio for different assays reduced the reliability of dye-binding assays. The regression equation (cP ˆ 0.301Db ‡ 8.18) for beef applies over a restricted range of protein content. The effect of a changing DBC is shown in the simulations reported in Fig. 9. One set of results are computed on the basis that the DBC is ®xed. Where DBC varies the dye/ protein ratio the simulated calibration graphs were nonlinear (Fig. 9). The curves are remarkably like actual calibration curves reported for ground pork and chicken (125). These samples showed a high dependence of DBC on protein content and large deviations from linearity. A linear equation did ®t the data but only over a highly restricted range of protein content. Ground chicken, pork loin, or cod ®llet having greater than 50% crude protein content should probably not be analyzed by Orange-G dye binding. It was on account of the dependence of DBC on protein content that Amido Black 10B was judged unsuitable for meat protein analysis.

* As Amido Black 10B was found to be unsuitable for raw meat analysis, the following discussion focuses on results obtained with Orange-G.

158

FIGURE 9

Chapter 5

The effect of a changing dye-binding capacity on calibration graphs for protein analysis in raw meat samples using Orange G binding. Shaded squares show normal response according to regression equations in Table 13. The open circles show simulated graphs for the assay response when DBC changes with sample protein content.

The Udy Method TABLE 14

159

Acid Orange 12 DBC of Egg and Meat Products

Meat product Egg (whole) Egg albumin (egg white) Chicken meat Chicken liver Beef or pork (ground) Beef liver Proteose peptone Gelatin

DBC (mg g

1

cP)a

410±440 390±410 460±480 360±390 430±440 420±440 90±145 310±350

a Ranges of values are given for analysis performed in the presence of excess of dye concentration of 0.4±0.6 mg mL 1. Source: Ref. 126.

C.

Egg, Chicken, and Meat Protein

Egg, chicken, and meat products were analyzed by Ashworth (126) (Table 14). As he was one of the ®rst investigators to apply dye-binding assays to foods, his approach merits attention. Reliable results were obtained provided that the free Acid Orange 12 concentration (after shaking with the protein sample) was kept within the range of 0.4±0.6 mg mL 1. To achieve this, the initial the dye/protein ratio was kept within a range of 0.64±0.92. Pork had the same DBC as beef, which was lower than the value of chicken. The DBC for mixtures of meat could be deduced from values for individual components. Dye binding was not affected by the presence of fat or by normal cooking (1608C, 40 minutes). It was concluded that dye binding is useful method for composition control in ground meats, eggs, and prepared mixes. D.

Sausage Protein

Seperich and Price (127) determined protein in model sausage emulsions and muscle components (myo®brillar protein, sarcoplasmic protein, and stroma) from which they were produced. The approach was modi®ed from Ref. 128.* These studies con®rmed that protein dye binding was not affected by sausage emulsion fat content from 20 to 40%. The DBC was a function of * Sausage emulsion samples (3.5 g) were homogenized with 51 mL of citrate (0.2 M)±phosphate (0.1 M) buffer (pH 5.5). Ten milliliters of the resulting homogenate was retained for Kjeldahl analysis. The remainder was shaken with 80 mL of Acid Orange 12 (0.56±3.64 mM; 0.2± 1.27 mg mL 1) in a 250-mL centrifuge tube for 30 minutes and then centrifuged (5.680g; 5 minutes).

160

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dye/protein ratio. At the highest dye concentration examined the DBC was of the order of 400 mg g 1 (cP), in line with values reported by other investigators. However, DBC decreased to about 33±34 mg g 1 (cP) at a dye concentration of 0.2 mg mL 1. 6.6.

Mushrooms

Nine strains of Agaricus bisporus (Lange) Imbach were analyzed by Weaver et al. (128) using dye binding, Kjeldahl, and quantitative amino acid analysis.*. The average protein content was 29.4 (+ 6.2)% by Kjeldahl analysis, 22.4 (+ 2.4)% by dye binding, and 28% (+ 3.4)% by amino acid analysis. Per wet weight basis, Agaricus had 2.6±2.8% protein. Quantitative amino acid analysis was more correlated with dye-binding analysis (R ˆ 0.74) than Kjeldahl analysis (R ˆ 0.4). Mushrooms are thought to contain high amounts of NPN, which could lead to errors in Kjeldahl analysis. Braaksma and Schaap (129) reported the protein content for Agaricus as 0.5% fresh weight or 7% per dry weight basis (Chapter 7).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

A Grollman. The combination of phenol red and proteins. J Biol Chem 64:141±160, 1925. WW Smith, HW Smith. Protein binding of phenol red, Diodrast, and other substances in plasma. J Biol Chem 124:107±113, 1938. HW Robinson, CG Hogden. The in¯uence of serum protein on the spectrophotometric absorption curve of phenol red in a phosphate buffer mixture. J Biol Chem 137:239±254, 1941. LM Chapman, DM Greenberg, CLA Schmidt. Studies of the combination between certain acid dyes and proteins. J Biol Chem 72:707±729, 1927. LMC Rawlings, CLA Schmidt. Studies on the combination of certain basic dyes and proteins. J Biol Chem 82:709±716, 1929. LMC Rawlings, CLA Schmidt. The mode of combination between certain dyes and gelatin granules. J Biol Chem 83:271±284, 1930. H Fraenkel-Conrat, M Cooper. The use of dyes for the determination of acid and basic groups in proteins. J Biol Chem 154:239±340, 1944. D Udy. Dye-binding capacities of wheat ¯our protein fractions. Cereal Chem 31:389±395, 1954.

* Mushrooms were diced, freeze dried, and then oven dried to a constant weight. A 100-mg portion of mushroom powder was mixed with 20 mL of dye solution. BSA was used as the standard protein. Amido Black 10B was used in conjunction with the Pro-Milk Mk II instrument.

The Udy Method 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28.

161

D Udy. Estimation of protein in wheat and ¯our by ion-binding. Cereal Chem 33:190±197, 1956. TCA McGann. Dye-binding procedures could lead to better utilization of world protein resources. Farm Food Res 4(6):138±139, 1973. AL Lakin. The estimation of protein and the evaluation of protein quality by dye-binding procedures. IFST Proc 6(2):80±83, 1973. AL Lakin. The estimation of proteins by dye binding: principles and experimental parameters. J Sci Food Agric 26:549±550, 1975. AW Hartley. Estimation of proteins by dye binding: applications to cereals and ¯ours. J Sci Food Agric 26:550±551, 1975. TCA McGann. The estimation of proteins by dye binding: applications to dairy products. J Sci Food Agric 26:551±552, 1975. RA Lowe. The estimation of proteins by dye binding. Applications to animal feed. J Sci Food Agric 26:552±553, 1975. TCA McGann. Automated physico-chemical methods for the analysis of milkÐa review of major advances (1960±1978). Ir J Food Sci Technol 2:141± 155, 1978. ER Cole. Alternative methods to the Kjeldahl estimation of protein nitrogen. Rev Pure Appl Chem 19:109±130, 1969. JW Sherbon. Recent developments in determining protein content of dairy products by dye binding. J Dairy Sci 61:1274±1278, 1978. AOAC International. AOAC Of®cial Method 967.12. Protein in milk. Dye binding method I. In AOAC International, ed. Of®cial Methods of Analysis of AOAC International. 16th ed. Vol II. Arlington, VA: Association of Of®cial Analytical Chemists, 1995, Chapter 33, p 14. US Ashworth. Determination of protein in dairy products by dye binding. J Dairy Sci 49:133±137, 1966. JW Sherbon. Rapid determination of protein in milk by dye binding. J Assoc Of®; Anal Chem 50:542±546, 1967. DC Udy. Improved methods for estimating protein. J Am Oil Chem Soc 48:29A±33A, 1971. DC Udy. A rapid method for estimating total protein in milk. Nature 178:314±315, 1956. US Ashworth, R Seals, RE Erb. An improved procedure for the determination of milk proteins by dye binding. J Dairy Sci 43:614±623, 1960. RM Dolby. Dye-binding methods for estimation of proteins in milk. J Dairy Res 28:43±55, 1961. US Ashworth, MA Chaudry. Dye-binding capacity of milk proteins for Amido Black 10B and Orange-G. J Dairy Sci 45:952±957, 1962. International Dairy Federation. MilkÐdetermination of protein content (routine dye-binding method using Amido Black). International IDF Standard 98:1980 (Provisional), 1980, 4 pp. International Organization for Standardization. MilkÐdetermination of protein contentÐAmido Black dye-binding method (routine method). International Standard; ISO 5542±1984,1984, 5 pp.

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29. International Dairy Federation Milk. Determination of protein contentÐ Amido Black dye-binding method (routine method). International IDF Standard; 98A:1985, 1985, 4 pp. 30. AOAC International AOAC Of®cial Method 975.17, Protein in milk. Dye binding method II. In AOAC International, ed. Of®cial Methods of Analysis of AOAC International. 16th ed. Vol II. Arlington, VA: Association of Of®cial Analytical Chemists, 1985, Chapter 33, p 15. 31. CM Wilson. Polyacrylamide gel electrophoresis of proteins: impurities in Amido Black used for staining. Anal Biochem 53:538±544, 1973. 32. JW Sherbon. Pro-Milk method for the determination of protein in milk by dye binding. J Assoc Of® Anal Chem 57:1338±1341, 1974. 33. JW Sherbon. Collaborative study of the Pro-Milk method for the determination of protein in milk. J Assoc Of® Anal Chem 58:770±772, 1975. 34. M A Al-Sketty, RKO Apenten. Modi®cations of protein dye binding assays. University of Leeds, 2001. 35. H Kuno, HK Kihara. Simple microassay of protein with membrane ®lter. Nature 215:974±975, 1967. 36. A Heil, W Zillig. Reconstitution of bacterial DNA-dependent RNApolymerase from isolated subunits as a tool for the elucidation of the role of the subunits in transcription. FEBS Lett 11:165±168, 1970. 37. W Schaffner, C Weissmann. A rapid, sensitive, and speci®c method for the determination of protein in dilute solution. Anal Biochem 56:502±514, 1973. 38. RS Kaplan, PL Pedersen. Determination of microgram quantities of protein in the presence of milligram levels of lipid with Amido Black 10B. Anal Biochem 150:97±107, 1985. 39. K Nakamura, T Tanaka, A Kuwahara, K Takeo. Microassay for proteins on nitrocellulose ®lter using protein dye-staining procedure. Anal Biochem 148:311±319, 1985. 40. S Bramhall, N Noack, M Wu, JR Lowenberg. A simple colorimetric method for determination of protein. Anal Biochem 31:146±148, 1969. 41. GS McKnight. A colorimetric method for the determination of submicrogram quantities of protein. Anal Biochem 78:86±92, 1977. 42. A Esen. A simple method for quantitative, semiquantitative, and qualitative assay of protein. Anal Biochem 89:264±273, 1978. 43. H Ahmad, M Saleemuddin. A Coomassie Blue-binding assay for the microquantitation of immobilized proteins. Anal Biochem 148:533±541, 1985. 44. RLM Allen. Color Chemistry. London: Thomas Nelson & Sons, 1971. 45. L Sandler, FL Warren. Effect of ethyl chloroformate on the dye binding capacity of protein. Anal Biochem 46:1870±1872, 1974. 46. RF Hurrell, P Lerman, KJ Carpenter. Reactive lysine in foodstuffs as measured by a rapid dye-binding procedure. J Food Sci 44:1221±1227, 1979. 47. IM Perl, MP Szakacs, A Koevago, J Petroczy. Stoichiometric dye-binding procedure for the determination of the reactive lysine content of soya bean protein. Food Chem 16:163±174, 1985.

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48. I MolnaÂr-Perl, M PinteÂr-SzakaÂcs, D Medzihradszky. Dye-binding ``stoichiometry'' and selectivity of cresol red with various proteins. Food Chem 35:69± 80, 1990. 49. D Racusen. Stoichiometry of the Amido Black reaction with proteins. Anal Biochem 52:96±101, 1973. 50. I MolnaÂr-Perl, M PinteÂr-SzakaÂcs, A KoÈvago, I PetroÂczy, UP KralovaÂnszky, I MaÂtyaÂs. Dye-binding stoichiometry of AO12, AB 10B and OG with etalon proteins, feed and feedingstuffs and its application for reactive lysine determination. Food Chem 20:21±38, 1986. 51. WAB McBryde. Spectrophotometric determinations of equilibrium constants in solution. Talanta 21:979±1004, 1974. 52. IM Klotz, FM Walker, RB Pivan. The binding of organic ions by proteins. J Am Chem Soc 68:1487±1490, 1946. 53. IM Klotz. Spectrophotometric investigations of the interactions of proteins with organic anions. J Am Chem Soc 68:2299±2304, 1946. 54. SE Sheppard, RC Houck, C Dittmar. The sorption of soluble dyes by gelatin. J Phys Chem 46:158±176, 1942. 55. M Pesavento, A Profumo. Interaction of serum albumin with a sulphonated azo dye in acidic solution. Talanta 38:1099±1106, 1991. 56. KE Lind, U Kragh-Hansen, JV Moller. Protein binding to small molecules. V. Binding of bromophenol blue by chemical modi®cations of human serum albumin. Biochim Biophys Acta 371:451±461, 1974. 57. Y-J Wei, K Li, S-Y Tong. The interaction of bromophenol blue with proteins in acidic solution. Talanta 43:1±10, 1996. 58. Y-J Wei, K Li, S-Y Tong. Spectral study of interaction of thymol blue with protein in acidic solution. Anal Chim Acta 341:97±104, 1997. 59. AN Glazer. On the prevalence of ``nonspeci®c'' binding at the speci®c binding sites of globular proteins. Proc Natl Acad Sci USA 65:1057±1063, 1970. 60. AG Mayes, R Eisenthal, J Hubble. Binding isotherms for soluble immobilized af®nity ligands from spectral titration. Biotechnol Bioeng 40:1263±1270, 1992. 61. J Hubble, AG Mayes, R Eisenthal. Spectral analysis of interactions between proteins and dye ligands. Anal Chim Acta 279:167±177, 1993. 62. GS Hartley. The effect of long-chain salts on indicators: the valency-type of indicators and the protein error. Trans Faraday Soc 30:444±450, 1934. 63. L Michaelis, S Granick. Metachromasy of basic dyestuffs. J Am Chem Soc 67:1212±1219, 1945. 64. SE Sheppard, AL Geddes. Amphipathic character of proteins and certain lyophile colloids as indicated by absorption spectra of dyes. J Chem Phys 13:63±65, 1945. 65. RW Congdon, GW Muth, AG Splittgerber. The binding interaction of Coomassie Blue with proteins. Anal Biochem 213:407±413, 1993. 66. DA Skoog, DM West. Fundamentals of Analytical Chemistry. 3rd ed. New York: Holt, Rinehart & Winston, 1976. 67. PJ Wood, RG Fulcher. Interaction of some dyes with cereal beta-glucans. Cereal Chem 55:952±966, 1978.

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68. RG Seals. Some aspects of dye binding of milk and milk powder proteins. PhD thesis, Washington State University, 1960. 69. A Conetta, L Stooker, H Zehnder. An automated system for the determination of milkfat, protein and lactose in milk. Advances in Automated Analysis. Technicon International Congress, 1970, 2:81±85, 1971. 70. DL Park, RL King. Evaluation of automated dye-binding determination of protein in milk. J Assoc Of® Anal Chem 57:42±46, 1974. 71. M Kroger, EE Katz, JC Weaver. Determining protein content of ice cream and frozen desserts. J Dairy Sci 61:274±277, 1978. 72. JW Sherbon, HA Luke. Collaborative study of the dye binding method applied to chocolate milk drinks, cultured buttermilk, and half-and-half. J Assoc Of® Anal Chem 51:811±816, 1968. 73. JW Sherbon, HA Luke. Comparison of the dye binding and Kjeldahl methods for protein analysis of non-fat dry milk and ice cream. J Assoc Of® Anal Chem 52:138±142, 1969. 74. JW Sherbon. Dye binding method for protein content of dairy products. J Assoc Of® Anal Chem 53:862±864, 1970. 75. AL Lakin. The estimation of protein and the evaluation of protein quality by dye-binding procedures. ISFT Proc 6:80±83, 1973. 76. AL Lakin. Comparison of the amounts of dyes bound by milk proteins under the conditions employed in dye-binding procedures. XIX International Dairy Congress 1E:277±278, 1974. 77. RF Wilkinson, GH Richardson. Continuous ¯ow analysis of milk proteins using ultra-violet spectroscopy. J Dairy Sci 58:798, 1975. 78. T Kristoffersen, KH Koo, WL Slatter. Determination of casein by the dye method for estimation of cottage cheese curd yield. Cult Dairy Prod J 9:12±14, 1974. 79. JW Sherbon, R Fleming. Comparison of two formulations of Acid Orange 12 for the determination of protein in milk. J Assoc Of® Anal Chem 58:773±776, 1975. 80. JC Bruhn, S Pecore, AA Franke. Measuring protein in frozen dairy desserts by dye binding. J Food Prot 43:753±755, 1980. 81. N Rawson, RR Mahoney. Effect of processing and storage on the protein quality of spray-dried lactose-hydrolyzed milk powder. Lebensm Wiss Technol 16:313±316, 1983. 82. N Rawson, RR Mahoney. A modi®ed method for determination of reactive lysine in milk powder using Remazol Brilliant Blue R. Lebensm Wiss Technol 16:1±4, 1983. 83. AH Luke. Collaborative testing of the dye binding method for milk protein. J Assoc Anal Chem 50:560±564, 1967. 84. RH Kleyn. Frozen desserts under protein analysis. Dairy Ice Cream Field 159(7):44, 46, 1976. 85. JC Bruhn. Protein determinations in ice cream. Am Dairy Rev 40(2):34B± 34D, 1978.

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86. JA O'Connell. Evaluation and modi®cation of the Pro-Milk Tester Mk II for protein estimation in milk. Lab Pract 19:1119±1120, 1970. 87. TCA McGann, JA O'Connell. Evaluation of the Pro-Milk automatic for rapid protein determination in milk. Dairy Ind 36:685±687, 1971. 88. TCA McGann, A Mathiassen, JA O'Conell. Applications of the Pro-Milk Mk II. IV. Monitoring the degree of denaturation of whey proteins in heat processing of milk, and the heat treatment classi®cation of milk powders. Lab Pract 21:865±871, 1972. 89. L Szijarto, DA Biggs, DM Irvine, DW Stanley. Mark II Pro-Milk Tester for estimation of protein percentage in plant milk supplies. J Dairy Sci 56:854± 857, 1973. 90. TCA McGann, JA O'Connell, R McFeely. Use of semi-automatic instruments for process and product control in the dairy-food industry. Routine assessment of total protein and heat treatment of milk powders by due binding. J Soc Dairy Technol 28:23±27, 1975. 91. R Grappin, VS Packard, RE Ginn, J Mellema. Precision of the Pro-Milk method in routine determination of protein in dairy testing laboratories. J Food Prot 43:52±53, 1980. 92. M Kroger, JC Weaver. Use of protein dye-binding values as indicators of the `chemical age' of conventionally made cheddar cheese and hydrolyzed-lactose cheddar cheese. J Food Sci 44:304±305, 1979. 93. H Lueck. The protein content of condensed milk as determined by the amino black dye-binding method. S Afri J Dairy Technol 5:77±79, 1973. 94. JC Radcliffe. Use of a recording spectrophotometer for Amido Black milk protein determinations. Aust J Dairy Technol 23:143, 1968. 95. JW Sherbon. Pro-Milk method for the determination of protein in milk by dye binding. J Assoc Of® Anal Chem 57:1338±1341, 1974. 96. M. Kroger. Techniques for milk protein testing. Food Prod Dev 6(7):68,77,1972. 97. M Uzonyi. Experiences with the Amido-Black 10B dye-binding method in the Hungarian dairy industry. Zesz Probl Postepow Nauk Roln 167:41±47, 1975. 98. MJ Patel, GK Patel, KC Patel, RD Patel. Protein determination in milk. Indian J Chem Educ 5:26, 1978. 99. KF Ng-Kwai-Hang, JF Hayes. Effects of potassium dichromate and sample storage time on fat and protein by Milko-Scan and on protein and casein by a modi®ed Pro-Milk Mk II method. J Dairy Sci 65:895±899, 1982. 100. A Reusel, CJ Klijn. Automated methods for routine analysis of raw milkÐthe dye-binding method for determination of the protein content of milk. Bull Int Dairy Fed 208:17±20, 1987. 101. J Roeper, RM Dolby. Estimation of the protein content of wheys by the Amido Black method. N Z J Dairy Sci Technol 6(2):65±68, 1971. 102. TCA McGann, A Mathassen, JA O'Connell. Rapid estimation of casein in milk and protein in whey. Ir Agric Creme Rev 26:17±22, 1973.

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103. E Renner, S Ando. Determination of the casein and whey protein contents of milk by Amido Black methods. XIX International Dairy Congress E:459±460, 1974. 104. EH Reimerdes, B Flegel. Casein micelles: heat-induced changes of the dyebinding capacity. XX International Dairy Congress E:2243±2244, 1978. 105. R Grappin, R Juenet, D Ale. Determination of the protein content of cows' and goats' milk by dye-binding and infrared methods. J Dairy Sci 62(Suppl 1):38±39, 1979. 106. RM Mabon, EY Brechany. The measurement of protein in fresh and stored goats' milk by a dye-binding technique. Lab Pract 31:26±27, 1982. 107. R Waite, GM Smith. Measurement of the protein content of milk from mastitic quarters by the Amido Black method. J Dairy Res 39:195±201, 1972. 108. WB Sanderson. Determination of undenatured whey protein nitrogen in skim milk powder by dye binding. N Z J Dairy Sci Technol 5:46±48, 1970. 109. JA O'Connell, TCA McGann. Rapid estimation of protein in skim milk powders. Ir Agric Cream Rev 25(110):17±19, 1972. 110. WT Greenaway. Comparisons of the Kjeldahl, dye binding, and biuret methods for wheat protein content. Cereal Chem 49:609±615, 1972. 111. Y Pomeranz, RB More. Reliability of several methods for protein determination in wheat. Bakers Dig 49:44±48, 58, 1975. 112. LC Parial, LW Rooney, BD Webb. Use of dye-binding and biuret techniques for estimating protein in brown and milled rice. Cereal Chem 47:38±43, 1970. 113. Y Pomeranz, RB Moore, FS Lai. Reliability of ®ve methods for protein determination in barley and malt. J Am Soc Brew Chem 35:86±93, 1977. 114. D Baker, WH Hunt. Pro-Meter evaluation. Cereal Foods World 20:246±247, 1975. 115. Y Pomeranz. Evaluation of factors affecting the determination of nitrogen in soya products by the biuret and Orange-G dye-binding methods. J Food Sci 30:307±311, 1965. 116. T Hymowitz, FI Collins, SJ Gibbons. A modi®ed dye-binding method for estimating soybean protein. Agron J 61:601±603, 1969. 117. L Sandler, FL Warren. Effect of ethyl chloroformate on the DBC of protein. Anal Chem 46:1870±1872, 1974. 118. IM Perl, MP Szakacs, A Koevago, J Petroczy. Stoichiometric dye-binding procedure for the determination of the reactive lysine content of soya bean protein. Food Chem 16:163±174, 1985. 119. I Molnar-Peal, M Pinter-Szakacs, D Medzihradszky. Dye-binding ``stoichiometry'' and selectivity of cresol red with various proteins. Food Chem 35:69± 80, 1990. 120. S Lin, AL Lakin. Thermal denaturation of soy proteins as related to their dyebinding characteristics and functionality. J Am Oil Chem Assoc 67:872±878, 1990. 121. CR Romo, AL Lakin, EF Rolfe. Properties of protein isolates prepared from ground seeds. I. Development and evaluation of a dye binding procedure for the measurement of protein solubility. J Food Technol 10:541±546, 1975.

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122. MB Medina, DH Kleyn, WH Swallow. Protein estimation in sesame seed and rapeseed ¯ours and meals by a modi®ed Udy dye binding method. J Am Oil Chem Assoc 53:555±558, 1976. 123. YK Goh, DR Clandinin. The estimation of protein in rapeseed meal by a dyebinding method. Can J Anim Sci 58:97±103, 1978. 124. G Bunyan. Orange-G binding as a measure of protein content. J Sci Food Agric 10:425±430, 1959. 125. J Torten, JR Whitaker. Evaluation of the biuret and dye-binding methods for protein determination in meats. J Food Sci 25:168±174, 1964. 126. US Ashworth. Proteins in meat and egg products determined by dye binding. J Food Sci 36:509±510, 1971. 127. GJ Seperich, JF Price. Dye binding procedure for the estimation of protein content of meat components and sausage emulsions. J Food Sci 44:643±645, 1979. 128. JC Weaver, M Kroger, LR Kneebone. Comparative protein studies (Kjeldahl, dye binding, amino acid analysis) of nine strains of Agaricus bisporus (Lange) Imbach mushrooms. J Food Sci 42:364±366, 1977. 129. A Braaksma, DJ Schaap. Protein analysis of the common mushroom Agaricus bisporus. Postharvest Biol Technol 7:119±127, 1996.

6 The Bradford MethodÐPrinciples

1. INTRODUCTION Proteins bind with Coomassie Brilliant Blue G250 (CBBG,* C.I. 42655) to produce a sparingly soluble complex (1). Protein-dye binding alters the absorption spectrum for CBBG. This is the basis of the assay developed by Bradford in 1976 (2). The Bradford assay has several advantages compared with the Lowry test: (a) four- to tenfold greater sensitivity, (b) tenfold greater speed, (c) decreased susceptibility to interferences, (d) requirement for a single reagent, and (e) lower cost. The Bradford assay is quicker than dye-protein precipitation (Udy assay) as no ®ltration step is required. Ready-to-use CBBG dye reagent is available from Bio-Rad Laboratory Ltd., Pierce Warriner Ltd., and the Sigma-Aldrich Chemical Company. Principles of the Bradford assay are described in this chapter. Applications for food protein analysis are discussed in Chapter 7. Coomassie Blue is the trade name for a group of dyes ®rst produced by Imperial Chemical Industries (ICI) Ltd. (UK). Weiler discovered CBBG in 1913. The 1971 edition of the Colour Index (3) lists 40 Coomassie dyes including Coomassie Blue FF (C.I. 42645), Coomassie Blue R (C.I. 42660), Coomassie Blue BL (C.I. 50315), Coomassie Brilliant Blue G (C.I. 42655), Coomassie Blue GL (C.I. 50320), and Coomassie Blue RL (C.I. 13390). The * Abbreviations: CBBG, Coomassie Brilliant Blue G250; CBBR, Coomassie Brilliant Blue R250.

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FIGURE 1 The structure of Coomassie Brilliant Blue G250 (CBBG). Coomassie Brilliant Blue R250 (CBBR) lacks two methyl groups.

G and R labels refer to dyes having a greenish blue or reddish blue hue. Samples of dye with 2.5 times greater purity than the standard grade (19± 22% purity) are labeled ``250.'' CBBG is also known as C.I. Acid Blue 90, Xylene Brilliant Cyanine G, or Brilliant Blue G (4). CBBR* was ®rst used as a protein stain in 1963 by Fazekas de St. Groth et al. (5). Cellulose acetate electrophoresis support was soaked in sulfosalicylic acid to ®x protein bands and then transferred to the CBBR solution (0.25% w/v in water). Blue protein bands form against a clear background. Nonspeci®c staining increased if CBBR dye was prepared with methanol rather than water. From a densitometric analysis of polyacrylaminde gels, the blue color was proportional to protein amount (0±20 mg). In later developments, polyacrylamide gels were stained with CBBR dissolved with a 5:1:5 mixture of methanol, acetic acid, and water or 12.5% (w/v) TCA (6). Diezel et al. (1) introduced CBBG as a protein stain for electrophoresis. CBBG has two methyl groups more than CBBR (Fig. 1) and is therefore less soluble in 12% TCA. This lowers dye penetration into polyacrylamide gels and reduces background staining. The sensitivity of

* CBBR (CI 42660) is also known as Acid Blue 83, Coomassie Blue R, Xylene Brilliant Cyanine 6b, or Supranolcyanin 6B.

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CBBG for protein is also signi®cantly greater than CBBR.* Reisner et al. (7) used perchloric acid (3.5%) as solvent for CBBG. The free dye exists as a colorless (leuco) molecule in perchloric acid solvent. Binding to protein leads to a blue protein-dye complex. There was virtually no background staining for polyacrylamide gels. Assay sensitivity was comparable to that obtained with Naphthylamine Black 10 (0.5% w/v) stain.{ 2. THEORY OF THE BRADFORD ASSAY Protein-protein variations in the sensitivity of the Bradford assay (8,9) led to interest in CBB-protein interactions. Coomassie Brilliant Blue binds with proteins by electrostatic interactions. The complex is also held together by van der Waals forces (5). The ®rst quantitative investigation of CBBR binding with proteins was reported by Tal et al. (10). Dye binding occurs only with polypeptides larger than about 3000 daltons. The number of dye molecules bound per molecule of protein (n) was strongly correlated with the total number of arginine, histidine, and lysine (Arg ‡ His ‡ Lys) residues. However, the average DBC was 100% greater than combined numbers of basic amino acid residues. Hydrophobic interactions may account for dye binding when charged protein sites were saturated. Rosenthal and Koussale (11) determined the critical micelle concentration (cmc) of nonionic surfactants with the Bradford reagent. Hydrophobic solubilization of CBBG within micelles led to marked increases in absorption at 620 nm. 2.1.

Overview of CBBG Protein Binding

Compton and Jones (12) assessed the effect of pH, protein, and surfactants on the absorption spectrum of CBBG. They concluded that CBBG exists in three ionized forms rather than two. The absorptivity of protein-CBBG complexes increased with protein molecular weight (13). Sign®cantly higher color yields were obtained for CBBG binding with polyarginine, polylysine, or polyhistidine. There was no dye binding with polyaspartic acid, polyglutamic acid, and polyproline. Lea et al. (14) recorded anomalous results with the Bradford assay for highly basic proteins. Chemical * A polyacrylamide slab gel (10 6 15 cm) is soaked in 40 mL of 12.5% TCA for 5 minutes to ®x protein. Then 2.5 mL of CBBG solution (0.25% w/v dissolved in water) is added and the gel is incubated for 15±30 minutes. Transferring the gel to a 5% (w/v) acetic acid solution for 12 hours increases protein band intensity and reduces background staining. { This dye is the same as Amido Black 10B (Chapter 5).

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modi®cation to lower the numbers of positive charges increased (rather than decreased) the assay response for polylysine and histone. The expected reductions in assay sensitivity occurred when a more concentrated dye reagent solution was used (15). Protein-CGGB binding parameters were reported by Chial and Splittgerber (16), who also characterized the Bradford assay at pH 1 and pH 7. Congdon et al. (17) reported the dissociation constant for CBBGprotein binding. Although not involving CBBG, the study of Cibracron blue binding to lysozyme (18) is a ®ne example of contemporary methods for studying protein-dye binding. Atherton et al. (19) apply these principles to the CBBG system. 2.2.

The Compton-Jones Scheme for CBBG Ionization

CBBG dye shows two lmax values at 470 and 650 nm at pH 0.8* (12). Adjusting the dye reagent to pH 1.2 produced the following changes: (a) decreased absorbance at 650 and 470 nm and (b) a new absorbance peak at 595 nm. There was no isobestic point, meaning that more than two interconverting dye species occurred over the pH range examined. In a different experiment, addition of 140 mg of BSA to CBBG dye reagent produced a difference spectra (dye ‡ protein versus dye) with lmax at 595 nm. Exposure of CBBG to excess SDS diminished the 470 peak and produced a new peak at 650 nm. Shareef and Shetty (20) identi®ed a fourth CBBG charged species. The purple bi-ionic form (CBBG2 , lmax ˆ 515 nm) appears at pH 11.5. With acidic conditions (pH 1.25) Coomassie Blue dye is a positively charged/ cationic/red (CBBGH2)1‡ species (lmax ˆ 475 nm). This is in equilibrium with other CBBG forms. (CBBGH2)1‡ is converted to the zero-charge/ neutral/green (CBBGH)0 species (lmax ˆ 650 nm) at about pH 1.6. Thereafter (CBBG)1 , which is the anionic blue form (lmax ˆ 595), is produced at pH 1.8±pH 7. The Compton-Jones scheme for CBBG ionization is summarized in Table 1. Crystal violet shows a similar three-state ionization as it changes color from violet to green to yellow at pH 8 to pH 2.4 and pH 0.6 (21); R1‡ …violet†?RH2‡ …green†?RH2 3‡ …yellow†

…1†

Similar transformations occur with many other triphenylamine dyes (rosaniline, para-rosaniline, malachite green, aniline blue). * Compton and Jones reported that the Bradford reagent had a pH of 0.8. Our own measurements found pH 1.25.

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173

TABLE 1 The Compton-Jones Scheme Showing the Three-State Ionization of CBBG Dye form

Anion

Structure Net charge Color lmax(nm) ‡ve charges ve charges pH*

(CBBG)1 1 Blue 595 1 2 1.8±7

2.3.

„

Neutral

„

(CBBGH)0 0 Green 650 2 2 1.6

Cation (CBBGH2) 1‡ ‡1 Red/leuco 470±475 3 2 1.25

Spectrophotometric Analysis of CBBG±Protein Binding

It is not easy to decide whether protein-bound CBBG is charged or not. Compton and Jones (12) stated, ``based on the identical lmax for the dyeprotein complex and the blue dye anion, . . . the bound dye species is in fact the dye anion. The (dye ionization) equilibrium shown above are forced to the left as the anion is bound by protein.'' CBBG binding to serum albumin leads to lmax ˆ 620 nm when the protein is present in excess (22). By contrast, lmax ˆ 595 nm with excess dye. A lmax value of 620 nm does not match the absorption maximum for any of the dye forms in solution (470, 595, or 650 nm; Table 2). Two possible models may be proposed for proteinCBBG binding. A.

Binding Scenario 1ÐExcess Protein

Just prior to binding there is the anionic form (CBBG)1 in solution. Dye binding is with the positively charged protein site (RNH3)1‡. Protein-bound CBBG has a net charge of zero. …CBBG†1 ‡ …RNH3 †1‡ ?…CBBG:RNH3 †0

…2†

Electrostatic intereactions will neutralize charges on the dye and protein molecule, forming (CBBG.RNH3)0. The analogous reaction occurs between (CBBG)1 and the hydrogen ion: …CBBG†1 ‡ H1‡ ?…CBBGH†0

…3†

Table 1 shows that lmax is 650 nm for the neutral dye species (CBBGH)0 in

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solution. For the protein-bound nuetral dye form (CBBG.RNH3)0 we ®nd lmax is 620 nm. To explain the hypsochromic shift from 650 to 620 nm, consider the different dye environments for (CBBG.RNH3)0 and (CBBGH)0. The lmax shifts from 650 to 600±620 nm when (CBBGH)0 is internalized within the nonpolar environment of micelles formed by nonionic detergents. One implication is that charged protein sites for dye binding are (within) nonpolar environments. With scenario 1, the Bradford assay can be performed at 620 nm. Eq. (2) partly explains the correlation between DBC and total numbers of basic amino acids per molecule of protein. However, excess protein leads to the occupancy of strong binding sites (Chapter 5). B.

Binding Scenario 2ÐExcess Dye

Just prior to binding, the free dye form is the blue (CBBG)1 species. Dye binding involves hydrophobic interactions with one of two types of neutral protein sites, Ro or (CBBGRNH3)0. Protein-bound CBBG has a net charge of 1. CBBG1 ‡ R0 ?…CBBGR†1 or CBBG1 ‡ …CBBGRNH3 †0 ?…CBBG2 RNH3 †1

…4†

Dye binding shifts the dye ionization equilibrium toward (CBBG)1 and produces an absorbance increase at 595 nm. Equation (4) accounts for the use of A595 readings for the Bradford assay. Dye binding with R0 does not account for the correlation between DBC and the number of positively charged basic amino acid residues. In contrast, the number of (CBBGRNH3)0 sites is determined by the number of basic amino acids. The Bradford assay can be monitored at either 620 or 595 nm. The alternative dye-binding scenarios are not mutually exclusive. As described in Chapter 5, dye binding involves both nonpolar and ionic sites. CBBR binds to proteins with a dye basic amino acid ratio ranging from 1:1.5 to 1:2 (10). Compton and Jones (12) suggest that 60% of the Gibbs free energy change for protein-CBBG binding (*40 kJ mol 1) is due to the nonpolar structure (benzene and methylene groups) of the dye. The remaining binding free energy (*30 kJ mol 1) arises from protein interactions with the sulfonate groups of CBBG. The metachromatic properties of crystal violet provide relevant insights (23). In contemporary terms, metachromasia is a change in the dye absorption spectra due to changes in the dye environment. The lmax

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175

may shift to shorter or longer wavelengths. Sometimes, the absorbance peak increases without lmax shifting. Crystal violet showed metachromatic behavior due to (a) increasing dye concentration, (b) addition of low concentrations of ammonium sulfate, or (c) binding to sites followed by optically signi®cant interactions between dye molecules. Low concentrations of ammonium sulfate induce the dimerization of crystal violet. The singly charged R‡ (violet) form [see Eq. (1)] binds to phosphate groups from nucleic acids with a ratio of 1:1. For this reaction, lmax remains unchanged but the absorption peak increases. Crystal violet binding to agar sulfonate groups apparently shifts lmax from 585 to 510 nm as the peaks at these wavelengths decrease and increase, respectively. Each agar sulfonate group binds several dye molecules. The shift in lmax was ascribed to ``stacking interactions'' as excess dye molecules adsorb to dye molecules initially bound by electrostic interactions. Dye stacking is essentially a nonpolar process like those leading to dimerization. Examples of metachromatic phenomena were reported for dyes with nonionizing quaternary nitrogen groups; the process does not require dye ionization. Evidence from spectral measurements also supports two modes of CBBG-protein binding. With excess protein lmax ˆ 620 and (CBBG)1 binds to positively charged sites. The formation of a neutral 1:1 protein-dye complex is accompanied by a metachromatic shift of lmax by *30 nm. By contrast, excess dye favors nonpolar interactions probably involving stacking. Eq. (4) shows two different hydrophobic binding sites; R0 sites include the side chains of tyrosine, tryptophan, phenylalanine, leucine, and isoleucine. (CBBGRNH3)0 is produced when (CBBG)1 binds to a charged protein group. The (CBBG)1 binding with nonpolar sites accounts for the lmax value of 595 nm. Perhaps lmax is the same for (CBBGR)1 and CBBG1 because R0 sites are highly hydrated. Given the amphipathic nature of the CBBG molecule (Fig. 1), it is likely that the principal binding sites (R0 and RNH4‡) are also amphipathic. 2.4.

Quantitative Analysis of CBBG Binding to Proteins*

Protein-dye binding parameters (Kd, n, and De) were reported by Klotz (24,25), Klotz et al. (26), and also Aizawa (27). From the law of mass action, Kd ˆ

…D

DA=De†…nP DA=De

DA=De†

The concentration of bound dye (Db) is DA/De while Df ˆ D * All symbols are de®ned in Chapter 5, Sections 4.3 and 4.4.

…5† Db and

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therefore Kd ˆ

Df …nP Db † Db

…6†

Db …Kd ‡ Df † ˆ nPDf

…7†

nPDf Kd ‡ Df

…8†

and Db ˆ

Eq. (8) describes protein-ligand binding with n independent sites. Linearization of this relation leads to Eqs (9)±(11) for extracting binding parameters (Kd and n). 1 Kd 1 ‡ ˆ Db Df nP nP

…9†

Multiplying this double-reciprocal equation with nPDb and rearrangement lead to Db n ˆ PDf Kd

Db PKd

…10†

Substituting G ˆ Db/P, we obtain the equation for the Scatchard plot. G n ˆ Df Kd

G Kd

…11†

The more familiar version of Eq. (11) (Db/Df ˆ nP/Kd Db/Kd) is usable where the protein concentration is kept constant. Most dye-binding studies employ a constant concentration of dye while the protein concentration is varied. Two studies of CBBG-protein binding have been published. Congdon et al. (17) segregated protein binding sites for CBBG into ``strong'' and ``weak'' sites. To characterize strong binding sites, different amounts of BSA (0±41 mM) were added to a ®xed concentration (20 mM)* of CBBG. From a graph of 1/DA versus 1/P the x ˆ 0 yields a reciprocal for the maximum * The concentration of CBBG was 0.0166% (w/v) or 200 mM. To a ®xed volume (100 mL) of dye reagent solution was added 0±2.66 mg of BSA. The ®nal volume of mixture was brought to 1 mL where needed with distilled water. Then DA620 was recorded for each protein concentration.

Bradford MethodÐPrinciples

177

absorbance change (1/DAmax) at an in®nite protein concentration. From this, De ˆ DAmax/D. The absorbance measurements were also used to estimate the number of strong sites. First, values for Db ( ˆ DA/De) and Df ( ˆ D Db) were determined for each protein concentration. Then results were analyzed using Scatchard and related equations. To determine the total number of binding sites, a ®xed concentration of CBBG (133 mM) was exposed to 0.5±100 mg of BSA (0.025±0.512 mM). The conditions (excess dye) are similar to those used for the normal Bradford assay. The number of binding sites was determined from the relation (16) d…DA† ˆ nDe dP

…12†

The left-hand side of Eq. (12) is supposedly the maximum gradient from the Bradford assay standard curve.* The results for CBBG binding to seven proteins are reported (Table 2). For BSA binding with T-azo-R, Eq. (11) gives a poor ®t to the results. The presence of two classes of binding sites (e.g., strong and weak sites) should lead to curvature in the Scatchard plot (28,29) although this has not been demonstrated. Because few graphs for CBBG-protein binding have been published, I have reexamined the data for BSA binding with T-azo-R TABLE 2 Parameters for Coomassie Brilliant Blue G250 Binding to Selected Proteinsa Protein BSA Alcohol dehydrogenase a-Lactalbumin Glutamate dehydrogenase Chymotrypsin Ovalbumin Carbonic anhydrase

Kd (mM)

n(ns)

18.6 40.0 110.0 8.9 80.0 23.0 35.0

105 (2.7) 30 (7) 14 (2.4) ? (2.2) 13 (2.3) 33 (1.9) 28 (2.8)

eb(M

1

cm 1)

48,000 57,700 55,400 57,900 55,700 41,900 51,900

a nS ˆ number of strong binding sites (in parentheses) and Kd are average values from the modi®ed Scatchard plot [Eq. (11)] and Hill plot [Eq. (15)]. Source: Based on results from Ref. 18.

* Eq. (12) is not an appropriate expression for the assay sensitivity. The right-hand expression should ˆ nDeD/Kd (Section 3 of this chapter). The consequence of using Equation (12) to estimate n is described in Section 5.

178

Chapter 6

(see Chapter 5, Figs 5 and 6 and associated text). The data from Ref. 30 were replotted using the Scatchard equation [Eq. (11)] or modi®ed Scatchard relations [Eqs (13)±(15)]. 1 n ˆ Df GKd 1 Kd ˆ G nDf G

1 G

ˆ

1 Kd

…13†

1 n n

…14† 1

n

Kd nDf

…15†

Equations for the straight lines and binding parameters are reported in Table 3. The results show a poor ®t to the Scatchard plot; the regression coef®cient (R) for the graph shown in Fig. 2 was 0.7310. From the equation of the straight line Kd ˆ 11.6 mM and nS ˆ 106. Other equations led to more gratifying transformations of the data. Using Eqs (13)±(15), R ˆ 0.9941. In Table 3, two data entries are shown for each graph. In the ®rst case, values for Kd and nS were assessed assuming that all data conform to a straight line for a single class of binding sites. Figs 2±5 show deviations from linearity at the extremes. The second data entry in Table 3 is derived from results ®tted to the main linear phase in each graph. Eqs (13) and (15) emphasize data collected at high protein TABLE 3 Parameters for T-Azo-R Binding to Bovine Serum Albumin Analyzed Using Scatchard and Modi®ed Scatchard Plots Linearization equation Eq. (11) Eq. (13) (strong sites)a Eq. (14) (weak ‡ strong sites)a Eq. (15) (strong sites)a a

Equation 6

4

Y ˆ 9.21 6 10 ± 8.62 6 10 X Y ˆ 1.06 6 107 X ‡ 164 6 105 Y ˆ 9.26 6 10

8

X ‡ 164 6 10

Y ˆ 0.984 ± 9.26 6 10

8

X

2

Kd (mM)

n

11.6 6.1 3.8 5.6 19.61 5.8 4.2

106 64 43 61 76 63 48

Different equations emphasize data collected at a high protein/dye ratio (strong sites) or a low protein/dye ratio (weak ‡ strong binding sites). Second data entries are calculated using the major linear phase of each graph.

Bradford MethodÐPrinciples

179

FIGURE 2 Scatchard plot for T-Azo-R binding with bovine serum albumin. Dye (10 mM) was titrated with 0±6 mM BSA. Study was performed at pH 2.3. Data from Ref. 31 plotted in accordance with Eq. (8).

concentrations and dominated by strong binding sites. Eq. (14) emphasizes both weak and strong dye binding sites. It appears there are 43±48 strong binding sites on the BSA molecule for T-azo-R (Table 3). By comparison, ns ˆ 26 for thymol blue and 6 for bromophenol blue (see Table 5, Chapter 5). The number of strong binding sites depends on the type of dye and also the reaction conditions. Counting the numbers of weak as well as strong binding sites using a single experiment may require the approach described by Rosenthal (28). Chial and Splittgerber (16) assessed protein-CBBG binding at pH 1 and pH 7. The total number of binding sites (n) was determined using Eq. (12). Detailed procedures are the same as reported by Congdon et al. (17). A summary of results is given in Table 4. The assay at pH 7 had decreased sensitivity. In a number of cases (BSA, a-lactalbumin, carbonic anhydrase) the sensitivity change from pH 1 to pH 7 could apparently be explained using Eq. (12). A 40-fold decrease in assay sensitivity for BSA at pH 7 compared with pH 1 could arise from 2-fold and 20-fold decreases in the values n and De, respectively. Eq. (12) predicts a sensitivity change of 2 6 20 ( ˆ 40-fold). The second footnote in this section suggests that this agreement is fortuitous.

180

Chapter 6

FIGURE 3 Modi®ed Scatchard plot for T-Azo-R binding with bovine serum albumin. Dye (10 mM) was titrated with 0±6 mM BSA. Study was performed at pH 2.3. Data from Ref. 31 plotted in accordance with Eq. (10).

FIGURE 4

Modi®ed Scatchard plot for T-Azo-R binding with bovine serum albumin. Dye (10 mM) was titrated with 0±6 mM BSA. Data from Ref. 31 plotted in accordance with Eq. (14).

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181

FIGURE 5 Modi®ed Scatchard plot for T-Azo-R binding with bovine serum albumin. Dye (10 mM) was titrated with 0±6 mM BSA. Data from Ref. 31 plotted according to Eq. (15).

In Table 4 the molar absorptivity for protein-bound CBBG at 620 nm (eb,620) was not affected by solvent pH. Hence eb,620 was 49 (+8.3) 6 103 M 1 cm 1 at pH 1 or 53 (+6.0) 6 103 M 1 cm 1 at pH 7.0. In contrast, the free dye had an extinction coef®cient (ef,620) of 43,700 M 1 cm 1 at pH 7.0 and 8800 M 1 cm 1 at pH 1.* Consequently, we have De ˆ 39.2 6 103 M 1 cm 1 at pH 1 and De ˆ 9.3 6 103 M 1 cm 1 at pH 7. The reaction time for protein-dye binding was about 60 minutes at pH 7 compared with about 5 minutes at the pH for the normal assay. 2.5.

Identifying CBBG Binding Sites on Proteins

The relative color yield for CBBG binding to different poly-L-amino acids was poly-L-lysine (1), poly-L-histidine (4.2), poly-L-tryptophan (4.4), poly-Ltyrosine (4.7), and poly-L-arginine (36) (12). With increasing dye concentration there was a more similar CBBG response to different basic amino acids

* It is not certain whether these calculations were corrected for changes in the dye ionization with pH. The low ef,20nm value at pH 1 could be due to a sixfold lower concentration of blue (neutral) dye species at pH 1 compared with pH 7.

182

TABLE 4 Protein

Effect of pH on Dye Binding Parameters and Sensitivity of the Bradford Assaya Sensitivityb (DA620 mg 1)6104

(pH 1.0) Alcohol dehydrogenase Bovine serumalbumin Carbonic anhydrase Chymotrypsin a-Lactalbumin b-Lactoglobulin Ovalbumin (pH 7.0) Alcohol dehydrogenase Bovine serumalbumin Carbonic anhydrase Chymotrypsin a-Lactalbumin b-Lactoglobulin Ovalbumin 1

cm

1

(pH 7) or 8800 M

De620(M

1

cm 1)

11,300 18,300 7,300 1,300 9,300 12,300 8,300

2.6 4.8 0.9 2.1 15.0 3.0 0.9

49,200 39,200 43,200 47,200 44,200 25,200 34,200

127.0 193.0 125.0 101.0 143.0 84.0 87.0

1

cm

1

n 28 100 25 16 14 18 33

Lys ‡ Arg 32 86 27 17 13 18 36

2 5 1 2 6 12 2

(pH 1).b Sensitivity as determined from the maximum slope of the calibration graph.

Chapter 6

a ef (620) ˆ 43,700 M Source: Ref. 17.

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183

(10); DA595 readings were 0.92, 1.2, and 1.5 for poly-L-lysine, poly-Lhistidine, and poly-L-arginine, respectively. These studies agree on the importance of poly-L-arginine as a CBBG binding site. Moreno et al. (13) reported a relative color yield from different polyamino acids as poly-Llysine (1), poly-L-tyrosine (1.9), poly-L-arginine (3), poly-L-histidine (>5.5). CBBG binding increased with the polypeptide molecular weight, but differences in color yield are less marked when absorbance changes are expressed per unit mass (microgram) of material analyzed. In summary, studies involving CBBG/R binding to poly-L-amino acids indicate that these dyes bind to basic groups. Nonpolar amino acid residues, notably tyrosine and tryptophan, are also important binding sites. Additional nonpolar sites for CBBG binding are created as the anionic dye molecule binds to positively charged protein sites. However, poly-L-amino acid results should be treated with caution. Proteins rarely feature the high density of sites associated with homopolymers. The correlation between CBBG binding and the number of basic amino acids occurs when ionic bonding predominates and the dye species is limiting (Chapter 5). The relations between protein-binding parameters (n, Kd, De) and characteristics of the Bradford assay are discussed in the next section. 3. EFFECT OF PROTEIN-DYE BINDING PARAMETERS ON THE BRADFORD ASSAY The plot of DA versus protein concentration leads to a hyperbolic calibration graph.* DA ˆ

nDePD Kd ‡ nP

…16†

Eq. (16) is a result of the equilibrium between free dye, protein, and the bound dye. At low protein concentrations nP 2% poultry or >3% meat. Examples include ham, sausages, sources, soups, stews, pizzas, and frozen dinners. From the preceding PAPI and net trade values, we may suppose that the U.S. meat consumer is overcharged by between US$250 million and US$6 billion annually. The most ef®cient techniques for detecting protein adulteration are immunological. Other well-established approaches include quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis (QSDS-PAGE), isoelectric focusing (IEF), and capillary electrophoresis (8). High-pressure liquid chromatography (9) and fast protein liquid chromatography (10) have also been employed for the differentiation of milk from different species. Finally, mention must be made of non±protein-based methods. Detection of amino acids, sugars, or fats associated with particular species can be a clue to adulteration. Species-speci®c DNA testing using hybridization probes or polymerase chain reaction (PCR) is also increasing. The interested reader is referred to papers by Hunt et al. (11), Fairbrother et al. (12), Lenstra and Buntjer (13), and Wolf et al. (14); the subject is reviewed by Meyer and Candrian (15).

Immunological Assay: General Principles

FIGURE 2

225

Methods for food protein immunoassay are divided into marker-free and marker-linked methods.

2. IMMUNOLOGICAL METHODS Immunoassays are based on the use of antibodies (Fig. 2). The range of techniques includes marker-free techniques in solution or agar, e.g., solution-phase precipitation, agar gel immunodiffusion, immunoelectrophoresis, and counterelectrophoresis. The marker-linked techniques involve enzyme immunoassay (EIA) or radioimmunoassay (RIA). Application of immunoassays in food analysis and authenticity testing are reviewed by Samarajeewa et al. (16), Gazzaz et al. (17), Allen and Smith (18), Barai et al. (19), Lee and Morgan (20), Hernandez et al. (21), Taylor et al. (22), Smith (23), and Mandokhot and Kotwal (24).

2.1.

Methods of Food Protein Immunoassay *

With marker-free immunoassays, antibody-antigen binding is monitored directly. Excess antibody is added to a sample suspected to contain antigen. * The term ``antiserum,'' although established in the literature, can lead to confusion. Descriptions such as rabbit antiserum for horse serum are not helpful. We will use antibody wherever possible.

226

Chapter 8

Antibody-antigen binding leads to a precipitate whose height and/or volume is proportional to the antigen concentration. Antibody-antigen binding can take place within an agar gel matrix (Section 3). Marker-free immunoassays are easy to perform and give qualitative (yes-no) answers in a relatively short time. They are, however, relatively insensitive. Marker-linked immunoassays provide more sensitive, although more expensive, analysis.

2.2.

Principles of Immunoassay

Antibody-antigen binding involves a reversible equilibrium reaction, Ig ‡ P „ B

…2†

where, Ig is the free antibody concentration and P the free concentration of antigen. From Eq. (2), the association constant (Ka) for antibody-antigen binding is Ka ˆ

B B ˆ ‰IgŠ‰PŠ …IgT B†P

…3†

where IgT is the total concentration of antibody and B the concentration of bound antibody. From this, one can readily write down the Scatchard equation: B ˆ IgT

B PKa

…4†

A graph of [B] plotted versus [B]/[P] yields a straight line with a slope of 1/Ka and the intercept IgT. Dividing the value of the intercept by the known molar concentration of antibody gives the number of antigen binding sites per Ig molecule; ordinarily, the answer should be 2. The Scatchard plot is usually nonlinear, showing the presence of different populations of antibodies. Adsorbing Ig to a solid support will also obscure some antigen binding sites, leading to a range of Ig-antigen binding af®nity. We turn to the effect of antibody-antigen binding parameters on the immunoassay characteristics. The various terms in Eq. (3) can be expressed in terms of the fraction of antigen bound, X: Ka ˆ

…IgT

X PX†…1

X†

…5†

where X ˆ B/PT and the term PT is total antigen concentration. Rearranging

Immunological Assay: General Principles

227

this relation gives the quadratic relation aX 2 ‡ bX ‡ C ˆ 0

…6†

with a ˆ 1, b ˆ [PT ‡ IgT 1/Ka]/PT, and c ˆ IgT/PT. Theoretical X vs. PT graphs showing the fraction of antigen bound versus concentration were simulated from known values for Ka, IgT, and PT (Figs 3 and 4). The ®rst graph shows a binding curve for an immunoassay using two antibody concentrations (IgT) of 10 8 and 10 9 M. The value of Ka is constant (1011 M 1). Fig. 3 shows that the analytically useful binding interactions occur over the antigen concentration where X ˆ 1. The concentration of antigen should not exceed the total concentration of antibody binding sites, otherwise the fraction bound decreases below 1. The linear dynamic range depends on the concentration of antibody. Simulations were also performed in which the concentration of antibody was kept constant while changing Ka from 1010 to 108 and 106 M 1. Fig. 4 shows that decreasing antibody-antigen binding strength decreases the sensitivity (slope) of the calibration graph. We expect that the LLD for antigen will increase with decreasing Ig-antigen binding af®nity.

FIGURE 3

Simulated binding curves for food protein immunoassay. A plot of the fraction of antigen bound versus the total antigen concentration.

228

Chapter 8

FIGURE 4 Simulated binding curves for food protein immunoassay. Effect of changing antigen binding af®nity constant. The Ka is given values of 1010, 108, or 106 M 1.

2.3.

Background Immunology

Antibodies are secreted by vertebrates in response to foreign matter or antigen. An antibody binds strongly to the antigen that triggered its formation. The antibodies are immunoglobins (Igs), which are proteins that dissolve in dilute salt solution and function in connection with immunity. The Igs are produced by blood cells called B lymphocytes. Each organism has at least 100 million different B lymphocytes. Each B lymphocyte has a distinct Ig protruding from its surface. One of the 100 million B lymphocytes is likely to recognize any antigen encountered in the environment. Binding of antigen to a cell-bound Ig leads to a proliferation of the recognizing B lymphocyte within the general population (an idea called the clonal selection theory). Antigen binding also triggers B-lymphocyte transformation into two new cell types. One cell type responsible for Ig production is called the plasma cell. The second cell type, B memory cells, primes the body to remember its encounter with the antigen. To induce Ig production, an antigen must possess a molecular weight >5000. Low-molecular-weight

Immunological Assay: General Principles

229

compounds (haptens) act as antigens when covalently attached to carrier proteins. Large antigens possess multiple sites (epitopes) for Ig binding and are said to be polyvalent. Each epitope elicits Ig production by a speci®c B lymphocyte. The heterogeneous mixture of antibody produced by a population of B lymphocytes is described as a polyclonal antibody (pAb). A sample of pAb contains a mixture of Ig speci®c for the different epitopes on a single polyvalent antigen. Two proteins sharing a common epitope, perhaps because of sequence or structural homology, will be recognized by pAb, leading to cross-reactivity. The diversity of antibodies arises from their structure and large numbers of encoding genes. Antibodies are glycoproteins with a molecular mass >150 kDa. Each antibody comprises two heavy (55±75 kDa) polypeptide chains and two light (25 kDa) chains joined by (three) intermolecular disul®de bonds. Each heavy (H) or light (L) chain has variable (v) amino acid sequences at the C terminal and constant (c) regions near the N terminal. There are ®ve different H chains (g, a, m, d, and e) giving rise to ®ve classes of antibody; IgG, IgA, IgM, IgD, and IgE. The different classes of Ig occur at plasma concentrations of about 12, 3, 1, 0.1, and 0.001 mg mL 1 and differ in their stability and ease of handling. There are two groups of L chains designated k and l. The three-dimensional structures of the H chain comprises ®ve domains whereas each L chain is organized into two domains. The Hc and Lc regions are produced by two gene alleles and Lv and Hv regions are each encoded by about 300 genes. In all, an organism is able to produce at least 3002 or 9 6 104 types of antibody. Further diversity comes from a process of somatic recombination and the translation of messenger RNA (mRNA) transcripts using different reading frames. As a result of such mechanisms, vertebrates produce up to 1 6 108 different antibodies and their corresponding B lymphocytes before exposure to an antigen. The pAbs for commercial immunoassays are produced through the agency of live animals. B lymphocytes have not been successfully cultured in vitro. Such restrictions make the production of pAbs dif®cult to standardize. Immunizing a second animal must lead to variations in the crude reagent, which must then be puri®ed and standardized in some way. The technology for producing monoclonal antibodies (mAbs) was developed by Kohler and Milstein (25) of the University of Cambridge, U.K. Tumor cell lines, which survive inde®nitely when grown in cell culture, produce mAbs naturally. To produce mAbs of de®ned speci®city, spleen cells from immunized mice are fused with tumor cells. The resulting spleen cell±myeloma hybrid (hybridoma) possesses traits from both parental cell lines, i.e., immortality and the ability to synthesis speci®c mAbs derived from mice spleen cells. The hybridoma can be grown in PVC microwell

230

Chapter 8

plates and the cell supernatant examined for the mAb. It was possible to isolate a single hybridoma cell line (or clone) that produces the required Ig. Hybridomas can be grown inde®nitely and large amounts of mAbs produced for analytical use. Only a few of the available genes for the H and L regions are expressed owing to a process called allelic exclusion (25). Fusion is needed for the production of mAbs. The mere mixing of myeloma cells and spleen cells (polyethylene glycol as a fusion agent) will not lead to new Ig. 3.

SPECIATION OF PROTEINS BY AGAR GEL DOUBLE IMMUNODIFFUSION ASSAY

Modern agar gel double immunodiffusion (AGID) assays were developed by Cutrufelli et al. (26,27). The technique is approved by the AOAC. The AGID assay is fast, requires minimal equipment, and can be performed by personnel with minimum training. The tests are based on the two-stage precipitin reaction. AGID comes in several forms (28): (a) linear diffusion, (b) double radial diffusion, (c) single radial immunodiffusion (SRID), (d) immunoelectrophoresis, and (e) counterimmunoelectrophoresis. With linear diffusion, antibody is added to molten agar and the mixture is allowed to set in a small tube. The sample antigen is placed on the column of gel and diffuses downward, forming an opaque precipitin phase. The distance of migration is a function of antigen concentration. For an SRID assay the antigen is placed in a circular well cut into the antibodyagar plate (29). The radius of diffusion, indicated by a precipitin phase, is related to the concentration of antigen. Double immunodiffusion assays involve two sets of circular wells cut into an agar gel. Into separate wells are placed antibody and analyte. They diffuse toward each other, combining to form an opaque precipitation band. With immunoelectrophoresis, the protein sample is placed within holes cut into the gel and forced to migrate under an electrical potential. Then antibody is placed along a rectangular trough parallel to the direction of electrophoresis. Each protein band reacts with the diffusing antibody, leading to a series of precipitin arcs (30). 3.1.

Procedures for Meat Protein Identi®cation by AGID Assay

AGID assays are an extension of the test tube precipitation test ®rst demonstrated by Uhlenauth in 1901 (cited in Refs. 5 and 31). Further work between 1902 and 1928 led to the production of less complex antigen (inoculation) mixtures designed to improve speci®city and reduce toxicity to

Immunological Assay: General Principles

231

immunized animals. Gel-phase immunodiffusion assays were described by Oudin around 1946 and then by Ouchterlony (32). The last study led to a simple in vitro assay for diphtheria toxin and for toxin-producing bacteria. Heating diphtheria toxin led to its denaturation and failure to produce a precipitin reaction. Early serological tests for meat and other food proteins are described by Oswarld (33). To produce pAb for analytical applications, a host animal is immunized with antigen at intervals ranging from several weeks to months. Mice, rats, rabbits, sheep, goats, or horses are used for antibody production. Larger animals are preferred by large-scale, commercial, producers. After about 4 weeks, blood is taken from the immunized animal and centrifuged to produce antiserum, i.e., blood serum containing antibody. Further booster injections may be administered and antibody production monitored until a high plasma concentration (antibody titer) is attained. To avoid cross-reactivity, pAb may be puri®ed by immunoadsorption. When the titer of antibodies reaches a high level, larger samples of blood are removed, allowed to coagulate, and centrifuged to produce crude antibody. Preservative is normally added before storing at 208C. Important variables for antibody production include the following: 1. Purity of the antigen. Puri®ed antigen is seldom required. However, more pure antigens have less toxic effects on the immunized animal. 2. Choice of host animal. A large animal such as a goat yields more serum than a mouse. To reduce cross-reactivity, pAb should be raised in a species similar to the species for which the antibody is intended to differentiate. Goats or sheep pAb is more able to differentiate between antigens from cattle, buffalo, and other bovine species (59). 3. Choice of adjuvant. Most investigators use Freund's complete adjuvant. This is an oil suspension of inactivated Mycobacterium tuberculosis. A muramyl peptide from the bacterial wall stimulates antibody production. Alternatively, antigen is coprecipitated or adsorbed on insoluble aluminum oxide, synthetic muramyl peptides, or carbohydrate (34). 4. Mode of immunization. Subcutaneous or intramuscular injection is common. Several investigators describe injections via the foot pads of rabbits. Intervals of injections are usually weekly. Larger animals may be injected at 30-day intervals. 5. Time and method for bleeding. Immunization schedules usually require trial bleedings at 4- to 7-day intervals after the injection of

232

Chapter 8

booster antigen. Blood may be collected from a vein in the ears or tail. 6. Puri®cation by immunoadsorption. To remove cross-reactivity with sheep, goat pAb may be exposed to sheep antigen and then centrifuged to remove any precipitate formed. The soluble pAb phase should not now react with sheep. Immunoabsorption can be performed with soluble sheep antigen or antigen immobilized on cyanogen bromide±activated support (35±39). AGID tests are performed according to the following steps: (a) extraction of antigen and immunization of host animal (see Method 1), (b) production of pAb (Method 1), (c) puri®cation of pAb by immunoadsorption (see Method 2), (d) preparation of agar gel plates (see Method 3), and (e) conduction of AGID test. Details of these steps are given next. The protocols are drawn from reference listed in Table 2 (Sec. 3.4). The inexperienced worker should seek expert advice regarding humane treatment and immunization of host animals. Commercial pAb should be used when available. The internet is a good source of addresses of companies currently supplying immunoassay reagents and ready-to-use kits. Method 1 Extraction of meat antigens and pAb production (40,59). Reagents 1. Phosphate-buffered saline (PBS; 0.01 M phosphate buffer, pH 6.8±7.2 ‡ 0.1 M KCl) 2. Muslin cloth 3. Freund's adjuvant Procedure Fresh meat antigen. Stir ground meat with 1 volume of PBS overnight at 48C. Filter through muslin cloth and centrifuge at 10,400g for 60 minutes. Filter the supernatant through Whatman No. 3 paper to remove ¯oating fat droplets. Freeze dry for prolonged storage. Immunization. Mix the antigen (5±10 mg mL 1 PBS) with an equal volume of Freund's complete adjuvant. Inject adult rabbits with 2 mL of antigen suspension at multiple sites. Reinject the animals with booster doses of antigen dissolved in Fruend's incomplete adjuvant at weekly intervals. With goats the ®rst injection can be 4 mL of antigen. The booster dose is injected every other week. Small amounts of blood should be removed in the intervening weeks and tested for pAb production. Treatment of blood and antibody production. Collect whole blood, allow to coagulate at room temperature for about 3 hours, and then

Immunological Assay: General Principles

233

store at refrigerator temperatures overnight. Centrifuge at 2000g to remove blood cells. To the supernatant (antibody) add 100 ppm of methiolate as a preservative and store at 208C until needed. Immunization schedules are ¯exible, ranging from 3±4 weeks for rabbits to several months for goats, sheep, or horses. The pAbs for ORBIT and PROFIT were produced by injecting goats with 10 mL of antigen coprecipitated with alum. The same antigen was injected 21±30 days later (5 mL per hind leg), then at 3-weekly intervals. Blood samples were taken *7 days after each injection to monitor pAb production. The immunization process is continued until a desired concentration of antibody is attained. Method 2 Antibody puri®cation by immunoadsorption. 1. A simple immunoadsorption procedure (31,35). To 1 mL of crude pAb for species A that shows cross-reactivity with species Y, add 20 mg of antigen from species Y. Incubate the mixture at room temperature for 4 hours. Refrigerate at 48C overnight and centrifuge (2500g for 20 minutes) to remove pAb-antigen complexes. The supernatant phase should be pAb with improved speci®city for species A. 2. Immunoadsorption using ethylchrolorformate cross-linked antigen. The original method was described by Avrameas and Ternynck (36,37). Adjust a solution of antigen (e.g., 500 mg of whole serum protein in 10 mL of 0.2 M acetate buffer) to pH 5.0 with dilute HCl. Add ethylchloroformate (*3 mL) dropwise. Maintain the acidity at pH 4.5±5 using 1 M NaOH. Allow to react for about 10± 15 minutes, redisperse the resulting gel in a further 10 mL of 0.2 M acetate buffer, and allow to react for another 60 minutes. Recover the gel formed by ®ltration or by centrifuging gently. Wash with distilled water and keep refrigerated until use. Shake antibody for species A and with cross-linked antigen from species Y overnight. Recover the soluble serum phase, which should now be monospeci®c for species A. This method has been demonstrated by Kang'ethe et al. (59). Immunosorption can be performed with the dried antigen, cross-linked antigen, or antigen immobilized on CNBr-activated support. Method 3 Standard agar gel double immunodiffusion assay. Reagents 1. Agar (Oxoid Ltd.)

234

Chapter 8

2. PBS buffer 3. 6.5-mm-diameter ®lter discsÐoptional Procedure Prepare 1% (w/v) molten agar in PBS by heating on a water bath. Pour about 4 mL of molten agar into a petri dish and allow to set at room temperature. Refrigerating for a few hours will harden the gel. Cut out several 6-mm-diameter holes or wells using a cork borer. Place one well in the center of the petri dish and then surround this with a quartet of wells each placed 6 mm from the central well. Remove the circular piece of gel using a Pasteur pipette attached to a vacuum line. Place a drop of molten agar into each well to act as a seal, thereby avoiding the migration of samples below the well. To perform a standard AGID assay, place pAb in the central well. In the surrounding wells, place one each of the test samples (*20 mL). Cover the petri dishes to avoid dehydration and allow to stand at room temperature for 24 hours. 3.2.

Agar Gel Double Immunodiffusion Assay for Uncooked Meat

The ®rst attempts to identify raw meat using the AGID assays were described by Warnecke and Saf¯e (38). They found that actomyosin was a poor antigen, with injections of 20±150 mg leading to only moderate pAb production in rabbits. Rabbit pAB for beef whole serum showed crossreactivity for lamb extract. No reactivity was seen for horse meat or pork. The crude pAb was rendered monospeci®c for beef by immunoadsorption. An AGID assay with immunoadsorbed pAb allowed the detection of beef. Many of the methods described in this report are still in use at the present time. Further developments in AGID assays for meat speciation occurred in the laboratories of the U.S. Department of Agriculture, Beltsville, MD. Fugate and Penn (39) used AGID assays to identify meat from beef, horse, pig, and sheep. Of 12 meat samples examined, 11 were correctly identi®ed. They recommended that the AGID test should be subjected to collaborative testing. AGID assays using pAb for residual serum albumin from meat were produced by Hayden (40). Hers was probably the ®rst dissertation on this subject. At the Department of Food Science, University of Georgia (Athens, GA), Helm et al. (41) compared the AGID assay and a simple test tube precipitin test using (rabbit) pAb for beef, horse, lamb, and pork. The AGID test detected adulteration at the 2% level.The solution precipitin test was *three times faster and four times more sensitive. In Australia, Swart

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235

and Wilks (42) differentiated beef, horse, kangaroo, and mutton by AGID assay. Species identi®cation ®eld tests (SIFTs) were developed in 1984 by Mageau et al. (43) at the USDA. Finally, Darwish et al. (44) also described an AGID assay for detecting beef adulteration with camel and pork. Some examples of the application of immunodiffusion assays include the detection of pork in beef mince meat (45), Alaska pollack surimi analysis in meat products (46), and the detection of various meat types (beef, pork, horse, poultry) in hamburger (47). 3.3.

Species Identi®cation Field Tests (SIFTs) for Uncooked Meat

SIFTs are designed for ®eld testing in abattoirs and meat inspection stations. The ®rst of these test kits is called ORBIT (43). The acronym stands for the Overnight Rapid Bovine Identi®cation Test. ORBIT was followed by PROFIT (Poultry Rapid Overnight Field Identi®cation Test) (48), SOFT (Serological Ovine Field Test) (49), PRIME (Porcine Rapid Identi®cation Method) (50), REST (Rapid Equine Serological Test) (51), DRIFT (Deer Rapid Identi®cation Field Test) (52), and MULTI-SIFT (Multispecies Identi®cation Field Test) (53). The last kit can simultaneously test for beef, poultry, pork, sheep, horse, and deer meat. After successful collaborative trials (26), ORBIT and PROFIT received approval from the AOAC. These methods can detect meat adulteration levels of about 10% or greater (27). SIFTs are not radically different from classical AGID assays. However, considerable design effort has gone into making SIFTs attractive and easy to use. Antibody and meat sample extract are adsorbed onto ®lter paper discs. Both are then freeze dried, providing stabilized reagent discs that can be stored for up to 12 months at refrigeration temperature. The antibody ®lter discs and all materials needed to perform SIFTs are available commercially. The tests are used by the USDA and meat inspection services in the United States. According to the team responsible for SIFTs, conventional methods for meat speciation (e.g., electrophoresis, chromatography) share many of the following disadvantages: (a) tests are usually performed within a formal laboratory, (b) relatively sophisticated equipment is needed, (c) high levels of staff expertise and training are necessary, (d) time delays arise due to offsite testing with an attendant need for transmitting samples and assay results to and from the analyst, and (e) relatively labile reagents are used. SIFTs were developed with the aim of avoiding such disadvantages. The assays are highly reliable, fast, easy to use, accurate, and sensitive. Little expertise or previous experience is needed for successful testing. Finally, SIFTs use

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stabilized reagents (antibody and gel plates) with a shelf life of up to 12 months. SIFTs use pAb speci®c for native serum albumin (40). The tests are therefore intended for raw meat speciation. A key feature is the use of lyophilized reagents that are more stable for storage. Commercial pAbs as well as those prepared in house proved usable. Assay results were affected by the distance of separation between ®lter discs, with a distance of 4±5 mm being optimal. The tests showed considerable temperature tolerance. Incubating agar gels plates at 25±378C had no ill effect. Higher temperatures led to gel dehydration. Lower temperatures prolonged the time needed for precipitin formation. Finally, SIFTs were suitable for the analysis of whole meats, ground meat, meat emulsions, and other formulated meat products, provided these were not heated. Table 1 summarizes some performance characteristics of SIFT assays. The MULT-SIFT assay allows simultaneous testing of an unknown meat sample (antigen) against six different reference pAbs (Table 1). 3.4.

Thermostable Meat Antigens for Cooked Meat Analysis

Ideal antigens for cooked meat analysis survive thermal treatment at 708C or autoclaving temperatures of 1208C for 15 minutes. Hayden (54) considered troponin as a heat-stable meat antigen (Table 2). Extensive research since 1977 has shown that thermostable meat antigens are usually troponin C, troponin I, or troponin T. Schweiger et al. (55) used puri®ed turkey troponin T for AGID assay. Thermostable antigens were initially developed from adrenal gland extracts (56,57) by Milgrom and Witebsky (58).* Rabbit pAb for boiling-resistant, ethanol-insoluble (BE) antigen from beef showed cross-reactivity with BE antigen from sheep. There was no reaction with heated adrenal extracts from pig, rat, guinea pigs, or humans. The method for BE antigen preparation has remained unchanged for nearly 40 years. Hayden (56,57) used BE antigen for detecting cooked beef sausage adulteration with horse, sheep, and pig meat. Radhakrishna et al. (62) showed that (buffalo, goat, oxen) muscle BE antigen had SDS-PAGE bands corresponding to troponin (C, I, and T) and tropomyosin. Bhilegaonkar et al. (67) found that the concentration of (buffalo, sheep, goat, pig) BE antigen was 21±130% higher in adrenal tissue * Boiling-resistant ethanol-insoluble (BE) antigen is produced by boiling an aqueous extract from bovine adrenal glands or muscle tissue followed by centrifugation. The supernatant is autoclaved at 1208C for 30 minutes and centrifuged. Then three volumes of 95% ethanol are added to the soluble fraction. A whitish precipitate forms after *12±14 hours at 378C and is then dissolved in normal saline for immunization.

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237

TABLE 1 Characteristics of Some Species Identi®cation Field Tests (SIFTs)a Test acronym and speci®city

Immunized host

ORBIT, beef (39)

Rabbit, goat, sheep

PROFIT, poultry (40)

Goat

PRIME, pork (42)

Goat

SOFT, sheep (41) REST, horse (43)

Calf

DRIFT, deer (44)

Goat

MULTI-SIFT, six different species (45)

Various

Sheep

Performance 70 samples. No false positives/negatives, Speci®city: bison ( ‡ ), bovine ( ‡ ), water buffalo ( ‡ ), deer ( ), elk ( ), goat ( ), horse ( ). 66 samples. No false ‡ / results. Speci®city: chicken ( ‡ ), turkey ( ‡ ), goose ( ‡ ), quail ( ‡ ), partridge ( ‡ ), bovine ( ), deer ( ), horse ( ), pig ( ), sheep ( ).LLD 3% or 5% in pork or beef 83 samples. No false ‡ / results. LLD 5% pork (in beef), 3% pork in lamb. 90 samples. No false ‡ / results. LLD 3% mutton in beef. 101 samples. No false ‡ / results. Speci®city: deer, ( ), donkey ( ‡ ), mule ( ‡ ), beef ( ), pork ( ), sheep ( ), chicken ( ), turkey ( ), kangaroo ( ). LLD 3% horse 100 samples. No false ‡ / results. Speci®city: mule deer ( ‡ ), elk ( ‡ ), moose ( ‡ ), reindeer ( ‡ ), beef ( ), horse ( ), sheep ( ), chicken/turkey ( ). No false ‡ / results. Speci®city (as above)

a

LLD, Lower limit of detection or minimum % weight of adulterant detectable. All antiserum ®lter discs are stable for 4±5 months at room temperature and 12 months at 48C. ( ‡ ) positive test, ( ) negative test.

than in muscle. SDS-PAGE analysis of adrenal BE antigens showed a single 35.5-kDa component (troponin T). Cow and buffalo muscle BE antigen had three components with molecular masses of 35.5 kDa (troponin T), 19 kDa (troponin I), and 16 kDa (troponin C). Sheep and goat muscle BE antigen had the 35.5- and 19-kDa components. Pork thermostable antigen showed three troponin bands and an extra 66-kDa protein.

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TABLE 2 Boiling-resistant Ethanol-Insoluble (BE) Protein as Thermostable Antigen for Agar Gel Double Diffusions Assay of Cooked Meat Analysis/comments Troponin T, as chicken muscle antigen BE antigen for sausage analysis Troponin T antigen for turkey analysis BE antigen for differentiating domestic meat sources (beef, goat, and sheep) from 14 game species BE antigen for detecting chicken adulteration by buffalo, sheep, goats, pig meat BE antigen identi®ed as troponin T; isolation from buffalo, cattle, sheep, goat, and pig adrenals and muscle tissue BE antigen for detection of pork in beef, buffalo, sheep, goat, chicken meat End-point temperature determination, myoglobin

Reference Hayden (54) Hayden (56,57) Schweiger et al. (55) Kang'ethe et al. (59) Sherikar et al. (60,61) Radhakrishna et al. (62); Bhilegaonkar et al. (67); Sherikar et al. (63) Reddy and Giridhar-Reddy (64); Saisekhar and Reddy (65) Levieux and Levieux (66)

Saisekhar and Reddy (65) isolated troponin T from raw beef and buffalo meat. An AGID assay based on native troponin T antigen from buffalo cross-reacted with cattle, goat, and sheep meat. There was no reaction with chicken or pork. The (rabbit) pAb for buffalo was rendered monospeci®c by immunoadsorption with cattle, goat, and sheep antigen. The AGID assay using monospeci®c buffalo pAb could detect beef or mutton adulteration with 1% of buffalo meat. Interestingly, (rabbit) pAb for bovine troponin T was monospeci®c for beef without prior immunopuri®cation. No cross-reactivity occurred with buffalo, goat, sheep, or chevon meat. The LLD was 10% beef addition to samples of buffalo, chevon, or mutton. These tests based on pAb for native troponin T did not detect cooked meat. In contrast, pAb for native troponin T from chicken or turkey was sensitive to (poultry meat ) antigen in fried sausages (54,55). Clearly, troponin T is not heat resistant. Heating may generate a denatured but soluble troponin T that functions as a BE antigen. Much evidence points to troponin T being the major BE antigen. However, other muscle proteins might also play this role. SDS-PAGE analysis of muscle proteins extracted by 0.6 M KCl shows bands for myosin (200 kDa), actinin, actin (42 kDa), and troponin T (37 kDa). In the 32±28 kDa region appeared bands for troponin I, tropomyosin, and myosin light

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chain (68). After comminution, troponin T was susceptible to proteolysis, forming a 30-kDa fragment. Proteolysis also occurred for meats up to 7 days old (69). After heating meat to 808C, only bands for troponin T, actin, and the myosin light chains remained. As actin is highly conserved between species (70), antibodies for this protein will not differentiate meat from different mammals (62,67). The pAb for actin could form a general test for animal tissue.

3.5.

Agar Gel Double Immunodiffusion Assays for Cooked Meat

Myoglobin was not wholly successful as a heat-stable antigen (31). To produce an AGID test for cooked meat, (rabbit) pAb was prepared using pure myoglobin preheated to 908C for 15 minutes. Cooking meat samples to 708C led to loss of sensitivity to meat myoglobin. There was cross-reactivity between (rabbit) pAb for lamb and beef myoglobin. However (goat) pAb for porcine myoglobin showed no cross-reactivity with beef. Thus 3% pork could be detected in unheated meat samples. Mild heating raised the LLD (lower limit of detection) to 10% adulteration of beef with pork. Hayden (31) proposed that (rabbit) pAb for heated myoglobin might be suited for developing AGID tests for mildly heated meat from bovine, horse, seal, or whale. Actually, pAb for heat-denatured pure myoglobin did not recognize myoglobin heated within a meat matrix. Apparently, the conformation of denatured myoglobin depends on the heating matrix. The denaturation of heme proteins involves an in-series mechanism. First, there is a conformational change and dissociation of the heme group. This binds covalently to the apo protein and other proteins or else forms heme oligomers (71); it is possible that some heme degradation products are antigenic. The apo protein then undergoes incorrect refolding and/or aggregation. The presence of other meat proteins can easily affect the end point for myoglobin denaturation. The AGID assay using (rabbit) pAb for chicken troponin T could detect the presence of 1±5% chicken meat in beef sausages cooked to an internal temperature of 70±908C (54). Using (rabbit) pAb for turkey troponin T, turkey sausages heated to an internal temperature of 708C and cooked for 10±15 minutes were also successfully analyzed. All assays were also highly sensitive to turkey meat in raw meat balls (mixture of pork and beef) and extract from poultry fried sausages (55). Thermostable BE antigen was the basis for the AGID tests for differentiating meat of domesticated species (cattle, goats, sheep) from 14

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wildlife game species.* The tests developed by Kang'ethe et al. (59) from the University of Nairobi used (goat) pAb. The crude pAb was surprisingly monospeci®c in most cases. Cross-reactivity was observed for some closely related species: buffalo and cattle, bushbuck and cattle, Grant's gazelle and sheep, and Grant's gazelle and Thomson's gazelle. After immunoadsorbtion, each pAb was rendered monospeci®c for thermostable antigen, cooked meat extracts, or fresh meat. The domesticated species were clearly distinguished from the game species. Differentiating between Grant's and Thomson's gazelle, kongoni and topi, and kongoni and wildebeest remained problematic. Using goat as the host for pAb production probably accounts for low cross-reactivity. By comparison, (rabbit) pAb shows lower speci®city and a greater likelihood of cross-reactivity between closely related species. Chicken meat is in high demand in parts of Asia owing to religious restrictions related to the consumption of beef. Sherikar et al. (61,63) produced an AGID assay for chicken meat adulteration with beef, buffalo, goat, mutton, or pork. BE antigen was prepared from heart, kidney, liver, spleen, or lung tissue. Crude (rabbit) pAb for pork was monospeci®c. The other (rabbit) pAbs were puri®ed by immunoadsorption. Partially puri®ed pAbs reacted only with homologous antigen from raw tissue or tissue mildly heated at 708C. Fully cooked meat could not be detected unless further processed to BE antigen (by autoclaving, centrifugation, and ethanol precipitation). Apparently, components in the relatively complex cooked meat extract interfered with antibody-antigen binding and/or precipitin formation. AGID assays were performed on samples with BE antigen. Adulteration of chicken with 10% (w/w) beef, buffalo, goat, or sheep meat was detectable. The LLD for pork was 5% (w/w). Reddy and Giridhar-Reddy (64) also produced an AGID assay for cooked pork. Porcine muscle BE antigen and the corresponding (rabbit) pAb were prepared as usual. The crude (rabbit) pAb was monospeci®c for pork. Samples for analysis were extracted from raw pork or after heating at 1208C for 30 minutes. The AGID assay detected 10% (w/w) pork in cooked meat from buffalo, cattle, chicken, goat, and sheep. The LLD for pork in raw meat mixtures was 20% (w/w). The frequency of undeclared meat substitutions was referred to earlier. Hsieh et al. (7) examined 806 raw meat and 96 cooked meat samples (mostly * The species include buffalo (Syncerus caffer), bushbuck (Tragelaphus scriptus), cattle (Bos indicus), eland (Taurotragus oryx), goat (Capra aegagrus hircus), Grant's gazelle (Gazella granti), impala (Aepyceros malampus), kongoni (Alcelaphus buselaphus cokii), oryx (Oryx spp.), sheep (Ovis ammonaires), Thomson's gazelle (Gazella thomsoni), topi (Damaliscis lunatus), waterbuck (Kobus spp.), and wildebeest (Connochaetes taurinus).

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241

beef and ground veal). The AGID tests involved commercially available pAb speci®c for raw sheep, pork, beef, and horse meat. Enzyme-linked immunosorbent assay (ELISA) kits utilizing pAb for thermostable antigen were used for the analysis of cooked meats. About 16% of raw meat samples were adulterated with meat from another species. The frequency of adulteration increased to 23% for cooked meat. The most frequent adulterants for ground veal or beef were sheep (47%), pork (42%), or poultry (31%). There were no substitutions involving horse meat. Crosscontamination via (improperly cleaned) processing equipment was not signi®cant. Ground lamb and pork had adulteration frequencies of 66.7% and 53%, respectively. So far, immunological methods cannot distinguish beef from veal or mutton from lamb.

3.6.

Agar Gel Double Immunodiffusion Assays for Other Food Proteins

AGID assays have not proved popular for the analysis of nonmeat proteins. The analysis of soy protein in meat is reviewed by Llewellyn (5). Hammond et al. (72) found that cross-reactivity with other legume proteins was widespread. The reliability of AGID tests for vegetable proteins is poor because of the effect of heat, extrusion, texturization, and other forms of processing. More promising are methods based on ELISA (Chapter 10).

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44. AM Darwish, MA Soliman, HA Aideia, TM Nouman. Evaluation of the agar-gel immunodiffusion technique for differentiation of meats. J Egypt Vert Med Assoc 51:739±745, 1991. 45. DR Martin, J Chan, JY Chiu. Quantitative evaluation of pork adulteration in raw ground beef by radial immunodiffusion and enzyme-linked immunosorbent assay. J Food Prot 61:1686±1690, 1998. 46. MS Dreyfuss, ME Cutrufelli, RP Mageau, AM McNamara. Agar-gel immunodiffusion test for rapid identi®cation of pollock surimi in raw meat products. J Food Sci 62:972±975, 1997. 47. ME Flores-Munguia, MC Bermudez-Almada, L Vazquez-Moreno. Detection of adulteration in processed traditional meat products. J Muscle Foods 11:319±325, 2000. 48. ME Cutrufelli, RP Mageau, B Schwab. Development of Poultry Rapid Overnight Field Identi®cation Test (PROFIT) J Assoc Off Anal Chem 69:483± 487, 1986. 49. ME Cutrufelli, RP Mageau, B Schwab, RW Johnston. Development of Serological Ovine Field Test (SOFT) by modi®ed agar-gel immunodiffusion. J Assoc Off Anal Chem 72:60±61, 1989. 50. ME Cutrufelli, RP Mageau, B Schwab, RW Johnston. Development of Porcine Rapid Identi®cation Method (PRIME) by modi®ed agar-gel immunodiffusion. J Assoc Off Anal Chem 71:444±445, 1988. 51. ME Cutrufelli, RP Mageau, B Schwab. Development of a Rapid Equine Serological Test (REST) by modi®ed agar-gel immunodiffusion. J Assoc Off Anal Chem 74:410±413, 1991. 52. ME Cutrufelli, RP Mageau, B Schwab. Development of a Deer Rabid Identi®cation Field Test (DRIFT) by modi®ed agar-gel immunodiffusion. J Assoc Off Anal Chem 75:74±76, 1992. 53. ME Cutrufelli, RP Mageau, B Schwab. Development of a multispecies identi®cation ®eld test by modi®ed agar-gel immunodiffusion. J Assoc Off Anal Chem Int 76:1022±1026, 1993. 54. AR Hayden. Detection of chicken ¯esh in beef sausages. J Food Sci 42:1189± 1192, 1977. 55. A Schweiger, S Baudner, HO Gunther. Isolation by free-¯ow electrophoresis and immunological detection of troponin T from turkey muscle: an application in food chemistry. Electrophoresis 4:158±163, 1983. 56. AR Hayden. Use of antisera to a heat-stable antigen from equine adrenals for detection of horse meat in cooked beef sausages. J Anim Sci 49(Suppl 1):221± 222, 1979. 57. AR Hayden. Use of antisera to heat-stable antigens of adrenals for species identi®cation in thoroughly cooked beef sausages. J Food Sci 46:1810±1813, 1819, 1981. 58. F Milgrom, E Witebsky. Immunological studies on adrenal glands. 1. Immunization with adrenals of foreign species. Immunology 5:46±66, 1962. 59. EK Kang'ethe, JM Gathuma, KJ Lindqvist. Identi®cation of the species origin of fresh, cooked and canned meat and meat products using antisera to

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thermostable muscle antigens by Ouchterlony's double diffusion test. J Food Sci Agric 37:157±164, 1986. AT Sherikar, JB Khot, BM Jayarao, SR Pillai. Differentiation of organs of meat animals and identi®cation of their ¯esh in chicken using anti-adrenal BE sera. Indian J Anim Sci 58:565±573, 1988. AT Sherikar, JB Khot, BM Jayarao, SR Pillai. Use of species-speci®c antisera to adrenal heat-stable antigens for the identi®cation of raw and cooked meats by agar gel diffusion and counter immunoelectrophoresis techniques. J Sci Food Agric 44:63±73, 1988. K Radhakrishna, DV Rao, TR Sharma. Characterization of the major component in thermostable muscle proteins. J Food Sci Technol India 26:32± 35, 1989. AT Sherikar, UD Karkare, JB Khot, BM Jayarao, KN Bhilegaonkar. Studies on thermostable antigens, production of species-speci®c antiadrenal sera and comparison of immunological techniques in meat speciation. Meat Sci 33:121± 136, 1993. PM Reddy, M Giridhar-Reddy. Immunochemical detection of pork using thermostable muscle antigens. J Food Sci Technol India 32:326±328, 1995. Y Saisekhar, PM Reddy. Use of troponin for species identi®cation of cattle and buffalo meats. J Food Sci Technol India 32:68±70, 1995. D Levieux, A Levieux. Immunochemical quanti®cation of myoglobin heat denaturation: comparative studies with monoclonal and polyclonal antibodies. Food Agric Immunol 8:111±120, 1996. KN Bhilegaonkar, AT Sherikar, JB Khot, UD Karkare. Studies on characterization of thermostable antigens of adrenals and muscle tissues of meat animals. J Sci Food Agric 51:545±553, 1990. C-S Cheng, FC Parrish. Heat induced changes in myo®brillar proteins of bovine logissimus muscle. J Food Sci 44:22±24, 1979. S Ebachi, T Wakabayashi, F Ebashi. Troponin and its components. J Biochem 69:441±445, 1971. L Stryer. Biochemistry, 3rd ed. New York: WH Freeman, 1988, pp 920±974. JL Forsyth, RKO Apenten, DS Robinson. The thermostability of puri®ed isoperoxidases from Brassica oleracea Var. gemmifera. Food Chem 65:99±109, 1999. JC Hammond, IC Cohen, B Flaherty. A critical assessment of Ouchterlony's immunodiffusion technique as a screening test for soya protein in meat products. J Assoc Public Anal 14:119±126, 1976.

9 Speciation of Meat Proteins by Enzyme-Linked Immunosorbent Assay

1. INTRODUCTION Enzyme-linked immunosorbent assay (ELISA) was invented by Eva Engvall during her Ph.D. studies in Stockholm in 1971 (1). A similar assay was produced by Van Weemen and Shuurs (2). Developments between 1971 to 1981 are reviewed in reference 3. ELISA is an example of an enzyme immunoassay (EIA). These are immunological tests ending with enzymatic analysis. Enzyme multiplied immunoassay tests (EMITs) are performed in solution. ELISA employs immunoglobins attached to a solid surface. EIA is further categorized as competitive or noncompetitive (Table 1). Secs 2±5 of this chapter describe authenticity testing for raw and cooked meat by ELISA. The meat antigens detected with such tests are mostly residual serum proteins from the blood or else muscle proteins. Sec. 6 covers ELISA for meat protein using monoclonal antibodies (mAbs). Seafood speciation is discussed in Sec. 7, followed by a comparison of the different ELISA formats in Sec. 8. Attempts to detect meat and bone meal in animal feeds are described in Sec. 9, as are some new ELISA tests for the bovine spongiform encephalopathy (BSE) agent. During EMIT an enzyme-antigen conjugate (Enz-antigen) reacts with antibody (Ig). Steric hindrance from Ig binding reduces enzyme activity. The 247

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TABLE 1 A Range of Enzyme Immunoassays (EIAs)a Solution-phase EIA EMIT Competitive Enz-antigen

Solid-phase EIA ELISA Competitive Enz-antigen, Enz-Ig Noncompetitive Direct, indirect, sandwich

a

Enz, enzyme; Ig, antibody. Other acronyms are de®ned in the general text.

added sample (with known concentration of antigen) competes with the Enzantigen for Ig. This process relieves steric inhibition of the Enz-antigen. EMITs are suitable for analyzing small molecular weight antigens. The measured enzyme activity is proportional to the added analyte concentration. To perform EMIT requires access to pure antigen and Enz-antigen conjugate. Competitive ELISA uses enzyme-labeled antibody (Enz-Ig or EnzIg0 )* or Enz-Antigen conjugate as reagent: 1. Adsorb pure antigen on microwell plates (Fig. 1) 2. Block excess adsorption sites with milk protein. 3. In a separate step, preincubate Enz-Ig with the sample (containing an unknown concentration of antigen). 4. Transfer the mixture to the antigen-coated microwell plate. 5. Wash to remove excess reagent. 6. Assay the microwell plate for bound Enz-Ig using enzyme substrate. The enzymatic activity is inversely related to the concentration of antigen in step 3. An alternative one-step competitive ELISA is performed as follows: 1. Coat microwell plates with antibody. 2. Block nonspeci®c sites (as described above). 3. Add Enz-antigen and sample and allow competition for the microwell-bound Ig. 4. Wash microwell plates and assay with enzyme substrate. The enzyme activity is inversely related to sample antigen concentration. * Enz-Ig ˆ enzyme linked to a primary antibody speci®c for a food protein. Eng-Ig0 ˆ enzyme conjugate with antibody (Ig0 ) speci®c for the primary antibody, e.g., goat antibody for (rabbit) Ig.

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FIGURE 1 Competitive ELISA using enzyme-labeled antibody or enzyme-labeled antigen. (Top) Direct competitive ELISA, bound antigen (1) and free antigen (3) compete for Enz-Ig (2). (Bottom) Enz-Antigen (2) competes with antigens (3) for bound antibody (1).

Noncompetitive ELISA is performed using a direct, indirect, or sandwich format. For indirect ELISA the order of reagent addition is sample, ®rst antibody, Enz-Ig0 conjugate, and enzyme substrate (Fig. 2). After each step, wash the microwell plates to remove excess reagents. The ``indirect'' pre®x refers to the use of a second antibody (see EnzIg0 ) to visualize the bound antigen. With direct ELISA, the antigen is visualized (directly) using one antibody (Enz-Ig). Although requiring two antibodies, indirect ELISA is a more ¯exible and ultimately cheaper. The second antibody (Ig0 ) is produced by immunizing goat or sheep with the primary rabbit Ig. Commercially available Enz-Ig0 can be used for visualization wherever (rabbit) pAb is used for analysis. By contrast, a distinct Enz-Ig has to be synthesized for every new direct ELISA. The sandwich ELISA format may be implemented by binding ``capture''

250

FIGURE 2

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Formats for noncompetitive ELISA. Meat antigen (1) adsorbed on the surface of a microwell plate, (2) species-speci®c rabbit antibody, (3) enzyme-antibody directed against the rabbit antibody.

antibody to the microwell plate, followed by antigen. Thereafter one may adopt either the indirect or direct visualization strategy. Applications of ELISA for food analysis were ®rst discussed at a symposium held at the University of Surrey in September 1983 (4). The same theme was addressed by Hitchcock (5) at the second symposium on the application of immunoassays in veterinary and food analysis. Potential targets for immunoassay are listed in Table 2. Reviews dealing with ELISA for food analysis and authenticity testing include those by Allen and Smith (6), Samarajeewa et al. (7), Gazzaz et al. (8), Barai et al. (9), Lea and Morgan (10), Hernandez et al. (11), Taylor et al. (12), Smith (13), Simpkins and Harrison (14), Mandokhot and Kotwal (15), and Luethy (16). Hitchcock and co-workers (17)* were ®rst to use ELISA for food protein speciation. Compared with ELISA, other immunological assays have some of the following disadvantages: (a) a requirement for large

* They were af®liated with Unilever Research Laboratories, Colworth House, Bedford (UK).

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TABLE 2 Potential Food Analytes (Antigens) for ELISA Major groups Trace components/contaminants Mycotoxins Bacterial toxins Hormones and anabolic agents Drugs and Antibiotics Antinutrients Vitamins Plant hormones Pesticide and residues Additives Low molecular weight High molecular weight Food protein speciation Meat and egg Milk Blood Bacterial and fungal Plant proteins

Examples Ochratoxin A1, a¯atoxins Clostridium, Staphylococcus, E. coli Natural, synthetic reproductive or growth-affecting hormones Solanine, trypsin inhibitor Indoleacetic acid, abscisic acid Colors, ¯avors Gums, stabilizers, emulsi®ers Beef, buffalo, camel, poultry Cow, ewe, goat's milk (speciation), speci®c milk proteins Serum protein speciation Single-cell proteins Soya, wheat gluten, pea, potato

Source: Adapted from Refs. 4 and 5.

quantities of antibody; (b) long assay times of between 18 and 24 hours, although 2±3 hours have been used for qualitative studies, and (c) high cost per analysis due to the cost of antibody. The advantages of ELISA compared with argar gel immunoassays include (18) (a) a 100- and 1000-fold lower requirement for antibody, (b) small sample size (1 mg of meat) per assay, (c) increased (10- and 100-fold higher) sensitivity, (d) more easily interpreted results, (e) screening for multiple adulterations possible, and (f) increased potential for partial automation. ELISA is a major technique for meat protein speciation (Table 3). Implementing ELISA involves the following stages: (a) production of antibody, (b) puri®cation of antibody by af®nity chromatography, (c) preparation of Enz-Ig conjugate, (d) extraction of meat antigen, and (e) ELISA. Depending on past experiences, one can perform all operations in house or use a commercially available ready-to-use kit. Some off-the-shelf components may be combined with reagents prepared in house. Essential equipment and accessories include a 96-well microwell plate reader, microwell plate washer, precision micropipettes, and microwell plates. A

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TABLE 3 Speciation of Raw Meat by ELISA with pAb Analysis/comments ELISA Indirect ELISA, horse, beef Indirect ELISA, beef, camel, horse, kangaroo, sheep Sandwich ELISA, beef, sheep, horse, kangaroo, pig, camel, buffalo, and goat Sandwich ELISA, pork, beef Sandwich ELISA, beef, buffalo Indirect ELISA, beef, pork, horse Meat identi®cation ELISA kits (UK) Sandwich ELISA,a beef, pork Sandwich ELISA,a chicken Indirect/competitive ELISA, direct/competitive ELISA, pork, beef Indirect ELISA, hand or mechanically recovered chickena meat Seafood (sardine, tuna, and crustacea)

Reference Engvall and Perlman (1) Kang'ethe et al. (19) Whittaker et al. (18,20) Patterson et al. (21) Jones and Patterson (22) Patterson and Spencer (23) Jones and Patterson (24) Pelly and Tindle (25) Martin et al. (26,27) Martin et al. (27) Ayob et al. (29) Stevenson et al. (30) Taylor et al. (31)

a

Studies based on muscle protein antigen. Indirect ELISAs are noncompetitive methods unless stated.

plate reader enables colorimetric readings from microwell plates in situ. The 96 wells can be read within a space of 1.5 minutes. Several precision micropipettes are necessary for dispensing reagents; most essential are 50-mL and 100-mL pipettes. A continually adjustable (50±200 mL) multiwell pipette is convenient for rapid dispensing. Polystyrene microwell plates appear to be the solid phase of choice. 2.

RAW MEAT SPECIATION BY INDIRECT ELISA

Noncompetitive indirect ELISA is probably the simplest EIA format. A reasonably comprehensive description of this technique is provided here to encourage those wishing to implement this assay for the ®rst time. This section also considers the limits and tolerances of this technique. Method 1 Analysis of uncooked meat by noncompetitive indirect ELISA. Reagents 1. Detection antibody (e.g., peroxidase-labeled antibody)

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253

2. Antigen standard 3. Coating buffer (0.1 M sodium carbonate buffer, pH 9) 4. PBST (phosphate-buffered saline with 0.05% Tween 20)Ðwash and diluent buffer 5. Enzyme assay buffer (citrate-phosphate buffer, pH 4.2) 6. Enzyme substrate (variousÐsee the following) 7. Enzyme stopping solution (variousÐsee the following) Procedure 1. Coat microwell plates. Add 100 mL of meat extract (diluted in PBST) to microwell plates and incubate for 60 minutes at room temperature. Wash with PBST (100 mL) three times. 2. First antibody. Add 100 mL of rabbit antibody (diluted in PBST). Incubate for 30±60 minutes and wash wells with PBST (100 mL) three times. 3. Detection antibody. Enzyme conjugate. Add 100 mL of Enzantibody conjugate. Incubate for 60 minutes. Wash with PBST (100 mL) three times. 4. Enzyme assay. Add 100 mL of enzyme substrate. Incubate for 30 minutes. Add stopping solution and record absorbency reading with the plate reader. Not counting the PBST washing steps, indirect ELISA involves four steps. Perform preliminary experiments to establish the optimum sample (antigen) dilution as well as the required concentrations of antibody and Enz-Ig conjugate. Incubation times ranging from 30 minutes to 3 hours have been employed. Horseradish peroxidase (HRP) is the most common enzyme label for ELISA. Suitable substrates for HRP are ABTS [2,20 -azino-di(3ethylbenzthiazoline sulfonate)] and hydrogen peroxide. Other HRP substrates are listed in Table 4. Alkaline phosphatase, urease, and glucose oxidase have also been used as labels. Kang'ethe et al. (19) developed indirect ELISA for horse meat. Polyclonal antibody (pAb) for horse serum albumin (HrSA) was raised by immunizing rabbits. Preliminary tests using AGID assay showed that (rabbit) pAb cross-reacted with BSA and sheep serum albumin (SSA). Therefore crude (rabbit) pAb for HrSA was puri®ed by column immunoadsorption. Antibody samples were diluted by about 100 1 and meat extracts diluted by 200 1 and 3200 1 before assay. Indirect ELISA was performed essentially as described in Method 1. Substitution of beef with 5± 80% horse meat produced the calibration response   1 DA492 K 1 %Horse ˆ ln …1† C K2

254 TABLE 4

Chapter 9 Enzymes and Substrates for ELISA

Enzyme Horseradish peroxidase

Horseradish peroxidase

Horseradish peroxidase

Horseradish peroxidase

Horseradish peroxidase

Alkaline phosphatase

Substrate and assay conditions ABTS (2 mM) ‡ H2O2 (2 mM), citratephosphate (0.1 M, pH 4.2), DA ˆ 414 nm o-Dinisidine (0.08%) ‡ H2O2 (0.006%), citratephosphate buffer (0.1 M, pH 5); DA ˆ 620 nm o-Diphenylenediamine ‡ H2O2 in citratephosphate buffer (0.1 M, pH 5), DA ˆ 492 nm o-Toluidine (2.5 mM) ‡ H2O2 (2.5 mM), citratephosphate (0.1 M, pH 4.5), DA ˆ 620 nm 3,30 ,5,50 -Tetramethylbenzidine (1 mM) ‡ H2O2 (3 mM) in 0.2 M citrate buffer (pH 3.95), DA ˆ 450 nm p-Nitrophenyl phosphate (1 mg/mL) in diethanolamine buffer, pH 9.8, DA ˆ 405 nm

Stopping solution 30 mL of NaCN (37 mM) or 50 mL citric acid (0.1 M) 50 mL of H2SO4 (4 M)

50 mL of H2SO4 (12.7 M)

50 mL of H2SO4 (12.7 M)

100 mL of H2SO4 (3 M)

25 mL of NaOH (0.4 N NaOH)

Source: Compiled from multiple sources in Table 3.

where C, K1, and K2 are constants and A492 is the absorbance reading. A simple straight-line equation applied for 0±60% substitution of beef by horse meat. The assay precision was 2.3±8%. Whittaker et al. (18,20) employed indirect ELISA for the identi®cation of uncooked meat from cattle, camel, horse, kangaroo, and sheep. To improve speci®city, (rabbit) pAbs for serum proteins were puri®ed via af®nity chromatography. Antigen adsorption to microwell plates was optimum at pH 5±6. The working range for analysis was 10±80% (w/w) adulteration.

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255

3. RAW MEAT SPECIATION BY SANDWICH ELISA The ®rst step for sandwich ELISA is coating microwell plates with capture antibody. Then meat extract is added followed by either Enz-Ig conjugate or the combination of second Ig ‡ Enz-Ig0 . Binding meat antigens to a ``bed'' of antibody introduces selectivity. Noncomplementary proteins do not bind to the ®rst antibody and can therefore be washed from the microwell plate. Further speci®city derives from the second antibody (Fig. 3). Patterson et al. (21) were the ®rst to develop a sandwich ELISA for meat speciation. Different ELISA tests were produced with speci®city for meat from buffalo (water buffalo),* camel, cattle, goat, horse, kangaroo, pig, or sheep. Capture pAbs were usually raised by immunizing sheep with whole serum protein from camel, cattle, goat, etc. Speci®c pAb for sheep was produced using cattle as host. The yield of pAb was greater if the host animal was phylogenetically different from the donor species. Sheep produced greater quantities of pAb when injected with kangaroo antigen as compared with beef antigen. Detection pAbs were raised using rabbits. Samples of crude pAb were puri®ed by af®nity chromatography using the complementary antigens immobilized on CNBr-activated Sepharose.

FIGURE 3

Sandwich ELISA format. (1) Microwell plate±bound ®rst (capture) antibody, (2) meat antigen, (3) second (usually rabbit) antibody, and (4) enzyme-conjugated goat antibody for rabbit IgG.

* The American plains bison is also buffalo. The buffalo referred to in this chapter is the water buffalo of Asia and Africa called simply buffalo in the literature.

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Tests with 8 pAbs versus 8 meat extracts (64 tests) showed three false positives. The (cow) pAb for sheep cross-reacted with goat meat. However (sheep) pAb for goat did not cross-react with sheep. The (sheep) pAb for beef cross-reacted with buffalo meat, but (sheep) pAb for buffalo did not react with beef. By choosing the host animal for antibody production carefully, speci®c pAb could be produced for sandwich-ELISA. The relatively high pAb speci®city was ascribed to the following factors: (a) choice of host species (sheep or goats produced more speci®c antibodies than rabbit or mice) and (b) choice of antigen. Using whole serum protein for immunization, rather than a single pure protein, introduces many antigenic determinants. The pAbs are produced that are more discriminating between species. Meat samples (1 g) were extracted by 10 mL of solvent and diluted by a factor of 10 1±5000 1 before analysis. The LLD was 1% (w/w) kangaroo meat substitution for beef or 1% (w/w) substitution of goat meat for sheep. Cross-reactivity between closely related species (beefbuffalo, goat-sheep, and donkey-horse) was evident. Patterson and Spencer (23) also produced monospeci®c pAbs for buffalo, goat, or donkey using cattle, sheep, or horse as host, respectively. Each pAb was then puri®ed by immunoaf®nity chromatography. Thus, (sheep) pAb for goat was puri®ed with a column of Sepharose±goat serum protein. Bound pAb was eluted with ammonium thiocyanate (2.5 M, pH 7.0), desalted by gel ®ltration with Sephadex G25, and concentrated by ultra®ltration. The pAb sample containing 8 mg mL 1 protein was divided into two portions; half was covalently conjugated to HRP for detection and the other half was used for capture. Enzymatic detection was via o-toluidine±hydrogen peroxide (Table 4). From visual inspection the LLD was 0.1% (w/w) donkey meat added to horse meat or 0.1% (w/w) goat meat added to mutton. Beef adulteration with > 1.0% (w/w) buffalo meat was detectable. Jones and Patterson (22) showed that it was possible to detect 0.5±1% (w/w) adulteration of beef by pork using a sandwich ELISA. The linear range of analysis was 1±3%. The capture antibody was (rabbit) pAb for porcine serum albumin (PSA). The detector pAb was produced with a sheep host. Both (rabbit) pAb and (sheep) pAb for PSA were puri®ed by a twostage af®nity procedure. The order of reagent addition was (rabbit) pAb for PSA, meat extract, (sheep) pAb for PSA, and HRP-pAb conjugate for sheep Ig. The (sheep) pAb for PSA was unstable when directly adsorbed on microwell plates.* Presumably, instability prevents the preparation of HRP

* Reagent stability is an important feature of sandwich ELISA. Once coated with capture antibody, microwell plates may be dried and stored at refrigerator temperatures for 6 months.

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257

conjugate with (sheep) pAb for PSA. Also important for assay design is the low speci®city of (rabbit) pAb for PSA. Using (sheep) pAb for PSA to complete the ``sandwich'' improved assay sensitivity. For details of the sample pretreatment see footnote.* The LLD was 1% (w/w) pork in minced beef, beef sausage mix, or beef burger mix. For 1±10% (w/w) substitution, the assay response was described by the relation A492 ˆ 0:301 ‡ 0:153 log…%Pork†

…2†

These results should be compared with the analysis of pork in commercial meat (ham, pork-soy sausages, pate) products (29) by indirect ELISA. Using af®nity-puri®ed (rabbit) pAb for pork, the linear range of analysis was 1±40% (w/w) substitution.

4. MUSCLE PROTEIN ANTIGENS FOR ELISA The ELISAs described so far were speci®c for residual blood proteins within meat. These assays are unsatisfactory. Cross-contamination by blood from another species gives a positive ELISA test (26,30). Furthermore, the amount of blood lost during the conversion of muscle to meat is variable (32). Attempts to identify different cuts of meat using ELISA for blood serum proteins were not successful. There were large variations in the blood content in different samples (33). Martin and co-workers (26) developed a sandwich ELISA for (porcine) muscle protein. Extracts of pork diluted by between 20 1 and 20,480 1 gave absorbance readings of < 1.0. Substitution of beef with 1±50% (w/w) pork led to the calibration response A492 ˆ 0:268 ‡ 0:114 ln…%Pork†

…3†

With experienced personnel, the assay precision was 2±3%. In another study, chicken muscle antigen was isolated using an af®nity column ®lled with Sepharose±protein A complex with (rabbit) pAb for chicken (28). Bound antigen was eluted with diethylamine buffer (0.05 M, pH 11.5). An * The assay was calibrated using samples of 0±10 g of pork added to 1±2 kg of lean minced beef. The mixture was blended with 900 mL of distilled water for 2 minutes, ®ltered through Whatman No. 3 paper, and stored at 208C. Other meat products of known formulation were also analyzed. Ten-kilogram amounts of beef burger and beef sausage mixtures were prepared according standard recipes. Then 40-g samples were extracted with 360 mL of water and ®ltered. The resulting extracts were diluted with PBST for immunoassay. Samples were diluted by 50 1 and 250 1 for assay.

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SDS-PAGE analysis followed by Western transfer and immunostaining showed that the chicken-speci®c antigen was probably pyruvate kinase. The antigen was used for sandwich ELISA with both the capture and detector antibody being (rabbit) pAb. Substitution of beef with 1±10% (w/w) chicken meat gave the following response: A492 ˆ 0:621 ‡ 0:161 ln…%Chicken†

…4†

The corresponding assay for pork adulteration with 1±10% (w/w) chicken meat led to the following performance. A492 ˆ 0:590 ‡ 0:154 ln…%Chicken†

…5†

Stevenson et al. (30) developed an assay for mechanically recovered chicken meat and hand-deboned chicken using indirect ELISA for bone marrow antigen. Crude (rabbit) pAb was puri®ed by ammonium sulfate precipitation and af®nity chromatography using Sepharose-immobilized bone marrow antigen extracted with 7 M urea. The ELISA was visualized using af®nity-puri®ed (rabbit) pAb for chicken bone marrow, followed by commercial HRP-labeled (goat) pAb for rabbit IgG. HRP was assayed with the substrate 3,30 ,5,50 -tetramethylbenzidine (TMB), which, unlike o-diphenylenediamine, is noncarcinogenic. The ®nal assay showed only slight selectivity toward MRM as compared with hand-deboned meat. There was no cross-reactivity with hand-deboned beef, mutton, or pork. Substitution of beef with MRM chicken could be detected at levels of 2±50%. The identity of muscle protein antigens has not been fully established. An SDS-PAGE analysis of soluble protein from horse meat revealed *20 proteins. Immunoaf®nity chromatography using immobilized (rabbit) pAb for horse muscle reduced the number of SDS-PAGE bands to nine. Three antigens (37, 70, and 96 kDa) increased in concentration after af®nity chromatography (34). SDS-PAGE also featured in the partial identi®cation of the bone marrow antigen from chicken (30). Proteins from bone marrow or muscle were separated by SDS-PAGE followed by Western blot transfer to a nitrocellulose membrane. The bound protein was visualized with species-speci®c (rabbit) pAb and commercially available HRP-labeled (goat) antibody for rabbit IgG. Immunostaining revealed three antigenic proteins with molecular sizes of 69, 45, and 96 kDa. The 69-kDa protein was tentatively identi®ed as chicken serum albumin. The identities of the 45- and 96-kDa proteins are not known. The case for adopting muscle protein antigens for ELISA is compelling. However, the extra effort involved in isolating muscle proteins may require further justi®cation. Fig. 4 shows results for sandwich ELISA using pAb speci®c for muscle soluble protein or blood serum albumin. These

Speciation of Meat Proteins

FIGURE 4

259

Blood versus muscle protein antigen for meat speciation using sandwich ELISA. Adulteration of beef with pork was analyzed using (rabbit) pAb for porcine muscle protein (open circles) or pAb for porcine serum albumin (closed circles).

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results are comparable. Furthermore, it is not certain that the quantity of soluble muscle proteins is the same for different meat tissues from any single species. Myoglobin levels vary with tissue type and levels of exercise.

5. 5.1.

COOKED MEAT ANALYSIS BY ELISA Boiling-Resistant Ethanol-Soluble (BE) Antigen

Kang'ethe and Lindqvist (35) found that BE antigen was not wholly suitable for indirect ELISA. The antigen showed irregular adsorption to microwell plates because of the presence of extraneous proteins (probably gelatin). Samples of BE antigen gelled at 48C. Notwithstanding partial puri®cation by size exclusion chromatography, indirect ELISA using BE antigen yielded poor sensitivity (36). Tests involved (goat) pAb for partially puri®ed BE antigen from 4 domesticated species (cattle, camel, pig, and sheep) and 14 games species.* With a total of 324 tests (18 antibody 6 18 meat samples), no cross-reactivity was observed using pAbs for water buffalo, camel, horse, topi, and pig. The (goat) pAb for cattle BE antigen cross-reacted with virtually every species tested. Using the appropriate species-speci®c (goat) pAb between 1 and 10% (w/w) adulteration of beef (with buffalo), pork (with warthog), or goat (with impala) was detectable. The sensitivity of meat tests using BE antigen was improved 100-fold by adopting the sandwich ELISA format (37). The higher performance was attributed to BE antigen binding to capture antibody rather than to a ``bare'' microwell plate. Direct binding to surfaces alters antigen conformation and can reduce antibody binding (38,39). In all, six sandwich ELISA tests were developed using (goat) pAb for BE antigen from buffalo, bushpig, camel, cattle, horse, and pig. Each assay was tested with meat samples from 14 species.* The ELISA test for beef showed cross-reactivity for buffalo, horse, and bushbuck meat. All other assays were species speci®c. Adulteration of beef or pork with 1±20% (w/w) buffalo or camel meat was readily detected using the appropriate sandwich ELISA for these adulterants.

* Buffalo (Syncerus caffer), bushbuck (Tragelaphus scriptus), cattle (Bos indicus), eland (Taurotragus oryx), goat (Capra aegagrus hircus), Grant's gazelle (Gazella granti), impala (Aepyceros malampus), kongoni (Alcelaphus buselaphus cokei), oryx (Oryx spp.), sheep (Ovis ammonaires), Thomson's gazelle (Gazella thomsoni), topi (Damaliscus linatus), waterbuck (Kobus spp.), and wildebeest (Connochaetes taurinus).

Speciation of Meat Proteins

5.2.

261

Thermostable Muscle Protein Antigen

Hsieh and co-workers produced thermostable muscle protein antigens for analysis of cooked poultry (40), pork (41,42), or red meat (43). Lean meat paste was blended with three volumes of water or 0.85% saline. The resulting meat slurry was heated at 1008C for 15 minutes, cooled to refrigeration temperatures, and shaken gently for 2 hours. The supernatant obtained after centrifugation (14,000g) was employed as thermostable antigen. SDS-PAGE and Western blot analyses revealed that thermostable antigens from chicken skeletal muscle had molecular masses of 22±25, 30±35, and 120 kDa. The proteins were not identi®ed but are likely to correspond to troponin C, troponin T, and myosin fragment. Analysis of the antigen from other animal sources showed bands with molecular sizes ranging from 14.5 to 26.5 kDa. Thermal-stable muscle protein antigens were the basis for developing mAb for ELISA (Sec. 6). Rencova and co-workers described indirect competitive ELISA tests for heat-processed meat from horse, kangaroo, poultry, or rat. The tests involved (rabbit) pAb for heat-stable muscle antigen. This antigen was prepared by heating meat sample extracts with PBS at 100±1208C for 30 minutes. The assay detected chicken or kangaroo meat within commercial meat products from the retail market in the Czech Republic (44). 5.3.

Native Thermostable Antigen

Native thermostable antigens (nTAs) are produced from raw tissue by ammonium sulfate fractionation and ion-exchange chromatography on carboxymethylcellulose (45). The isolation process, although technically straightforward, lasts several days. The yield of nTA was 25 mg per kg of meat. Sandwich ELISA for using nTA for chicken cross-reacted with turkey (Fig. 5). No cross-reactivity occurred with red meat (beef, deer, horse, kangaroo, or sheep). Both cooked and uncooked poultry meat could be analyzed. This is understandable because a genuinely heat-resistant meat antigen should be unaffected by thermal treatment. A sandwich ELISA using nTA for pork was also developed (Fig. 6). The nTA-based tests can detect chicken or pork in a wide range of cooked meat products: (a) frankfurters (horse, beef, pork, sheep, deer, chicken or turkey); (b) bologna (beef, pork, chicken, or turkey); (c) chopped, pressed, and sliced meats (beef, ham, chicken, turkey); (d) canned baby foods (beef, pork, lamb, chicken, or turkey); and (e) canned meat spreads (beef, ham, chicken). In every case, product varieties containing poultry or pork were correctly identi®ed. The LLDs for chicken and pork were 126 and 250 ppm, respectively. The sensitivity, ascribed to the biotin-streptavidin

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FIGURE 5 Speci®city of sandwich ELISA for chicken native thermostable antigen. (Drawn using results from Ref. 45.)

ampli®cation system, was more than adequate to detect meat adulterations of practical signi®cance. Assay performance was not affected by the nature of the meat matrix. The nTA was also the basis for sandwich ELISA tests for beef, deer, horse, and mutton (46). The nTAs were routinely puri®ed by immunoadsorption (47,48). Even then some cross-reactivity occurred with the following samples: beef/American bison, goat/sheep, donkey/horse, whitetail deer/mule deer±caribou. With cooked meat adulterants the LLD was 0.16% (w/w). Apparently the assays were less ef®cient for raw meat. A commercial ELISA kit utilizing nTA was used by Hsieh et al. (49) to survey cooked meat adulteration in Florida. Commercial kits, said to be based on the USDA protocol, are available for cooked beef, pork, poultry, sheep, horse, and deer meat (ELISA Technologies, Alachua, FL). In the United Kingdom similar kits are available form Cortecs Diagnostics Ltd (Newtech Square, Deeside Industrial Park, Flintshire, CH5 2NT, UK). Potential dif®culties arising from the analysis of cooked meat samples containing high amounts of gelatin using commercial ELISA kits were described (50).

Speciation of Meat Proteins

FIGURE 6

263

Speci®city of sandwich ELISA for cooked pork based on antibody for native thermostable antigen. (Drawn using results from Ref. 45.)

The identity of the 50-kDa protein associated with nTA has not been established. Antibody for nTA did not react with a-acid glycoprotein (also called a-HS-glycoprotein) (45,46). The list of blood serum proteins includes immunoglobins (160 kDa), transferrin (85 kDa), albumin (66 kDa), Ig fragment (45 kDa), a1-antitrypsin (45 kDa), orosomucoid (44 kDa), GC globulin (51 kDa), a-HS-glycoprotein (49 kDa), g-globulin (25 kDa) and b2-microglobulin (11.8 kDa). The a-HS-glycoprotein is probably the most heat-stable serum protein. However, several serum proteins have molecular sizes around the region 45±50 kDa (51). The thermal stability characteristics of nTAs are unlike those of other muscle proteins (Table 5, Fig. 7). Levieux et al. (52) heated readily extractable muscle proteins and analyzed the residual soluble proteins by QSDS-PAGE. The order of thermal resistance was albumin > myoglobin ˆ lactate dehydrogenase (M4) > IgG > transferrin. All proteins were completely denatured by heating at > 808C for 30 minutes. I estimate that the half-life (t1/2) of chicken nTA is 213.6 or 52 minutes at 100 or 1258C, respectively. Turkey or beef nTAs were relatively less heat resistant by comparison with t1/2 of 51.3 or 103 minutes at 1008C. These t1/2 values were reduced to 35 minutes (turkey nTA) or 68 minutes (beef nTA) at 1208C. The heat deactivation mechanism for nTA was also different from

264 TABLE 5

Chapter 9 Thermal Inactivation Parameters for Some Soluble Muscle Proteins DH# (kJ mol 1)

DS# (J mol 1 K 1)

IgG

184.3

280

LDH (M4)

340.7

740

Myoglobin

455.7

1040

Albumin

475.3

1130

Muscle protein

Chkn-nTA

86

49

Trk-nTA or pork-nTA

22

201

Arrhenius equation ln k ˆ 58.6 (1/T) ln k ˆ 113.0 (1/T) ln k ˆ 136.4 (1/T) ln k ˆ 150.6 (1/T) ln k ˆ 17.9 (1/T) ln k ˆ 1.25 (1/T)

2.23 6 104 4.05 6 104 4.90 6 104 5.3 6 104 1.03 6 104 2.67 6 103

Source: Based on data from Refs. 45, 46, and 60.

FIGURE 7 Simulated thermal inactivation pro®les for bovine muscle proteins and native-thermostable antigen from chicken (Chkn-nTA) or turkey (Trk-nTa). Samples are held at 20±958C for 30 minutes. (Calculated from data in Table 5.)

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265

that for the other muscle proteins. The activation enthalpy change (DH#) and entropy change (DS#) for heat deactivation were large and positive for most of the muscle proteins. This is indicative of large conformational changes being the rate-limiting step during heat denaturation. For nTA we ®nd that DH# < 100 and DS# was negative. Low transition state parameters are characteristic of conformationally plastic proteins (53). Such proteins survive heat treatment owing to the ability to refold once the thermal stress is removed. A low DH# can also arise where bioactivity (antigenicity) resides in lower order (primary or secondary) protein structure. Simulated thermal inactivation pro®les for nTA and some other muscle proteins are compared in Fig. 7. I have assumed that (a) the Arrhenius equation (Table 5) applies over a temperature range of 20±1208C and (b) thermal deactivation kinetics are ®rst order (54). Caution is also warranted because the initial data (45,60) for modeling came from a limited temperature range of 54±668C for muscle proteins and 100±1208C for nTA. 5.4.

End-Point Temperature Determination

According to USDA guidelines, meat imported into the United States should be heated to a certain minimum end-point temperature (EPT) to ensure that it is free from pathogens and viruses. Compliance with the USDA guidelines on meat EPT can be assessed by ELISA (55±59). For example, Denise Smith and co-workers developed a sandwich ELISA for chicken or turkey skeletal muscle lactate dehydrogenase (LDH). Heart LDH was detected with 1000-fold lower af®nity. No cross-reactivity occurred with porcine or bovine skeletal muscle or heart muscle LDH. The limit of detection was 1 ng mL 1 or 1 ppb. Heating to an EPT of 68.3±728C inactivated muscle LDH. Didier and Annie Levieux (60) also developed immunological EPT indicators.

6. MONOCLONAL ANTIBODIES FOR MEAT SPECIATION The production and use of pAbs for protein analysis has several disadvantages: (a) the process involves live animals and results in batchto-batch variation in pAb, (b) there is a requirement for reagent standardization with respect to pAb concentration and af®nity for antigen, (c) there is limited pAb production from a single animal host, and (d) crude pAb requires purifying to lessen cross-reactivity. This process is technically demanding, slow, and expensive. Such disadvantages can be overcome with hybridoma technology for monoclonal antibody (mAb) production.

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Between 1989 and 1999 two research groups explored the use of mAbs for meat speciation. One group (from Spain) utilized af®nity-puri®ed (unheated) meat antigen for immunization. Hsieh's group at the Department of Nutrition and Food Science, Auburn University initially used (crude) thermostable meat antigens. The comparatively small number of publications describing ELISA with mAbs are summarized in Table 6.

6.1.

Poultry (Chicken and Turkey)

Af®nity-puri®ed chicken muscle antigen (28) (Section 4) was used for mAb production. Three hybridoma cell lines (designated AH4, BC9, and CF2) were identi®ed that produced mAb for chicken muscle antigen. SDS-PAGE and immunoblotting revealed that mAb-CF2 was speci®c for chicken muscle pyruvate kinase (58 kDa). Both mAb-AH4 and mAb-BC9 bound to a 47-kDa protein tentatively identi®ed as 3-phosphoglycerate kinase. Martin et al. (62) puri®ed mAb-BC9 by ion-exchange chromatography with a Mono-Q column for use as capture antibody. The detection antibody for their sandwich-ELISA was (rabbit) pAb for chicken muscle antigen. Visualization was by commercial HRP-labeled (goat) antibody for rabbit IgG. A sandwich ELISA using mAb-BC9 was selective for raw poultry (chicken and turkey) meat. No cross-reactivity was observed with beef, horse, pork, rabbit, or mutton. There was also no reaction with puri®ed proteins such as casein, gelatin, or soy protein. The sandwich ELISA test TABLE 6 Monoclonal Antibody for the Speciation of Raw or Heated Meat Proteins by ELISA Analysis, commentsa Chicken Chicken Porku,c Horse Chicken LDHc Turkey LDHc Poultryc Meat (beef, pork, etc.)c Central nervous system tissue a

Reference MartiÂn et al. (61) Martin et al. (62) Morales et al. (63), Chen et al. (41), Chen and Hsieh (42) Garcia et al. (64) Abouzied et al. (55) Wang and Smith (57) Sheu and Hsieh (40) Hsieh et al. (43), Chen et al. (41) O'Callaghan (65), Schmidt et al. (66)

c, Cooked or autoclaved meat, all other samples, and u, unheated samples.

Speciation of Meat Proteins

267

responses were described by A405 ˆ 0:455 ‡ 0:47 ln…%Chicken†

…6†

A405 ˆ 0:404 ‡ 0:46 ln…%Chicken†

…7†

for beef or pork adulteration by chicken, respectively. The linear dynamic range for analysis was 1±100% (w/w). The preceding assays are for raw meat samples only. Notice that the sandwich ELISAs using mAb are more sensitive than those with pAb [Eqs (4) and (5)]. 6.2.

Pork and Horse Meat

A pork-speci®c mAb-DD9 did not react with beef, chicken, horse, casein, soy, gelatin, or BSA (63). The mAb-DD9 was prepared from unheated muscle protein antigen puri®ed by immunoaf®nity chromatography (26,27). Indirect ELISA utilizing mAb-DD9 detected beef or chicken adulteration by 1±100% (w/w) pork. The calibration response was described by A405 ˆ 0:0692 ‡ 0:7989 ln…%Pork†

…8†

A405 ˆ 0:0745 ‡ 0:7612 ln…%Pork†

…9†

for beef and chicken samples, respectively. The LLD was 0.1% (w/w), which is below levels probably economically advantageous to the retailer. The assay sensitivity compares favourably with results expressed in Eq. (3). Heating meat samples to 658C for 30 minutes had no adverse on the assay reponse, but autoclaving samples at 1208C for 20 minutes led to loss of assay sensitivity. Chen and Hsieh (42) have recently described an mAb-based ELISA for detecting the presence of pork within cooked or processed meat products. The assay employs porcine thermostable antigen (Sec. 5.3). The LLD for pork was 0.5% (w/w) with intrassay and interassay precision of 5.8% and 7.9%. The highly accurate method was able to identify pork in 45 commercial processed meat samples. Sawaya and co-workers (67) also produced an ELISA test sensitive to cooked pork although they used pAb. Horse meat±speci®c mAb-DD3 (64) showed no cross-reactivity for beef, chicken, pork, soy proteins, casein, gelatin, or BSA. Addition of 0±50% (w/w) horse meat to beef led to the following ELISA response. A405 ˆ 0:4626 ‡ 0:0314 …%Horse†

…10†

The LLD for horse meat was 2% (w/w). The Spanish group suggest that

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antigen puri®cation by af®nity chromatography may be necessary for mAb production. 6.3.

Cooked Red Meat

The mAb-2F8 had selectivity for beef, sheep, or lamb and reduced sensitivity for deer meat (43). There was no cross-reactivity with raw pork, beef, lamb, mutton, or deer. Chicken or turkey was not detected in either the raw or cooked state. Indirect ELISA using mAb-2F8 was speci®c for red meat. Adulteration of poultry (chicken and/or turkey) with 0.5±15% (w/w) beef, horse, sheep, or pork was easily detectable. The LLD for deer meat was * 5% (w/w) addition to poultry. Multiple adulterants produced a cumulative signal. Red meat is often more expensive than chicken. However, the preceding test will be useful for detecting the substitution of chicken meat with less valuable beef or pork trimmings (49). Chicken is also in higher demand in some parts of Asia where the consumption of beef is restricted by religious stricture. 7.

FISH AND SEAFOOD IDENTIFICATION BY ELISA

Seafood adulteration may be growing in importance for three main reasons. First, there is a trend away from red meat toward the consumption of ®sh and seafood. Fish and seafood are perceived as healthy because of their higher content of unsaturated fatty acids. Second, world ®sh stocks continue to decline, increasing the economic incentive for adulteration. Third, improvements in processing technology have led to larger markets for comminuted ®sh products (68) and ®sh protein concentrates (69). These products are dif®cult to identify visually and offer signi®cant scope for adulteration. Speciation by immunological methods is seen as complementary to traditional methods such as isolectric focusing and SDS-PAGE (70). Suzuki (71) used an agar gel diffusion assay to identify tuna. Fish protein authentication using ELISA is discussed in this section. 7.1.

Sardine and Tuna

Taylor and Jones (72) and also Taylor et al. (73) described an indirect ELISA using (rabbit) pAb for soluble antigens from canned sardine, bonito, yellow®n tuna, or skipjack tuna. Crude (rabbit) pAb for canned ®sh antigen was nonspeci®c. High responses were obtained for most canned ®sh samples (Fig. 8). To improve the speci®city, pAb was puri®ed via (a) batch immunoadsorption, (b) immunoadsorption using antigens immobilized on

Speciation of Meat Proteins

269

FIGURE 8 Noncompetitive indirect ELISA for the identi®cation of canned ®sh species. Graph shows the speci®city of crude (rabbit) antibody for different canned ®sh samples. (Based on data from Ref. 64.)

magnetic beads, and (c) immunoaf®nity chromatography using the antigen immobilized on CNBr-activated Sepharose. Tuna species (albacore, yellow®n, and skipjack) were dif®cult to differentiate from each other or from bonito (a potential tuna substitute). Attempts to increase the speci®city of pAb for tuna species (by immunoadsorption) led to large reductions in the net concentration of free pAb. The indirect ELISA for canned ®sh was able to differentiate between ®sh and crustacea (prawn and scampi). The assay is potentially useful for detecting the adulteration of prawn, scampi, and other crustacean meat with cheaper ®sh (74). 7.2.

Rock Shrimp

SDS-PAGE analysis of seafood tissue extracts showed that protein M was unique to rock shrimp (Sicyonia brevirostris). The electrophoresis band corresponding to protein M was excised, homogenized with buffer, and centrifuged. The supernatant was dialyzed overnight and utilized as antigen for mAb production. Indirect ELISA using mAb-4H2±10D3 successfully

270

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identi®ed rock shrimp from three geographic locations in the United States. These samples were also correctly differentiated from 23 other seafood samples including white shrimp (from Colombia, Ecuador, Honduras, and Peru) and blue shrimp (from Ecuador). The speci®city for rock shrimp was attributed to the use of puri®ed antigen for mAb production. The test was more sensitive for heated samples probably because protein M was heated during isolation by SDS-PAGE. The antigen had molecular size of 17.7±18.5 kDa and made up *20% of the total water-soluble protein (75). 7.3.

Red Snapper

Adulteration of red snapper (Lutjanus campechanus) appears widespread. Approximately 14 out of 15 (91%) ®sh samples branded as red snapper by U.S. retailers were inaccurately labeled. Another survey involving 24 samples found 70% mislabeling. The high level of adulteration indicates genuine error on the part of retailers. Huang et al. (76) developed mAb-C1C1 and mAb-A1B1 for red snapper. The species-speci®c antigen (protein A, pI ˆ 5.93 ) was isolated by isoelectric focusing of red snapper muscle extract. Both mAb-C1C1 and mAb-A1B1 were applied for indirect ELISA. The detection system was (rabbit) pAb for mouse IgG conjugated to alkaline phosphatase. In tests involving 24 ®sh, seafood, and meat samples, indirect ELISA with mAb-C1C2 gave positive results for red snapper samples of vermilion snapper, lane snapper, mutton snapper, and yellowtail snapper. Using both mAb-C1C2± and mAb-A1B1±based ELISA tests simultaneously allowed a clear differentiation of red snapper samples from most other seafoods. 8.

PERFORMANCE CHARACTERISTICS FOR DIFFERENT ELISA FORMATS

Factors affecting assay performance include (a) the ELISA format, (b) the type of enzyme label, (c) antibody characteristics, and (d) the nature of the antigen used. Calibration graphs for sandwich ELISA [Eqs (6) and (7)] or indirect ELISA [Eqs (8) and (9)] show that the indirect format is more sensitive. Using pAb for capture can lead to loss of assay response for sandwich ELISA. Multiple interactions involved in pAb-antigen binding may leave few epitopes for the detector antibody. Poor assay sensitivity also arises when a sandwich ELISA utilizes the same mAb for both capture and detection. Under such circumstances, highly speci®c epitopes become occupied by the capture mAb with few left for binding with the (same) mAb for detection. Comparing the sandwich ELISA tests for muscle antigen

Speciation of Meat Proteins

271

shows that using mAb for capture enhances assay sensitivity by about an order of magnitude compared with the use of pAb. Finally, whether puri®ed antigens are necessary for mAb production is contentious. With mAb for unheated samples it seems necessary to use puri®ed antigen. However, thermostable antigens appear to induce speci®c mAb formation (see earlier). 9. MEAT TESTING FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AGENTS 9.1.

Meat and Bone Meal

Bovine spongiform encephalopathy (BSE) or ``mad cow disease'' is linked with feeding contaminated meat and bone meal to cattle. The ®rst recognized cases of BSE appeared in the United Kingdom in 1985±1986. New-variant Creutzfeldt-Jakob disease (nvCJD), thought to be a human form of BSE, was detected 10 years after the ®rst BSE cases (77). Transmission to other animals was demonstrated in laboratory studies with cats, pigs, goats, and sheep. The origin of BSE and other transmissible spongiform encephalopathies (TSEs) remains uncertain. One hypothesis highlights changes in meat rendering procedures in the late 1970s and early 1980s. The discontinuation of meat rendering processes involving hydrocarbon solvent±steam treatment may have led to a critical (even if slight) increase in the infectivity of BSE-contaminated feed. The theory, although compelling, is not necessarily accepted by all (78). The government of the United Kingdom banned the use of meat and bone meal in cattle feed in 1988. The incidence of BSE in the United Kingdom declined as a result of this ban, which was later extended to feed materials for all farm animals. However, large numbers of cattle remain affected with BSE. Infected cattle have also been discovered in the European Community countries including France and Belgium. Public health concerns about BSE seem likely to continue because the projected incubation period for nvCJD could extend from 4 to 30 years (79). Methods are being developed to monitor compliance with the legislation excluding meat and bone meal from feeds. Adequate heat treatment can also turn out safe meat and bone for use and/or disposal (80). The inactivation characteristics of the BSE agent were studied by Taylor and co-workers (79). BSE survives irradiation or boiling. Heating at 1218C (15 psi) leads to partial inactivation and thermal treatment at 1348C for 60 minutes produces complete inactivation. Meat-rendering plants in Germany heat treat samples at 1338C for 20 minutes at 3 bars of pressure. Klaus Hofmann used the Cortecs ELISA test kit for pork to test for bone and meat meal. Similar ELISA test kits exist for cooked beef or

272

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mutton. Samples of meat and bone meal heated to a rendering temperature of 1338C for 20 minutes were fully degraded and undetectable by ELISA (81,82). In the near future such tests might be used for routine monitoring of meat-rendering plants in Germany. Collaborative trials involving 21 laboratories from 12 countries (83) showed that these tests had suf®cient reliability as judged by a precision between 11 and 12%. Understerilized meat and bone meal were clearly distinguishable by a 10-fold greater assay signal. 9.2.

Central Nervous System Tissue

Concentrations of the BSE agent are higher in the central nervous system (CNS) tissue compared with peripheral nerves. A sandwich ELISA for CNS tissue was developed using mAb for GFAP (glial ®brillary acidic protein) (66). This protein is found only in astrocyte cells in the CNS.* The ELISA for GFAP had an approximate LLD of 1 ng (GFAP) with a linear range extending to 40 ng. The within-assay precision was 3.25±4%. While assay sensitivity remained constant for different matrices, the LLD increased in the order ground beef ‡ brain tissue > ground beef ‡ spinal cord tissue > puri®ed GFAP standards > brain > spinal cord. The ELISA gave the concentrations of GFAP in spinal cord tissue (55,000±220,000 ng mg 1 tissue), brain tissue (9000±55,000 ng mg 1 tissue), and cerebral cortex (17,000 ng mg 1 tissue). Neck muscle and ground beef were free of GFAP. The antigen is not very stable; therefore CNS tissue could be detected for only up to 8 days when samples were stored at 48C. CNS tissue was also analyzed using immunoblot analysis (84). The tests were directed at two antigens: GFAP and neuron-speci®c enolase. Immunoblot analysis did detect CNS tissue if samples were subjected to extreme temperature processing. 9.3.

Direct Immunological Detection of the BSE Agent

As of December 2000, the European Commission accepted ®ve direct tests for the BSE agent for further evaluation (85). The tests were produced by * The details of the sandwich ELISA for GFAP were essentially as described elsewhere. (a) Coat microwell plates with a commercial pAb for GFAP (supplied by Dako Corporation, Carpentaria, CA) at 378C for 1 hour or at 48C overnight. (b) Block nonspeci®c microwell plate sites with PBST±powdered milk protein. (c) React pAb-coated microwells with GFAP standards or samples for 60 minutes. (d) Add diluted mAb for GFAP (supplied by Boehringer Mannheim, Indianapolis, IN). (e) Add enzyme-labeled antibody, i.e., alkaline phosphatase± labeled (rabbit) pAb for mouse IgG. Assay for enzyme activity.

Speciation of Meat Proteins

273

ID Lalyated, Netherlands Imperial College of Science and Technology, UK Institute of Neurodegenerative Diseases, University of California, San Francisco Perkin Elmer Life Sciences, UK Prionics AG, Switzerland The BSE test produced by Prionics AG, Switzerland (Prionics AG, University of Zurich, 8057 Zurich, Switzerland) appears to be the favorite. Prionics-checkTM employs immunoblot analysis. Brain tissue extract is ®rst exposed to a protease solution followed by SDS-PAGE and then transferred to a nitrocellulose membrane by Western transfer. The membrane-bound proteins are detected immunologically using mAb speci®c for prion particles. The test is able to differentiate between the benign prion protein (PrPC) and the disease-causing PrPSc because the former is susceptible to protease attack but the latter is not. According to the advertising literature, Prionics-check is intended for (a) identi®cation of suspected BSE cases, (b) diagnostic testing in abattoirs and slaughterhouses, and (c) general monitoring for scrapie and BSE. Validation of the Prionics-Check tests has been documented (86). Prionicscheck will be used for mandatory BSE testing in the European Union from 2001. Some important characteristics of the Prionic-checks include (a) high selectivity and speci®city, (b) ability to distinguish cattle with BSE from those with other neurological disease states, (c) detection of subclinical cases of BSE, (d) ease of use and availability in a kit form, (e) suitability for ®eld use, (f) high sample throughput (the time of analysis is reportedly 6±8 hours from tissue extraction to ®nal test results), and (g) current use for Swiss BSE surveillance for all sick and falling cattle. At this time, Prionics AG manufacture at least two prion-speci®c mAbs (6H4 and 34C9) as well as pAb (RO29). The antibodies are suitable for developing ELISA. The company also has available the full kit for BSE detection. The life science diagnostics company Bio-Rad Ltd. currently manufactures an ELISA test for BSE (PLATELIA2 BSE test). This test is commercially available in the United Kindom, France, Germany, Belgium, Luxembourg, Norway, Sweden, Switzerland, Italy, and Spain. At this time the test is not being sold in the United States (87). The list of companies now entering the BSE diagnostics market is growing rapidly as shown by the following list (88).

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Abbeymoy Ltd. Bayer Boehringer-Ingelheim AG Commissariat a l'EÂnergie Atomique Genesis Biventures Mary Jo Schmerr New York State Basic Research Institute for Neurological Disorders Prionics AG

Altegen Inc. Caprion Pharmaceuticals Inc. Centre Suisse d'EÂlectronique et de Microtechnique SA Enfer Scienti®c Ltd.

Anonyx Inc. Bio-Rad Inc.

IGEN International Paradigm Genetics Inc.

Microsens Biohase Ltd. Nen Life Science Products Inc. Prion Developments Laboratory

Proteome Sciences Ltd. Q-One Biotech Ltd.

Celcus Inc. Disease Sciences Ltd.

V.I. Technologies Inc.

For further discussions of immunological tests for the BSE agent see Refs. 89 and 90.

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41. FC Chen, YHP Hsieh, RC Bridgman. Monoclonal antibodies to porcine thermal-stable muscle protein for detection of pork in raw and cooked meats. J Food Sci 63:201±205, 1998. 42. FC Chen, YHP Hsieh. Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA. J Assoc Off Anal Chem Int 83:79±85, 2000. 43. YHP Hsieh, S-C Sheu, RC Bridgman. Development of a monoclonal antibody speci®c to cooked mammalian meats. J Food Prot 61:476±481, 1998. 44. E Rencova, I Svoboda, L Necidova. Identi®cation by ELISA of poultry, horse, kangaroo and rat muscle speci®c proteins in heat-processed meat samples. Vet Med (Prague) 45:353±356, 2000. 45. RG Berger, RP Mageau, B Schwab, RS Johnston. Detection of poultry and pork in cooked and canned meat foods by enzyme-linked immunosorbent assays. J Assoc Off Anal Chem 71:406±409, 1988. 46. CD Andrews, RG Berger, RP Mageau, B Schwab, RW Johnston. Detection of beef, sheep, deer, and horse meat in cooked meat products by enzymelinked immunosorbent assay. J Assoc Off Anal Chem Int 75:572±576, 1992. 47. A Avrameas, T Ternynck. Biologically active water-insoluble protein polymers. I. Their use for isolation of antigens and antibodies. J Biol Chem 242:1651, 1967. 48. T Ternynck, S Avrameas. Polymerization and immobilization of proteins using ethylchloroformate and glutaraldehyde. Scand J Immunol Suppl 3:29± 35, 1976. 49. Y-HP Hsieh, BB Woodward, S-H Ho. Detection of species substitution in raw and cooked meats using immunoassays. J Food Prot 58:555±559, 1995. 50. K Hofmann, K Fishcher, E Mueller, W Babel. ELISA-test for cooked meat species identi®cation on gelatine and gelatine products. Nahrung 43:406±409, 1999. 51. GH Grant, JF Kachmar. The proteins of body ¯uids: plasma and serum proteins. In NW Tietz, ed. Fundamentals of Clinical Chemistry. London: WB Saunders, 1976, p 356. 52. D Levieux, A Levieux, A Venien. Immunochemical quanti®cation of heat denaturation of bovine meat soluble proteins. J Food Sci 60:678±684, 1995. 53. RKO Apenten, K Mahadevan. The heat resistance and conformational plasticity of Kunitz soybean trypsin inhibitor. J Food Biochem 23:209±224, 1999. 54. RKO Apenten. The effect of protein unfolding stability on their rates of irreversible denaturation. Food Hydrocolloids 12:1±8, 1998. 55. MM Abouzied, CH Wang, JJ Prestka, DM Smith. Lactate dehydrogenase as safe endpoint cooking indicator in poultry breast rolls: development of monoclonal antibodies and application to sandwich enzyme-linked immunosorbent assay (ELISA). J Food Prot 56:120±124, 1993. 56. CH Wang, JJ Pestka, AM Booren, DM Smith. Lactate dehydrogenase, serum protein, and immunoglobulin G content of uncured turkey thigh rolls as

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59. 60. 61. 62. 63. 64.

65. 66.

67. 68. 69. 70.

Chapter 9 in¯uenced by endpoint cooking temperature. J Agric Food Chem 42:1829± 1833, 1994. CH Wang, DM Smith. Lactate dehydrogenase monoclonal antibody immunoassay for detection of turkey meat in beef and pork. J Food Sci 60:253±256, 1995. DM Smith, LD Desrocher, AM Booren, CH Wang, MM Abouzied, JJ Pestka, J Veeramuthu. Cooking temperature of turkey ham affects lactate dehydrogenase, serum albumin and immunoglobulin G as determined by ELISA. J Food Sci 61:209±212, 1996. DS Smith, LD Desrocher. Immunoassays for determination of endpoint processing temperatures in poultry and beef products. J Muscle Foods 7:335± 344, 1996. D Levieux, A Levieux. Immunochemical quanti®cation of myoglobin heat denaturation: comparative studies with monoclonal and pAb. Food Agric Immunol 8:111±120, 1996. R MartiÂn, RJ Wardale, SJ Jones, PE HernaÂndez, RLS Patterson. Production and characterization of monoclonal antibodies speci®c to chicken muscle soluble proteins. Meat Sci 25:199±207, 1989. R MartiÂn, RJ Wardale, SJ Jones, PE HernaÂndez, RLS Patterson. Monoclonal antibody sandwich ELISA for the potential detection of chicken meat in mixtures of raw beef and pork. Meat Sci 30:23±31, 1991. P Morales, T GarciÂa, I GonzaÂlez, R MartiÂn, B Sanz, PE HernaÂndez. Monoclonal antibody detection of porcine meat. J Food Prot 57:146±149, 1994. T GarciÂa, R MartiÂn, P Morales, AI Haza, G Anguita, I GonzaÂlez, B Sanz, PE HernaÂndez. Production of a horse-speci®c monoclonal antibody and detection of horse meat in raw meat mixtures by an indirect ELISA. J Sci Food Agric 66:411±415, 1994. JP O'Callaghan. Quantitation of glial ®brillary acidic protein: comparison of slot-immunoassay with a novel sandwich ELISA. Neurotoxicol Teratol 13:275±281, 1991. GR Schmidt, KL Hossner, RS Yemm, DH Gould, JP O'Callaghan. An enzyme-linked immunosorbent assay for glial ®brillary acidic protein as an indicator of the presence of brain and spinal cord in meat. J Food Prot 62:394±397, 1999. WN Sawaya, MS Mameesh, E El-Rayes, A Husain, B Dashti. Detection of pork in processed meat by an enzyme-linked immunosorbent assay using antiswine antisera. J Food Sci 55:293±297, 1990. V Venugopal. Mince from low cost ®sh species. Trends Food Sci Technol 3:2± 5, 1992. SR Tannenbaum, BB Stillings, NS Scrimshaw, eds. The Economics, Marketing, and Technology of Fish Protein Concentrate. Cambridge, MA: MIT Press, 1974. W Sidwell. Fish speciation by immunochemical techniques. In: MRA Morgan, CJ Smith, PA Williams, eds. Food Safety and Quality Assurance. Applica-

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71. 72. 73. 74. 75. 76.

77. 78. 79. 80. 81. 82. 83. 84. 85. 86.

87. 88.

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tions of Immunoassay Systems. Barking, UK: Elsevier Science Publishers, 1992, pp 49±54. A Suzuki. Recovery of species-speci®c antigenicity from heat denatured serum protein of tuna. Bull Jpn Soc Sci Fish [Nihon Suisan Gakkai Shi] 41:373, 1975. WJ Taylor, JL Jones. An immunoassay for verifying the identity of canned sardines. Food Agric Immunol 4:169±175, 1992. WJ Taylor, NP Patel, J Leighton-Jones. Antibody-based methods for assessing seafood authenticity. Food Agric Immunol 6:305±314, 1994. WJ Taylor, JL Jones. An immunoassay for distinguishing between crustacean tailmeat and white ®sh. Food Agric Immunol 4:177±180, 1992. H An, PA Klein, K-J Kao, MR Marshall, MWS Otwell, C We. Development of monoclonal antibody for rock shrimp identi®cation using enzyme-linked immunosorbent assay. J Agric Food Chem 38:2094±2101, 1990. T-S Huang, MR Marshall, K-J Kao, WW Otwell, C Wei. Development of monoclonal antibodies for red snapper (Lutjanus campechansus) identi®cation using enzyme-linked immunsorbent assay. J Agric Food Chem 43:2301±2307, 1995. RG Will, JW Ironside, M Zeidler, SN Coursens, K Estibeiro, A Alperovitch, S Poser, M Pocchiary, A Hofman, PG Smith. A new variant Creutzfeldt-Jakob disease in the UK. Lancet 347:921±925, 1996. P Brown. On the origins of BSE. Lancet 352:252±253, 1998. D Taylor. BSE: our future in the balance. Chem Ind (June):444±447, 1998. P Brown. BSE: the ®nal resting place. Lancet 351:1146±1147, 1998. K Hofmann. Proof of proper heating at meat-and-bone meals. Fleischwirtschaft 76:1037±1039, 1996. K Hofmann, K Fischer, E Mueller, V Kemper. Experiments to demonstrate the effectiveness of heat treatments applied to canned meats and meat-andbone meals. Fleischwirtschaft 76:920±923, 1996. C von Holst, KO Honickel, W Unglaug, G Kramer, E Anklam. Determination of an appropriate heat treatment of animal waste using the ELISA technique: results of a validation study. Meat Sci 54:1±7, 2000. EH Luecker, E Eigenbrodt, S Wenisch, R Leier, M Buelte. Identi®cation of central nervous system tissue in retail meat products. J Food Prot 63:258±263, 2000. European Commission. DG24. Directorate B, Unit B3. The evaluation of tests for the diagnosis of transmissible spongiform encephalopathy in bovines (8 July 1999). http:/europe.eu.int./comm/dg24/health/bse. O Schaller, R Fatzer, M Stack, J Clark, W Cooley, K Bif®ger, S Egli, M Doherr, M Vandevelde, D Heim, O Oesch, M Moser. Validation of a Western immunoblotting procedure for bovine PrPSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathol 98:437±443, 1999. http://www.fda.gov/oc/opacom/hottopics/bse.html#3anchor. Prion Disease Diagnostics. http://www.mad-cow.org/00/feb01_last.html.

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10 Speciation of Soya Protein by Enzyme-Linked Immunoassay

1. INTRODUCTION There are usually guidelines for adding plant and other nonmeat protein to meat products. Food technologists use such ingredients legitimately to enhance functional properties such as water holding, fat binding, and gelation. Nevertheless, levels of nonanimal proteins in meat should be monitored. Much research has appeared in connection with soybean protein, this being the most important nonmeat protein ingredient. This chapter describes immunological methods for detecting bulk quantities (>0.5% w/w) of soybean protein in meat and meat products. The topic is dominated by methods of sample pretreatment designed to ensure accurate results no matter the sample processing history.

2. SAMPLE PRETREATMENT AND ANALYSIS OF SOY PROTEIN Hitchcock et al. (1) were ®rst to use ELISA for soy protein analysis. The assay was designed for a wide range of commercial soy samples including ¯our, protein isolates, and texturates. To correct for variable (heat) processing history, samples are pretreated with 8 M urea. Denatured soy 281

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protein is then renatured before analysis by competitive indirect ELISA. Details of this pretreatment regime are given later (Method 1). Developments leading to the eventual commercialization and of®cial approval for soybean protein ELISA tests are summarized in Table 1. With indirect ELISA (1),* the ®nal absorbance measurement is inversely related to the concentration of soy protein. The assay was highly speci®c with negligible responses toward beef, milk, ®eld bean, or wheat proteins. Speci®city was for conglycinin, which is the 7S soybean globulin (Fig. 1). The precision for soy protein determination was 10.5%. Grif®ths et al. (3) found that a commercial pAb for native and/or heat-denatured soy protein was as effective as the laboratory-developed (rabbit) pAb for renatured soy protein (Fig. 2). Collaborative testing of a commercial ELISA kit for soya protein involved 13 laboratories from the United Kingdom (4,7). The kits were supplied by Biokits Ltd. (Newtech Square, Deeside Industrial Park, Deeside, Clwyd CH5 2NU, UK), who also organized a 1-day workshop to familiarize trial members with the test procedures. For the actual trial, TABLE 1 Determination of Soybean Proteins by ELISA Analysis and comment First ELISA for soya protein ingredients (¯our, isolates, concentrates, and extrudates) Commercial pAb for soy protein ELISA Commercial pAb for soy protein in ELISA, collaborative study Europe-wide collaborative study ELISA of soya protein by immunoblotting Collaborative study of ELISA kit AOAC approval for ELISA for soy proteins Detection of soy milk in bovine milk

Reference Hitchcock et al. (1), Grif®ths et al. (2) Grif®ths et al. (3) Crimes et al. (4) Olsman et al. (5) Ravestein and Driedonks (6) Hall et al. (7) McNeal (8) Hewedy and Smith (9)

* The following steps are involved: (a) Pretreat soy sample and standards by denaturationrenaturation protocol. (b) React soy samples or standards with (rabbit) pAb for soy protein. (c) Add mixture to a microwell plate precoated with bound (renatured) soy protein. (d) Incubate to allow antibody-antigen reaction. (e) Wash thoroughly with PBST. (f) Add enzyme-labeled pAb for rabbit IgG. (g) Wash thoroughly with PBST. (h) Add enzyme substrate. (i) Stop reaction after a ®xed incubation time and record absorbance readings.

Speciation of Soya Protein

FIGURE 1

283

Competitive indirect ELISA for soya bean protein using commercially available (rabbit) polyclonal antibodies (1) or experimental (rabbit) polyclonal antibodies for renatured soya bean protein. (Drawn from Refs. 1 and 3.)

FIGURE 2 Effect of cooking temperatures on soy protein analysis in beefburgers using competitive indirect ELISA. (Drawn from Refs. 1 and 3.)

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participants analyzed duplicate samples of raw beefburger mix (three samples), raw sausages (two samples), cooked pate (three samples), and a trial sample. The average LLD was 0.7% (w/w) soy protein. The percent protein detected (accuracy) was 89 (+6.1)% (7). A soybean ELISA kit was also evaluated by Rittenburg et al. (10). The accuracy depended on the type of soy additive, type of food matrix, and subsequent processing. Protein recovery was 93 (+15.5), 75 (+7.5), 81 (+8.5), or 82 (+8.8)% for soy isolate, ¯our grit, concentrate, or texturate, respectively. For 72 retail meat products (including beef and/or pork sausages, bacon and ham loaf, and mince), the average recovery for soy protein was 91 (+12.3)% when added at levels of 1.2±1.6% (soy protein isolate), 2.4±5% (soy ¯our), or 4.8% (soy texturate). Autoclaving (1218C, 15 psi; 20 minutes) produced a signi®cant decline in accuracy (Fig. 2). Overall, the performance of the commercial ELISA kit was deemed acceptable. ELISA results agreed with the soy protein levels declared by most manufacturers. Method 1 Denaturation-renaturation sample treatment for soy protein antigens (1,4,7). Samples or soy protein standards are dissolved with hot ureamercaptoethanol* solution. This unfolds proteins and destroys S22S bonds. The sample is then transferred to a renaturation buffer. This treatment encourages protein refolding and restores soy proteins to a baseline conformation regardless of their previous thermal history. Reagents 1. Urea 2. Dithiothreitol (DTT) 3. Tris-HCl buffer (0.25 M, pH 8.6) Procedure Denaturation-extraction buffer (urea 10.6 M, DTT 18.8 mM in *25 mM Tris-HCl, pH 8.6). To a 250-mL volumetric ¯ask add 80 g of urea, 20 mL of Tris-HCl buffer (0.25 M, pH 8.6), and warm to dissolve. Add 30 mg of DTT to the hot solution, dissolve, and keep solution in a 1008C water bath. Add 60 mL of distilled water. Renaturation buffer (0.06 M NaCl with 7.5 mM L-cystine). Dissolve 1.8 g of L-cysteine with sodium hydroxide (1.0 M, 20 mL). Add the solution to 900 mL of 0.06 M NaCl solution. Adjust to pH 9 with 1 M HCl and make up to 1000 mL. * Mercaptoethanol (2-ME) was later replaced with the less odorous dithiothreitol (DTT).

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Sample extraction. Homogenize 12 g of ®nely chopped (meat) sample with 48 mL of Tris-HCl buffer (0.05 M, pH 8.6) using an UntraTurax homogeneizer. Add 2.5 g of meat homogenate or 40 mg of soy protein standard to a 50-mL ¯ask. Add 7.5 mL of urea-DTT solution, mix, and heat at 1008C for 60 minutes. Transfer the mixture to a 508C water bath. Renaturation. Add renaturation buffer (20 mL, prewarmed to 508C), mix, and then allow to cool to room temperature. Bring the ®nal volume to 100 mL and ®lter through Whatman No. I paper. Collect the ®rst 10 mL of ®ltrate for ELISA. Meat samples suspected of containing undeclared soy protein were extracted with acidic ethanol, followed by acetone, and then air dried to produce acetone powder. These samples and soy antigen (for immunization or precoating microwell plates) were dissolved using urea-DTT solvent and renatured as just described. In theory, the renaturation treatment transforms all soy protein to a baseline renatured state with reconstituted antigenic determinants. In practice, the recovery of antigenicity is found to be about 20% for 11S soy globulin and 70% for the 7S soy protein. Reasons for this are twofold. First, severe heat treatment leads to covalent modi®cation of protein side chains. The chemical changes (lysinoanaline formation, cross-link formation, deamidation, etc.) cannot be reversed by the renaturation procedure. Second, protein refolding is usually less than 100% ef®cient. Competing side reactions result in the formation of protein aggregates and misfolded structures with improperly aligned S22S bonds. The ranaturation procedure reverses protein sulfhydryl/-disul®de exchange and noncovalent interactions produced during food processing.

3. STRUCTURE, DENATURATION, AND RENATURATION OF SOYBEAN PROTEINS 3.1.

Soy Protein Structure

Soya beans contain between 40 and 50% protein by dry weight. The major protein groups are (a) storage proteins (70±80% total), (b) enzymes (notably lipoxygenase, lactate dehydrogenase), (c) protease inhibitors (notably the Bowman-Birk and the Kunitz inhibitors), and (d) other storage proteins (e.g., lectin). The foremost storage protein (*40% total protein) is 11S glycinin. It is a member of the leguminin family of proteins. The second storage protein (*30% total protein) is the 7S conglycinin. Glycinin (molecular mass of 350 kDa) is a hexamer of A-B subunits. The six acidic (A) and basic (B) subunit pairs are each joined by a disul®de

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bond. The A-B subunit is synthesized as a single polypeptide chain containing an intermolecular disul®de bond and a linker sequence. Posttranslational proteolysis removes the linker sequence, leaving the A and B subunits linked by a disul®de bond. Isoelectric focusing reveals ®ve Asubunit isoforms (A1, A2, A3, A4, A5). There are at least four B-chain isoforms (B1, B2, B3, B4). The structural characteristics of the glycinin were reviewed by Peng et al. (11), Nielsen (12), and Fukushima (13). Conglycinin (170 kDa) is a trimer. From the structural model proposed by Thanh and Shibasaki (14), conglycinin has six isomers. They are produced from the random combination of four subunits; a, a0 , b, and g. Conglycinin contains about 5% carbohydrate but is virtually devoid of cysteinyl residues or S2 2S bonds (15).

3.2.

Thermal Denaturation

The effects of heating on soy proteins were extensively investigated by nonimmunological techniques (16±21). Turbidimetric measurements showed that heating glycinin at 1008C (0.5% w/v in potassium phosphate buffer, I ˆ 0.5) led to aggregation. The rate of aggregation increased (while the net aggregation decreased) in the presence of 2-ME as a source of free sulfhydryl groups. The kinetics of aggregation conformed to a reaction order of 1.2. The Arrhenius plot for the reaction was biphasic between 70 and 908C (16). Studies using turbidity and UV difference measurements do not differentiate between intermolecular and intra-intermolecular effects such as dissociation, unfolding, and aggregation. Precise thermal stability data were obtained with differential scanning calorimetry (DSC). Glycinin (7±10% w/v) had a denaturation temperature (TD) of 85±948C, whereas the TD for conglycinin was 10±128C lower. The TD increased by a maximum of 308C with increasing salt (0±2 M NaCl) concentration for both glycinin and conglycinin (22±25). The ratio of calorimetric and Van't Hoff enthalpies was *1, implying that seed globulins denature via a one-step (all or nothing) process (24,25). By contrast, the kinetics of conglycinin and glycinin thermal denaturation is biphasic with separate dissociation and protein unfolding steps (26,27). Apparently, DSC studies do not register changes in soybean protein quaternary structure. The thermal dissociation of 11S glycinin leads to a 7S trimer and then to individual (3S) A-B subunits. Glycinin trimer has a sedimentation number equal to that of native 7S conglycinin and is therefore designated 7sÏ . Further heating produces disul®de bond lysis and the separation of the A and B subunits. The hydrophobic B subunit forms aggregates, leaving the A units in solution. From the many studies of glycinin denaturation comes the

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287

following commonly accepted scheme below. 11S „ 7s „ 3S ‰A subunits ‡ B subunitsŠ ; B Subunits …sol-aggregates†

…1†

; B Subunits …solid-aggregates† Processes in Equation (1) will be modi®ed by soy protein interactions with muscle proteins, lipid, rusk, and other ingredients. Indeed, TD for soy protein depends on the moisture level, ionic strength, pH, and the presence of sulfhydryl compounds including other meat proteins. Within a defatted ¯our matrix containing about 60% (w/w) moisture, TD for glycinin was 908C. The TD value increased to 1608C for absolutely dry ¯our (28,29).

3.3.

Effect of Heat Treatment on Glycinin Structure and Antigenicity

Preheating soy protein for 60 minutes in a high-ionic-strength medium (0.035 M phosphate buffer, pH 7.6 with 0.4 M NaCl) at 30±808C did not impair pAb binding with glycinin* at room temperature (30,31). By contrast, the thermal treatment of glycinin dissolved in low-salt solvent (0.035 M phosphate buffer, pH 7.6, with 0.15 M NaCl) produced a total loss of antigenicity (32). Apparently, pAbs for native glycinin bind to the nondissociated protein [Eq. (1)]. The antibody recognizes discontinuous epitopes formed by the intact protein. High salt concentrations stabilize glycinin. Thermal treatment at 1008C (5 minutes) led to a total loss of glycinin antigenicity in the presence of 0±0.7 M salt. However, the residual antigenicity increased in the presence of 0.7±2.0 M NaCl. Glycinin retained 90% antigenicity at the highest salt concentrations tested (33). Exposure to extremes of pH (pH < 2 and pH > 10) also diminished glycinin antigenicity. High acidity or basicity is known to cause the dissociation of glycinin (34). The order of anitgenicity for soy protein preparations (with a rabbit host) was 11S 4 A1a*A2 > A3 * The effect of heating on glycinin antigenicity was examined by agar gel double immunodiffusion (AGID) or single radial immunodiffusion (SRID). The tests involved crude (rabbit) pAb for native glycinin, puri®ed by immunoadsorption using ethylchloroformate cross-linked glycinin (see Chapter 8 for a description of this method). AGID assay for soy proteins showed speci®city for glycinin with no cross-reactivity for whey proteins or conglycinin.

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* B1 (35). No antibodies formed against A4, B3, and B4 glycinin subunits despite multiple injections. Gel immunodiffusion assays con®rmed that pAb for native glycinin did not react with isolated A or B glycinin subunits. 3.4.

Effect of Heat Treatment on Conglycinin Structure and Antigenicity

Immunoanalytical investigations reveal that b-conglycinin is more heat resistant than glycinin. Heating conglycinin (in 0.035 M K-phosphate buffer with 0±0.1 M NaCl) at 1008C for 5 minutes produced 50±75% retention of antigenicity (compared with zero for glycinin). Conglycinin subunits and soluble aggregates produced by thermal treatment or native subunits (a, a0 , and b) isolated by ion-exchange chromatography are able to react with pAb for the native protein (36). These results are consistent with one or more of the following conclusions: (a) antigenic sites for conglycinin are continuous, i.e., involve a contiguous sequence of amino acids formed from lower order (18 and 28) protein structure; (b) thermal dissociation of conglycinin does not involve large changes in conformation of subunits; and (c) antigenic sites for conglycinin (subunits) are surface located and unaffected by the protein association-dissociation transition (37). Even though it has a lower TD, b-conglycinin is apparently less vulnerable to thermal processing than glycinin. 3.5.

Renaturing Ef®ciency of Glycinin and Conglycinin

The antigenicity of soy protein is dominated by the 7S protein (Fig. 1). This is the result of a more ef®cient renaturation of conglycinin compared with glycinin. After exposure to urea, extreme pH or high temperature, glycinin dissociates into its constituent subunits (37±39). German et al. (19) showed that heating then disrupts S22S bonds between A-B subunits. Disul®de bond cleavange is catalyzed by indigenous free sulfhydryl compounds associated with soy protein. Isolated glycinin B subunits then aggregate via SH/S22S exchange while the A chains remain soluble (20). On the other hand conglycinin does not aggregate extensively upon heating. This protein lacks S22S bonds and also has low levels of SH groups. Extensive heating eventually produces soluble conglycinin aggregates although SDS-PAGE analysis shows little irreversible damage. Conglycinin and glycinin refold with *80% and *70% ef®ciency after exposure to 8 M urea and dialysis, respectively. However, treatment with urea±2-ME (which disrupts A-B disul®de bonds) leads to a glycinin renaturing ef®ciency of *20%. Breaking the A-B disul®de linkage produces a marked reduction in glycinin renaturation ef®ciency and enhanced

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aggregation. A small proportion of glycinin molecules also form incorrectly refolded soluble monomers with altered antigenic properties. Reconnecting S2 2S bonds in a correct orientation is the limiting step for glycinin renaturation (39). According to Berkowitz and Webert (40), the renaturation procedure for soybean products, although long and laborious, is inescapable. They proposed using SDS-PAGE analysis and immunoblotting as a more rapid ELISA format. During immunoblotting, soybean protein is denatured, separated by SDS-PAGE, and transferred to a nitrocellulose membrane before immunoassay. There is no distinct sample renaturation step, which reduces the assay time.

4. SOLVENT-EXTRACTABLE SOYBEAN PROTEIN There may be other more convenient methods for preparing soy protein for analysis. Medina (41) ultrasonicated soy protein standards (1±2 mg) with 10±20 mL of coating buffer (3.2 mM sodium carbonate, pH 9.8 ‡ 0.1% thimerosol and 0.05% Tween 20). Samples of cooked sausage (1 g) were similarly homogenized with 10 mL of carbonate buffer and subjected to untrasonication and (1000-fold) dilution followed by ®ltration. Results using soy protein fractions showed pAb binding to glycinin A subunits. Pretreating commercial soy protein isolate with reducing agent increased antibody binding. Assay precision was improved by increasing the time for pAb coating on microwell plates. The linear dynamic range was 0±0.2 mg soy protein per well, or 0±5% (w/w) soy protein in cooked sausages. For laboratory-prepared sausages the amount of soy protein found (Y, %) was described by the relation Y…%† ˆ 1:22 ‡ 1:09X

…2†

where X is the amount of soy protein known to be present in samples. From the gradient in Equation (2) there was a quantitative recovery of soy protein antigen from the cooked sausage matrix.

5. THERMOSTABLE ANTIGENS FOR SOYBEAN PROTEIN ANALYSIS ELISA for soy products would be improved by using thermostable antigens that are unaffected by cooking and other forms of processing. Possible

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thermostable antigens include protease inhibitors, peptide antigens (natural or synthetic), and conglycinin. 5.1.

Protease Inhibitors

The Bowman-Birk inhibitor is a possible thermostable antigen for ELISA of soybean additives (42,43). Kunitz soybean trypsin inhibitors (KSTIs), which rival the Bowman-Birk inhibitor in thermal resistance (44), are another potential thermostable antigen (45). 5.2.

Peptide Antigens

Peptide antigens with continuous epitopes are potentially useful thermostable antigens. Yasumoto, et al. (46) developed a noncompetitive indirect ELISA for soybean protein predigested with trypsin. The assay was highly speci®c with no cross-reactivity for pork, beef, egg, or azuki bean proteins. Soya protein (10.4±20.8% w/w) was quantitatively determined for pork sausages cooked at 808C for 20 minutes. The LLD was 0.4%. The pAbs used for this assay were raised by immunizing rabbits with intact glycinin. As a form of pretreatment, glycinin standards were autoclaved at 1208C for 180 minutes and then digested with trypsin for 24 hours. Microwell plates were coated with the peptide digest and blocked with BSA to avoid nonspeci®c binding. Food samples, e.g., cooked sausages, were pretreated by homogenization with acidic ethanol, followed by acetone precipitation and drying. The powders were suspended in buffer, autoclaved for 1208C for 180 minutes, and digested with trypsin for 24 hours at 378C. Proteolysis was terminated by boiling brie¯y. The supernatant was removed for indirect ELISA. Carter et al. (47) used glycinin peptides as antigens for pAb and mAb production. The absence of discrete protein bands during SDS-PAGE analysis con®rmed a complete hydrolysis of glycinin (1 mg mL 1) by treatment with 20 mg of subtilisin (in ammonium bicarbonate buffer, 50 mM, pH 8) overnight. The (rabbit) pAbs for glycinin peptides were nonspeci®c (Fig. 3). Avidity for intact glycinin was attributed to surfacelocated antigenic determinants. There was cross-reactivity with other seed globulins (b-conglycinin, pea 11S globulin), probably due to partial amino acid sequence homology. Three mAbs (designated mAbs IFRN024, IFRN025, and IFRN026) were also produced with interesting speci®city characteristics (Fig. 4). The mAb-IFRN024 was nonspeci®c and recognized an epitope that was susceptible to denaturation by SDS treatment. Both mAb-IFRN025 and mAb-IFRN026 were speci®c for both intact glycinin and glycinin peptides. The former mAb showed a 100-fold greater avidity

Speciation of Soya Protein

291

FIGURE 3

Indirect ELISA for soya bean and other seed globulins using polyclonal antibodies for glycinin peptides. (Drawn from Ref. 47.)

FIGURE 4

Indirect ELISA for soybean and other seed globulins using mAbs for glycinin peptides. Results for mAb-IFRN0024 and mAb-IFRN0026 were multiplied by 10 before plotting. (Drawn from Ref. 47.)

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for these samples. The epitopes recognized by mAb-IFRN025 and mAbIFRN026 were resistant to combined SDS and thermal treatment. One should expect that peptide antigens will be resistant to processing effects owing to their small size. 5.3.

Conglycinin

Structural changes for thermally treated conglycinin were monitored using mAb-IFRN089 (48). The antigenicity of b-conglycinin increased after preheating to 658C, which is the TD for this protein. Huang et al. (49) also monitored heating effects on glycinin using mAb-IFRN025.* Thermal treatment led to progressive ``denaturation'' at temperatures above 908C and an increase in protein antigenicity (49). The insoluble protein precipitate was not analyzed. In summary, the use of mAbs for continuous epitopes should lead to ELISAs that are suitable for processed foods. Continuous epitopes comprising consecutive amino acids (protein 18 structure) are more thermoresistant than conformational or discontinuous epitopes formed from higher order protein (48, 38, or 28) structure. Beyond these considerations, sample solubility may still be a limiting factor. Ef®cient strategies are needed for resolubilizing proteins that have undergone severe processing. 6.

OTHER NONMEAT PROTEINS

Nonmeat protein additives from cereal sources, milk, and egg have been analyzed by ELISA. However, there is far greater interest in the detection of trace amounts of these proteins in relation to their ability to cause allergic reactions. The analysis of protein allergens is discussed in Chapter 11. REFERENCES 1. 2.

CHS Hitchcock, FJ Bailey, AA Crimes, DAG Dean, PJ Davis. Determination of soya proteins in food using an enzyme-linked immunosorbent assay procedure. J Sci Food Agric 32:157±165, 1981. NM Grif®ths, MJ Billington, W Grif®ths. A review of three modern techniques available for the determination of soya protein in meat products. J Assoc Public Anal 19:113±119, 1981.

* Glycinin (4 mg mL 1 in 0.35 mM K-phosphate buffer ‡ 0.1 M NaCl) was heated and then centrifuged to remove insoluble aggregates. The soluble fraction was analyzed by ELISA.

Speciation of Soya Protein 3.

4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

293

NM Grif®ths, MM Billington, AA Crimes, CHS Hitchcock. An assessment of commercially available reagents for an enzyme-linked immunosorbent assay (ELISA) of soya protein in meat products. J Sci Food Agric 35:1255±1260, 1984. AA Crimes, CHS Hitchcock, R Wood. Determination of soya protein in meat products by an enzyme-linked immunosorbent assay procedure: collaborative study. J Assoc Public Anal 22:59±78, 1984. WJ Olsman, S Dobblelaere, CHS Hitchcock. The performance of an SDSPAGE and an ELISA method for the quantitative analysis of soya protein in meat products: an international collaborative study. J Sci Food Agric 36:499± 507, 1985. P Ravestein, RA Driedonks. Quantitative immunoassay for soya protein in raw and sterilized meat products. J Food Technol 21:19±32, 1986. CC Hall, CHS Hitchcock, R Wood. Determination of soya protein in meat products by a commercial enzyme immunoassay procedure. Collaborative trial. J Assoc Public Anal 25:1±27, 1987. JE McNeal. Semi-quantitative enzyme-linked immunosorbent assay of soy protein in meat products: summary of collaborative study. J Assoc Anal Chem 71:443, 1988. MM Hewedy, CJ Smith. Modi®ed immunoassay for the detection of soy milk in pasteurized skimmed bovine milk. Food Hydrocolloids 3:485±490, 1990. JH Rittenburg, L Adams, J Palmer, JC Allen. Improved enzyme-linked immunosorbent assay for determination of soy protein in meat products. J Assoc Off Anal Chem 70:583±587, 1987. IC Peng, DW Quassa, WR Dayton, CE Allen. The physicochemical and functional properties of soybean 11S globulinÐa review. Cereal Chem 61:480±489, 1984. NC Nielsen. The chemistry of legume storage proteins. Philos Trans R Soc Lond Ser B 304:287±296, 1984. D Fukushima. Structures of plant storage proteins and their functions. Food Rev Int 7:353±381, 1991. VH Thanh, K Shibasaki. Beta-conglycinin from soybean proteins. Isolation and immunological and physical properties of the monomeric forms. Biochim Biophys Acta 490:370±384, 1977. MC Garcia, M Torre, ML Marina, F Laborda. Composition and characterization of soyabean and related products. CRC Rev Food Sci Nutr 37:361± 391, 1997. WJ Wolf, T Tamura. Heat denaturation of soybean 11S protein. Cereal Chem 46:331±402, 1969. K Hashizume, T Watanabe. In¯uence of heating temperature on conformational changes of soybean proteins. Agric Biol Chem 43:683±690, 1979. VH Thanh, K Shibasaki. Major proteins of soybean seeds. Reversible and irreversible dissociation of b-conglycinin. J Agric Food Chem 27:805±809, 1979.

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19. B German, S Damodaran, JE Kinsella. Thermal dissociation and association behavior of soy proteins. J Agric Food Chem 30:807±811, 1982. 20. S Damodaran, JE Kinsella. Effect of conglycinin on the thermal aggregation of glycinin. J Agric Food Chem 30:812±817, 1982. 21. S Iwabuchi, H Watanabe, F Yamauchi. Observations on the dissociation of bconglycinin into subunits by heat treatment. J Agric Food Chem 39:34±40, 1991. 22. AM Hermansson. Physico-chemical aspects of soy proteins structure formation. J Texture Stud 9:33±58, 1978. 23. S Damodaran. Refolding of thermally unfolded soy proteins during the cooling regime of the gelation process: effect on gelation. J Agric Food Chem 36:262±269, 1988. 24. AN Danilenko, EK Grozav, TM Bikbov, VY Grinberg, VB Tolstogozov. Studies of the stability of 11S globulin from soybeans by differential scanning microcalorimetry. Int J Biol Macromol 7:109±112, 1985. 25. VY Grinberg, AN Danilenko, TV Burova, VB Tolstoguzov. Conformational stability of 11S globulins from seeds. J Sci Food Agric 49:235±248, 1989. 26. S Iwabuchi, H Watanabe, F Yamauchi. Thermal denaturation of bconglycinin. Kinetic resolution of reaction mechanisms. J Agric Food Chem 39:27±33, 1991. 27. K Watanabe. Kinetics of heat insolubilization of soybean 11S protein in phosphate buffer system. Agric Biol Chem 52:2095±2096, 1988. 28. DJ Sessa. Hydration effects on the thermal stability of proteins in cracked soybeans and defated soy ¯our. Lebensm Wiss Technol 25:365±370, 1992. 29. DJ Sessa. Thermal denaturation of glycinin as a function of hydration. J Am Oil Chem Soc 70:1279±1284, 1993. 30. N Catsimpoolas, EW Meyer. Immunochemical properties of the 11S component of soybean proteins. Arch Biochem Biophys 125:742±750, 1968. 31. N Catsimpoolas, TG Campbell, EW Meyer. Association-dissociation phenomena in glycinin. Arch Biochem Biophys 131:577±569, 1969. 32. N Catsimpoolas, J Kenney, EW Meyer. The effect of thermal denaturation on the antigenicity of glycinin. Biochim Biophys Acta 229:451±458, 1971. 33. S Iwabuchi, K Shibasaki. Immunochemical studies of the effect of ionic strength on thermal denaturation of soybean 11S globulin. Agric Biol Chem 45:285±293, 1981. 34. A Demonte, IZ Carlos, EJ Lourenco, JE Dutra de Oliveira. Effect of pH and temperature on the immunogenicity of glycinin (Glycine max L.). Plant Foods Hum Nutr 50:63±69, 1997. 35. M A Moreira, WC Mahoney, BA Larkins, NC Nielsen. Comparison of the antigenic properties of the glycinin polypeptides. Arch Biochem Biophys 210:643±646, 1981. 36. S Iwabuchi, K Shibasaki. Immunochemical studies of the effects of ionic strength on thermal denaturation of soybean 7S globulin. Agric Biol Chem 45:1365±1371, 1981.

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37. VH Thanh, K Shibasaki. b-Conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms. Biochim Biophys Acta 490:370±384, 1977. 38. RC Roberts, DR Briggs. Isolation and characterization of the 7S component of soybean globulins. Cereal Chem 42:71±85, 1965. 39. K Kitamura, T Takagi, K Shibasaki. Renaturation of soybean 11S globulin. Agric Biol Chem 41:833±840, 1977. 40. DB Berkowitz, DW Webert. Determination of soy in meat. J Assoc Off Anal Chem 70:85±89, 1987. 41. MB Medina. Extraction and quantitation of soy protein in sausages by ELISA. J Agric Food Chem 36:766±771, 1988. 42. D Brandon, AH Bates, M Friedman. Monoclonal antibody±based enzyme immunoassay of the Bowman-Birk proteinase inhibitor of soybeans. J Agric Food Chem 37:1192±1196, 1989. 43. H Frokiaer, L Horlyck, V Barkholt, H Sorensen, S Sorensen. Monoclonal antibodies against soybean and pea proteinase inhibitors: characterization and applications for immunoassays in food processing and plant breeding. Food Agric Immunol 6:63±72, 1994. 44. CM DiPietro, IE Liener. Heat inactivation of the Kunitz and Bowman-Birk soybean protease inhibitors. J Agric Food Chem 37:39±44, 1989. 45. RE Oste, DL Brandon, AH Bates, M Friedman. Effect of Maillard browning reactions in the Kunitz soybean trypsin inhibitor on its interaction with monoclonal antibodies. J Agric Food Chem 38:258±261, 1990. 46. K Yasumoto, M Sudo, T Suzuki. Quantitation of soya protein by enzyme linked immunosorbent assay of its characteristic peptide. J Sci Food Agric 50:377±389, 1990. 47. JM Carter, HA Lee, EN Mills, H Lambert, HW-S Chan, MRA Morgan. Characterization of polyclonal and monoclonal antibodies against glycinin (11S storage protein) from soya (Glycine max). J Sci Food Agric 58:75±82, 1992. 48. GW Plumb, N Lambert, EN Clare Mills, MJ Tatton, CCM D'Ursel, T Bogracheva, MRA Morgan. Characterization of monoclonal antibodies against b-conglycinin from soya bean (Glycine max) and their use as probes for thermal denaturation. J Sci Food Agric 67:511±520, 1995. 49. L Huang, EN Clare Mills, JM Carter, MRA Morgan. Analysis of thermal stability of soya globulins using monoclonal antibodies. Biochim Biophys Acta 1388:215±226, 1998.

11 Determination of Trace Protein Allergens in Foods

1. INTRODUCTION Food allergy is one of a number of adverse reactions to foods (Table 1). The nontoxic adverse reactions are mediated by the immune system (allergy). Non±immune system mediated adverse reaction is termed a food intolerance. Some allergic reactions to food, for example, anaphylactic shock, may be severe and even fatal. General symptoms of food allergy are reviewed by Anderson (1), Blades (2), Jones (3), and also Burks and Sampson (4). Allergenic foods are discussed by He¯e et al. (5) and also Taylor et al. (6). Food allergens are reviewed by Matsuda and Nakamura (7), Bush and He¯e (8), and He¯e (9). This chapter describes the analysis of trace amounts of protein allergens in food. Allergens are associated with eight or nine major food groups (Sec. 1.1). Soybean, peanut, and gluten allergies are described in Secs 2, 3, and 4. In each instance we also consider the structure of the protein allegens and the effect of processing on assay results.

1.1.

Food Allergens and Labeling Regulations

Articles by Campbell (10) and Amor (11) provide summaries of current food labeling regulations related to allergens. It is believed that food may be rendered safe by providing information related to allergen content. 297

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TABLE 1 Adverse Reactions to Foods Class Toxic reactions Nontoxic reactions Immune mediated IgE mediated

Non-IgE mediated Non-immune mediated Food intolerance Food aversion

Compounds

Adverse effects

Natural food components, food additives

Range of effects at high concentrations

Protein allergens

Anaphylaxis, oral allergy syndrome, atopic dermatitis, gastrointestinal effects, etc. Celiac disease and related gastroenteropathy Various effects

Protein allergens Lactase de®ciency, inborn errors of Metabolism, pharmacological compounds Psychological avoidance

Therefore, manufactures are not to consider allergens as incidental additives that are exempt from declaration. On the contrary, voluntary declaration of allergens is encouraged even where they are obviously parts of proprietary formulations. Of major concern is the presence of hidden allergens. As of 1996, manufacturers are required to take all necessary steps to prevent, remove, or otherwise arrange not to have inadvertent allergens in foods. Steps should be taken to avoid the transfer of allergens from one food product to another. Firms must make all reasonable efforts to provide adequate labeling describing the allergenic status of their product. According to the Food and Drug Administration (FDA), which is concerned with food regulations in the United States, food allergens are associated with the following eight commodities: (a) legumes (soybean, peanut), (b) milk, (c) eggs, (d) ®sh, (e) crustacean, (f) mollusks, (g) wheat, and (h) tree nuts. The United Nations Codex Alimentarius Commission also provide a list of the major sources of food allergens. The Codex list is similar to the that provided by the FDA although (allergenic food groups are) de®ned in a slightly broader manner: (a) peanuts, soybean, and products made from these; (b) milk and milk products (including lactose); (c) eggs and egg products; (d) ®sh and ®sh products; (e) crustacea and derived products; (f) tree nuts and nut products; (g) cereals containing gluten

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(wheat, rye, barley, oats) and derived products; and (h) sul®te in concentrations of 10 mg kg 1 or greater. Future FDA and Codex Commission policy may require the declaration of foods containing allergenic proteins produced through genetic engineering. The concern is that genetic modi®cation may result in the inadvertent transfer of allergenic proteins from one food source to another via genetic modi®cation (12). Food regulations and mandatory labeling requirements related to allergens are also discussed in articles by Nestle (13) and others (14,15).

1.2.

Detecting Allergens and Allergies

Some of the major protein allergens in foods have been isolated and partially characterized. Many are heat resistant and survive common food processing operations. They also tend to be resistant to digestion. The presence of antibodies in the circulation suggests that allergens pass through the digestive tract (16). Allergenic peptides and perhaps whole proteins may be absorbed intact, especially in infants. Some well-characterized food allergens are listed in Table 2. The detection of food allergens plays a vital role in the management of diets for susceptible subjects. Two groups of analyses are employed for food allergens: (a) in vivo tests using live patients, in which mild symptoms are induced in the subject during a skin prick test or the double-blind, placebo-controlled food challenge (DBPCFC) test, and (b) in vitro detection, which covers three types of tests involving isolated pAbs. A well-known procedure is the radioallergoadsorbent test (RAST) for circulating antibody. The food sample containing antigen is mixed with serum (collected from the allergic subject). The mixture is then added to a ®xed amount of antigen immobilized on CNBr-activated Sepharose. After washing the support, any bound pAb is determined by reacting with iodine-125-labeled (rabbit) antibody for human IgE. The amount of radioactivity bound to the solid phase, measured with a scintillation counter, is inversely related to the concentration of circulating pAb as well as to the food allergen content. RAST is essentially a competitive immunosorbent assay using iodine-125labeled (rabbit) pAb for ``visualization'' (17,18). The second in vitro assay for food allergens involves immunoblotting (19,20); examples of these studies are listed in Table 3. The food sample is analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to serum from the allergic subject. The bound IgE is visualized using iodine-125-labeled (rabbit) pAb for human IgE followed by radiography (Table 3). The ®nal group of in vitro tests for food allergens involves classical ELISA. Included in this group are tests described in Chapter 10 for bulk food proteins.

300 TABLE 2

Chapter 11 Some Food Allergens Involved in IgE-Mediated Adverse Reactions

Food group Milk Egg white Soybean Peanut Castor bean Cod®sh Shrimp and other crustacea

Major allergen, comments aS1-casein (23 kDa, heat stable) b-lactoglobulin (18.4 kDa, heat stable) Ovalbumin (43 kDa) Ovamucoid (23-kDa glycoprotein trypsin inhibitor, heat stable) 30-kDa Gly m Bd 30 (oil body protein, papain analogue) 28-kDa allergen, KSTI-16 protein allergen protein in all Ara h 1. (65-kDa allergen, conarachin) Ara h 2. (17±21 kDa allergen) 12-kDa allergen (amylase/trypsin inhibitor, heat stable) Parvalbumin or allergen M (12-kDa Ca2 ‡ binding protein, heat resistant) Tropomyosin (70-kDA muscle protein), heat stable

Source: Refs. 7 and 8.

The merits and disadvantages of in vivo or in vitro tests for food allergens were described by Nordlee and Taylor (49) and also Taylor and Lehrer (50). One advantage of using human pAb for immunoblotting is that the results are related to functional allergens (49). The test can be done in

TABLE 3 Analysis of Food Allergens by SDS-PAGE and Immunoblotting with Human pAb Allergen source Soybean Peanut Shrimp Brazil nuts Almonds Egg white Milk proteins

References Shibasaki et al. (21), Bucks et al. (22), Bush et al. (23), Herian et al. (24), Ogawa et al. (25), E-F Ebabiker et al. (26) Sachs et al. (27), Barnett et al. (28), Bucks et al. (29), Bucks et al. (30), Uhlemann et al. (31), He¯e et al. (32), de Jong et al. (33) Hoffmann et al. (34), Nagpal et al. (35), Lehrer et al. (36), Reese et al. (37) Hide (38), Morgan et al. (39), Nordlee et al. (12), Borja et al. (40) Bargman et al. (41), Hlywka et al. (42) Hoffman (43), Langeland (44), Leduc et al. (45) Ball et al. (46), Restani et al. (47), Rosendal and Barkholt (48)

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the absence of the allergenic individuals. Immunoblotting is the default method for detecting allergens in food materials. The main disadvantages are that (a) care is needed in the selection of human donors for pAb and (b) a requirement for human pAb increases costs. Once a major allergen is identi®ed and isolated, less expensive sources of pAb or mAb may be developed. 2. SOYA BEAN PROTEIN ALLERGENS 2.1.

Structure and Characteristics of Soya Bean Allergens

Soybean allergy is common in children. The allergic reaction is induced by soy protein, with soybean oil being comparatively hypoallergenic (51). The identities of soybean protein allergens were established by SDS-PAGE and immunoblotting with serum pAbs from allergic patients. The same approach enabled the identi®cation of food allergens associated with peanut, shrimp, almonds, Brazil nut, and egg white (Table 3). Research leading to the identi®cation, characterization, and quantitative assays for the major soybean allergens is described in this section. Sixteen soybean allergens were identi®ed SDS-PAGE analysis, Western transfer to a nitrocellulose membrane, and immunoblotting using pAb pooled from 361 patients exhibiting atopic dermatitis (eczema). Protein bands were visualized using iodine-125-labeled (rabbit) pAb for human IgE and autoradiography (25). Approximately 20% of patients with atopic dermatitis showed IgE production for soy protein. The major soybean allergens were associated with the 7S protein fraction (7SF). Of the subset of 69 patients showing sensitivity to soybeans, two thirds produced pAb for a 30-kDa protein thereafter named Gly m Bd 30K. About 23% of patients had circulating pAb for a 70-kDa protein designated Gly m Bd 70 (Table 4). Only 1.4% and 2.9% of patients produced pAb for the A subunit of glycinin and the Kunitz soybean trypsin inhibitor, respectively. It has been suggested that the pattern of IgE speci®city can vary in different populations depending on (a) the age of the subjects, (b) history of sensitization to the allergen, (c) route of sensitization (airways vs. digestive tract), and (d) method of sample pretreatment for immunoblotting. It remains to determined whether Gly m Bd 30K is the major soy allergen in other clinical populations. The foremost soybean allergen (Gly m Bd 30K) seems identical to a 34-kDa oil body protein from soybean seeds. Ogawa et al. (52) found that native Gly m Bd 30K forms a 350-kDa oligomer that elutes ahead of glycinin during gel ®ltration column chromatography. Treatment with SDS and 2-ME produced a dissociated 30-kDa protein with a pI of 4.5. The

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TABLE 4 Major Soybean Allergens Detected by Immunoblotting with pAb from 69 Patients (average age 6 years) Protein size (kDa) 30 68±70 28 63±67 52±55 47±50 43±45 33±35 35 20

Identity 7SF, Gly m Bd 30K b-Conglycinin asubunit 7SF, Gly m Bd 28K 7SF 7SF 7SF 7S globulin, b-unit 7SF 11S globulin, a-unit 2S, KSTI

Patients with antibody (%) 65.0 23.3 23.3 18.8 14.5 10.1 10.1 15.9 1.4 2.9

Source: Ref. 25.

N-terminal 15-amino-acid sequences for Gly m Bd 30K and 34-kDa oil body protein were the same. The mAb for either protein cross-reacted with the other. Following established convention, Gly m Bd 30K, being the ®rst soybean allergen identi®ed, was designated Gly m 1. Kalinski et al. (53) independently characterized the 34-kDa oil body protein. Within intact cells, the protein was a vacuolar protein designated P34. Like other storage proteins, protein P34 undergoes post-translational glycosylation and proteolysis. During processing, P34 appears along the endoplasmic recticulum, Golgi bodies, and eventually within vacuoles or protein bodies. Protein P34 had partial sequence homology with cysteine proteases from the papain superfamily. It is not certain that P34 has proteolytic activity (54). Its association with soybean oil bodies was an experimental artifact produced by cell disruption. A survey of a large number of soybean strains shows that P34 is widespread. The possibility of eliminating P34 from soybean lines by breeding seems doubtful (55). The next important soybean allergen (Gly m Bd 70K) is the 70-kDa b-conglycinin a-subunit. Antibody speci®c for the a-subunit did not crossreact with the other two (a0 or b) subunits of b-conclycinin despite some sequence homology between these polypeptides (56). The third soybean allergen (Gly m Bd 28K) is a 26-kDa glycosylated protein with a pI of 6.1 (57). It is apparently unstable and present in very small quantities in defatted soy ¯our (*15 ppm). Perhaps for these reasons, Gly m Bd 28K was dif®cult to detect in processed foods containing soy protein. Finally, pAbs from some soybean-sensitive individuals recognized the A-chain residue

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303

from glycinin. The epitope consisted of a 114-amino-acid residue fragment (58). The epitope for Gly m Bd 30K recognized by mouse mAb was also identi®ed by Hosoyama et al. (59).

2.2.

Quantitative Analysis of Soybean Allergens

Quantitative ELISA for Gly m Bd 30K was developed by Tsuji et al. (60) using mAb produced by conventional means. Eventually, two cell lines producing mAb (mAb-F5 and mAb-H6) were isolated. Immunoblot analysis showed that mAb-F5 and mAb-H6 were both speci®c for Gly m Bd 30K. Sandwich ELISA for Gly m Bd 30K employed mAb-H6 as the capture antibody and an HRP conjugate with mAb-F5 as detector. The linear range for analysis was 2±200, 5±500, or 10±500 ng protein for reduced/ carboxymethylated-allergen, SDS/2-ME±treated sample, or native soy protein, respectively. Ten soybean products and ®ve meat products containing soy protein isolate (SPI) were analyzed by a sandwich ELISA for Gly m Bd 30K (61). Results from this study are summarized in Table 5.* From such data it may be surmised that (a) high concentrations of Gly m Bd 30K are detectable within a range of processed soybean products, (b) fermented soy products (miso, soyu, and natto) are free of allergen, (c) Gly m Bd 30K can be accurately determined in cooked meat products known to contain soy protein isolate, (d) sandwich ELISA results were corroborated by immunoblot analysis using pAbs from soybean-sensitive patients, and (e) Gly m Bd 30K is heat resistant, accounting for its detection in cooked products. The thermal stability of soybean antigens is discussed in Chapter 10. The allergen was also resistant to digestion by chymotrypsin and trypsin. Levels of Gly m Bd 30K declined to negligible values in fermented soy products, probably as a result of digestion by microbial (acid) proteases. Treating soybean samples with added microbial proteases reduced the level of allergen, leading to hypoallergenic soybean products (62). Clearly, the preceding assay has potential uses for monitoring soybean allergen in processed foods. The ELISA test could also ®nd use as a quality control tool for hypoallergenic soybean products.

* As pretreatment each (5-g) sample was homogenized with Na- phosphate buffer (50 mM, pH 8.0 with 1% w/v SDS and 20 mM 2-ME). The extract was centrifuged and SDS was precipitated with KCl (1 M ®nal concentration). After centrifugation, the SDS-depleted sample was analyzed by sandwich ELISA.

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TABLE 5 Determination of the Major Allergen (Gly m Bd 30K) in Soybean-Containing Products using Sandwich ELISA

Product Soybean Soymilk Tofu (kinugoshi) Tofu (momen) Kori-dofu Kinako Abura-age Yuba Miso Shoyu Natto Meatballs Beef croquettes Fried chicken Fish sausages Hamburger

Sandwich ELISA results (mg allergen g 1 N) 126 106 89 65 64 29 59 66 npb np np 17 21 9 np np

Immunoblot analysisa ‡ ve ‡ ve ‡ ve ‡ ve ‡ ve ‡ ve ‡ ve ‡ ve

‡ ve ‡ ve ‡ ve

a

SDS-PAGE/immonoblotting with IgE from soy-sensitive patients. ‡ ve ˆ positive, ( ) negative results. b np, no allergen present. Source: Adapted from Ref. 61.

2.3.

Indirect Monitoring of Soybean Allergens by ELISA

ELISA for bulk soy protein analysis (Chapter 10) may be useful for monitoring soybean allergens indirectly. Yeung and Collins (63) described a competitive ELISA using (rabbit) pAb for whole soybean extract. The assay, which was virtually identical to those used by Hitchcock and coworkers (64±67), was intended to detect bulk soy protein as opposed to trace allergen. The antigen for immunizing rabbits was prepared from soybean ¯our defatted with cold acetone.* The competitive ELISA for soy protein (63) had the following characteristics:

* Soy ¯our was extracted with cold Tris-HCl buffer (10 mM, pH 8, with 1% w/v SDS and 10 mM 2-ME) at 48C for 16 hours.

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1. Speci®city. There was no cross-reactivity with 10 other legumes, 8 varieties of nuts, 11 common food ingredients, and 2 phytoestrogens. 2. Acuity. The concentration of soy proteins that produced 50% inhibition of pAb-antigen binding (IC50) was 35 ng mL 1 and the linear range was 3±117 ng mL 1. For various real foods, the detection limit was 2 ppm. 3. Recovery. For processed tuna ®sh having 13.5±54 ppm of soy protein, the recovery was 77±95%. Similar recoveries (63±96%) were observed with hamburger matrix. 4. Precision. For model solutions and hamburger matrix containing soy protein, the interassay precision was 3.5±4.2% and 3.6±8%, respectively. The corresponding intra-assay precision 2.4±5.1% or 1.5±2.6%. Features such as assay speci®city and acuity were not routinely reported for ELISA designed to quantify soy protein in meat samples (Chapter 10). 3. PEANUTS 3.1.

Peanut Allergy

Peanut (groundnut) allergy occurs in about 1 in 200 of the general population in the United States. The incidence rate is probably similar in Western Europe. Emmett et al. (68) found a partial association between peanut allergy and allergy to tree nut. Peanut allergy also persists for life. Allergic subjects appear in all age groups and with no gender bias (69). About 25±28% of all cases of food allergy involve peanuts. The ®gures for tree nuts are Brazil nut (10.2%), almond (7.8%), and hazelnut (7.1%). The incidence of peanut allergy also appears related to the frequency of exposure (70). In a study of 868 children in Singapore, 27% of subjects showed sensitivity to bird's nest soup. The incidences of other food allergies were 24% (crustacean), 11% (eggs and cow milk), and 7% (traditional Chinese herbs). There was no recorded adverse reaction to peanuts or tree nuts. The low incidence of peanut allergy was attributed to the low per-capita consumption of peanuts and other nuts in Singapore (71). Symptoms of anaphylactic shock arise in approximately 6% of allergic reactions to peanuts. The onset of fatal and near-fatal anaphylaxis can be very rapid. The ®rst reaction may appear within 5±30 minutes of contact with the allergen (72). More frequent clinical effects include atopic dermatitis or eczema (40%), angioedema (37%), and asthma (14%); digestive symptoms occur more rarely (1.5%). The quantity of peanut necessary to produce an adverse reaction ranges from a few micrograms to 1 g (73). The

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anaphylactic response can arise from topical contact with peanut residue on another individual, by inhalation (74), or by exposure to peanuts in vegetarian ``beef '' burgers (75). Small amounts of peanut protein associated with peanut oil can also induce an allergic episode. Peanut protein was found in some infant formula prepared from peanut oil (76,77). Highly re®ned protein-free oil apparently poses little threat (51). Reported fatalities due to peanut allergy are of the order of 125 persons per year in the United States (84). Figures from Pumphrey et al. (78) suggest that there were 21 deaths from peanut and tree nut allergy in the United Kingdom during the 5- to 6-year period following 1992. Of 541 patients showing sensitivity to nuts, 90% had serum IgE speci®c for peanut. About 67% of patients showed serum IgE for another nut besides peanut, while 34% of patients had immunoglobins for all nuts tested. The threshold quantity necessary to induce allergy varies for different subjects. Exposure to peanut allergens in early infancy ( b-gliadin >, g-gliadin > o-gliadin. Glutelin is highly polymerized by S22S bonds. The sulfur-rich prolamins possess N-terminal repetitive amino acid sequences containing high numbers of three amino acids: glutamine (Q), proline (P), and phenylalanine (F) (Figure 1). The C-terminal areas have variable sequences bearing a number of half cysteine residues normally involved in intramolecular S22S bonding. Also noteworthy is the rare occurrence of SH groups in o-gliadin. Regularities are also evident in the 28 structure of cereal proteins (126,127). Thus, a-gliadin, b-gliadin, and g-gliadin have 36±37% a-helix, 11±12% b-sheet, and 52±53% aperiodic structure plus b-turns. The precise quantity of b-turn or hairpin loop structure is uncertain because this does not produce a distinct circular * Stir 1 kg of ¯our with water-saturated n-butanol to remove lipids. Next, extract with four volumes of 70% (v/v) aqueous ethanol. Dry the extract by rotary evaporation and redissolve the protein in 0.1 M acetic acid solvent. Dialyze against the same solvent and fractionate with a CMC-cellulose column (10 cm 6 22 cm, containing 1.7 kg support) equilibrated with sodium acetate buffer (5 mM ‡ 1 M dimethylformamide and adjusted to pH 3.5 with acetic acid). Elute the bound protein using stepwise changes in NaCl concentration gradients.

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FIGURE 1 The primary structure of cereal prolamins. (Adapted from Refs. 124 and 126.)

dichroism absorbance peak. However, numerical calculations using amino acid sequence data suggest that the repetitive sequences are b-turns. Celiac toxic peptides have been identi®ed at the N-terminal regions of a-gliadin and b-gliadin (117,128,129). The epitopes recognized by intestinal T cells appear to be two tetrapeptides, PSQQ and QQQP. The ®rst occurs in a-gliadin at amino acid residues 13±16, 50±53, and 213±216. QQQP occurs at residues 16±19, 33±36, and 188±191. Four out of six potentially toxic sequences appear within the ®rst 56 amino acids from the N-terminal. The epitope bound by mAb-WC2 is probably QQQP, which is apparently common to all celiac-active proteins (129). Tanabe et al. (130), using

Determination of Trace Protein Allergens

317

antibody from celiac sufferers, identi®ed the sequence QQQPP as being celiac active. Bromelain digests proline-rich peptides leading to wheat ¯our that may be useful for producing hypoallergenic bread (131). Ginger protease has high speci®city for proline residues (132).

4.3.

Thermal Denaturation of Wheat Allergens

The thermal denaturation of gliadin was examined using 70% (v/v) ethanol as solvent. Heating g-gliadin led to a conformational change at 20±608C as monitored by far-UV circular dichroism.The spectral changes showed an isobestic point implying that denaturation was a two-state (helix-coil type) transition. At elevated temperatures the proportion of a-helix declined by 7% and the structure of gliadin loosened to expose phenylalanyl residues to the solvent. Puri®ed a-, b-, and o-gliadin behaved similarly. Conformational changes produced by the relatively short (5-minute) thermal treatments were reversible (126,127). Heating wheat ¯our matrix revealed that o-gliadin is more heat resistant than the sulfur-rich gliadins.* The levels of a/b-gliadin decreased substantially after 20 minutes of heating. The g-gliadin was resistant for up to 50 minutes of heating at 1008C. There was no change in the quantity of o-gliadin recovered after 0±100 minutes of heating. This result is due to the virtual absence of half cystine residues in o-gliadin. This protein can not undergo sulfhydryl-disul®de exchange during heating (133).

4.4.

Determination of Wheat Allergens

Immunological assays for cereal proteins are generally concerned with the detection of celiac-active peptides. Some attempts to assess grain varietal differences by EIA were reported (134). Also of interest is the identi®cation of (bread making) quality-related polypeptides by immunoassay (135,136); literature in this area was reviewed by Skerritt et al. (137). Sections 4.4 through to 4.10 consider ELISA tests for trace (nanogram to microgram) quantities of cereal proteins. ELISAs for wheat allergens and bulk protein adulterants are not fundamentally different in their design. However, the former techniques possess greater acuity. SDS-PAGE±immunoblot analysis is another ELISA format. * A suspension of wheat ¯our in water was heated at 1008C for 0, 10, 20, 30, 50, and 100 minutes. Flour proteins were then extracted with 1 M urea solution and analyzed by gradient gel electrophoresis.

318

4.5.

Chapter 11

Sandwich ELISA Tests for Gluten Using pAb

McKillop et al. (141) provide a useful introduction to ELISA of wheat gliadin. Sandwich ELISA and competitive indirect ELISA formats were implemented using (rabbit) pAb for unfractionated gliadin. The twoantibody format was selected for full evaluation. The assay characteristics were as follows: 1. Linear range. 0.05±6 mg (gliadin) mL 1. 2. Detection limit. A concentration of 23 ng (gliadin) mL 1 produced absorbances of 2 SD above the reagent blank. 3. Speci®city. Gliadin and gliadin-containing foods. Wheat ¯our had 5.7% (w/w) gliadin equivalent protein. The values for oats, rice, and corn ¯our were 240, 46, and 47 mg %, respectively. 4. Precision. The within-assay reproducibility for analysis ranged from 5 to 40%. Samples with > 330 ng (gluten) mL 1 were determined with below + 10% error. The preceding assay enabled the identi®cation of ``gluten-free'' ingredients. No tests were performed on heated samples. Table 8 summarizes other ELISAs for wheat proteins. Fritschy et al. (140) used (rabbit) pAb for total gliadin as the basis for a gluten-sensitive ELISA. Wheat ¯ours (20 different types) were found to contain 2.9±6.7% (w/w) gliadin equivalent proteins. The gliadin contents of many commercial gluten-free foods were insigni®cant. Rice contained 57.5 mg % (w/w) and sorghum 12.5 mg % (w/w) gliadin on a dry weight basis. Food samples were extracted with 70% (v/v) ethanol with a recovery of 77±107%. The LLD was 10 ng (gliadin) mL 1 sample extract. The assay was speci®c for cereal proteins. It was possible to assay rice, corn, oats, and barley protein when 1±2% BSA was included in samples to avoid nonspeci®c protein binding to microwell plates. Troncone et al. (145) puri®ed (rabbit) pAb by af®nity chromatography using Sepharose 4B±immobilized gliadin. They then implemented a conventional sandwich ELISA using (rabbit) pAb for capture. The linear dynamic range for gliadin analysis was 5±400 ng mL 1 with an LLD of 0.5 ng (3 ng for the competitive assay). There was cross-reactivity with maize and oat prolamins, although these were detected with 1000-fold lower sensitivity. The response for gliadin was 104-fold higher than obtained for rice prolamin. Rye and barley were not assayed. Interference from maize, rice, or oat prolamins could be avoided by using high protein dilution.* * Equal amounts of prolamin from different cereals yield 1000- to 10,000±fold differences in assay sensitivity. The ELISA response for wheat protein (cf. maize, oats, and rice proteins) measures ``gluten''-like structure and functionality. ELISA results do not necessarily show the absolute amounts of prolamins present in the different cereals.

Determination of Trace Protein Allergens TABLE 8

319

Analysis of Gliadin and Other Prolamins by ELISA

ELISA format and antibody

Antigen

Sandwich, pAb

a-Gliadin, gliadin

Sandwich, Competitive, pAb dot blotting, mAb

Gliadin

Sandwich, Competitive, pAb Sandwich, mAb Immunoblotting Sandwich, pAb Competitive, pAb

Gliadin

o-Gliadin, gliadin

Gliadin Gliadin Gliadin Gliadin

Sandwich, mAb Sandwich, mAb AOAC approved laboratory test

a-Gliadin, g-gliadin o-Gliadin, glutenin subunits

Sandwich, mAb Home kit

o-Gliadin, glutenin subunits Gliadin

Sandwich-ELISA Sandwich, mAb Competitive

Gliadin peptides

References Windemann et al. (138), Meier et al. (139), Fritschy et al. (140) McKillop et al. (141) Skerritt and Smith (133), Skerritt (142), Skerritt et al. (143), Freedman et al. (144) Troncone et al. (145) Freedman et al. (146) Janssen et al. (147) Ayob et al. (148) Friis (149), Chirdo et al. (150) Mills et al. (151) Hill and Skerritt (152), Skerritt et al. (153), Skerritt and Hill (154,155) Skerritt and Hill (156) Hekkens and Twist de Graaf (157) Ellis et al. (158), DeneryPapini (159), Ellis et al. (160), Nicolas et al. (161)

Gliadin was accurately determined in human milk. Eleven of 18 lactating mothers who had consumed gluten meals 2 hours prior to testing showed gliadin-reactive material in their breast milk. Gliadin was detectable in human plasma after heat treatment to denature circulating human antibodies. Thermal treatment (1218C, 5 minutes) had no adverse effect on assay sensitivity. There was apparently no immunoreactive material in blood plasma from celiac patients (145). By contrast, Lane et al. (162) found

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signi®cantly high levels of gluten in the serum of patients suffering from dermatitis herpetiformis and celiac disease (compared with normal controls). Different assay conditions used in these studies may explain the lack of agreement. It is perhaps signi®cant that Lane and co-workers calibrated their assay by adding gliadin to human plasma. The performance of a low-cost sandwich ELISA test for gluten was compared with two commercial gluten tests produced by Cortecs Ltd. (UK) and R-Biopharm (Germany) (163). Results for the in-house test were highly correlated (R ˆ 0.0.9967) with both commercial tests. The former was more sensitive than the Cortecs gluten test (see the following). The in-house test had the advantage of a 10-fold lower cost. The linear range for the in-house assay was 50±150 ng mL 1 compared to 20±80 ng mL 1 for the R-Biopharm test. The LLD values of the two tests were 0.04 and 0.1% mg %, respectively. 4.6.

Competitive ELISA for Gluten Using pAb

A competitive ELISA test for gliadin (149) had a working range of 10±250 ng mL 1 with an LLD of 1 ng mL 1. The highly accurate test had an interassay precision of 33%. There was speci®city for a-, b-, g-, and ogliadin as well as barley, oats, and rye prolamins. No responses were obtained for maize, soya, or millet protein. Buckwheat gave a low response. The competitive ELISA test for gluten was not tested for heat-processed samples. Chirdo et al. (150) used (rabbit) pAb for competitive ELISA for gluten in a range of processed foods including cake ¯our, breakfast cereal, processed meat, soup, and chocolate. The working range for gliadin standards was 0.6 ng mL 1 to 10 mg mL 1. The LLD of 1 ng mL 1 (0.002 mg %) was the same as reported by Friss (149). The average within-assay precision was about 9%. It was claimed that this test was useful for thermally processed foods. 4.7.

The AOAC-Approved ELISA Test for Gluten

The AOAC-approved test for gluten employs mAbs for capture and detection (154). Skerritt and Smith (133) and Skerritt (142) found two mAbs (mAb-401/21 and mAb-304/13) with speci®city for o-gliadin, HMW and LMW glutenin subunits. Because o-gliadin is heat resistant (Section 4.3), the preceding tests may be suitable for the analysis of heat-processed foods (see later). Either mAb-401/21 or mAb-304/13 could be used for capture or detection, leading to similar results. Preliminary tests (153) showed speci®city for wheat (bread or durum) > rye*barley 4 maize > oats. No responses were observed for oat, maize, or rice protein at levels comparable

Determination of Trace Protein Allergens

321

to those for wheat gluten. Sorghum was not tested. Assay performance depended on these factors (152,153,154): 1. Extraction solvent. The optimal solvent for gluten extraction from a range of food samples was 40 % (v/v) ethanol. Using 70% (v/v) ethanol, 1 M urea, or 1 mM HCl as solvent led to underestimation or overestimation of the gluten content. 2. Form of extraction. Homogenization of ¯our samples using an omnimixer or Ultraturrax mixer for about 30 seconds led to accurate analysis. Vortexing for 30 or 60 seconds duration (four times per hour) led to inaccuracy, probably due to shear-induced precipitation of gluten. 3. Choice of gluten standard. Normal gluten is a suitable standard. This is relatively soluble, stable in the freeze-dried form, and usable over a wide concentration range (dilutions of up to 10,000 1 were used for analysis). 4. Choice of solid phase. Polystyrene microwell plates were preferable to PVC plates, which produced high nonspeci®c binding. Background adsorption of gluten was not ameliorated by a range of blocking solutions including PBST with 0.05±5% BSA, 0.65 M NaCl, or 2% Tween. 5. mAb characteristics. The nature and concentration of the capture and detector antibody and coating conditions (time and temperature of coating) affected the assay. Following optimization, the linear dynamic range of the AOACapproved ELISA test was 0.0075±5 mg mL 1 (15 mg % to 10% w/w gluten in actual foods). The LLD was 0.1±0.2 mg mL 1, which is equivalent to 0.2±0.38-mg gliadin per 100 g of ¯our (0.2 ±0.38 mg %). A partial list of food types successfully analyzed includes starches (wheat, maize), cake ¯ours (plain, self-raisin, bread crumb, cookie), gluten-free bread mixes, processed meat (cooked beef, ham sausages, salami), baby foods (beef or chicken based with and without ¯our thickener), breakfast cereals, baked goods (bread crumb mix, sweet cookies, crisp bread), soups, confectionery (caramel, chocolate), and others (lentils, eggs, milk powder).

322

4.8.

Chapter 11

Collaborative Testing of AOAC-Approved ELISA Test for Gluten

Prior to AOAC approval, the previous ELISA was subjected to a collaborative trial by 15 laboratories* (155). Eighteen samples including ®ve prestudy samples (three wheat starches and two meat-gluten blends) with known amounts of added gluten were tested. Participating laboratories familiar with ELISA methodology were supplied with commercial versions of the Skerritt-Hill test. The linear dynamic range for gluten was 16 mg % to 11% (w/w) basis. Assay reproducibility was 24±33% with a repeatability of 19±22%. Gluten was accurately determined in a range of foods (164). The accuracy of the Skerritt-Hill ELISA test is indicated by its high precision. Gluten levels reported by collaborators were less than +12% different from expected values. The collaborative study led to AOAC approval for the Skerritt-Hill ELISA test for gluten. Potential limitations of the AOAC assay for gluten have been suggested (165). A minicollaborative trial using a limited number of gluten test kits was organized by the Celiac Society of Great Britain. Participating groups were major UK clinical laboratories with longstanding interest in celiac disease.{ Results from ®ve laboratories agreed with respect to four gluten-free foods and nine gluten-containing foods (spaghetti bolognese, egg and bacon breakfast, crispbread, malted drink, porridge, barley, plain ¯our, and wheat starches). By contrast, large disparities (®ve- to sixfold differences) were reported for gluten levels in 11 foods including egg and bacon, beer, low-alcohol lager, stout, gluten-free ¯our, gravy powder, corn¯our, and rice pudding. An mAb-based gluten test developed by Mills et al. (151) showed a linear range comparable to that of the AOAC test. They used pAbs from chicken{ for capture while mAb-IFRN 033 * The list of participants includes M. Billington (City of Birmighman Public Analysts, Birmingham, UK), A. Crimes, (Unilever, Bedford, UK), C. Cuncliffer, (Somerset County Council, Taunton, UK), M. Cutrufelli, (USDA, Betsville, MD), T. McKenny (Cerestar, Manchester, UK), M. Murlry, (Kraft Research Center, Glenview, IL), N. Paterl, (Campden, Food and Drink Research Association, Chipping Campden, UK), J. Rhodes, and D. Lord, (Lancashire County Lab., Preston, UK), B. Ritter, (ABC Research, Gainesville, FL), M. Scooter, (Food Science Division, MAFF, London, UK), M. Smith, (Avon County Scienti®c Services, Bristol, UK), C. Stanley, (Laboratory of Government Chemists, Teddington, UK), P. Sutton, and S. Cooper, (British Food Manufacturing Industries Research Association, Letherhead, JK), and B. Taylor and D. Ansell, (Greater Manchester Public Analyst, Manchester, UK). { Participating institutions were St James University Hospital (Leeds), St Bartholomew's Hospital (London), Western General Hospital (Edinburgh), Radcliff In®rmary (Oxford), and Bristol Royal In®rmary (Bristol). { The pAb is recovered from the eggs of immunized chicken.

Determination of Trace Protein Allergens

323

functioned as the detector antibody. The mAb-IFRN 033 was speci®c for agliadin and g-gliadin, which together constitute *85% of gliadin. The linear range for gluten was 0.25±2.5 mg mL 1 with the LLD being 0.1 mg mL 1. The preceding tests may be compared with the 1987 sandwich ELISA test developed by Freedman et al. (146) from Guys and St Thomas's Hospital, London. They used (rabbit) pAb for capture and mAb for unfractionated gliadin for detection. The bound mAb was visualized with alkaline phosphatase±labeled (goat) pAb for murine IgG/IgM. The linear dynamic range for this assay was 10 ng mL 1 to 1 mg mL 1. Strong wheat ¯our contained 3.7% (w/w) gliadin-equivalent proteins. Several brands of ``gluten-free'' ¯our produced from wheat starch had 1.9±3.3 mg % of gliadin. The day-to-day precision of the assay was better than 5%. 4.9.

A Cocktail mAb-Based Sandwich ELISA Test for Gluten

Gluten tests should be equally sensitive to all celiac-active proteins from wheat, barley, or rye. However, proteins from celiac-negative cereals (maize, rice, millet, etc.) should not interfere. Single mAbs had different sensitivities for different celiac toxic prolamins (166). Combining different mAbs is one way to ensure cross-reactivity for a range of celiac-active prolamins. A cocktail antibody test for gluten was developed by mixing two capture mAbs (mAb-13B4 and mAb-Rye5) with speci®city for barley and rye (167). A third mAb for rye protein (mAb-Rye3) was chosen for detection. Results compared favorably with the commercial AOAC-ELISA test for gluten. The cocktail sandwich ELISA produced comparable calibration graphs for gliadin, hordein, and secalin with a linear range of 3±100 ng mL 1 and an LLD of 1.5 ng (gliadin) mL 1, 0.05 ng (hordein) mL 1, 0.15 ng (secalin) mL 1, or 12 ng (avenin) mL 1. The acuity toward barley and rye prolamin was higher than obtained for wheat. The response toward avenin was 10±100 times lower, which is as expected from the lower toxicity of oats. Compared with the AOAC test, the cocktail mAb test had an LLD for hordein or gliadin that was *25-fold and 4- to 10-fold lower. The mAbRye3 used for detection in the cocktail ELISA had low speci®city for promlamins from wheat, barley, and rye. This feature probably explains the relatively high response for barley and rye. 4.10.

A Home Test for Gluten

The Skerritt and Hill (156) home test kit for gluten performed well with ordinary citizens, who successfully identi®ed gluten-free foods with 82± 100% accuracy. The home test agreed closely with results from the AOACapproved laboratory test kit. The home test kit can be usefully compared

324

Chapter 11

with the home pregnancy test kits, which are now commonplace. Similar self-use kits are possible for other food allergens. The home-gluten test consists of the following components: 1. Polystyrene test tubes (Nunc, Denmark) precoated with mAb-401/ 21 and blocked with 1% BSA. The antibody-coated test tubes are stable at 48C for 12 months or at 208C for 6 months. 2. Graduated tubes for sample extraction. 3. Enzyme-labeled mAb-402/21 for detection. 4. Substrate solution (TMB/hydrogen peroxide). The kit was ®eld tested in eastern Australia by 5 dieticians or food technologists and 47 ordinary citizens registered with the Celiac Society. Participants were 10- to 77-year-old urban and rural dwellers of varied educational background. The testers were given six food samples to grade as having ``low,'' ``borderline,'' or ``high'' gluten. Samples classed in the borderline (40 mg %) or high (>150 mg %) gluten categories were not acceptable as ``gluten-free'' foods, whereas those with soy isolate > whole wheat protein. The level of protein required for maintenance is also a sensitive index of PNV. A feeding trial using one level of nitrogen failed to identify egg protein as possessing superior quality.

* Where B ˆ Nitrogen balance and I ˆ Nitrogen intake as protein (see Table 1).

348

Chapter 12

TABLE 3 Estimation of Protein Nutrient Value from N Balance Studies with Young Adult Mena Protein source, levels of N Single test Egg Soy isolate Whole wheat Slope method Egg Soy isolate Whole wheat

N utilization ef®ciency (slope)*

N required for maintenance (intercept)**

0.63 0.54 0.65

81 94 78

0.50 0.43 0.27

88 107 131

a

Protein quality was determined as the (*) the dimensionless slope or (**) intercept [mg N kg (body weight) day 1] of the N balance curve.

3.

1

SMALL ANIMAL BIOASSAYS FOR PROTEIN NUTRIENT VALUE

Bioassays using humans or large mammals are time consuming and expensive for three reasons: (a) larger animals grow slowly, (b) there is a requirement for special and more exacting accommodation, and (c) people (even students) may require remuneration. In contrast, small animals such as rats exhibit higher growth rates than adult humans and are cheaper to house. The use of model animals cuts cost and speeds up analysis. Protein bioassays using weanling rats have received of®cial approval in Canada and the United States. Many proposed chemical methods for assessing PNV are routinely calibrated using the rat bioassay. Nevertheless, the use of small animal models (as opposed to humans, livestock, etc.) represents a compromise. Rats and chicks have different amino acid requirements than people and farming livestock. 3.1.

The Rat Bioassay

Before 1994 there was a statutory requirement in the United States and Canada for food labeling information based on the PER. Protein quality is now measured by a new technique described in Chapter 14. The classic PER assay remains important. This is usually determined using 4-week-old rats. The PER technique was thoroughly discussed in the proceedings from the 38th Annual Meeting of the Institute of Food Technologist Dallas, Texas

Tests for Protein Nutrient Value

349

(1978) titled Protein Quality Testing: Industry Problems, Needs, Approaches. Articles by the following authors provide a comprehensive overview of the topic: Rosen®eld (49), Staub (50), Hackler (51), Anderson (52), Burnette and Russoff (53), and Hsu et al. (54). The proceedings from the symposium Measuring Protein Quality for Human Nutrition (American Association of Cereal Chemist, New Orleans, 1976) likewise provides informative summaries by Steinke (55), Bodwell (56), and also Hackler (57). The following discussion is based on materials from these sources and a number of other general reviews (4±16). Osborne et al. (58) introduced PER as an index for measuring the growth-promoting effects of proteins. To determine the PER, cohorts of 10 rats were fed a diet containing 10% protein for 28 days. Their weights were then measured. PER is the ratio of weight gained (or lost) to the weight of protein consumed. The procedure was later standardized with respect to test animals (strain, gender, age) and nonprotein dietary factors by Chapman et al. (59). They adopted casein as a standard protein. A casein standard diet is assessed along with the test protein. Results are then adjusted such that the PER for casein is 2.5. The casein-corrected PER value (cPER) is calculated from the relation cPER ˆ

2:5PERSAMPLE PERCASEIN

…2†

Values for cPER allow more reliable comparisons of assay results from different laboratories. Bender and Doell (60) incorporated weight loss for rats fed on a protein-free diet into the PER equation. The resulting index, called the net protein ratio (NPR), is a measure of the protein requirement for growth and maintenance. In contrast, PER measures only the protein requirement for growth. Dividing the NPR by the corresponding value for a standard protein (casein or lactalbumin) yields the relative NPR value (RNPR). PER, cPER, NPR, and RNPR represent increasingly robust indices for protein quality determined using the rat bioassay. In Europe, the preferred rat bioassay involves the measurement of carcass nitrogen. To determine net protein utilization (NPU), rats are fed for 10 days on a protein-free diet. Another group of rats is fed the same basal diet supplemented with 10% protein. Both groups of rats are sacri®ced and their carcasses are dried at 1058C or dissolved in concentrated sulfuric acid. Whole-body nitrogen is determined by Kjeldahl analysis or else estimated from the known body moisture content. Rats have a ®xed water/nitrogen ratio (61). The PER, NPR, and NPU assays are single-point assays. These tests involve a single level of N intake. A slope method or multiple-level tests

350

Chapter 12

involving diets with different levels of protein yield more reliable estimates of quality. PER measurements based on multiple levels of N intake were introduced by Hegsted and Chang (62) and Hegsted et al. (63). Feeding multiple levels of nitrogen led to two slope measurements for protein quality called the relative nutritive value (RNV) and the relative protein value (RPV). 3.2.

Sources of Error

During PER determination, the test sample should contribute a dietary protein level of about 10%. Provided that the sample and casein standard diets have similar proximal composition (moisture, protein, fat, crude ®ber, minerals), some leeway is permitted in the amounts of nonprotein dietary constituents. Values for PER change with the amount of dietary water, ®ber, and mineral mix as well as net food intake. The PER test is also more suited for simple foods or protein concentrates (milk powder, infant formula, wheat ¯our, soy ¯our). With complex foods (hamburgers, processed meat, sausages, bread, rice, and ready-to-eat meals), measuring PER requires a great deal of attention to detail. Samples having one or more of the following characteristics may pose dif®culty: (a) high moisture content, (b) high fat (greater fat content than protein content), (c) high moisture and fat content, and (d) low ( 79% the true protein value was only 21±28%. At least one of the samples appeared to be adulterated with feather meal judging from the low methionine content and high amounts of gelatin. The NPR value was 0.4±2.2 for most meat meals with a value of 1.6 signifying weight loss during the rat bioassay.

4.2.

Orange G Binding

Soybean and ®sh meal samples were heated in steel pans or autoclaved at 1208C (20). The DBC was positively correlated with PER determined using TABLE 3 Summary Characteristics of Concentrates and Feedstuffs Sample and characteristics

Meat meal (n ˆ 26)

Whale meat meal (n ˆ 16)

Fish meal (n ˆ 13)

Crude protein (%) NPU (%) TDC (%) BV (%) Total Lys (g 16 g 1 N) Reactive Lys (g 16 g 1 N) Methionine (g 16 g 1) Correlations identi®ed a Dye binding (mg g 1) 1. Crude protein (Y1)

40±90 9±38 70±90 Ð 5.4±7.8 3.0±4.6 0.9±2.6

25±95 17±62 75±88 34±69 Ð 4.0±7.3 1.6±2.6

60±73 22±66 74±90 27±80 Ð 3.4±6.9 1.3±2.5

Y1 ˆ 0.278Db ‡ 30

Y1 ˆ 0.261Db ‡ 30

2. True protein (Y2) 3. NPU (Y3) Reactive Lys (X1)

Y2 ˆ 0.239Db ‡ 27 Y3 ˆ 3.86Db±0.43 None

Y2 ˆ 0.287D ‡ 17 Y3 ˆ 6.37Db±19.8 BV ˆ 7.52X1 ‡ 4.0

0.325 Db ‡ 24 Ð Ð None

a Y1 ˆ crude protein, Y2 ˆ true protein, Y3 ˆ NPU, Db ˆ amount of dye bound (acid equivalents of dye bound/104 grams of protein), X1 ˆ reactive Lys.

390

Chapter 13

a chick bioassay. Signi®cant decreases in DBC occurred within the ®rst 15 minutes of heat treatment. The quality of raw soybean meal was lower than expected from its DBC, probably due to the presence of protease inhibitors in the sample. The overall impression is that the measurement of DBC is a useful for monitoring heat process damage. Despite this, the DBC for heattreated ®sh meal was high even though samples showed low levels of cysteine and low PNV; lysine does not appear to be the limiting essential amino acid in ®sh meal. The results from Choppe and Kratzer (21) also show that Orange G binding is positively correlated with protein quality results from the chick growth assay.

4.3.

Acid Orange 12 Binding

The thermal processing damage suffered by ®sh meal, groundnut meal, blood meal, freeze-dried beef steak, and lactalbumin was evaluated using Acid Orange 12 binding by Hurrell and Carpenter (22). The DBC was also determined for Remazol Brilliant Blue and Cresol Red. The results were compared FDNP-reactive lysine and total basic amino acids. With severely heated samples (1218C,  28 hours), Acid Orange 12 binding capacity agreed closely with the concentration of histidine ‡ arginine ‡ reactive lysine (or the HARL value). Proteins that were subjected to mild heat treatment (378C for  30 days) and/or experienced early Maillard reactions exhibited little change in DBC even after a 90% decline in available lysine. The early Maillard products were dye reactive. Carpenter and Opstvedt (23) collated results from a collaborative study of eight commercial ®sh (capelin) meals. Protein quality was evaluated from the measurement of Acid Orange 12 binding, chick bioassay and FDNB-reactive lysine. Interlaboratory variations in results were generally as large as differences between samples. Outlying results were identi®ed and those samples reanalyzed. A correlation matrix was established indicating a to strong relationship between the indices of quality. Much of the variation between samples was dependent on one sample. Omitting this sample led to the collapse of all correlation below signi®cant levels. The numbers of samples used in this trial should, perhaps, have been larger. The effect of commercial processing and laboratory heat treatment on 126 rapeseed meal samples was evaluated by Goh et al. (24±26). Heat damage was measured via Acid Orange 12 binding and as FDNB-reactive lysine. The DBC was 124.4 mg g 1 (sample) or 349±356 mg g 1 (cP) for unheated rapeseed meal. The lysine content for unheated Brassica seed protein is about 5.45 (+ 0.53) g per 100 gram (27). Autoclaving produced changes in the DBC at 45±120 minutes of heating. The concentration

Effect of Processing on Protein Nutrient Value

391

FIGURE 3 Effect of autoclaving on rapeseed meal protein quality assessed from Acid Orange 12 binding, FDNB-reactive lysine, and total levels. (Drawn using data in Refs. 24±26.)

of FDNB-reactive lysine declined steadily over the entire heating time (Fig. 3). The mismatch between DBC and available lysine values occurs because DBC is dependent on the total basic amino acid concentration. I have performed multiple regression analysis of DBC versus the concentration of lysine, histidine, and arginine yielding the following relations: DBC ˆ 26 …+2:3† Lys ‡ 203 …+11:8†

…1†

DBC ˆ 1 …+0:067† TBAA

…2†

DBC ˆ 17 …+4:7† Lys ‡ 74 …+37† His ‡ 5 …+6:4† Arg ‡ 30 …+80†

…3†

These relations were generated from data in Table 5 of Ref. 26. The ranges of values used were 2.54±5.9 g (lysine) 100 g 1 protein, 2.43±2.74 g (histidine) 100 g 1 protein, and 2.98±5.65 g (arginine) 100 g 1 protein. The total basic amino acid range is therefore 7.95±14.3 g per 100 g 1 protein. The regression coef®cients for Eq. (1), Eq. (2), and Eq. (3) were 0.9201, 0.9533, and 0.9532, respectively. The nonzero intercept from Eq. (1) suggests that Acid Orange 12 binds to sites other than lysine. Eq. (2) con®rms that the DBC is strongly

392

Chapter 13

correlated with TBAA and that Acid Orange 12 binds no sites other than the three basic amino acids. Eq. (3) con®rms the previous results. The coef®cient for arginine, histidine, and lysine shows their relative contributions to DBC. Apparently, the order of dye binding to basic amino acids is histidine > lysine 4 arginine. Nevertheless, heating produced the greatest decline in lysine with histidine being the least heat susceptible. The extent of heat damage to rapeseed meal is also a function of the sample moisture content (Fig. 4). The DBC was reduced to a greater extent by heating rapeseed meal at 10±20% moisture as compared with 2% or 40% moisture. Reactions producing quality loss are limited by lack of reagent mobility in low-moisture systems and by dilution effects in high-moisture systems. Changes in available lysine show a more complex dependence on moisture levels. With prolonged heating, losses in available lysine were higher for samples having 2% moisture as compared with 40%. The assessment protein heat damage from DBC is described further by Hook (28,29), Peal et al. (30), Randall et al. (31), and also by Kratzer et al. (32). Protein samples investigated include wheat, soybean, defatted milk, whole egg, and whole blood proteins. In general, changes in DBC tended to

FIGURE 4

Effect of sample moisture content and autoclaving time on the Acid Orange 12 binding capacity for rapeseed meal.

Effect of Processing on Protein Nutrient Value

393

lag behind PER. Quality loss was strongly correlated with decreases in available lysine. Lin and Lakin (33) reported disparities between the DBC and FDNB-reactive lysine for heated soy meal samples. Steaming soy meal at atmospheric pressure led to the progressive loss of the nitrogen solubility index (NSI) due to protein denaturation and insolubilization by covalent (sulfur-disul®de exchange) and noncovalent aggregation. Urease activity decreased because of enzyme inactivation after 60±80 minutes of heating. In vitro protein digestibility increased, probably due to the inactivation of soybean trypsin inhibitors. The level of unreactive lysine increased gradually from 0.14 g (lysine) g 1 protein and leveled off at 0.26 g (lysine) g 1 protein after heating of soybean meal for 120 minutes. Assuming an initial lysine content of 6.3 g per 100 g, then 95±98% of lysine residues remained available after heating. Steam treatment led to an increase in DBC, probably due to heat denaturation of soy protein. 4.4.

Hot Water±Soluble Protein and Quality

Meat and bone meal samples had high levels of gelatin measured as the amount of hot water±soluble protein. There was a considerable difference in the PNV for individual samples owing to their varied thermal history. For 20 different samples, Choppe and Kratzer (21) found a strong (negative) correlation between the amount of hot water±soluble bone meal gelatin and PNV. El (34) suggested a regression equation for predicting PER values for meat or ®sh based on the collagen content. Calculated PER values agreed closely with experimental values for sardine, lamb, bovine liver, chicken meat, or beef sausages. Collagen content may provide a rapid and inexpensive assay for estimating PNV for meat. 5. LEGUMES AND OILSEEDS The structure and characteristics of legume proteins and effect of processing on their quality were reviewed by Vanderstoep (35), Chang and Satterlee (36), Sathe et al. (37), Friedman (38), and de Lumen and Uchimiya (39). Common processing operations for legumes include baking/roasting, dehulling, cooking, canning, extrusion cooking, fermentation, germination, and hydrothermal treatment. A list of some legumes for which the protein quality has been investigated over the last decade is given in Table 4. Most of these processes improve the quality of legume proteins through the reduction of protein antinutritional factors (trypsin inhibitors, hemagglutinins/lectins) and chemical antinutrients (oxalates, phytic acid, and tannins).

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TABLE 4 Determination of Protein Nutritional Value for Legumes Commodity Acacia farnesiana, Cercidium microphyllium, Cercidium sonorae, Mimosa grahamii, Olneya tesota, Parkinsonia aculeata, and Prosopis juli¯ora. African locust bean Alfalfa protein concentrate Bauhinia purpurea L Canavalia brasiliensis Egyptian legumes; faba beans, lentils (Lens culinaris), common beans (Phaseolus vulgaris), cowpea, and soybeans (Glycine max L.). Lupin varieties; Lupinus polyphyllus Lindl var., L. angustifolius, Lupinus albus cv. Multolupa Mung beans (Phaseolus aureus), black gram, and wild beans (Vigna sublobata) Pea protein Pigeonpea (Cajanus cajan) Velvet bean (Mucuna pruriens L.) Soybean varieties Sudanese legumes: lupin (Lupinus terminis), pigeon pea (Cajanus cajan), two types of cowpea (Vigna sinensis and Vigna unguiculata), bonavist bean (Dolichos lablab), faba bean (Vicia faba), and soybean

5.1.

References Ortega-Nieblas et al. (40)

Kapu et al. (41) Hernandez et al. (42) Karuppanan et al. (43) Oliveira et al. (44) Youssef and Abdel-Gawad (45)

Aniszewski (46), Egana et al. (47) Khalil and Khan (48) Wang et al. (49) Singh et al. (50) Permal et al. (51) Vibha and Simlot (52) Ahmed and Nour (53)

Available Lysine

Reductions in legume available lysine levels in occur during domestic cooking with little nutritional consequence. Lysine is not normally the limiting essential amino acid in legumes; methionine is. Heating also produces bene®cial reductions in antinutritional factors. Available lysine levels from dhal (split pulses) of ®ve legumes (Lens esculenta, Phaseolus mungo, Lathyrus sativus, Cicer arietinum, and Pisum sativum) was 5.55± 9.12 g 100 g 1 (54). Cooking each legume for 30 minutes produced a maximum of 25% reduction in available lysine (range 4.21±6.79 g 100 g 1).

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395

Pressure cooking for 10 minutes produced a 35% reduction in available lysine (3.89±5.87 g 100 g 1). A similar study involving ®ve types of beans (white kidney beans, black eye beans, crab eye beans, butter beans, red kidney beans) found available lysine levels of 6.34 (+ 0.11) g 16 g N 1 in the raw beans (55). Pressure cooking (30 minutes at 15 lb in 2) produced a mean available lysine of 5.42 (+ 0.36) g 16 g N 1, a reduction of about 14.5% compared with the raw beans. The available lysine values were 5.48 (+ 0.48) g 16 g N 1 and 4.78 (+0.16) g 16 g N 1 after cooking for 2 and 8 hours. These changes did not produce net declines in PNV according to rat bioassays. 5.2.

Steaming and Hydrothermal Treatment

Subjecting whole rapeseed to steam treatment for 10 minutes inactivated the enzyme myrosinase, decomposed glucosinolates, and improved protein quality (56). The bene®ts of hydrothermal treatment are also observed with other legumes. Steam treating beans (Phaseolus vulgaris L) at 102, 119, and 1368C resulted in a loss of lectin and trypsin inhibitor activity. There was also a decline in the available lysine (57). Inactivation of trypsin inhibitor involved biphasic ®rst-order kinetics. Effects of steaming temperature on rate constants followed Arrhenius-type relations. Both total lysine and available lysine were reduced to differing extents. Steam treatment at 1198C for 5 or 10 minutes was proposed as a compromise for inactivating antinutritional factors while avoiding high degrees of protein damage as measured by total and available lysine. 5.3.

Soaking, Dehulling, and Cooking

Dehulling before cooking leads to greater improvements in protein quality as compared with cooking alone. The effects of dehulling, sprouting, and/or steam cooking on rice beans and mung beans were evaluated by Mehta, et al. (58). As well as the in vitro protein digestibility, they measured proximate composition, sugars, starch, and trace minerals for the raw and processed beans. The maximum protein content was 23.5% for rice beans and 26.5% for mung beans. After a combination of sprouting, dehulling, and cooking, in vitro protein digestibility increased by 35% for rice beans and 30.8% for mung beans. Soaking a range of legume [soybean, lupin, bean (Phaseolus vulgaris)] seeds in 0.5% sodium bicarbonate reduced the level of antinutritional factors (phytic acid, tannin, trypsin inhibitor, and hemagglutinin activity). Interestingly, protein extractability (in distilled water, NaCl, or sucrose solutions) was also reduced although in vitro digestibility and available lysine were improved (59).

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Seeds of perilla (Perilla frutescens Linn, Britton) have concentrations of essential amino acids above FAO/WHO/UNU recommendations for infants. Lysine was limiting. Longvah and Deosthale (60) found that dehulling and cooking perilla seeds further increased the net protein ratio (NPR), NPU, and TPD as determined by a rat bioassay although values were lower than for a casein-based control diet. Therefore, perilla seed represents a good source of protein for human and animal nutrition, particularly after dehulling and cooking. In conclusion, legume protein quality can be improved by a number of inexpensive processing methods. These studies show that weaning foods and food supplements may be produced from locally available foodstuffs.

5.4.

Roasting and Irradiation

Roasting and malting led to signi®cant improvements in the protein quality for a range of local legumes and cereals examined by Gupta and Sehgal (61), Dahiya and Kapoor (62), and Gahlawat and Sehgal (63). Plahar et al. (64) evaluated the effect of roasting (preceded by dehulling) on four varieties of cowpea (Vigna unguiculata). Dehulling reduced the tannin content by up to 98%. With cowpea having a highly pigmented coat, dehulling improved the protein quality. Roasting signi®cantly improved digestibility and more than doubled the PER. It was suggested that dehulled and roasted cowpea may be useful as a protein supplement in cereal-based weaning foods. The bene®cial effects of roasting have also been con®rmed with chickpeas (Cicer arietinum) and peanut (Arachis hypogaea), where cooking led to a decrease in the trypsin inhibitor activity and an increase in the true digestibility, relative nitrogen utilization, and PER values. Hira and Chopra (65) found that roasting chickpea or peanuts decreased the trypsin inhibitory activity, available lysine, and BV (P < 0.05). However, the true digestibility, relative nitrogen utilization, and PER values were all improved. Microwave heating presoaked legume seeds (faba beans, peas, chickpeas, soybeans, lentils, common beans) reduced levels of protein antinutritional factors (hemagglutinins and inhibitors) comparable with conventional cooking. In consequence, PER values were increased. Microwave processing of dried seeds had less effect. Antinutrients from common bean were destroyed by microwave heating (66). These effects are in agreement with more general ones showing that effects of both industrial and domestic microwave cooking on nutritional characteristics including proteins are comparable to those of conventional cooking (67,68).

Effect of Processing on Protein Nutrient Value

5.5.

397

Multiple ProcessingÐWeaning Foods

Weaning foods are produced from mixtures of cereal and legumes (and occasionally milk). The multiple protein sources provide complementary supplies of essential amino acids cysteine/methionine and lysine. A recent emphasis is on the production of low-cost weaning foods using materials locally available in developing countries. Gupta and Sehgal (61), Dahiya and Kapoor (62), Gahlawat and Sehgal (63) from Haryana University (Hisar State, India) describe a number of weaning food formulations based on local cereals and legumes including wheat, pearl millet (bajra), Bengal gram, green gram (mung beans), groundnuts, peal millet, rice, kangini (Setaria italica), and sanwak (Echinochloa frumentacea). Formulations containing two or three components were generally subjected to a range of processing techniques including sprouting, roasting, and malting. For commodities such as bajra (peal millet), barley, green gram, amaranth grain (Amaranthus sp.), and jaggery, malting and/or roasting led to protein quality indices comparable to those for a commercial weaning food; PER ˆ 2.04±2.13, BV ˆ 79.56±80.68, NPU ˆ 66.75±67.86, NPR ˆ 2.13±2.76, and PRE (protein retention ef®ciency) ˆ 34.18±44.18. Dahiya and Kapoor (62) produced food supplements for preschool children using malted and/or roasted bajra, Bengal gram, green gram (mung beans), groundnuts, jaggery, or amaranth leaves. Bajra-based food supplements had quality indices (PER, food ef®ciency ratio, BV, NPU, NPR, and PER) signi®cantly higher than those of wheat-based supplements (P < 0.05). The authors suggest that the quality of their formulations was equal to that of Cerelac2, a commercial supplement. However, PNV was found to be lower (P < 0.05) than the value for casein (standard protein). Rats fed on bajrabased supplements showed an excellent growth pattern throughout the feeding trial. Santos et al. (69) prepared extruded weaning foods using a mixture of rice, mung bean, and milk (70:25:5). Protein quality was determined by a rat bioassay. The quality of the weaning food was signi®cantly improved if rice and mung bean were extruded ®rst before milk was added. The optimal PER was 2.25, which is comparable to the growth-promoting effect of casein. Extrusion cooking the complete mixture led to a PER value of 1.93. Supplementation with lysine increased the PER value to 2.10. Obviously, extrusion cooking destroyed some lysine. Fermentation and supplementation of a traditional Ghanaian cornmeal weaning food with soybean meal improved its protein quality (70). Studies also suggest that roasted cowpea, widely grown in the Sahel, may be suitable as a weaning food supplement (64,71).

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Mahgoub (72) produced ®ve weaning formulations comprising 8.5% skim milk powder and/or sorghum, groundnuts (peanuts), sesame seeds, and chickpeas (various concentrations) also along with 5% sugar and a vitamin and mineral mixture. The formulations were processed by a twinroller drum dryer. Protein digestibility and available lysine PER, NPR, and NPU were determined for the different formulas. Formulation F3 (60% sorghum, 20% chickpeas, 5% sesame, 8.5% skim milk powder, 5% sugar, and 1.5% vitamins ‡ minerals) and F2 (55% sorghum, 15% chickpeas, 5% groundnuts, 10% sesame, 8.5% skim milk powder, 5% sugar, and 1.5% vitamins ‡ minerals) had compositions and properties comparable to those of Cerelac. In summary, cosupplementation using different protein sources is a key feature of weaning formulations. The choice and order of processing applied are also important, with some forms of processing (sprouting, fermentation, malting) sometimes able to compensate for others (autoclaving) (73).

6.

CEREAL AND CEREAL PRODUCTS

Cereal products (bread, biscuits, cooked rice, noodles, pasta, etc.) are part of the staple diet in much of North and South America, North Africa, and Asia. Cereals also provide proteins indirectly when used as animal feedstuffs. We now consider the nutritional quality of cereal proteins. Bread, biscuit, and pasta making quality etc. are not discussed. The reader should refer to the following reviews for information on protein functional quality (74±76). The effect of extrusion cooking on protein quality was reviewed by Bjoerck and Asp (77), Cheftel (78), and Mercier (79). Salunkhe et al. (80) reviewed the nutritional quality of cereal proteins in general. Lorenz (81) considered the effect of sprouting on cereal protein quality. Dixon-Philip (82) discussed the consequences of milling, baking, extrusion, hydrothermal processing, and fermentation on the quality of cereal products. Examples of processing effects on PNV in cereal-based products are described here. 6.1.

Amaranth

Protein nutrient value for amaranth grain (Amaranthus cruentus) was determined using a nitrogen balance study with 12 adult men over three periods of 9 days each (83). There was no signi®cant difference in digestibility of popped and extruded amaranth. The nitrogen balance index (NBI) was 0.97, 0.98, and 0.96 for cheese, extruded amaranth, and popped

Effect of Processing on Protein Nutrient Value

399

amaranth, respectively. These results indicate that amaranth is a good source of high-quality protein. 6.2.

Maize

Bressani et al. (84) examined the effect of processing maize into tortillas on their nutritional characteristics. Eleven ordinary maize cultivars and one variety of quality-protein maize (QPM) called `Nutricta' were processed into cooked maize or tortillas according to methods used in rural Guatemala. Protein quality was signi®cantly higher (P < 0.03) in tortillas than in raw maize. The QPM cultivar had superior PNV both as raw grain and as tortilla. Gupta (85) reported increased protein quality for maize after sprouting provided that radicals and plumules were removed from corn kernels. The true digestibility was unaffected by sprouting although BV, NPU, and utilizable protein increased. Gupta and Eggum (86) developed a process for transforming the by-product from corn oil production into a food-grade protein meal. Commercial oil cake was extracted with hexane and 80% ethanol and then sieved to remove undesirable materials. The defatted maize germ oil cake had 24.7% protein. Albumin, globulin, and zein decreased while glutelin and residue protein fraction increased. The meal protein had higher levels of lysine and tryptophan than whole maize grain. Protein digestibility and BV were improved as compared with the starting material. 6.3.

Rice, Millet-Sorghum

Eggum and Juliano (87) found that rice protein quality was not adversely affected by simple cooking or parboiling. Extrusion cooking led to adverse effects on protein quality. Lysine is the limiting essential amino acid in millet (88). The digestibility of all cultivars was high (Table 5). Autoclaving led to a 19±25% decrease in the digestibility and an overall increase in BV. Forti®cation with lysine led to greater increases in BV as compared with the effect of heating. Geervani (89) reviewed effects of processing on protein quality in millet sorghum and other cereals important for developing countries. Changes in amino acid composition and protein quality characteristics for millet (as well as barley, oats, wheat, rye, and maize) before and after boiling are discussed. Dry heat processing (e.g., as used for baking biscuits and bread), frying, fermentation (especially sorghum and millet products), and germination are also discussed. Pawar et al. (90) obtained a PER increase from 2.14 to 2.32 for pearl millet by soaking in 0.2 N HCl for 15 hours. Cooking for 20 minutes led to further improvements

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TABLE 5 Protein Quality in Millet Cultivars Index

Value

Protein (%) Lysine (g 16 g 1 N) True digestibility (%) Biological value (%)

10.78±17.3 0.995±1.39 95±99.3 48.3±56.5

Range of samples: Italian millet (Setaria italica), French millet (Panicum miliaceum), barnyard millet (Echinochloa colona), Kodo millet (Paspalum scrobiculatum), and Little millet (P. miliare) were investigated. Source: Summarized from Ref. 88.

in protein quality. The TPD, BV, NPU, and utilizable protein were also increased for soaked and/or scari®ed pearl millet.

6.4.

Storage and Insect Infestation

Insect infestation produced a signi®cant decline in protein quality in wheat grain. Jood and Kapoor (91) examined the effect on wheat grains of 25, 50, and 75% infestation by mixed populations of Trogoderma granarium Everts and Rhizopertha dominica Fabricius. Changes in PNV were evaluated using the rat PER assay. With a diet containing insect-infested wheat grain (at 50 and 75% infestation) there was a decrease in food intake, body weight gain, PER, nitrogen absorption, BV, NPU, and dry matter digestibility. Virtually all protein nutrient quality parameters showed a negative association with infestation levels. Below 25% grain infestation, protein quality was not affected signi®cantly. The mechanism by which protein quality is reduced by insect infestation is uncertain. Infestation may elicit physiological changes associated with plant defense (92). Infested seed samples have increased levels of polyphenols, protease inhibitors, and other plant defense metabolites. Infestation of wheat, maize, and sorghum produced signi®cant decreases in amino acid scores for all essential amino acids. Levels of nonessential amino acids were also reduced. Large reductions were found in methionine, isoleucine, and lysine concentrations for infested wheat, maize, and sorghum grains, respectively. Insect infestation did not change the order of ®rst (lysine) and second (isoleucine) most limiting amino acids.

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7. IMPROVING CEREAL PROTEIN QUALITY BY SCREENING Much effort is being devoted to breeding high-lysine cereal varieties. Developments in this ®eld are discussed by Johnson et al. (93), Whitehouse (94), Bressani (95), Mertz (96), and also de Lumen and Uchimiya (97). The breeding programs require methods for the rapid identi®cation of highlysine seeds. Suitable methods should enable the evaluation of hundreds of seed varieties quickly and cheaply. Many investigators used dye-binding assays to detect high-lysine cereal cultivars. A high correlation is reported between DBC and lysine concentration determined by amino acid analysis. The correlation between lysine content and DBC is signi®cantly improved by normalizing results for Kjeldahl protein (98±100). Example of breeding programs evaluated using dye binding are listed in Table 6. There is generally a negative correlation between lysine content and other grain quality indices. Grain yield, size, and crude protein content

TABLE 6 Identi®cation of High-Lysine Cereal Cultivars Using Dye Binding Cereal Barley Barley Barley Barley Barley Barley, ®eld beans, and wheat Oats Peal millet Rice Rice Rice and maize Rice Sorghum Triticale Wheat Wheat Wheat

References

Country

Doll et al. (98) Bhatty and Wu (104), LaBerge et al. (105), LaBerge et al. (106) Lekes and Rozkosna (107) Saastamoinen (108) Bansal et al. (101) Gullord (99)

Denmark Canada

Finland India Norway

Young et al. (109) Rabson et al. (110) Kaul et al. (111,112) Juliano et al. (113) Le Thi Xuan et al. (114) Chutima et al. (115) Jambunathan et al. (116) Knoblauch (117) Mossberg (118) Sharma and Kaul (119) Iqbal-Khan (120)

USA USA India Philippines Vietnam Thailand India USA Sweden Germany Pakistan

Czechoslovakia

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decreased as the lysine content increased (98,101). Chatterjee and Abrol (102) reported that high-lysine barley varieties were less resistant to damage. Increased lysine content is probably a consequence of the increased synthesis of water-soluble protein (albumin and globulins) at the expense of less soluble proteins (prolamins and glutelins). The soluble protein fractions have a high total basic amino acid content. This increases the avarage protein hydrophilicity, increases their DBC, and increases their electrophoretic mobility. Lawrence et al. (103) examined DBC for seven wheat protein fractions separated by polyacrylamide gel electrophoresis. PAGE lanes were stained with Amido Black 10B. Matching protein bands were excised and analyzed for their amino acid pro®le. Electrophoretic mobility and dye-binding strength are positively correlated with the arginine and lysine content and negatively correlated with the glutamate/ glutamine levels. Total basic amino acid content varied with electrophoretic mobility from the top to the bottom of the gel: *2.5±5% (gliadins), 18±19% (albumins), and 28% (globulins). The genetic linkage between the high-lysine phenotype, impaired endosperm development, and decreased yield is not well understood. Horvatic et al. (121) noted that lysine, methionine, and tryptophan levels in nine cultivars of wheat were negatively correlated with total crude protein and with the gluten content. In high-lysine opaque (o2) maize the endosperm was ¯oury, chalky, and soft. Kernel hardness was also low, leading to reduced resistance to insect damage and low processability. Quality maize o2 hybrids had kernels with increased density, vitreosity, and increased grinding time. Paulis et al. (122) found a positive correlation between kernel hardness and the content of prolamins. Perhaps prolamins somehow enhance protein-starch interactions. Clore and Larkins (123) reported high levels of a protein (designated EF-1alpha) in high-lysine maize varieties. Grain lysine content was highly correlated with concentrations of protein EF-1alpha. Using immunocytochemistry and confocal microscopy, they showed that EF-1alpha was associated with the cytoskeletal network within the developing endosperm. The network of proteins they suggested might be necessary for the formation of protein bodies. It is feasible that increasing EF-1alpha and other lysine-rich proteins may have adverse effects on the cohesiveness of the starchy endosperm. REFERENCES 1.

MA Burnette III, II Rosof. GMA test protocol for protein quality assays. Food Technol 32(12):66±68, 1978.

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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

403

KM Henry, SK Kon, CH Lea, JCD White. Deterioration on storage of dried milk. J Dairy Res 15:292±356, 1948. CH Lea, RS Hannan. Studies of the reaction between proteins and reducing sugars in the ``dry'' state. I. The effect of activity of water, of pH and of temperature on primary reactions between casein and glucose. Biochim Biophys Acta 3:313±325, 1949. CH Lea, RS Hannan. Studies of the reaction between proteins and reducing sugars in the ``dry'' state. II. Further observations on the formation of caseinglucose complex. Biochim Biophys Acta 4:518±531, 1950. CH Lea, RS Hannan. Studies of the reaction between proteins and reducing sugars in the dry state. III. Nature of the protein groups reacting. Biochim Biophys Acta 5:433±454, 1950. AMR van den Bruel, PJ Jenneskens, JJ Mol. Availability of lysine in skim milk powders processed under various conditions. Neth Milk Dairy J 26:19± 30, 1972. M E®genia, B Povoa, T Moraes-Santos. Effect of heat treatment on the nutritional quality of milk proteins. Int Dairy J 7:609±612, 1997. E Renner. Content of available lysine in heat-treated milk products. Kiel Milchwirtsch Forschungsber 35:313±314, 1983. SN El, A Kavas. Available lysine in dried milk after processing. Int J Food Sci Nutr 48:109±111, 1997. A Burvall, N-G Asp, A Dalhqvist, R Oste. Nutritional value of lactosehydrolyzed milk: protein quality after some industrial processes. J Dairy Res 44:549±553, 1977. N Rawson, RR Mahoney. Effect of processing and storage on the protein quality of spray-dried lactose-hydrolyzed milk powder. Lebensm Wiss Technol 16:313±316, 1983. N Rawson, RR Mahoney. A modi®ed method for determination of reactive lysine in milk powder using Rezamol Brilliant Blue R. Lebensm Wiss Technol 16:1±4, 1983. A Burvall, N-G Asp, A Bosson, CS Jose, A Dahlqvist. Storage of lactosehydrolyzed dried milk: effect of water activity on the protein nutritional value. J Dairy Res 45:381±389, 1978. GV Mitchell, E Grundel. Nutritional value for proteins in powdered infant formula. In vitro and in vivo methods. J Agric Food Chem 34:650±653, 1986. G Sarwar, RW Pearce, HG Botting. Effect of amino acid supplementation on protein quality of soy-based infant formulas fed to rats. Plant Foods Hum Nutr 43:259±266, 1993. R Nagendra, V Mahadevamma, V Baskaran, S Venkat-Rao. Shelf-life of spray-dried infant formula supplemented with lactulose. J Food Process Preserv 19:303±315, 1995. E Ferrer, A Alegria, R Farre, P Abellan, F Romero. Effects of thermal processing and storage on available lysine and furfural compounds contents of infant formulas. J Agric Food Chem 48:1817±1822, 2000.

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18. AW Boyne, JJ Carpenter, AA Woodham. Progress report on an assessment of laboratory procedures suggested as indicators of protein quality in feedingstuffs. J Sci Food Agric 12:832±848, 1961. 19. J Bunyan, SA Price. Studies on protein concentrates for animal feeding. J Sci Food Agric 11:25±37, 1960. 20. ET Moran Jr, LS Jensen, J McGinnis. Dye binding by soybean and ®shmeal as an index of quality. J Nutr 79:239±244, 1963. 21. W Choppe, FH Kratzer. Methods for evaluating the feeding quality of meatand-bone meals. Poultry Sci 42:642±646, 1963. 22. RF Hurrell, KJ Carpenter. The use of three dye-binding procedures for the assessment of heat damage to food proteins. Br J Nutr 33:101±115, 1975. 23. KJ Carpenter, J Opstvedt. Applications of chemical and biological assay procedures for lysine to ®shmeals. J Agric Food Chem 24:389±393, 1976. 24. YK Goh, DR Clandinin, AR Robblee. Application of the dye-binding technique for quantitative and qualitative estimation of rapeseed meal protein. Can J Anim Sci 59:181±188, 1979. 25. YK Goh, DR Clandinin, AR Robblee. The application of the dye-binding method for measuring protein denaturation of rapeseed meals caused by autoclave or oven heat treatments for varying periods of time. Can J Anim Sci 59:189±194, 1979. 26. YK Goh, AR Clandinin, AR Robblee. Protein quality evaluation of commercial and laboratory heat-damaged rapeseed meals by the dye-binding technique and by biological assay with chicks. Can J Anim Sci 59:195±201, 1979. 27. FW Sosulski. Rapeseed protein for food use. In: BJF Hudson, ed. Developments in Food ProteinsÐ2. London: Applied Science Publishers, 1983, pp 109±132. 28. SCW Hook. A test for heat-damaged wheat using the RAPID-meter. Milling Feed Fertilizer 162:20±21, 29, 1979. 29. SCW Hook. Dye-binding capacity as a sensitive index for the thermal denaturation of wheat protein. A test for heat-damaged wheat. J Sci Food Agric 31:67±81, 1980. 30. IM Peal, MP Szakacs, A Kowvago, J Petroczy. Stoichiometric dye-binding procedure for the determination of the reactive lysine content of soya bean protein. Food Chem 16:163±174, 1985. 31. PG Randall, L Stone, R Jacobs, AEJ McGill. Detection and quanti®cation of heat damage in wheat. Food Rev 15(Suppl 2):45±47, 1988. 32. FH Kratzer, S Bersch, P Vohra. Evaluation of heat-damage to protein by Coomassie Blue G dye binding. J Food Sci 55:805±807, 1990. 33. S Lin, AL Lakin. Thermal denaturation of soy proteins as related to their dyebinding characteristics and functionality. J Am Oil Chem Soc 67:872±878, 1990. 34. SN El. Evaluating protein quality of meats using collagen content. Food Chem 53:209±210, 1995.

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35. J Vanderstoep. Effect of germination on the nutritive value of legumes. Food Technol 35(3):83±85, 1981. 36. KC Chang, LD Satterlee. Chemistry of bean proteins. J Food Process Preserv 6:203±225, 1982. 37. SK Sathe, SS Deshpande, DK Salunkhe. Dry beans of Phaseolus. A review. I. Chemical composition: proteins. CRC Crit Rev Food Sci Nutr 20:1±46, 1984. 38. M Friedman. Nutritional value of proteins from different food sources. A review. J Agric Food Chem 44:6±29, 1996. 39. BO de Lumen, H Uchimiya. Molecular strategies to enhance the nutritional quality of legume protein: an update. AgBiotech News Info 9(3):53N±58N, 1997. 40. M Orgega-Nieblas, L Vazquez-Moreno, MR Robles-Burgueno. Protein quality and antinutritional factors of wild legume seeds from the Sonoran desert. J Agric Food Chem 44:3130±3132, 1996. 41. MM Kapu, KM Shehu, RAI Ega, HO Akanya, GO Obodo, DJ Schaeffer. Protein quality of tamarind and African locust bean seed meals. Lebensm Wiss Technol 23:260±261, 1990. 42. T Hernandez, C Martinez, A Hernandez, G Urbano. Protein quality of alfalfa protein concentrates obtained by freezing. J Agric Food Chem 45:797±802, 1997. 43. V Karuppanan, S Perumal, J Karnam. Chemical composition, amino acid content and protein quality of the little-known legume Bauhinia purpurea L. J Sci Food Agric 73:279±286, 1997. 44. JTA Oliveira, IM Vasconcelos, MJL Gondim, BS. Cavada, RA Moreira, CF Santos, LIM Moreira. J Sci Food Agric 64:417±424, 1994. 45. MKE Youssef, AS Abdel-Gawad. Protein quality and trypsin inhibitors in some common Egyptian legume seeds. Assiut J Agric Sci 23:3±18, 1992. 46. T Aniszewski. Nutritive quality of the alkaloid-poor Washington lupin (Lupinus polyphyllus Lindl var. SF/TA) as a potential protein crop. J Sci Food Agric 61:409±421, 1993. 47. JI Egana, R Uauy, X Cassorla, G Berrera, E Yanez. Sweet lupin protein quality in young men. J Nutr 122:2341±2347, 1993. 48. IA Khalil, S Khan. Protein quality of Asian beans and their wild progenitor, Vigna sublobata (Roxb). Food Chem 52:327±330, 1995. 49. N Wang, PR Bhirud, RT Tyler. Extrusion texturization of air-classi®ed pea protein. J Food Sci 64:509±513, 1999. 50. U Singh, R Jambunathan, K Sexana, N Subrahmanyam. Nutritional quality evaluation of newly developed high-protein genotypes of pigeonpea (Cajanus cajan). J Sci Food Agric 50:201±209, 1990. 51. S Permal, V Karuppanan, J Karnam. Chemical composition and protein quality of the little-known legume, velvet bean (Mucuna pruriens L. DC.). J Agric Food Chem 44:2636±2641, 1996. 52. D Vibha, MM Simlot. Effect of trypsin inhibitor on protein quality of blacksoybean and mothbean meals. J Food Sci Technol India 34:208±211, 1997.

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53. AH R Ahmed, AAAM Nour. Protein quality of common Sudanese leguminous seeds. Lebensm Wiss Technol 23:301±304, 1990. 54. S Datta, SC Datta. Available lysine in cooked pulses. Indian J Nutr Diet 15:128±130, 1978. 55. AE Bender, H Mohammadiha, K Almas. Digestibility of legumes and available lysine content. Qual Plant Plant Foods Hum Nutr 29:219±226, 1979. 56. H Kozlowska, M Piskula, D Rotkiewicz. Steaming whole rapeseeds to improve protein and oil quality. In: Proceedings of the World Conference on Oilseed Technology and Utilization. American Oil Chemists Society, Champaign, Ill pp 458±460, 1993. 57. TFB Van der Poel, J Blonk, DJ van Zuilichem, MG van Oort. Thermal inactivation of lectins and trypsin inhibitor activity during steam processing of dry beans (Phaseolus vulgaris) and effects on protein quality. J Sci Food Agric 53:215±228, 1990. 58. U Mehta, P Verma, I Singh. Effect of cooking, sprouting and dehulling on the nutritional quality and protein digestibility of rice bean (RBL-1) Vigna umbellata in comparison to mung bean (Phaseolus aureus). J Dairy Food Home Sci 12:165±172, 1993. 59. TA El-Adawy, EH Rahma, AA El-Bedawy, TY Sobihah. Effect of soaking process on nutritional quality and protein solubility of some legume seeds. Nahrung 44:339±343, 2000. 60. T Longvah, YG Deosthale. Effect of de-hulling, cooking and roasting on the protein quality of Perilla frutescens seed. Food Chem 63: 519±523, 1998. 61. C Gupta, S Sehgal. Protein quality of developed homemade weaning foods. Plant Foods Hum Nutr 42:239±246, 1992. 62. S Dahiya, AC Kapoor. Biological evaluation of protein quality of homeprocessed supplementary foods for pre-school children. Food Chem 48:183± 188, 1983. 63. P Gahlawat, S Sehgal. Protein quality of weaning foods based on locally available cereal and pulse combination. Plant Foods Hum Nutr 46:245±253, 1994. 64. WH Plahar, NT Annan, CA Nti. Cultivar and processing effects on the pasting characteristics, tannin content and protein quality and digestibility of cowpea (Vigna unguiculata). Plants Foods Hum Nutr 51:343±356, 1997. 65. CK Hira, N Chopra. Effects of roasting on protein quality of chickpea (Cicer arietinum) and peanut (Arachis hypogaea). J Food Sci Technol India 32:501± 503, 1995. 66. M Hernandez-Infante, V Saousa, I Motalvo, E Tena. Impact of microwave heating on hemagglutinins, trypsin inhibitors and protein quality of selected legume seeds. Plant Foods Hum Nutr 52:199±208, 1998. 67. K Lorenz. Microwave heating of foodsÐchanges in nutrient and chemical composition. CRC Crit Rev Food Sci Nutr 7:339±370, 1976. 68. GA Cross, DYC Fung. A review of the effects of microwave cooking on foods. J Environ Health 44(4):188±193, 1982.

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69. RV Santos, MA Udarbe, CC Marcado, JM Gonzales. Effects of extrusion on the protein quality of a milk supplemented rice-mungbean weaning food. ASEAN Food J 8:61±65, 1993. 70. K Addo, S Lykins, C Cotton. Indigenous fermentation and soy forti®cation: effects on protein quality and carbohydrate digestibility of a traditional Ghanaian corn meal. Food Chem 57:377±380, 1996. 71. CA Nti, WA Plahar. Chemical and biological characteristics of a West African weaning food supplemented with cowpea (Vigna unguiculata). Plant Foods Hum Nutr 48(1):45±54, 1995. 72. SEO Mahgoub. Production and evaluation of weaning foods based on sorghum and legumes. Plant Foods Hum Nutr 54(1):29±42, 1999. 73. S Mbithi-Mwikya, W Ooghe, J van Camp, D Ngundi, A Huyghebaert. Amino acid pro®les after sprouting, autoclaving, and lactic acid fermentation of ®nger millet (Eleusine coracan) and kidney beans (Phaseolus vulgaris L.). J Agric Food Chem 48:3081±3085, 2000. 74. NE Pogna, P Tusa, G Boggini. Genetic and biochemical aspects of dough quality in wheat. Adv Food Sci 18:145±151, 1996. 75. CY Lui, KW Sheperd, AJ Rathjen. Improvement of durum wheat pasta making and bread making qualities. Cereal Chem 73:155±166, 1996. 76. SK Bhupendar, JD Scho®eld. Molecular and physico-chemical basis of bread makingÐproperties of wheat gluten proteins: a critical appraisal. J Food Sci Technol India 34:85±102, 1997. 77. I Bjoerck, NG Asp. The effects of extrusion cooking on nutritional valueÐa literature review. J Food Eng 2:281±308, 1983. 78. JC Cheftel. Nutritional effects of extrusion cooking. Food Chem 20:263±283, 1986. 79. C Mercier. Nutritional appraisal of extruded foods. Int J Food Sci Nutr 44(Suppl 1):S45±S53, 1993. 80. DK Salunkhe, SS Kadam, JK Chavan. Nutritional quality of proteins in grain sorghum. Qual Plant Plant Foods Hum Nutr 27:187±205, 1997. 81. K Lorenz. Cereal sprouts: composition, nutritive value, food applications. CRC Crit Rev Food Sci Nutr 14:353±385, 1980. 82. R Dixon-Philip. Nutritional quality of cereal and legume storage proteins. Food Technol 51:62, 64±66, 1997. 83. R Bressani, ECM de Martell, CM de Godinez. Protein quality evaluation of amaranth in adult humans. Plant Foods Hum Nutr 43:123±143, 1990. 84. R Bressani, V Benavides, E Acevedo, MA Ortiz. Changes in selected nutrient contents and in protein quality of common and quality-protein maize during rural tortilla preparation. Cereal Chem 67:515±518, 1994. 85. HO Gupta. Protein quality evaluation of sprouted maize. Plant Foods Hum Nutr 46:85±91, 1994. 86. HO Gupta, BO Eggum. Processing of maize germ oil cake into edible food grade meal and evaluation of its protein quality. Plant Foods Hum Nutr 52:1± 8, 1998.

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87. BO Eggum, BO Juliano. Properties and protein quality in growing rats of a low-glutelin content rice mutant. Cereal Chem 74:200±201, 1997. 88. P Geervani, BO Eggum. Nutrient composition and protein quality of minor millets. Plant Foods Hum Nutr 39:201±208, 1989. 89. P Geervani. The in¯uence of home processing on the quality of cereal and millet proteins. In: International Association of Cereal Chemistry, ed. Amino Acid Composition and Biological Value of Cereal Proteins. 1985, pp 495±519. 90. VD Pawar, MV Khandagale, NF Quadri. Assessment of protein quality of depigmented pearl millet. J Food Sci Technol India 27:109±110, 1990. 91. S Jood, AC Kapoor. Biological evaluation of protein quality of wheat as affected by insect infestation. Food Chem 45:169±174, 1992. 92. S Jood, AC Kapoor, R Singh. Amino acid composition and chemical evaluation of protein quality of cereals as affected by insect infestation. Plant Foods Hum Nutr 48:159±167, 1995. 93. VA Johnson, PJ Mattern, JW Schmidt. The breeding of wheat and maize with improved nutritional value. Proc Nutr Soc 29:20±31, 1970. 94. RNH Whitehouse. The prospects of breeding barley, wheat and oats to meet special requirements in human and animal nutrition. Proc Nutr Soc 29:31±39, 1970. 95. R Bressani. Protein quality of high-lysine maize for humans. Cereal Foods World 36:806±811, 1991. 96. ET Mertz, ed. Quality Protein Maize. St. Paul, MN: American Association of Cereal Chemists, 1995. 97. BO de Lumen, H Uchimiya. Molecular strategies to enhance the nutritional quality of legume protein: an update. AgBiotech News Info 9:53N±58N, 1997. 98. H Doll, B Koie, BO Eggum. Induced high lysine mutants in barley. Radiat Bot 14:73±80, 1974. 99. M Gullord. The dye binding capacity method (DBC-method) for determination of basic amino acids in protein. Meld Nor Landbrukshogsk 53, 21 pp. 100. TR Sharma, AK Kaul. Rationale of using dye-binding capacity (DBC) for the evaluation of protein content and quality in segregating lines of wheat. Z P¯anz 76:204±214, 1976. 101. HC Bansal, RR Singh, S Bhaskaran, IM Santha, BR Murty. Hybridization and selection for improving seed protein in barley. Theor Appl Genet 58:129± 136, 1980. 102. SR Chatterjee, YP Abrol. Protein quality evaluation of popped barley grains (sattu). J Food Sci Technol India 14:247±250, 1977. 103. JM Lawrence, SC Liu, DR Grant. Dye-binding capacity and amino acid content of wheat-protein gel-electrophoresis bands. Cereal Chem 47:110±117, 1970. 104. RS Bhatty, KK Wu. Lysine screening in barley with a modi®ed Udy dyebinding method. Can J Plant Sci 55:685±689, 1975. 105. De LaBerge, DR Metcalfe, R Tkachuk. Comparison of methods for lysine screening in barley. Can J Plant Sci 56:25±30, 1976.

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106. DE LaBerge, AW MacGregor, DR Metcalfe. Screening for high-lysine cultivars in a barley breeding program. Can J Plant Sci 56:817±821, 1976. 107. J Lekes, A Rozkosna. A contribution to the breeding of productive spring barley varieties with higher biological value of the protein. Z P¯anzenzucht 74:199±210, 1975. 108. M Saastamonien. On the DBC protein content and on the amino acid contents in F5 lines of the barley line Hiproly. J Sci Agric Soc Finl 51:40±50, 1979. 109. VL Young, KD Gilchrist, DM Peterson. Protein the current emphasis in oat quality. Cereal Sci Today 18:409±411, 1973. 110. R Rabson, WW Habbam, H Axman. Potential for improving the protein content of pearl millet grain using induced mutation. In: Seed Protein Improvement in Cereals and Grain Legumes, Vol 11. Vienna: International Atomic Agency, 1979, pp 367±376. 111. AK Kaul, RD Dhar, P Raghaviah. The macro and micro dye-binding techniques of estimating the protein quality in food samples. J Food Sci Technol Mysore 7:11±16, 1970. 112. AK Kaul, RD Dhar, MS Swaminathan. Microscopic and other dye binding techniques of screening for proteins in cereals. Plant Foods Hum Nutr 2:113± 117, 1972. 113. BO Juliano, AA Antonio, BV Esmama, Effects of protein content on the distribution and properties of rice proteins. J Sci Food Agric 24:295±306, 1973. 114. Le Thi Xuan, KC Nguyen, HT Nguyen, VU Nguyen. Use of the dye binding method (DBC) for estimating protein and lysine content in rice and maize. Acta Agron Acad Sci Hung 25:391±394, 1976. 115. K Chutima, BR Jackson, S Duangratana, R Boonduang, N Kongseree, K Suwantaradon. Results of multi-location tests over several years for yield and seed protein content of indigenous. Thai rice varieties. In: Seed Protein Improvement in Cereals and Grain Legumes, Vol 11. Vienna: International Atomic Agency, 1979, pp 279±291. 116. R Jambunathan, NS Rao, S Gurtu. Rapid methods for estimating protein and lysine in sorghum (Sorghum bicolor (L.) Moench). Cereal Chem 60:192±194, 1983. 117. CJ Knoblauch. Early generation selection in triticale for factors contributing to protein quality. Dissertation Absracts International B; 36 (1) 31: Order no. 75-14769. 118. R Mossberg. Practical problems concerning the marketing of cereals with improved protein value. Proc Nutr Soc 29:39±48, 1970. 119. TR Sharma, AK Kaul. Correction between some quality characters and protein determinants in bread wheat. Curr Sci 40:150±152, 1971. 120. M Iqbal-Khan. Lysine estimation with the modi®ed Udy-dye binding method in hexaploid wheat. Experientia 34:711±712, 1978. 121. M Horvatic, M Gruener, N Mulalic. Relationship between protein nutritive quality and technological property parameters of wheat ¯our. Nahrung 33:901±903, 1989.

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122. JW Paulis, AJ Peplinski, JA Bietz, TC Nelsen, RR Berquist. Relation of kernel hardness and lysine to alcohol-soluble protein composition in quality protein maize hybrids. J Agric Food Chem 41:2249±2253, 1993. 123. AM Clore, BA Larkins. Protein quality and its potential relationship to the cytoskeleton in maize endosperm. J Plant Physiol 152:630±635, 1998.

14 Protein Digestibility±Corrected Amino Acid Scores

1. INTRODUCTION The protein digestibility±corrected amino acid score (PDCAAS) was adopted as the AOAC-approved index for protein quality in 1993±1994. The PDCAAS is the amino acid score (AAS) multiplied by protein digestibility. Alterations in either parameter changes protein nutrient value (PNV). PDCAAS is discussed in this chapter. In Sec. 2 considration is given to digestibility and its relation to protein structure. In Secs 3 and 4 is a review of protein denaturation and chemical deterioration during food processing and their effect on the PDCAAS. There is also increasing realization that moisture-temperature-time relations affect the food matrix and protein quality. Some possible links between Tg (glass transition temperature) and PNV are explored in Sec. 5. The determination of PDCAAS for a range of foods is discussed in Sec. 6.

2. PROTEIN DIGESTIBILITY True protein digestibility (TPD) measured using a rat bioassay agrees with in vitro protein digestibility (Chapter 13). This results has two implications: 411

412

Chapter 14

1. Absorption across the intestinal membrane does not limit (bio)availability. Exceptions to this rule appear in Section 6.7, where availability-corrected amino acid scores (AvCAAS) are discussed. 2. Fundamental research on protein structure and susceptibility to proteolysis can be marshaled to help make sense of the PDCAAS.

2.1.

Protein Conformation, Molten Globules, and Digestibility

Binding interactions between a protease and its substrate follow the approach of two molecules of comparable sizes. An exposed region on the substrate protein then enters the protease active site. Linderstrom-Lang et al. (1) described enymatic digestion via a two-stage reaction. First, the native protein (N) conformation changes to an unfolded (U) state that exposes peptide bonds to the external solvent [Eq. (1)]. N„U

…1†

Protein unfolding increases within the stomach. The low-pH, high-chloride environment transforms globular proteins into a molten globule state. This is an expanded, more ¯exible structure compared with the N state (2,3). At low pH, ®brous proteins (myosin, collagen) exhibit changes in proteinprotein interactions followed by unfolding. Second, the U state is hydrolyzed by an enzymatic reaction that leads to the irreversibly modi®ed (I) state [Eq. (2)]. U!I

…2†

The rate of proteolysis (v) is determined by the fraction of protein substrates unfolded (FU) and the rate constant for the U ! I reaction (ki): v & ki PFU ˆ

ki PKeq 1 ‡ Keq

…3†

where P is the protein concentration. The preceding relation applies to the whole protein or to speci®c segments.* * Protein unfolding is often described as a two-state (all or nothing) process. In this idealized model all peptide bonds are either inaccessible or fully accessible to the external solvent. In contrast to this global (un)folding process, a non±two-state unfolding event leads to the localized exposure of bonds.

Corrected Amino Acid Scores

2.2.

413

Protein Structure, Stability, and Digestibility

In vitro digestibility is a sensitive probe for protein structure. Under nondenaturing conditions Keq 5 1 and FU &Keq [see Eq. (3)]. The value of Keq is ®nitely large or small but never ``zero.'' Proteases attack the U state readily and therefore slight changes in the N/U equilibrium [Eq. (1)] alter protease susceptibility. Proteolytic attack on the N state conformation occurs at surfaces, exposed peptide loops, beta-turns, and at random (aperiodic) sequences.* Digestion is more likely between protein domains. Oligomeric proteins are digested following dissociation into subunits. The rate of digestion is low within areas of regular secondary structure. Protein stability is inversely related to digestibility (4). Matthyssens et al. (5) monitored the thermal denaturation of lysozyme at pH 2 by measuring the rate of proteolysis by pepsin. With increasing temperatures, lysozyme unfolds and the rate of proteolysis increases. Imoto et al. (6,7) monitored the thermal unfolding transition for lysozyme from the kinetics of proteolysis with pronase. Church et al. (8) used immobilized pronase activity as a probe for normal or chemically modi®ed lysozyme, betalactoglobulin, and casein. Daniel et al. (9) established an inverse correlation between protein stability and susceptibility to proteolysis. Ueno and Harrington (10) monitored structural changes in myosin at 5±408C using papain, chymotrypsin, or trypsin. A relationship between protein digestibility, ¯exibility, surface hydrophobicity, and physical functional properties (foaming, emulsi®cation) was proposed by Kato et al. (11). Kato et al. (12) reported a similar relation for heat-denatured lysozyme and ovalbumin. The sites of autolysis for thermolysin had high segmental mobility as detected by temperature factors (B values) from X-ray crystallography (13). Unstable proteins for which the N/U equilibrium lies toward the U state are more susceptible to proteolytic degradation. Acid-stable proteins (e.g., betalactoglobulin) are resistant to unfolding and hence to protease attack. The relationship between protein stability and digestibility is altered within the stomach. The majority of food proteins become destabilized at low pH at 378C. Using the ¯uorescent dye anilino-naphthalene-8-sulfonic acid (ANS). Folawiyo and Apenten (14) showed that oilseed proteins undergo a conformational change at pH 1±2. The degree of ANS binding at low pH was 10-fold greater than obtained by heating alone. Preheating had little effect on ANS binding sites at low pH.

* Under nondenaturing conditions, the small proportion of the U state in equilibrium with the native state is attacked by proteases. It is a moot point whether the N state itself undergoes proteolytic attack.

414

2.3.

Chapter 14

Enzyme Speci®city

The rate of proteolysis is also determined by protease speci®city. Schechter and Berger (15) supposed that proteases have up to seven active sites (. . . S3, S2, S1, S10 , S20 , S30 ) with speci®city for six peptidyl sites (P3, P2, P1, P10 , P20 , P30 ). The peptide bond subjected to cleavage occurs between P1 and P10 . Different proteases recognize different P1, P10 residues for bond hydrolysis. For trypsin, chymotrypsin, and pepsin, P1 ˆ lysine or arginine, an aromatic amino acid, or a hydrophobie residue, respectively. The effect of enzyme speci®city on the rate of proteolysis appears in the ki term of Eq. (3). In accordance with Michaelis-Menten formalism, ki ˆ Vmax =Km (ratio of the maximum velocity to the Michaelis constant). 3.

PROTEIN DENATURATION

Changes in protein structure affect digestibility and PNV. The important variables are temperature, pressure, pH, ionic strength, presence of surfaces, and shear rate (Table 1). Changes in protein structure follow a two-stage process [Eq. (1)±Eq. (2)]. Unfolding is either a global process or is restricted to speci®c segments of the protein. Exposure to chemical denaturing agents (urea, guanidine hydrochloride) leads to a U state that is approximately a random coil. Extremes of pH, extremes of temperature, and/or high pressure produce the partially unfolded (molten globule) state (Table 1, row C). The N/U transition [Eq. (1)] is formally reversible. Removing the protein from stress restores the N state. Irreversible U ! I reactions occur after prolonged exposure to extreme conditions [Eq. (2)]. Besides LinderstromLang and co-workers (1), Equations (1) and (2) were also developed by Eyring and Lumry (16), who applied them to describe protein denaturation. Proteolysis is an irreversible denaturation process where the U ! I reaction is peptide bond hydrolysis catalyzed by a protease. Nonenzymatic proteolysis occurs at low pH and at temperatures of about 1008C. Structural changes of practical signi®cance are irreversible. Charles Tanford's treatment of this subject is still unsurpassed after 30 years (17). Ahern and Klibanov (18,19) have also discussed denaturation at length. Kinsella (20) reviewed the chemistry of food protein reactions. On the relatively long time scale associated with aging and development, there is a constant leakage from the U state to the I state. The postharvest state is characterized by increased formation of the I state. Table 1 (row B) lists factors that are likely to support reversible or

Corrected Amino Acid Scores

415

TABLE 1 Forms of Food Protein Denaturationa A. Reversible denaturation

A*. Irreversible denaturation

N„U

N!I

B. Conditions for the N „ U transition Low protein concentration (5% w/v) Solvent pH&pI Extremes of pH (pH < 4, pH > 8) Prolonged exposure to denaturant

Low ionic strength C. Some N „ U processes Transient unfolding, expansion Semimolten globule Molten globule Domain denatured

High intensity of denaturant/severe process conditions High ionic strengh C0 . Some N ! I processes Covalent aggregation Noncovalent aggregaration Peptide bond lysis Racemization Cross-link formation Carbonylamine reactions Deamidation

D. Processing variables High temperatures (sterilization, cooking, drying) Low temperature (cold storage, freezing) Moisture control (dehydrated storage, drying) High pressure (sterilization, low-temperature gelation) High or low pH (solubilization, texturization) Procesing chemicals (alcohols, alkali, nitrate, reducing compounds, etc.) Exposure to interfaces (emul®cation, foaming, etc.) High shear treatment a

Notes: A, Two forms of protein denaturation; B, conditions that facilitate reversible and irreversible denaturation; C, types of reversible and irreversible denaturation; D, forms of food processes leading to denaturation.

irreversible denaturation. Irreversible changes in protein conformation arise from covalent aggregation via sulfhydryl-disul®de exchange or sulfhydryl oxidation. Physical denaturation processes such as gelation will be reversed in the low-pH gastric environment.

416

4.

Chapter 14

CHEMICAL DETERIORATION OF PROTEIN INGREDIENTS

Denaturation was initially de®ned only in operational terms by reference to the changing physical appearances of proteins. Protein precipitation, aggregation, gelation, or increases in viscosity were taken as signs of denaturation. These changes in appearance can be induced (by salting-out, exposure to organic solvents) without denaturation. After the publication of the 3D structure for myolglobin in 1963, denaturation was rede®ned in terms of changes in the 28, 38, or 48 structure. Alterations in protein 18 structure were not included in the de®nition of denaturation. It seemed unlikely that changes in 18 structure could occur under normal physiological  conditions (atmospheric pressure, T ˆ 0±37 C). There is now much sympathy for the idea that proteins from archaebacteria evolved at temperatures of 80±1208C. Many organisms persist under ``extreme'' physiological conditions of temperature and pressure where changes in protein 18 structure are conceivable. The conventional de®nition of denaturation is also too restricting in view of recent advances in protein chemistry and technology. Proteins exhibit 18 structure changes during food processing. The range of deteriorative changes includes peptide bond hydrolysis, carbonyl-amine reactions, racemization to form D-amino acids, and formation of covalent cross-links. Deamidation of glutamine and asparagine transforms these residues into glutamate and aspartate, respectively. The SH-disul®de exchange produces cystine cross-links from cysteine. Loss of hydrogen sul®de and dehydration generate dehydroalanine. Heating in the presence of sugars leads to glycation. Most deteriorative processes are accelerated by water. Addition of water to intermediate moisture foods encourages deterioration by lowering the glass transition temperature (Tg). With dissolved proteins, deterioration follows protein unfolding to expose reactive amino acid residues to the external solvent. 4.1.

Carbonyl-Amine Reactions

Within a polypeptide all except three essential amino acids are protected from the Maillard reaction. Luise Maillard (21) discovered the reactions between amino acids and sugars. Nonenzymatic browning leads to the formation of melanoidins and ¯avor precursors. The reactive protein residues are lysine (e-NH2), methionine (22S22CH3), and tryptophan (indole group). With high-carbohydrate (plant) foods, lysine is the most reactive essential amino acid and the most limiting. Methionine or tryptophan is usually limiting for animal proteins.

Corrected Amino Acid Scores

417

Carbonyl-amine reactions take place in four stages: (a) addition of the e-NH2 group of lysine to a carbonyl group of an aldose, (b) elimination of water to form a cationic Schiff base, (c) proton loss to form an enol, and (d) enol-keto rearrangement to form 1-amino-1-deoxy-2-ketose. Reactions (c) and (d) together constitute the Amadori rearrangement. For a keto-sugar the corresponding reaction is the Heyens reaction leading to 2-amino-2deoxyaldose. The effect of carbonyl-amine reactions on protein nutrient value (PNV) is reviewed by Dworschalk (22), Labuza and Saltmarch (23), Friedman (24,25), Saltmarch and Labuza (26), Feeney and Whitaker (27), Hurrell (28), and Feather (29). 4.2.

Controlling Factors for Carbonyl-Amine Reactions

The effect of pH, water activity (Aw), temperature, and different sugars on the rate of the carbonyl-amine reaction was studied by Labuza and Saltmarch (23,26). Maillard browning shows a bell-shaped pH dependence with a maximum at about pH 8. Extremes of pH lead to a decline in the rate of browning by (de)protonating the carbonyl and amino functions. The dependence on AW is also bell shaped. Browning decreases with decreasing moisture content due to the high medium viscosity and diffusion restrictions. With increasing AW the reaction rate increases until suf®cient water is available for monolayer coverage. With still higher moisture content the rate of reaction decreases due to the dilution of reactants. The temperature dependence of the Maillard reaction was a described with linear Arrhenius equation. The activation energy for lysine loss was about 80 kJ mol 1 (Section 5.3). The relative rates of reaction with different sugars follow the order lactose > D-ribose > D-fructose > D-glucose; pentose > hexose > disaccharide. 4.3.

Impact on Protein Quality

The products of the carbonyl-amine reactions have two principal effects on PNV: (a) the bioavailability of lysine is reduced and (b) digestibility is reduced by the presence of protein-bound sugar residues. The Amadori compound (1-amino-1-deoxy-2-ketose) is biologically unavailable (30). After ingestion by rats, a signi®cant proportion is excreted in the urine. The nonabsorbed fraction appears in the feces and/or is degraded by intestinal bacteria. Peptide bonds in the vicinity of glycated lysine residues are not susceptible to protease attack. Maillard reaction products also inhibit the absorption of other amino acids. Suggested physiological effects include increased protein allergenicity, mutagenicity, and effects on the reproductive capacity of rats. Normal cooking does not affect PNV.

418

Chapter 14

Microwave cooking also had no adverse effects (31,32). Maillard reaction products form during baking that have a detrimental effect on protein quality. The effects of the Maillard reaction on available lysine is also discussed in Chapter 12 and 13. 4.4.

Racemization and Cross-Linking

Cysteine, serine, and phosphothreonine residues are converted to dehydroalanine at high pH. The process involves the elimination of hydrogen sul®de, water, or the phosphate group, respectively. Dehydroalanine then reacts with a range of protein groups forming cross-links (Fig. 1). Racemization also takes place under alkaline conditions (33). The two reactions proceed via the following common steps: (a) abstraction of a proton from an a-C atom by a base (B) produces a carbanion ion, (b) readdition of H‡ to the a-C then generates the corresponding D-amino acid, (c) b-elimination from the carbanion ion produces dehydroalanine, and (d) addition of protein side chains (histidine, cysteine, lysine) to dehydroalaninie forms cross-links. The subject is reviewed by Hurrell (28), Maga (34), Otterburn (35), and Swaisgood and Catignani (36). 4.5.

Factors Controlling the Rate of Racemization and Cross-Linking

Friedman and Liardon (37) found that the rate of racemization is sensitive to ( ‡ ) or ( ) inductive effects. For simple amino acids the rate of racemization was predicted using linear free energy relationships. Free amino acids are *10-fold less reactive than protein-bound amino acids. The presence of an adjacent carboxyl group destabilizes the intermediate carbanion ion. A high pH (>9) increases the rate of racemization by facilitating the initial proton abstraction. Proline residues (especially when next to aspartate or aparagine) are more reactive than other amino acid residues. Polypeptide sequences containing serine and cysteine (e.g., Ser/ Cys22X22Lys or Ser/Cys22X22X) are also highly reactive. Racemization and cross-linking can be observed by heating model proteins in alkaline solution (38,39). Similar reactions take place in foods when proteins are exposed to high pH and temperature. A traditional method for producing tortilla involves steeping corn in 1% lime solution at 808C for 20±45 minutes. The preparation of ®sh protein concentrates, texturization of soy protein, recovery of residual protein from bone, alkaline extraction of plant or yeast proteins, and lye peeling also exposes food proteins to high pH values. Cross-linking and racemization reduce in vitro protein digestibility (40,41) as well as PNV (42).

Corrected Amino Acid Scores

419

FIGURE 1 A scheme for amino acid racemization, dehydroalanine formation, and protein cross-linking. Based on a scheme proposed by Masters and Friedman (33). B ˆ base.

420

5.

Chapter 14

MATRIX EFFECTS ON THE RATE OF DETERIORATION OF PROTEIN INGREDIENTS

The glass transition temperature (Tg) is an important index for molecular mobility and diffusivity within food matrices (43). Studies of the relationship between Tg and the rate of deterioration of protein ingredients are just beginning. Matrix-based models are also currently being formulated and tested in relation to freeze-dried (pharmaceutical) proteins. These studies and the small number of investigations involving food protein ingredients are discussed here. 5.1.

The Glass Transition Temperature

Changes within highly concentrated, glass-forming, food systems were described using a polymer chemistry approach by Slade and Levine (44,45). Key elements of glassy state and its relation to food quality deterioration can be summarized as follows: 1. Foods and food materials can be treated as classic polymer systems. 2. The glass transition temperature (Tg) for a glass-rubber transition is a critical parameter that determines processability, quality, stability, and safety. 3. At temperatures below Tg the viscosity of a food matrix is extremely high such that physical and chemical changes are inhibited. 4. Above the Tg the viscosity of a food matrix decreases and various relaxation process (or chemical changes) become possible. 5. Food stability may be assured by storing at temperatures below T g. 6. Water is a ubiquitous plasticizer for both natural and fabricated food ingredients and products. The plasticizing effect of water reduces Tg. 7. At temperatures above the Tg materials are in a disordered (rubbery or amorphous) state. 8. William-Landel-Ferry (WLF) kinetics apply at Tg < T < Tg ‡ 1008C. The kinetics of food change does not conform to the linear Arrhenius equation. 9. Nonequilibrium glass/rubbery state transitions affect all timedependent structural and mechanical properties in real-world foods; in contrast, thermodynamic concepts such as water activity are inapplicable.

Corrected Amino Acid Scores

5.2.

421

Models for Food Matrices

Food matrices are described using the fringed-micelle model or the folder-chain lamella model. According to the fringed-micelle model, food materials are composed of crystalline and amorphous phases. Strong intermolecular associations between polymer chains account for regions of high order. Amorphous or disordered regions arise from the lack of strong polymer-polymer interactions. Heating leads to consecutive orderdisorder transitions involving a glassy/rubber state (at Tg) followed by a crystalline/amorphous transition at the melting temperature (Tm). At temperatures below the Tg the system possesses a viscosity of about 1012 Pa s. Most physical and chemical processes (microbial growth, enzymatic activity, protein deterioration) are severely inhibited. For pure biopolymers Tg is generally about 150±2008C. As Tg 5 Tm a solid may undergo decomposition before the Tm is reached. According to the folded-chain lamella model (46), crystalline regions are formed by intramolecular associations involving a single polymer chain folded back on itself. Amorphous regions occur at interruption zones or regions of the polymer possessing defects, kinks, and other irregularities in the structure. The plasticizing action of water leads to Tg decreasing with increasing moisture content. At high moisture levels (>20% w/w) Tg decreases to below the freezing point for water. Cooling produces a freeze-concentrated system as pure solvent water solidi®es into ice. The freeze-concentrated component then undergoes a liquid/glass phase transition at characteristic temperature designated Tg0 . Solutes for which the Tg0 value is high exhibit greater preserving action (see later). The polymer science approach provides satisfactory explanations for a wide range of food-related phenomena including microbiological stability, enzymatic activity at low Aw, inhibition of collapse, and improved freeze drying, cooking, and frying processes. For low or intermediate moisture foods, the new approach leads to an integrated discussion of moisture, temperature, and time relations during the processing or storage. Speci®c process end points can be achieved using different time-temperature, moisture-temperature, and moisture-time combinations. 5.3.

The Glass Transition and Protein Quality

The Tg for gluten was reported by several workers (47±52). The Tg has also been reported for beef proteins (53), caseinate (54), and gliadin (55±57). Noel et al. (58) determined the Tg for fractionated wheat gluten proteins (a-, g-, and o-gliadins and HMW glutenin) using the Perkin-Elmer DSC2

422

Chapter 14

microcalorimeter ®tted with a liquid nitrogen cooling accessory (Fig. 2). Temperature scans from 270 and 370 K showed an exothermic peak at about 320 K (or 478C) for o-gliadin equilibrated with 12% water. For a moisture content < 20%, Tg decreased in accordance with the GordonTaylor equation, Tg ˆ

w1 Tg 1 ‡ w2 Tg 2 k w1 ‡ w2 k

…4†

where Tg is the observed glass transition temperature, Tg1 is the glass transition temperature for the pure protein, Tg2 is the glass-transition temperature for the pure plasticizer ( 138 K for water), w1 and w2 are the weight fractions of protein and moisture, respectively, and k is a constant whose value increases with increasing plasticization. Nonlinear regression analysis lead to prediction of Tg1 for dry proteins based on the

FIGURE 2 Effect of moisture on the glass transition temperature for fractionated glutenin proteins. High-molecular-weight glutenin subunits (HMW) a-gliadin, g-gliadin, and o-gliadin. Pro®les were generated according the Gordon-Taylor relation, Eq. (4), using values for Tg1 and k given in Refs. 55 and 56.

Corrected Amino Acid Scores

423

observed Tg values at different moisture levels. Values of Tg1 ranged from 397 K (1248C) to 417 K (1448C) for the four gluten proteins. In the presence of 20% moisture, Tg decreased to between 270 K ( 38C) and 280 K (78C). Morales and Kokini (59) measured Tg for 7S and 11S soy globulin fractions using DSC and mechanical spectrometry. There were two Tg values due to cross-contaminating amounts of the 7S globulin in the sample of 11S globulins and vice versa. Single Tg values were obtained for highly puri®ed soy globulins. With 7S soy protein Tg was 387 K (1148C) to 206 K (678C) for moisture contents of 0 to 35%, respectively. For the puri®ed 11S fraction Tg ranged from 433 K (1608C) to 256 K ( 178C) for moisture contents from 0 to 40%. Soy globulins behave as polymers that are highly plasticizable by water. The effect of moisture on the Tg and caking properties of ®sh protein hydrolysate was examined by Aguiliera et al. (60). Increasing the relative vapor pressure from 0 to 0.64 reduced Tg from 352.1 K (79.18C) to 230.2 K ( 42.88C). The plasticizing action of water was described by the GordonTaylor equation [Eq. (4)]. At a ®xed temperature of 198C (room temperature) collapse was initiated at a relative vapor pressure of 0.44, corresponding to the T Tg value of 35.88C. Above a relative vapor pressure of 0.55 the following quality defects occurred: nonenzymic browning, collapse, shrinkage, and setting into a sticky, high-viscosity brown liquid. Using the WLF equation, the viscosity of the matrix at the onset of collapse was estimated as 105±107 (Pa
s). To preserve protein samples from deterioration it is necessary to employ storage temperatures below the Tg. However, the glassy-rubbery state transition takes place over a ®nite temperature interval. Peleg (61±63) modeled mechanical changes during the glass-rubber transition using Fermi's equation: Yˆ

Yg 1 ‡ exp…T Tc=A†

…5†

TC ˆ TC;0 exp… kw†

…6†

A ˆ A0 exp…k00 w†

…7†

where Y is an apparent stiffness parameter; Yg is the stiffness in the glassy state; T is temperature; TC is the in¯exion temperature, which is not necessarily coincident with the Tg; and A is a parameter that measures the slope of the Y-temperature graph. Both TC and A are dependent on the %

424

Chapter 14

moisture content (w). We have replicated these simulations (Fig. 3) with the following two results: (a) the in¯ection temperature decreases with increasing moisture, and (b) the gradient of each graph increases with increasing moisture. Therefore, sensitivity to temperature increases with increasing moisture. To preserve high-moisture foods requires a low storage temperature and improved temperature control. Drying allows higher storage temperatures and increased tolerance with respect to temperature variations. So far, only a few investigators have applied the polymer science approach to food protein deterioration. The physical-mechanical basis for glass-liquid transitions for proteins has not been extensively discussed. According to Ferry (64), Tg is the temperature below which wriggling motions and conformational rearrangements within a polymer cease. Liquids and polymers have a constitutive volume (determined by van der Waals contacts) and free volume arising from packing irregularities or defects. During cooling the free volume changes in accordance with the

FIGURE 3

Effect of temperature and moisture on the apparent stiffness of gliadin near the glass transition temperature. The moisture content (%) is shown in the boxed legend. The in¯ection temperature for each transition is 138C, 38C, 148C, 288C, and 258C at a moisture content of 11.25%, 13%, 19.25%, 24.4%, and 27.2%. Simulations were performed using equation parameters from Peleg (56,61±63).

Corrected Amino Acid Scores

425

thermal expansion coef®cient. The Tg is a narrow temperature region in which the thermal expansion coef®cient, heat capacity, adiabatic compressibility, and speci®c volume show discontinuity. What can undergo a glass transition in a protein solid? In answer to this self-posed question, Morozov and Gevorkian (65) suggested that the glass-liquid transition in globular proteins involves surface groups and packing defects. These structural components may or may not be hydrated. There are also aperiodic or random sequences in most proteins. 5.4.

Studies on Freeze-Dried Proteins

The storage stability of pharmacologically active proteins is related to Tg. Freeze-dried high-value proteins undergo so-called moisture-induced deteriorative reactions. Most of these reactions have been identi®ed by accelerated testing (Table 1). Liu et al. (66) found that the storage stability of freeze-dried proteins was increased by adding excipients, mainly polyhydroxy alcohols. Costantino et al. (67) reported similar ®ndings. For proteins in the dry state additives act as platicizers that reduce Tg in accordance with the Gordon-Taylor equation. The destabilizing action of added excipients is inversely related to the Tg of the pure additive. Added excipients are stabilizing as compared with the equivalent weight of water. To achieve stabilization the pure additive should have Tg2 higher than water. Excipients also increase the denaturation temperature (Tm) in direct proportion to their effect on Tg (68±70). Indeed, for some synthetic polymers the two transition temperatures are strongly correlated: Tg …K† ˆ 0:66 …+0:04† Tm

…8†

The glass-liquid transition and the crystalline-amorphous transition appear to be subject to similar constraints (46). 5.5.

Protein Conformational Stability, Tm, and Quality

Protein solutions have Tg values below room temperature although Tm remains high (30±908C). Regular 28 structure yields regions of order and crystallinity within the N state. The net conformational (dis)order determines the peak temperature (Tm) and enthalpy change (DH) measured by differential scanning calorimetry (DSC). Equation (8) suggests that a high Tm (compare Tg) will produce lower rates of protein deterioration in solution. Protein stability-function relations were discussed by Apenten and Berthalon (71) and Apenten (72) using the two-stage denaturation scheme. Reactive amino acid groups are buried in the N state and become accessible

426

Chapter 14

to the solvent after protein unfolding. High conformational stability therefore attenuates U ! I reactions [see Eqs (1)±(3)]. The magnitude of DE # provides useful information about the mechanism of deterioration. The N/U transition is a highly cooperative process with a large temperature coef®cient (Q10 > 2). Where the ratelimiting step for deterioration is a conformational change [Eq. (1)] the value of DE # should be 250±700 kJ mol 1. In contrast, when the U ! I reaction determines the rate of deterioration then DE # is small (20±80 kJ mol 1). The magnitude of DE # is not only determined by the rate-limiting step for protein deterioration because 1. Deterioration is a multistage reaction [Eqs (1)±(3)]. Therefore, DE # is the sum of values for several reactions. 2. Quality loss might not conform to the N ! U ! I scheme. Multiple U states (U1, U2, U . . . Ui) and/or U ! I reaction occur. 3. DE # is temperature dependent (71,72). Labuza and Saltmarch (73) investigated the effect of moisture and temperature on available lysine and nonenzymatic browning in whey powder. Labuza et al. (74) examined similar reactions for pasta. The loss of FDNB-lysine and nonenzymatic browning followed ®rst- and zero-order kinetics, respectively. The rates of lysine and PNV losses were two to three orders of magnitude greater than the rate of browning. The reaction temperature dependence ®tted a linear Arrhenius equation at 35±558C with a squared regression (R2) coef®cient of *0.601. The DE # for lysine loss and browning ranged from 52 to 85 kJ mol 1 depending on the prevailing AW. Adding data from 258C produced curved Arrhenius plots. I have reexamined these results using a higher order Arrhenius equation that allows for variations in the value for DE # with temperature. The simple Arrhenius equation is ln k ˆ ln k0

DE # RT

…9†

where k is a rate constant and DE # is the energy required to form an activated complex. A semilog arithmic plot of ln k versus 1/T leads to a straight-line graph having a slope equal to DE # =R and an intercept value of ln k0 . A more comprehensive discussion of reaction rates is based on the transition-state theory. Here the activated complex from the Arrhenius model is replaced by a transition state, and ``activation energy'' becomes DG# (the Gibbs free energy change for producing the transition state). Comparing the Arrhenius and transition state reaction rate models leads to the realization that DE # is essentially equal to DH # (enthalpy change for

Corrected Amino Acid Scores

427

producing a transition state) where DH # ˆ DE # 2RT. Reactions taking place ``in water'' can be accompanied by signi®cant heat capacity change; DCp# ˆ d…DH # †=dT.* To allow for a temperature-dependent DH # value, the Arrhenius equation is expanded to a second-order polynomial. ln k ˆ a ‡ bx ‡ cx2

…10†

where a, b and c are constants and x ˆ 1=T. From general thermodynamic principles DE # ˆ Rd ln …k†=dx & DH # and d…DH # †=dT ˆ DCp# and therefore DH # ˆ

2c ‡b T 1

…11†

DCp# ˆ

2Rc T 2

…12†

and

Fig. 4 and Table 2 show results for the deterioration of pasta (74). The loss of FNDB-lysine and browning reactions were adequately described by Equation (10) (R2 ˆ 0:98 0:99). Close to the Tg (1208C) for a dry protein the magnitude of DH # is large, implying that quality loss may be limited by conformational transitions. The low DH # value near room temperature is a consequence of the temperature dependence of this parameter.

6. PROTEIN DIGESTIBILITY±CORRECTED AMINO ACID SCORES (PDCAAS) There has long been dissatisfaction with the rat PER assay. In 1991 an FAO/WHO Expert Consultative Group (75) agreed that the PER test should be replaced by a new method with the following characteristics: (a) greater accuracy and reproducibility than the PER test, (b) shorter time for assay, (< 48 hours per test), (c) incorporation of digestibility, (d) wider applicability to samples without extensive prepretreatment, (e) simplicity

* The heat capacity (Cp; J g 1 K 1) is the amount of heat required to raise the temperature of 1 g of material by 1 K. The reaction A ‡ B ! C ‡ D will lead to a heat capacity change (DCp ) if the reactants and products interact differently with the solvent.

428

Chapter 14

FIGURE 4 Arrhenius plot for protein deterioration in pasta at different temperatures. Relative vapor pressure is 0.44. (Based on data from Ref. 74.)

TABLE 2 Activation Parameters for Protein Deterioration in Pasta; Analysis by a Nonlinear Arrhenius Equation Parameter a b c R2 DH# (kJ mol 1) DS# (J mol 1K 1) DG# (kJ mol 1) DCp# (J mol 1 K 1) a

Lys loss (RVP ˆ 0.44)

Browning (RVP ˆ 0.44)

212.3 1.328 6 105 1.968 6 107 0.991 42.2a (272.2) 202.8 (419.1) 104.79 (107.6) 4395

192.7 1.22 6 105 1.825 6 107 0.980 40.1 (252.6) 229.1 (264.5) 110.7 (146.6) 2359

The ®rst and second values are for temperatures of 378C and 1208C.

Corrected Amino Acid Scores

429

and suitability for routine use, and (f) low cost not exceeding $200 per test at 1991 prices. The panel of eminent nutritionists selected a new test involving protein digestibility-corrected amino acid score (PDCAAS). They further agreed that PDCAAS should be based on the FAO/WHO/UNU 1985 list showing the essential amino acid requirements for 2±5 yr. preschool children (76). The values are given in Chapter 12 (p. 355). In 1993 the FDA (USA) adopted the PDCAAS procedure for the routine evaluation of PNV and for food labeling purposes. Developments leading to the adoption of the PDCAAS test are reviewed by Sarwar and McDonough (77), Boutrif (78), Madi (79), Henley and Kuster (80), and also Kuntz (81). The basic principles for evaluating PDCAAS and examples of its use are discussed in Section 6.5. First, however, we review older methods for PNV evaluation and how the PDCAAS index evolved (Sections 6.1±6.4). 6.1.

Protein Chemical Score

Mendel (82) stated that a protein's quality is related to its minimum quantity of essential amino acids. In 1946 Mitchell and Block (83) de®ned the chemical score for protein quality by calculating the de®cit of essential amino acids as compared with essential amino acids from whole egg. Samples with equal amounts of crude protein (%N 6 6.25) were analyzed for their essential amino acid content. The results were each divided by the essential amino acid content for whole egg. The result having the largest de®cit compared with egg protein shows the limiting essential amino acid. Chemical score ˆ

EAASAMPLE EAAWHOLE EGG

…13†

The chemical score for 28 proteins was positively correlated BV determined using the rat bioassay (Table 3). Thermal processing produced signi®cant changes in BV without affecting the essential amino acid content of proteins (Fig. 5). Knorr (84) found that potato protein concentrates prepared in different ways possessed high chemical scores and may be useful for human nutrition. Based on their chemical scores, immature durum wheat has a higher nutrient value than mature wheat (85). Chemical scores for eight peanut cultivars were highly correlated with RNV (R ˆ 0:98) and PER (R ˆ 0:88) (86). Ruales and Nair (87) found that the limiting amino acids in quinoa ¯our were tyrosine and phenylalanine, yielding a chemical score of 0.86. The chemical score has two shortcomings with regard to protein quality determination. First, adopting a Kjeldahl factor of 6.25 for two protein sources with equal nitrogen will lead to error if the samples vary

430

Chapter 14

TABLE 3 Chemical Scores for a Range of Food Proteins Determined Using Whole Egg Standard

Protein source Beef muscle Beef liver Egg albumin Cow's milk Lactalbumin Beef kidney Beef heart Casein Sun¯ower seed Soybean (heated) Rolled oats Yeast (average) White rice Corn germ Sesame seed Wheat germ Whole wheat Cottonseed Whole corn White ¯our Peanut Pea Gelatin Human milk Blood serum Hemoglobin Flax seed Alfalfa

Limiting EAA

Chemical score

Cys ‡ Met Ile Lys Cys ‡ Met Met Cys ‡ Met Ile Cys ‡ Met Lys Met Lys Cys ‡ Met Lys Met Lys Ile Lys Lys Lys Met Met Met Trp Met Ile Ile Lys Ile

0.71 0.70 0.69 0.68 0.66 0.65 0.65 0.58 0.53 0.49 0.46 0.45 0.44 0.39 0.39 0.38 0.37 0.37 0.28 0.28 0.24 0.24 0.0 0.86 0.44 0.10 0.35 0.45

BV (%)

Digestibility (%)

Chemical score 6 Dig/100

76 77 82 90 84 77 74 73 65 75 66 69 66 78 71 75 70 61 60 52 58 48 25

100 97 100 95 98 99 100 99 94 96 93 93 78 85 92 95 91 90 94 100 97 91 95

71.0 67.9 69.0 64.6 64.7 64.4 65.0 57.4 49.8 47.0 42.8 41.9 34.3 33.2 35.9 36.1 33.7 33.3 26.3 28.0 23.3 21.8 0.0

Source: Adapted from Ref. 78.

greatly in the amount of nonprotein nitrogen (Chapter 1). Second, changes in protein characteristics unrelated to their essential amino acid content are not measured by the chemical score. Protease inhibitors will affect PNV although such effects are not re¯ected by changes in the chemical score.

Corrected Amino Acid Scores

FIGURE 5

431

The chemical score as an index for protein quality. (Top) Chemical score is strongly correlated with the biological value. (Bottom) Showing the correlation between biological value and ``chemical score corrected for digestibility'' (open circles). A low correlation is observed between BV and digestibility (see closed symbols).

432

6.2.

Chapter 14

Pepsin Digest Residue Index

Sheffner et al. (88) introduced the pepsin digest residue (PDR) index in 1956. The PDR is determined by combining in vitro digestibility with the essential amino acid pattern. The food sample (containing 1 g of protein) was incubated with pepsin (25 mg) in 30 mL of sulfuric acid (0.1 N) solution for 24 hours.* Precipitating with sodium tungstate and 0.66 M sulfuric acid separated undigested protein from the products. The essential amino acid pro®les for the substrate and soluble digest (products) are determined by microbiological assay. Subtraction of these values gave the essential amino acid pattern for the nonhydrolysed protein residue. For both the digest and residue, each essential amino acid is expressed as a percentage of the total content of essential amino acids. The two columns of results were then divided by the corresponding values for egg protein. Next, the geometric mean was determined for the ``egg ratios'' and the resulting values were multiplied by a factor that takes into account the relative amounts of digest and residue formed by the action of pepsin on the sample and the egg protein. The PDR index was not easy to calculate. Sheffner et al. noted that any process that decreases pepsin digestibility will also lower protein nutritional value.

6.3.

Pepsin Pancreatic Digestion Index

Akeson and Stahmann (89) introduced the pepsin pancreatin digestion (PPD) index in 1964. Samples of proteins were digested with pepsin at low pH, neutralized, and then treated with pancreatin. The soluble products were concentrated, freeze dried, and subjected to amino acid analysis by ionexchange chromatography. The PPD index was then calculated in the same way as the PDR index. The PPD index showed an excellent correlation (R ˆ 0.99) with BV determined using the rat bioassay. Kennedy et al. (90) modi®ed the PPD index by performing the pepsin digestion within a dialysis cell. The pepsin digest dialysate (PDD) index for a range of protein ingredients (soy ¯our, gelatin, gluten, casein, whole egg powder, cows' milk forti®ed with carbohydrates, proteins, and vitamins) was highly correlated with BV. Some suggested advantages of the PDD index compared with the PDR and PPD indices include (a) use of simpler apparatus, (b) requirement * Some experiments were performed with multiple enzyme digestion using trypsin, pancreatin, and erepsin. However, the ®nal method used a single enzyme digestion by pepsin. It was suggested that the proportion of egg protein digested as chyme leaves the duodenum was about 30%. The in vitro study of Sheffner et al. employed conditions designed to ensure about 30% digestibility of their samples.

Corrected Amino Acid Scores

433

for only one enzyme, (c) use of modern amino acid analysis instrumentation, (d) higher reproducibility, and (e) use of computerized calculations.

6.4.

Computed-PER Index

Satterlee et al. (91) introduced a computed-PER (c-PER) index in an attempt to correct AAS for digestibility in 1977. First the essential amino acid pattern for a food sample was expressed as a percentage of the FAO/ WHO pattern for humans (1973). Then each ``%FAO value'' was multiplied by protein digestibility determined with a rat bioassay or in vitro. The digestibility-corrected AASs were assigned a statistical weighting inversely related to the abundance of each essential amino acid. The sum of the weighted values was divided by the corresponding value for casein. Finally, the results were transformed by one of four mathematical functions to generate c-PER values. A collaborative study involving seven laboratories found that the c-PER gave the same rankings of protein quality as the PER assays. The c-PER test could be completed in 72 hours compared with 28 days required for the PER determination.

6.5.

Available Amino Acid Score and PDCAAS

The PDCAAS is considered an excellent index for protein quality. Particularly appealing is the ease of calculation. It combines protein digestibility and AAS in a simple manner. With 20-20 hindsight the PDCAAS could have been anticipated earlier. Chemical scores were ®rst criticized for not taking digestibility into account in 1946 (92). Satterlee et al. (91) pretty much calculated the PDCAAS value in 1977 and then proceeded to transform such data to give the c-PER. In 1984 Sarwar (93) introduced the term ``available amino acid score'' to describe protein scores* corrected for protein digestibility. His formula was identical to that used later for calculating PDCAAS (Table 4). Published lists for TPD may be used to calculate PDCAAS. Digestibility data are also available from the FDA. Manufacturers should note that processing can lead to signi®cant deviations in the TPD compared with published values. Where a food source contains several proteins (Pi) * The protein score is calculated in the same way as the chemical score except that the reference essential amino acids is the pro®le for humans.

434

Chapter 14

TABLE 4 Stages for the Calculation of Protein Digestibility± Corrected Amino Acid Score (PDCAAS) for a Food Protein Procedure 1. 2. 3. 4.

Determine sample nitrogen content Calculate protein content (N 6 6.25 or speci®c conversion factor) Analyze sample for essential amino acids Determine the amino acid score ASS =; mg EAA per 1 g protein mg EAA per 1 g …FAO=WHO=UNU†

5. Determine digestibility 6. Calculate PDCAAS ˆ Lowest AAS 6 digestibility

the TPD is replaced by the weighted average (TPDav) value: P …TPDi 6Pi † P TPDav Pi

…14†

All proteins having a PDCAAS in excess of 100% (or > 1) are assigned a value of 100%. A protein with a PDCAAS value above 100% does not provide further bene®t as excess amino acids are utilized for energy. According to Henley and Kuster (80), the PDCAAS method provides a measure of protein quality that is directly correlated with human requirements. The PDCAAS method also has considerable ¯exibility. Manufacturers and diet planners can provide larger quantities of lower quality dietary protein in order to meet the recommended daily requirements. 6.6.

Applications

Sarwar et al. (94) showed that a number of rat bioassays (PER, PER, RPER, NPR, or RNPR) ranked 20 food products in the same order of protein quality. The correlations between different rat assays were highly signi®cant (r ˆ 0.98±0.99). Chemical scores were also correlated with the results of rat bioassays. Correcting the chemical score for the digestibility improved the observed correlation. Carnovale et al. (95) compared PNV for wild and cultivated species of Vigna. The wild type had a signi®cantly higher protein content, trypsin inhibitory activity, and tannin content. Protein digestibility was lower although PNV assessed in terms of the PDCAAS was not signi®cantly different.

Corrected Amino Acid Scores

A.

435

Quinoa

Quinoa (Chenopodium quinoa Wild) seed protein quality was evaluated using amino acid analysis and animal feeding trials. The ®rst limiting amino acids were tyrosine and phenylalanine with a chemical score of 0.86. Quinoa protein (14% w/w of the seed) had levels of lysine, methionine, and cysteine superior to those found in most other plant proteins. From animal feeding experiments NPU was 75.7, BV was 82.6, and digestibility was 91.7%. These results yield an estimated PDCAAS of 79%. This value is higher than the value for meat (87). B.

Rice

The quality of protein associated with cooked milled rice and a typical ricebased menu for Filipino preschool children and adults was assessed by Eggum et al. (96). Digestibility, BV, and NPU were assessed with growing rats. Digestibility was 88.8% for the preschool child diet, compared with a BV of 90.0 and NPU of 79.9. For the adult diet digestibility was 87.3%, BV was 86.6, and NPU was 75.5. On its own, cooked rice had a digestibility of 90.0%, BV equal to 82.5, and an NPU value of 74.3. The availability of lysine (the limiting essential amino acid) was 95.4% for the preschool child diet, 95.7 for the adult diet, and 100.0 for rice. For whole diets the chemical scores were 1.00 for the preschool child diet, 0.92 for the adult diet, and 0.62 for rice. From such ®gures the PDCAAS may be estimated as 88.8% for the preschool diet, 80.4% for the adult diet, and 56.0% for cooked rice. C.

Maize

The quality of protein from Canadian maize cultivars adapted to Northern latitudes (greater than 458 N) was assessed by Zarkadas et al. (97). The cultivars designated Dent CO251, Flint CO255, and Pioneer 3953 compared favorably with quality protein maize inbred (QPM-C13). Total protein levels for maize meal were 7.95% (QPM), 8.2% (Pioneer), 10.5% (Dent), and 11.79% (Flint). Compared with ordinary maize, QPM protein had double the amount of lysine and arginine, increased levels of tryptophan and cystein, and lower levels of leucine. QPM protein had a good balance of essential amino acids limited only in lysine. The PDCAAS was 67% (QPM), 28.5% (Pioneer), 31.0% (Dent), or 33.0% (Flint). PNV for a white ¯oury maize variety from the Indian Agricultural Program of Ontario (98) was also evaluated. Maize (IAPO-13) harvested over 1992, 1993, and 1994 seasons had an average protein content of 10.14 (+0.1)%. The limiting essential amino acid was lysine with an AAS of 0.414. The human digestibility of maize protein is reportedly 89%, leading to an estimated

436

Chapter 14

PDCAAS for IAPO-13 of 37±38%. It was concluded from such studies that Canadian QPM maize variety had higher quality than common maize and that breeding maize for high protein quality showed a great deal of promise. D.

Oats

Hull-less or naked oats (Avena sativa var. nuda) from temperate zones in Asia were studied by a Canadian group for their potential use for both human and animal nutrition. Zarkadas et al. (99) determined the protein content for three high-yielding and rust-resistant naked oat cultivars. The protein levels were 13.67 (+ 0.60)%, 13.93 (+ 0.53)%, and 14.40 (+ 0.55)% for varieties AC Percy, AC Hill, and AC Lotta. All cultivars had a good balance of nine essential amino acids. Lysine was limiting, followed by threonine. Assuming a human protein digestibility of 86%, estimates for PDCAAS are 54.9% (AC Hill), 56.3% (AC Lotta), and 59.3% (AC Percy). The PDCAAS values for other protein sources are mechanically dehulled oats (62%), maize (29%), soybean (86%), and egg (95%). Zarkadas et al. (100) also evaluated the PNV of two newly released Canadian oat cultivars (Newman and AC Stewart). Oat wholemeal had a total protein content of 10.75 (+ 0.23)% and 11.92 (+ 0.06)% for strains Newman and AC Stewart, respectively. The corresponding total protein content for the oat groats (dehulled grains) was 13.27 (+ 0.24)% and 12.61 (+ 0.94)%. The limiting amino acid was lysine for oat wholemeal and lysine plus threonine for groats. Values for PDCAAS based on the FAO/WHO/ UNU pattern (2-year-old preschool children) were reportedly 58±62.3% (strain Newman) or 66.7±62.3% (strain AC Stewart). Dehulling had no easily predictable effect on the PNV. E.

Collagen

Connective tissue protein (from skin, rind, tendon, or bone) is potentially useful for livestock feed supplements. The low PNV of collagen was inferred from feeding trials conducted in the 1960s. Meat meal protein quality is also inversely related to the hot water±soluble protein (collagen) content. Feather meal had a distinctly low PNV during an ARC collaborative study of animal foodstuffs reported in Chapter 13. A contemporary evaluation of PNV for collagen-containing meat by-products is reported by Zarkadas et al. (101). Three commercial batches of demineralized beef bone powder were subjected to quantitative amino acid analysis to determine total protein content and PDCAAS. The amino acid pro®les were consistent with the type I collagen being the main protein constituent. Glycine, proline, and hydroxyproline each constituted 20% of the total amino acids. Alanine (10% total amino acids) and basic amino acids (12.5% total) were also relatively

Corrected Amino Acid Scores

437

abundant. Comparatively low amounts of cysteine (*0.2%), methionine (*1.24±1.33%) and threonine (1.17±2.24%) were found. The protein content of bone meal ranged from 18 to 18.6%. Taking the average digestibility of collagen as 90%, the PDCAAS value was reportedly 14.4± 16.4%; it is not clear how such ®gures were determined. Table 5 lists the essential amino acids pro®le for the three batches of demineralized bone powder. Allowing for rounding-up errors, threonine is the limiting amino acid for one sample with a chemical score of 0.344. Luecine and isoleucine are the next limiting amino acids with a score of 0.510±0.550. Following the normal rules, the PDCAAS is between 31.0% and 49% for bone meal products. F. Beans Changes in the protein quality of red kidney bean (Phaseolus vulgaris L) due to autoclaving, domestic cooking, or canning were examined by Wu and coworkers (102) from Clemson University, South Carolina. A large number of quality indices were considered: available lysine, c-PER, mean chemical score for lysine, methionine-plus-cysteine levels, and the essential amino acids index. In vitro protein digestibility was determined with a multienzyme method. Cysteine plus methionine was limiting with a chemical score of 0.928. In vitro digestibility was 43.2% for uncooked kidney beans and 79±82% for heat-treated beans. The PDCAAS was 40% for raw beans compared with 70±75.4% for cooked beans. Other quality indices showed

TABLE 5 Estimation of the PNV for Bone Meal Products Amino acids Histidine Isoleucine Leucine Lysine Methionine ‡ cystine Phenylalanine ‡ tyrosine Threonine Tryptophan Valine Total EAA Total nonessential AA a

EAA for collagen

FAO/WHO/UNUa (mg/1 g protein)

11 16 35 335 15 23 12±22 11 27 184±195 805±816

19 28 66 58 25 63 34 11 35 339 661

From FAO/WHO/UNU (1985); [Refs. 75±81]; see Table 6 of Chapter 12.

438

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sensitivity to heating when corrected for digestibility. For example, raw and home-cooked beans had available lysine contents of 6.21 and 6.19 g per 100 g protein, respectively. This yields corresponding in vitro protein digestibility±corrected available lysine scores 2.68 and 5.1 g per 100 g protein. After normalization with the FAO/WHO/UNU (1985) pattern, the protein digestibility±corrected available lysine score (PDCALS) was 40% for raw beans and 74% for cooked beans. Apparently, lysine as well as cysteine plus methionine may be limiting for red kidney beans. 6.7.

Beyond Protein Digestibility±Corrected Amino Acid Scores

Amino acid availability (AAAv) is a more precise measure of digestibility than TPD (Chapter 12). Calculations of PDCAAS using TPD assume that all essential amino acids are released from proteins with equal ease. The rates of absorption are also assumed to be equal. Batterham (103) has reviewed the signi®cance of AAAv. Kuiken and Lyman (104) showed that availability of essential amino acids from a single protein source can differ. The AAAv was determined for a range of protein sources (egg, liver extract, roast beef, cotton seed, peanut, and wheat ¯ours) using a rat feeding trial. Employing a microbiological assay for amino acids, they determined the dietary and fecal concentrations of individual essential amino acids. With roast beef, egg, or liver protein values for AAAv were the same for all essential amino acids. The amino acids from cottonseed meal protein fell into three groups: (a) arginine, histidine, and tryptophan with high availability (>90%); (b) isoleucine, phenylalanine, and threonine with intermediate availability (70±90%); and (c) lysine with low availability (65%). Sarwar (105) con®rmed that AAAv was up to 25% lower than the TPD for legume protein. The AAAv and TPD were determined for 17 protein sources using male rats. Diets and feces were freeze dried, ground, and analyzed for amino acid pro®les and for crude protein. The AAS values for the different diets were expressed as percentages of human requirements speci®ed by the FAO/WHO (1985). Finally, these results were corrected for AAAv. The resultant index for protein quality was termed ``available amino acid score''; I have renamed this quantity availability-corrected amino acid score (AvCAAS).* Correcting the chemical scores for TPD led Sarwar to generate what is probably the ®rst list for PDCAAS. Both AvCAAS and the * Sarwar used AAS as an abbreviation for the available amino acid score. I have changed this to AvCAAS (availability-corrected amino acid scores). AAS is used by many investigators to mean amino acid score. Some investigators use AASTPB to describe amino acid scores corrected for true protein digestibility.

Corrected Amino Acid Scores

439

notional PDCAAS were highly correlated with the RNPR for 17 diets (R ˆ ‡ 0.92). However, values for PDCAAS were consistently higher than AvCAAS by between 2% and 8% (Fig. 6). Wu and co-workers (106,107) found much more signi®cant differences in PNV after correcting amino acid scores for TPD and AAAv. With raw kidney beans, cysteine plus methionine was limiting with a chemical score of 0.944. The TPD value was 15.7% for raw beans, increasing to 72±87% for heat-processed beans. By comparison, AAAv was negative ( 18.6%) for raw bean protein and positive (39.8±68.0%) for heat-processed beans. Rats fed with raw beans had higher concentrations of cysteine plus methionine in their feces as compared with amounts initially present in their diets. The AAAv values for several other amino acids (alanine, proline, valine, luecine, and threonine) were also negative. The AvCAAS for raw kidney bean protein was therefore 17.6% compared with a PDCAAS of 13.9%. The relative merits of the PDCAAS and AvCAAS are discussed by Darragh et al. (108). Estimating TPD using a rat assay is a slow process, requiring about 8 days for completion. In view of the high correlation between TPD and in vitro protein digestibility, correcting AAS using in vitro protein digestibility is more cost effective. Rozan and co-workers (109) considered AAS for soybean, lypine, and rapeseed meal proteins using the FAO/WHO/UNU (1995) pattern for 2- to 5-year-old preschool children. The AAS values were then corrected using in vitro protein digestibility measured as the degree of

FIGURE 6

Protein nutrient value estimates based on amino acid scores corrected for true protein digestibility (PDCAAS) or amino acid availability (AAS). [Drawn from the data of Sarwar (93).] Y-axis shows PDCAAS minus AAS.

440

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hydrolysis (DH) or/and nitrogen digestibility index (ND). Degree of hydrolysis±corrected AAS (DHCAAS) or nitrogen digestibility±corrected AAS (NDCAAS) values were highly correlated with values of PDCAAS (p < 0.001). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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104. KA Kuiken, CM Lyman. Availability of amino acids in some foods. J Nutr 36:359±368, 1948. 105. G Sarwar. Available amino acid score for evaluating protein quality of foods. J Assoc Anal Chem 67:623±626, 1984. 106. W Wu, WP Williams, ME Kunkel, JC Acton, Y Huang, FB Wardlaw, LW Grimes. True digestibility and digestibility-corrected amino acid score of red kidney beans (Phaseolus vulgaris L.). J Agric Food Chem 43:1295±1298, 1995. 107. W Wu, WP Williams, ME Kunkel, JC Acton, Y Huang, FB Wardlaw, LW Grimes. Amino acid availability and availability corrected amino acid score of red kidney beans (Phaseolus vulgaris L.). J Agric Food Chem 44:1296±1301, 1996. 108. AJ Darragh, G Schaarfsma, PJ Moughan. Impact of amino acid availability on the protein digestibility corrected amino acid score. Bull Int Dairy Fed 336(Dairy Foods Health):46±50, 1998. 109. P Rozan, R Lambhari, M Linder, C Villaume, J Fanni, M Parmentier, L Mejean. In-vivo and in-vitro digestibility of soybean, lupine and rapeseed meal proteins after various technological processes. J Agric Food Chem 45:1762± 1769, 1997.

Index

AAAvÐamino acid availability, 438±440 AASÐamino acid score, 354±355, 433 Absorptivity, 171 Accuracy, de®nition, 3 Acetylacetone, Kjeldahl assay and, 22 Acid Orange 12, 125, 127±130, 136 feedstuffs protein and, 390 rice protein and, 153 Acid Red 1, 134±135 Activation energy, browning, 427±428 Actomysin, antigens and, 234 Acylation, 363 Adjuvant, 231 Adulteration, 221 commerce and, 223 health and, 222 protein prices and, 223 economics and, 222 ELISA tests for, 255±257 Halal food and, 223 Kosher food and, 223

[Adulteration] PCR analysis and, 224 US meat trade and, 224 Advanced glycation products, Bradford assay and, 190 Adulteration frequency, 240 Adverse reaction, food and, 298 Agar gel double immunodiffusion assay, 230±246 Agaricus, Udy assay, 160 Aggregated protein, Bradford assay, 217 AGIDÐagar gel double immunodiffusion assay, 230±246 Albumin, biuret assay, 48±49 Alkali treatment, digestibility and, 373 Alkaline copper reagent, 47 Alkali-phenol reagent, Kjeldahl assay, 18 Allergens, 299±300 castor bean, 300 egg-white, 300 447

448 [Allergens] milk, 300 peanuts, 305±312 shrimp, 300 soybean, 301±305 wheat, 312±329 Allergy infants and, 306 pregnancy and, 306 Amaranth, 398±399 Amido Black 10B, 130±131 Amino acid analysis (see Quantitative amino acid analysis, QAAA) Amino acid availability (AAAv), 368, 438 Amino acid availability, protein quality and, 438±440 Amino acid score, 354±355, 433 errors in, 355 Ammonia determination, 18±23 Anaphylactic shock, 297, 305 Anilino-naphthalene-8-sulfonic acid, 413 ANS ¯uorescence, 413 Antigen actomysin, 234 binding curves, 227, 261 boiling and ethanol resistant, 236, 239, 240, 260 cooked meat and, 236, 238 peptides as, 324±327 thermostable proteins as, 236, 261 troponin T as, 236, 238, 239 AOAC, Association of Of®cial Analytical Chemists digestibility, 369 Dumas assay, 30, 34,36 dye binding assay, 149, 151 gluten ELISA, 320±321, 324±325 guidelines, rat bioassay and, 351±353 immunodiffusion assay, 230 Kjeldahl assay, 15 factors and, 10 Udy assay, 149

Index Applications biuret assay, 57±67 Bradford assay, 195 Udy assay, 147 Ara h 2, peanut allergens and, 308 Arachin, 306 Arginine, dye binding and, 135, 139, 212 Arrhenius equation, 384, 426±427 PNV and, 384 Assay performance, 5 Association of of®cial Analytical chemists (see AOAC) Atmospheric error, Dumas assay, 33 Atopic dermatitis, 305 Authenticity, proteins and, 222 Autoanalyzer, 21, 16 Autoclaving, effects on digestibility, 368 Automated, Kjeldahl assay, 15±16 Availability corrected amino acid score, 438 Available amino acid score (see AAS) Available lysine, 356±366 chromatographic analysis, 360±361 comparison of assays, 365 cottonseed, 360 interferences, 359, 362 lactalbumin, 365 legumes, 394±395 moisture and, 426 ovalbumin, 365 reagents for, 356 sodium borohydride and, 365±366 temperature and, 426±427 AvCAAS (see Availability corrected amino acid score) Azo dyes, 133±134,137 B lymphocytes, 228±229 Baby foods Dumas assay, 38 meat protein detection in, 262 peanut allergens in, 311 Baby formula, 37±38 Bacteria, feedstuffs quality and, 388

Index Baker's asthma, 312 Barley biuret assay, 55 Dumas assay, 36 dye binding assay, 153 Kjeldahl analysis, 8 protein analysis, 7, 8 rapid assays for analysis, 7 Udy assay, 152 BCA assay, 99±122 algae protein, 121 animal carcass protein, 119±121 automated, 113 calibration features, 109 copper analysis, 100 ¯ow injection analysis, 114±116 interferences, 110±111 lysine and, 119 mechanisms, 105 metal ion catalyzed oxidation, 107 method, 104 microwell plate format, 113±115 phenolic compounds effects, 118 reducing compounds and, 106 sample pretreatment for, 112 serum copper, 102 solid phase assay, 117 sugars and, 103 TCA-DOC precipitation and, 112 BCA, derivatives, 101 BE antigen (boiling and ethanol resistant antigen), 236, 239, 240, 260 Bean protein, Bradford assay, 213 Beans, PDCAAS value, 437 Beef analysis, ORBIT, 237 croquettes, soy protein detection in, 304 protein, dye binding assay, 157 Beer Bradford assay, 190, 204±206 celiac disease and, 312 Dumas analysis, 30, 36 Kjeldahl analysis, 8, 14 Lowry assay, 88

449 b-Lactoglobulin, 149, 182, digestibility, 309 dye binding, 149 Binding constant, protein-CBBG, 177 Binding sites, dyes, 140 Binding, T-azo-R, 177±181 Bioassay, 387, 388 chick, 354 human, 341 protein quality and, 346±354 rat 348±353 Biological value (BV), 342 Bio-Rad LTD, 207 Biotin-streptavidin detection, 261±262 2,20 -Biqinoline, Lowry assay and, 76 Birds nest soup, allergy, 305 Bitter, Bradford assay, 207 Biuret assay, 47 barley analysis, 55 casein determination, 49 cereal proteins, 57 protein solubility determinations by, 63 Biuret, structure, 47 Boiling and ethanol resistant antigen, 236, 239, 240, 260 Bone meal, 271 Bovine spongiform encephalopathy (see BSE agent) Bradford assay advantages, 169 beer protein, 204±205 beverages and, 190 bread crumb, 212 calibration features, 201 carotenoids and, 189 Chardonnay wine, 211 compatible solutes, 186, 187 effect of SDS, 187 interferences, 186 legumes, 213 lipids and, 191 mechanisms, 172 microassay format, 196 modi®ed, 196

450 [Bradford assay] monosaccharides and, 190 mungbean ¯our, 213 mushrooms, 216 pinot noir, 211 polypeptide selectivity, 171 polyphenols and, 209±210 polysaccharides and, 186, 191, 199 reagent pH, 172 sensitivity, 202±203 solid phase, 185 standard method, 196 wavelength of, 173±175 Bran protein, 55 Bread crumb, Bradford assay and, 212 Bread making quality, 212±213 Bread mix, gluten free, 321 Bread wheat, 57 Brest milk, 318 Brewing grains, Dumas assay, 30, 33, 36 British beers, Bradford assay, 208 BSA binding, CBBG, 185 BSE agent, 271±273 bone meal and, 271 immunological detection, 272 test kits for, 271±273 Buckwheat, celiacs and, 320 Buffalo, antigen, 238 Buffer effects, Lowry assay, 81 Butter milk, dye binding assay, 148 BV (see Biological value) Cake ¯our, gluten free, 321 Calibration, 3±7 ammonia analysis and, 19, 20, 23 BCA assay and, 109±110, 113 biuret assay for rice, 58±59 Bradford assay and, 201 de®nitions, 3 Dumas assay and, 35 dye binding assay, 129, 131, 154, 155 ELISA format and, 270 gliadin ELISA, 323 gluten ELISA, 323 horse meat ELISA, 267

Index [Calibration] Lowry assay, 72, 77±80 meat ELISA, 253 pork ELISA, 257, 267 soy ELISA, 284 Udy assay, 154 Camel, ELISA, 252, 254, 255, 260 Canned baby foods, ELISA and, 262 Canned ®sh, ELISA, 268±269 Canned foods digestibility, 370 Kjeldahl analysis, 16 Canned milk, 150 Canned tuna, 268 Carbethoxylation, ®sh meal, 363 Carbohydrates available lysine determination and, 359, 362, 366 Lowry assay and, 81 Carbonyl-amine reaction, (see also Maillard Reaction), 416±418 Caroteinoids, Bradford assay and, 189 Casein biuret assay, 49 digestibility, 370 dye binding assay, 126 PER value, 349 protein quality, 344 Udy assay, 138, 149 Catalysts, Kjeldahl assay and, 9 CBBC sources, Bradford assay and, 197±198 CBBG, bi-ionic form, 172 Celiac disease, 312±313 Celiac Society of Great Britain, 322 Celiac-negative cereals, 323 Cereal products Bradford assay, 212 PNV, 398±401 Cereal proteins, classi®cation, 314 Cereals allergens, 312±329 biuret assay, 57 Lowry assay, 88 Udy assay, 151

Index Cerelac, 397 Chardonnay wine, Bradford assay, 211 Cheddar cheese, dye binding, 150 Cheese, 151 Chemical deterioration, proteins, 416±420 Chemical score, 429±431 egg protein and, 354 Chemistry biuret assay, 50 Lowry assay, 77 Udy assay, 133±147 Chick bioassay, protein quality, 353±354 Chick pea, Udy assay, 153 Chicken antigen for, 238 determination amino acid analysis, 27 dye binding assay, 157, 159 immunodiffusion assay, 240 Chlorophyll, Lowry assay and, 81±82 Chocolate bars, ELISA, 310 gluten ELISA and, 320, 321 milk, dye binding assay, 148 peanut allergens in, 310±311 Chromatographic determination, available lysine, 360±361 Circular dichroism, 317 Citations off, Lowry assay, 71 CNS tissue in feed, 272 Cod ®llet, dye binding assay, 157 Codex Alimentarius Commission, allergens, 298 Collaborative testing Bradford assay, 205±206 Dumas assay, 30, 34, 36 gluten ELISA, 322 ice cream analysis dye binding, 150 PNV, 390 soya protein ELISA, 282 three-enzyme assay for digestibility, 370 Collagen Lowry assay, 93 PDCAAS value, 436±437

451 [Collagen] quantitative amino acid analysis, 27±28 Colorimetric assay, ammonia, 18±23 Colorimetric Kjeldahl analysis, 18±25 Combustion analysis, milk products, 37 Combustion nitrogen analyzer (see Dumas assay) Commodities, Udy assay, 128 Compatible solutes, Bradford assay, 186, 187 Compton-Jones scheme, CBBG, 172±173 ComputedÐPER index (see c-PER) Conarachin, 306 Condensed milk, 151 Confectionary, gluten ELISA, 322 Confectionary products, ELISA, 311 Conformational stability, 425 Conglycinin, 285±289, 292 renaturing ef®ciency, 288 Conversion factor, Kjeldahl assay, 10, 12 Cooked beef, ELISA, 260 Cooked meat antigens for, 236, 238 immunoassay, 239, 247, 260, 268 soy allergens and, 303 Cooked poultry, ELISA, 261, 262 Cooking oil, peanut allergens and, 310 Cooking, gluten analysis and, 326 Coomassie Brilliant Blue G250, 169 Copper analysis, BCA assay and, 101 Copper complex, Biuret assay, 50±52 Copper concentration, Lowry assay and, 82 Copper hydroxide, biuret assay, 48 Corn ¯our, gluten detection in, 322 Corn meal, BCA assay, 117 Corn protein, Bradford assay, 212, 213 Correlation coef®cient, de®nition, 5 Cortecs, 262, 271 gluten ELISA kit, 319 peanut ELISA kit, 311

452 [Cortecs] pork ELISA kit, 271 Costs Dumas assay, 32 PNV tests, 429 Cottage cheese, dye binding, 150 Cottonseed, available lysine, 360 Cowpea, Udy assay, 154±155 c-PER, 433 Creutzfeldt-Jacob disease, 271 Cross reactivity (see Speci®city) Cross-linking, 418±419 Crystal violet, protein binding and, 172 Crystalline-amorphous transition, 425 Cyoprotectants, Lowry assay and, 82 Cysteine, racemization, 418 Dairy products Dumas assay, 30 dye binding, 147 Dairy proteins, Biuret assay, 63 DBC (see Dye binding capacity) d DBC, differential dye-binding capacity available lysine determination and, 362±365 legumes and, 364 soybean meal and, 363 total lysine and, 364 Deer analysis, DRIFT, 237 Defatting, meat, 61 Degree of hydrolysis, 373 Dehulling, PNV and 395 Dehydrogenation, Lowry assay, 75 Denaturation, 414±419 meat antigens, 263±265 peanut allergens, 308±309 processing and, 415 Denaturation temperature (TD), soy proteins, 286±287 Denaturation-renaturation, soy antigens, 284 Denaturing agents, 414 Dent CO251 (see also Maize), 435 Design, Lowry assay, 69

Index Detergent effect, Bradford assay, 187±188 Dialysis assay, digestibility, 373 Differential dye-binding capacity (d DBC), 362±365 Differential scanning calorimetry (see DSC) Dif®culties, Udy assay, 130 Digestibility de®nitions of, 367±368 peanut allergens, 309±310 three enzyme method for, 369±372 Dimethyl sulfoxide and Lowry assay, 82 Diphtheria toxin, 231 Disul®de bonding gluten and, 315 in soybean protein, 285, 288 Donkey, ELISA, 256, 262 Double immunodiffusion, 230 Dried milk, 344 Kjeldahl assay, 13 PNV for, 382±384 DRIFT, 235 DSC, soy protein, 286 DSC2, Tg measurements, 422 Dumas assay, 29 Advantages of, 31, 36 atmospheric error and, 33 beer protein, 30, 36 feedstuffs, 34 instrumentation, 31±33 ketchup, 30 malt, 30 materials, 32 milk products, 37 oilseed protein, 33 semolina, 38 Dye binding assay beef protein, 157 calibration with Kjeldahl, 150, 152±155 chicken meat, 157, 159 cod ®llet, 157 Dye binding lysine, 362 Dye equivalent weight, 129

Index Dye purity, Bradford assay and, 197±198, 200 Effect of dye volume, Bradford, 201 Egg albumin, 344 biuret assay, 48, 63 Bradford assay, 217 Egg allergens, 305, 309 Egg products, dye binding assay, 159 Egg protein, 290, 292, 346, 347, 392 Udy assay, 159 Egg ratio, 354 Egg white allergens, 300 Egg yolk, Bradford assay and, 201 Egyptian legumes, PNV, 394 Electrophoresis (see also SDS-PAGE) CBBG, 170 samples, Bradford assay, 198, 199 ELISA a¯atoxins, 251 bacterial toxins, 25 boiling resistant antigen, 260 BSE agent, 271±273 buffalo, 256 meat, 260 cattle, 256 commercial, 282 competitive, 248 confectionary products, 311 cooked beef, 262 meat, 260±265 pork, 262 poultry, 262 enzyme substrates, 254 format, 248±250, 255 game meat, 260 gliadin, 317±329 gluten, 317±329 horse meat, 252±256, 258, 260±263, 266±268 muscle antigen for, 257±260 Ochratoxin, 251 performance characteristics, 270 peroxidase assay and, 254 pork, 260

453 [ELISA] analysis, 256, 257 pyruvate kinase as antigen, 258 raw meat, 252, 255 red snapper, 270 reviews, 250 rock shrimp, 269±270 sardine, 268 sea food, 268±270 sheep, 256 soy protein, 281±296 ELISA test kit BSE, 273 deer meat, 262 gluten, 323 meat, 262 allergens, 310 peanut, 311 pork, 262 soybean, 283±284 tuna, 268 working range, 254, 320 Emulsion proteins, Lowry assay, 90 End-point temperature, meat ELISA, 265 End-point temperatures, Biuret assay, 62 Enzyme linked immunosorbent assay (see ELISA) Enzyme, protein digestibility and, 366 Equations, Udy assay, 141±142 Equilibrium, Udy assay, 138, 156 Errors chemical score, 355 PER determination, 350 Essential amino acids, 355 Ethyl vinyl sulfone (EVS), 356 Evaporated milk, protein analysis, 149 FAO/WHO/UNU, 433 Fatalities, peanut allergy, 306 FDA, 298 FDNB, 1-Fluoro-2,4-dinitrobenzene assay, 356

454 [FDNB] available lysine determination, 356±359 correction factor, 359 Fecal nitrogen, 367, 368 Feed supplements, 386 Feedstuffs and concentrates, 386±393 Feedstuffs, 387 Dumas assay, 30, 33, 34, 35 dye binding assay, 156, 390 Kjeldahl analysis, 16 Orange G binding, 388±390 PNV evaluation, 346, 387±393 Udy assay, 156 Fermented soybean products, allergenicity, 304 Fertilizer, Dumas assay, 30 FIA, BCA assay using, 114 Ficol, Lowry assay, 82, 83 Fish, allergens, 298 Fish and seafood identi®cation, 268±271 Fish gelatin, 311 Fish meal, 387, 388, 390 Acid Orange 12 binding, 390 DBC, 389 Kjeldahl analysis and, 15 Udy assay, 138, 156 Fish protein, Tg, 423 Fish protein concentrate, 268 Fish sausages, soy protein detection in, 304 Flint CO255 (see Maize), 435 Flow injection analysis (see FIA) Flower bud protein, quantitative amino acid analysis, 27 1-Fluoro-2,4-dinitrobenzene (see FDNB) Folin-Ciocalteu reagent, 71 Food allergy, 297 intolerance, 297 matrix, protein quality and, 420±425 Foods, Dumas assay, 30 Freeze dried proteins, 425 Fringe-micelle model, 421 Frozen dessert, 151

Index Fruit juice, Bradford assay, 211 Game meat, ELISA, 260 Gel immunodiffusion assay, cooked meat, 239±241 Gelatin BCA assay, 105 Biuret assay, 49, 56 complexes with Amido Black 10B, 147 feedstuffs and, 156 Lowry assay, 93 Gelatin granules, 126 Gel-®ltration, beer, 206 Glass transition temperature (see also Tg), 420±425 Gliadin, 314±317 in human milk, 318 Tg value for, 424 Gluten, 315 available lysine, 360 confectionary products and, 322 ELISA, 317±329 home test, 323±324 preparation, 315 standards for ELISA, 321 Tg value for, 421 Gluten-free foods, 313, 318, 322, 323 Gly m Bd 30k, as soybean allergen, 301±304 Glycation, Bradford assay, 190 Glycinin, 285±289 Gordon-Taylor equation, 422±425 GPV (Gross protein value), 342 Grain and cereal, Kjeldahl assay, 13 Grains, Dumas assay, 33 Grape juice, Bradford assay, 210 Graphical analysis, Udy assay, 144±145 Gross protein value (see GPV) Ground rice, biuret assay, 59 Half and half milk, protein assay, 149 Hamburger, meat identi®cation and, 235 Heat effects, soy antigens, 287 Heat resistance, conglycinin, 288 Histidine, dye binding and, 135, 139

Index Home test, gluten, 323±324 Honey Bradford assay, 217 Kjeldahl analysis, 217 Horse antigen production using, 231 ELISA, 267 immunoassay, 234 immunodiffusion assay, 239 meat, 252±256, 258, 260±263, 266±268 meat protein, immunoassay, 252±256, 258, 260±263, 266±268 muscle antigen, identity, 258, 261 muscle protease, biuret assay and, 62 protein assay, 235 Horseradish peroxidase, 253±254 Human milk, gliadin detection in, 318 Hybridoma, 229 Hydrogen peroxide Biuret assay, 55 Kjeldahl assay and, 9 Hypoallergenic bread, 316 Ice cream dye binding, 148 protein, 150±157 Udy assay, 148, 150±151 Immunization schedule, 231, 233 Immunoabsorption, 232 Immunoassay, principles, 226 Immunoblotting, 299±300, 307 Immunodiffusion assay chicken meat, 240 meat, 230 Immunological assays, disadvantages, 250 Immunology, 228 Imperial Chemical Industries (ICI), 169 In vitro digestibility, 368±374 protein stability and, 413 protein structure and, 413 Incidental additives, 298 Indanetrione assay, Kjeldahl assay, 23 Indophenol reagent, Kjeldahl assay, 18 Infant formula, 384±386 amino acid supplementation, 386

455 [Infant formula] Dumas assay, 37±38 peanut protein detection in, 306 PER, 350 PNV bioassay, 385 rat bioassay, 385 Infrared analysis, cereal proteins, 7 Insect protein, Bradford assay, 199 Insoluble protein, Bradford assay, 217 Instant breakfasts, Kjeldahl assay analysis, 7 Interferences available lysine, 362, 363, 366 BCA assay list for, 111 biuret assay, 53±55 Bradford assay, 186±191 d DBC, 363 Lowry assay, 80 ninhydrin assay, 23 plant dyes as, 57, 58, 59 Udy assay, 147 Isobestic point, 140, 172 IVPD (see In vitro digestibility) Kangaroo meat, immunoassay, 234, 235 Ketchup, Dumas assay, 30 Kinetics, 395 Lowry assay, 77 proteolysis, 412±414 Kiwi fruit juice, Bradford assay, 211, 212 Kjeldahl analysis, 1, 7±25, 344 barley, 8 beer, 8, 14 catalysts, 9 colorimetric, acetylacetone and, 22 comparison with dye binding assay, 150, 152, 153 honey, 217 gliadin, 327 Kjeldahl factor and, 10, 12 nitrogen-protein conversion factor, 10, 12 PER values and, 349 PNV determination, 349

456 [Kjeldahl analysis] reactions of, 9 reliability, 7 sausages, 17 Kjel-foss, Instrument, 15 Labeling, food allergens, 297 Lactalbumin, PER value, 349 Lactose, protein quality and, 382, 385 Larger, Bradford assay, 208 Leaf protein, Lowry assay, 88, 90 LECO FP-2000, Dumas assay, 31 LECO FP-228, Dumas assay, 33 LECO FP-428, Dumas assay, 31±32 Legumes available lysine, 394±395 baking and PNV of, 395 Bradford assay, 213 dehulling and PNV, 395 dye biding, 153±156 ELISA and, 312 Lowry assay, 88 PNV, 393±398 roasting and PNV, 396 sprouting and PNV, 395 steaming and hydrothermal treatment, 395 Lesions, celiac disease, 312±313 Linear dynamic range, 5 Linear free energy relations, amino acids, 418 Linearity, Bradford assay, 201 Lipid Bradford assay and, 191, 200 interferences by, 84 Lower limit of detection (LLD) de®nition, 3, 6 ELISA, 265 gluten ELISA, 320, 324 soy bean ELISA, 284, 290 Udy assay, 152 Lowry assay buffers and, 81 carbohydrates and, 81

Index [Lowry assay] cereals, 88 chlorophyll and, 81±82 citations of, 71 cryoprotectants and, 82 design, 69 emulsion proteins, 90 leaf protein, 90 legumes, 88 mechanisms of, 77 nondairy creamers, 90 sample pretreatments for, 86±87 single cell protein, 91 Lysine BCA assay and, 119 bioassay and, 344 CBBG binding and, 171 determination, 356±366 dye binding and, 135, 138 glycation and Bradford assay, 190 loss, Arrhenius plot 385 Maillard reaction, 382, 416±418 available lysine, 366 pH and, 417 Maize available lysine, 360 cultivars, 435 Dumas analysis, 29 PNV, 399 Malt Dumas assay, 30, 36 dye biding, 152 Materials, Dumas assay, 32 Matrix effects, protein deterioration and, 420 Mean residue weight, tables of values, 27 Meat, 247±281, 284, 303 analysis for soybean, 285 antigenic components, 232, 251 BCA assay and, 119±121 biuret assay, 61±63 Dumas analysis, 29 dye binding assay, 157 gel immunodiffusion assay, 230

Index [Meat] quantitative amino acid analysis, 27 sample pretreatment for ELISA, 284 soy protein detection in, 303 Udy assay, 156±157 Meat analogue, Kjeldahl analysis, 16 Meat and bone meal, PNV, 393 Meat and meat products, Kjeldahl analysis, 15 Meat antigen, 231 Meat rendering plants, 272 Meat speciation, 247±280 Meatballs, soy protein detection in, 304 Mechanism biuret assay, 50 Lowry assay, 73, 77 Udy assay, 133±147 Melanoma, 229 Metachromasia, Crystal violet, 174 Metal ion catalyzed oxidation, Lowry assay, 73 Michaelis-Menten kinetics, 414 Microassay, Bradford, 196 Microbiuret assay, 56 Micro-Kjeldahl analysis, 21, 13 Microwave heating, 396 Microwell plate, BCA assay using, 113 Milk, 282 allergens, 298, 300 ELISA, 282 powder, 381 powder, PER, 350 prices and protein content, 125 products, Dumas assay, 37 protein, dye binding assay, 147, 148 protein, ELISA plate coating, 248, 272 protein, Udy assay, 147±151 soymilk detection in, 281 Millet PNV, 399±400 protein, biuret assay, 60 Mince, soy protein detection in, 284 Model wine solution, Bradford assay, 211

457 Moist heating, allergen stability and, 308 Moisture food deterioration and, 416 peanut allergens and, 308 PER values and, 350 PNV and, 383±384 rapeseed heat damage and, 392 Moisture-temperature-time relations, 411 Molten globule and digestibility, 412±415 Monoclonal antibodies development for ELISA, 265±268 gluten ELISA and, 325±327 meat analysis and, 265±267 pork ELISA and, 267 soy bean ELISA and, 290±292 turkey analysis, 266 Monosaccharides, Bradford assay and, 190 Multiple processing, legumes, 397 Mungbean, Bradford assay, 213 PNV, 394 Muscle lactate dehydrogenase, ELISA, 264, 265 Mushrooms Bradford assay, 216 Kjeldahl analysis, 160 quantitative amino acid analysis, 27 Udy assay, 160 Myoglobin, thermostable antigen and, 238, 239 Naphthylamine brown, 126 NB, nitrogen balance, 342, 346±347, 367 NDI, nitrogen digestibility index, 373 Nessler's reagent, Kjeldahl method and, 21 Net protein utilization (NPU), 342 Ninhydrin reagent, Kjeldahl method and, 23±24 Nitrogen balance (NB), 342, 346±347, 367 Nitrogen digestibility index (NDI), 373

458 Nondairy creamers, Lowry assay, 90 Nonanimal protein ingredients, 281 Nonfat milk, 149 NPU, net protein utilization, 342 Nucleic acid, Bradford assay and, 191 Nuts allergens and, 300±301 ELISA, 310±312 Oats Ac Hill, 436 Ac Lotta, 436 Ac Percy 436 biuret assay, 60 PDCAAS value, 436 Oilseeds, Dumas assay, 30, 33 OPA (see o-Phthaldehyde) o-Phthaldehyde, 356 Orange G binding, PNV, feedstuffs, 389±390 structure of, 136 Udy assay using, 130 ORBIT, 235 Ovalbumin, 182 pAb, for detection of donkey muscle protein, 256, 262 PAGE, 81, 402 Pavalbumin, allegen and, 300 PDCAAS, 411, 427, 429, 433±438 applications, 434±439 beans, 437 bene®ts, 434 calculation, 434 collagen, 436±437 maize, 434 oats, 436 rice, 434 Vigna, 434 PDR index (see Pepsin, digest residue index) Pea globulin, ELISA, 290, 291 Peanut allergens, 300, 306±308 cooking oil and, 310 preparation, 307

Index Peanut allergy, 305±312 average fatalities, 306 in infants, 306 in pregnancy, 306 Peanut protein, in confectionary, 311 Peanuts, chemical score, 429 Pepsin digest dialysate index (PDD index), 432 digest residue index (PDR index), 432 pancreatin digestion index (PPD index), 432 Peptide antigens gluten ELISA and, 324±327 soybean ELISA and, 290 Peptidyl sites, digestibility and, 414 PER (Protein ef®ciency ratio), 342, 348±351 AOAC guidelines, 351 complex foods and, 350 rat acclimation period, 350 sweetened foods and, 350 Perilla, 396 Peterson's Lowry assay, 72 Phenol red, 125 Phenol Bradford assay and, 188 BCA assay and, 119 Phospholipids, Lowry assay, 84 Phosphothreonine, racemization, 418 Pierce Warriner LTD, 207 Pinot Noir, Bradford assay, 211 Pioneer 3953 (see Maize), 435 Plant colors, Biuret assay and, 55 Plant metabolites, Bradford assay, 188±189 Plant proteins, 221 Amido Black 10B binding, 130 Plasticizer, water as, 420 PNV (protein nutrient value), 341±380, 411 amino acid losses and, 344±345 animal tests, 348±354 assessment by PDCAAS, 434 bioassays, 346±354

Index [PNV (protein nutrient value)] consumer factors and, 343 factors affecting, 343 food labeling and, 344 human assays and, 346±348 hygiene and, 343 indicators for, 342 infant formulas and, 384 insect infestation and, 400 Kjeldahl analysis and, 349 legumes, 393±398 literature, 345, 348±349 meat and bone meal, 393 pregnant and lactating women and, 344 processing and, 381±402 quality control and, 344 rat bioassay, 348 soaking and, 395 Polyacrylamide gels, CBBG stain, 170 Polyamino acids, dye binding and, 171, 181, 183 Polyclonal antibody production, 232 Polyethylene glycol, 132 Polymer chemistry, 420 Polymer science approach, 424 Polymerase chain reaction, 224 Polyols, 133 Polypeptide selectivity, Bradford assay and, 171 Polyphenols, Bradford assay and, 209±210 Polysaccharide interference, Bradford assay, 186, 191, 199 Polyvalent antigen, 229 Porcine rind protein, 27 Pork analysis, PRIME, 237 dye binding assay, 157 ELISA, 267±268 thermostable antigen, 238 Potato protein Bradford assay, 214±216 chemical score, 429 Kjeldahl analysis, 13

459 [Potato protein] Lowry assay, 88 Poultry analysis, PROFIT, 237 Poultry, ELISA for, 266±267, 268 PPPD index, 432 Precision, protein assays and, 5 Preservatives, milk, 148 Prionics AG, 273 Procedure biuret assay, 49 Bradford assay, 195 Lowry assay, 70 PDCAAS and, 433±434 soy protein antigen preparation, 284 Udy assay, 128 Processed foods, 2 digestibility, 370±371 gluten detection in, 310, 320 interferences in, 80 Kjeldahl analysis, 16 PROFIT, 235 Pro-meter instrument, Udy assay, 153 Propionic anhydride, acylation, 363 Protein analysis, signi®cance, 2 assay, characteristics, 2 binding, CBBG, 173, 175, 177 binding, dyes, 135 concentrates, 386±393 digestibility, 366, 411 digestibility corrected amino acid score, (see PDCAAS) ef®ciency ratio (see PER) haze, 209 immunoassay, 225 M, shrimp antigen, 269 molecular weights, 57 nutrient value (see PNV) precipitation, Bradford assay, 199 carbonyl-amine reaction and, 417±418 factors in¯uencing, 343 milk, 381 segmental mobility, digestibility and, 413

460 [Protein] stain, CBBR, 170 Protein-protein-variation, BCA assay, 105 Bradford assay, 197, 201, 212 Protein-quinone interactions, 210 Proteolysis, Lowry assay, 90±91 Puri®cation, Coomassie Brilliant Blue, 200 QPM (see Maize), 435 QSDS-PAGE, 7 Quantitative amino acid analysis, pork rind, 27 Quantitative sodium dodecylsulfate polyacrylamide gel electrophoresis (see QSDSPAGE) Racemization amino acids and, 418±419 cysteine, 418 factors controlling, 418±419 rate prediction, 418 Radcliff In®rmary, 322 Radioallergo absorbent test (see RAST) Rapeseed heat damage, 392 meal, PNV, 390±392 protein, Udy assay, 154±155 RAST, 299 Rat bioassay, 411 Raw meat, ELISA, 252, 255 R-Biopharm, ELISA test kit, gluten, 320 Reactive lysine (see also Available lysine), 356 Red snapper, 270 Red wheat, Udy assay, 153 Relative nutritive value (see RNV) Relative protein value (see RPV) Reliability Kjeldahl assay, 7 Udy assay, 152 Renaturing buffer, 284

Index Reverse phase high pressure chromatography (see RP-HPLC) Reviews celiac disease, 313 dye binding, 126±127 peanut allergy, 306 protein quality, 345 Rice, 402, 435 beans, protein, 395 protein biuret assay, 59 Udy assay, 153 PDCAAS value, 435 PNV and, 399 RNV, 342 Rock shrimp, 260±270 RP-HPLC available lysine, 361 gliadin analysis, 327 RPV, 342 Salt effects, Bradford assay, 201 Sample calibration, 4, 7 Sample pelleting, Dumas assay, 33 Sample pretreatment beer, Bradford assay, 205 cereals, 55 corn meal, BCA assay 117 Lowry assay and, 86±87 meat, 261 Biuret analysis, 61 ELISA, 285 Plasma, 49 soy bean product, 284 Sardine, 268 Sausages dye binding, 159 gluten and, 313, 321 Immunodiffusion assay, 239 Kjeldahl analysis, 17 soy protein, 284, 289, 290 Udy assay, 159 Scenario, dye binding, 141±143 Schecter and Berger scheme, 414 Screening, high-lysine cereals, 401±402

Index SDS, Bradford assay, 187 SDS-PAGE, 300, 307, 309 antigen analysis, 237 peanut allergens, 309 wheat allergen, 315±317 Seal meat, immunodiffusion, 239 Secondary structure, Gliadin, 317 Seed globulins, ELISA and, 291 Seharawi, celiac disease and, 312 Semolina, Dumas assay, 38 Sensitivity, 1, 5 BCA assay of copper, 102 biuret assay, 55, 56 Bradford assay, 177, 179, 183±185, 197, 201±204 colorimetric Kjeldahl assay, 18 dye binding, 131,132, 133 gluten ELISA, 329 immunoassay, 227 Lowry assay for wheat protein, 89 meat ELISA, 260 protein analysis, 1, 2, 5±6 Udy assay, 137 Serine, racemization, 418 Sesame ¯our, Udy assay, 155 Sheep antigen, 231 Sheep, ELISA, 249, 252, 255±256, 257, 260, 261, 262, 268 Sheep serum albumin (SSA), 253 Shrimps, 269, 300, 301 Single cell protein, Lowry assay, 91 Skimmed milk dye binding assay, 151 Kjeldahl factor for 12 Slope assay, protein quality, 347 Small animal bioassay, 348 Soaking, PNV and, 395 Sodium borohydride, available lysine, 365±366 Sodium dodecyl sulfate (see SDS) Solid phase assay Bradford assay, 185 Udy assay, 131±133, 143 Solubility relations, Udy assay, 143 Soluble complexes, Udy assay, 138±140

461 Sorghum, 399±400 biuret assay, 60 Soup, gluten detection in, 322 Soy bean allergens, analysis, 303±305 ELISA cooking and, 283 speci®city, 283, 291 ¯our, Udy assay, 153 products, ELISA, 289 protein, 285±286 in sausages, 289 thermal denaturation, 286±287 protein antigen, 284, 285 Soybean ELISA, 281±296 7S protein, 285±289 Udy assay, 138 Soybean meal, Orange G binding, 389 Soybean protein, available lysine, 360 Soymilk, 282 Species identi®cation ®eld test (see SIFTS), 235 Speci®city de®nition, 3 gluten, ELISA, 318, 325, 326 meat ELISA, 256, 263 peanut ELISA, 311 soya bean ELISA, 283 Spray dried milk, 382±383 dye binding, 129, 130,147 PNV, 382 Udy assay, 129 Sprouted cereals, protein quality, 345 St. Bartholomew's Hospital, 322 St. James University Hospital, 322 Standard assay, Bradford, 196 Starch interferences, 55 Statistical principles, 4 Steaming, PNV and, 395 Stiffness parameter, 423 Stout, Bradford assay 207 Structure Acid Orange 12 dye, 136 Acid Red 1, 135

462 [Structure] Amido Black 10B, 137 bicinchoninic acid, 100 biuret, 47 CBBG, 170 CBBR, 170 copper-biuret complex, 47 dehydropeptide, 75 iminopeptide, 75 Orange G, 136 T-azo-R, 135 Sudanese legumes, PNV, 394 Sulfhydryl compounds BCA assay and, 118 Lowry assay and, 84±85 Sulfhydryl group, dye binding, 126 Sulfhydryl-disul®de exchange, 317, 415 Symptoms celiac disease, 312±313 food allergy, 298 peanut allergy, 305 T-azo-R, 134 TCA-DOC precipitation Bradford assay and, 118, 195, 199±200 Lowry assay and, 72 TDC (total digestibility coef®cient), 342 Tectator heating block, 21 Test kits, species identi®cation and, 235±236 Tg (glass transition temperature), 420±425 ®sh protein hydrolysate, 423 gluten, 421 moisture and, 422 protein quality and, 420±425 proteins and peptides, 421 Theory (see Mechanism) Thermal conductivity detectors, 30 denaturation, peanut allergen, 308±309 stability, meat antigens, 263±265 Thermostable antigen, 238 meat and, 261

Index [Thermostable antigen] soybean and, 289±292 Three enzyme assay, in vitro digestibility, 369±372 TNBS assay, 119, 356, 361±362, 365, 369 Tomato protein, Dumas assay, 30 Tomato seed, Lowry assay, 88 Tortillas, 399 Total digestibility co-ef®cient (see TDC) TPD (see True protein digestibility) Trace allergens, 297 Trinitrobenzene Sulfonic acid, (see TNBS) Troponin, meat antigens and, 236, 238±239 Troponin T, ELISA antigens and, 261 True protein digestibility, 367, 433 Trypsin inhibitors, protein digestibility, 372 Tuna, 268 PER value for, 352 soy protein in, 305 Turkey sausages, 239 Two-state denaturation, 412, 414±415, 425 Tyrosinase, copper detection in, 99 Udy assay, 125±126, 138 barley, 152 cereal proteins, 151 dif®culties, 130 dye-protein solubility and, 143 equations for, 141±142 ®sh meal, 138, 156 mechanisms of, 133±137 milk protein, 147±151 reliability, 152 sausages, 159 various commodities, 128 UHT milk, protein quality and, 382 Ultra®ltration, 211 Uncooked meat, ELISA, 254±257 immunoassay, 234

Index Water ammonia determination in, 19±20 PER and, 350, 352 Water activity, 383±384 Maillard reaction and, 383±384, 417 Water holding, 281 Water supply, Kjel-Foss instrument and, 15 Weaning foods, PNV, 397±398 Western transfer, 273 Whale meal, 387 Wheat, 345 allergens of, 314±317 allergy, 312±313

463 [Wheat] biuret assay, 53, 57±60 dye binding assay, 151 reliability of analysis, 7 Udy assay, 151 Whey protein, Bradford assay, 217 Whole milk, dye binding, 149 William-Landel-Ferry kinetics, 420 Wine, Bradford assay, 190, 209±212 Yeast protein, Lowry assay, 63±64, 91 Zein, biuret assay, 49